CN1571677A - Hookworm vaccine - Google Patents

Abstract

Preparations which elicit an immune response to hookworm antigens and which may be uitilized as hookworm vaccines are provided. In addition, a method of increasing the effectiveness of vaccinations against infectious diseases in patients infected with hookworm is provided. The method involves chemically treating the hookworm infestation prior to administering the vaccine.

Description

Hookworm vaccine

Background of invention

Invention field

The present invention relates generally to the vaccine of ancylostome.Especially, the invention provides the antigenic vaccine of originating based on parasite.

Background of invention

Hookworm infection is an important public health problem of developing country in the world wide, and it causes enteritis, loses blood anemia, hypoevolutism and malnutrition in the intestinal.Have human hookworm infection case according to estimates in the world wide, 100,019,400 case (people such as Hotez, 1997 years) is only just arranged in China more than 1,000,000,000.Certain areas in China, such as in the Hainan Province at the South Sea, the crowd more than 60% carries ancylostome (people such as Gandhi, calendar year 2001).

The pathology symptom that great majority are caused by ancylostome results from the parasite in maturation period in the human intestine.Ripe Ancylostoma (Ancylostoma) ancylostome is the example that concerns between a kind of specific definition host-parasite in all parasitology to the mucosa of vertebrates small intestinal and submucosal absorption.In parasitic mouthful of sheath, comprise several cubic millimeters host's mucosa and submucosa tissue, can in the process of autopsy or thanatopsy, really touch host-parasitic association (Kalkofen, 1970; Kalkofen, 1974).

Ancylostoma caninum (Ancylostoma caninum) is all over the world morbidity of dog class and the main causes of death that comprise the subtropical zone, North America.Cause that serious anemia even the dead ischemia relevant with ancylostome can occur in the Canis familiaris L. single and infect (Soulsby, nineteen eighty-two between 2 and 3 weeks of back at first; Jones and Hotez, 2002).It should be noted that ancylostoma caninum (A.canium) has been accredited as a kind of important human pathogen recently.Infection has the parasitic zoonosis of ripe ancylostoma caninum can cause acidophilia's enteritis syndrome, the inflammatory conditions (Prociv and Croese, nineteen ninety) that promptly a kind of intestinal is attacked in response to parasite.The pathogeny that ancylostoma caninum (Ancylostoma canium) infects is relevant with the interior ischemia of intestinal, and described ischemia can occur in that ripe parasite is adsorbed and (Kalkofen, 1970 of feeding stage in the mammal small intestinal; Kalkofen, 1974).

The treatment that ancylostome is spread and the effort of control at present concentrates on anthelmintic from the ripe ancylostome of patient body's intercycle removal.This method has several limitation, comprises the quick repeated infection after the treatment, needs repeatedly to go to a doctor, and finally develop the anti-anthelmintic strain that ancylostome (people such as Savioli, 1997 after using the anthelmintic treatment for many years in a large number; Geerts and Gryseels, 2000).Therefore, will be very useful if having available other method of treatment and prevention mammal hookworm infection.For example, will be very useful if having the useful vaccine of treatment or prevention hookworm infection.

Summary of the invention

The invention provides the preparation of initiation at the immunne response of ancylostome.Described preparation contains multiple ancylostome antigen, and these antigens have been proved and have can be used for causing immunne response.These preparations can be used as the vaccine resisting mammal, for example human intravital ancylostome.After the administered formulation, the mammal of immunity inoculation can be developed the immunne response of anti-ancylostome, produces the immunity to parasitic infection, perhaps can show less worm carrying capacity, the alleviation of ischemia, the perhaps reduction of Ji Sheng ancylostome size.For this reason, but the invention provides and contain from reorganization of ancylostome or the compositions of synthetic antigen or its fragment and a kind of pharmaceutically suitable carrier.Recombinate or synthetic antigen can show and such as antigen as described below at least 80% homogeneity: Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1 be arranged, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API or Ay-TTR.In a preferred embodiment, antigen is Ac-TMP, Ac-MEP-1 or Ac-MTP-1.Antigen can derive from such as Necator americanus (Nector americanus), ancylostoma caninum (Ancylostoma canium), Ancylostoma ceylonicum (Ancylostoma ceylancium), and the ancylostome of Ancylostoma duodenale (Ancylostoma duodenale) kind.

The present invention also provides and has caused the method for mammal to the ancylostome immunne response.Described method comprises the step to the compositions of administration effective dose, and described compositions comprises reorganization or synthetic antigen (perhaps antigenic fragment) and the pharmaceutically suitable carrier that derives from ancylostome.The reorganization or synthetic antigen with show about at least 80% homogeneity: Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1 such as antigen as described below, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API and Ay-TTR.In preferred embodiments, antigen is Ac-TMP, Ac-MEP-1 or Ac-MTP-1.Antigen can derive from such as Necator americanus, ancylostoma caninum, the ancylostome of Ancylostoma ceylonicum and Ancylostoma duodenale kind.

The present invention further provides the method for the anti-ancylostome of immunity inoculation mammal.Described method comprises the step to the compositions of administration effective dose, and described compositions comprises reorganization or synthetic antigen (perhaps antigenic fragment) and the pharmaceutically suitable carrier that derives from ancylostome.Reorganization or synthetic antigen show and such as antigen as described below about at least 80% homogeneity: Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1 are arranged, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API and Ay-TTR.In preferred embodiments, antigen is Ac-TMP, Ac-MEP-1 or Ac-MTP-1.Antigen can derive from such as Necator americanus, ancylostoma caninum, the ancylostome of Ancylostoma ceylonicum and Ancylostoma duodenale kind.

The present invention further provides a kind of compositions, it comprises reorganization or the synthetic antigen (perhaps antigenic fragment) that derives from ancylostome.Reorganization or synthetic antigen show and such as antigen as described below about at least 80% homogeneity: Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1 are arranged, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API and Ay-TTR.Compositions further comprises a kind of pharmaceutically suitable carrier.In preferred embodiments, antigen is Ac-TMP, Ac-MEP-1 or Ac-MTP-1.Antigen can derive from such as Necator americanus, ancylostoma caninum, the ancylostome of Ancylostoma ceylonicum and Ancylostoma duodenale kind.

The present invention further provides a kind of vaccine, it comprises reorganization or the synthetic antigen (perhaps antigenic fragment) that derives from ancylostome.Reorganization or synthetic antigen show and such as antigen as described below about at least 80% homogeneity: Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1 are arranged, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API and Ay-TTR.Described vaccine further comprises a kind of pharmaceutically suitable carrier.In preferred embodiments, antigen is Ac-TMP, Ac-MEP-1 or Ac-MTP-1.Antigen can derive from such as Necator americanus, ancylostoma caninum, the ancylostome of Ancylostoma ceylonicum and Ancylostoma duodenale kind.

The present invention further provides a kind of compositions that causes immunne response, described compositions comprises reorganization or the synthetic antigen (perhaps antigenic fragment) that derives from ancylostome.Reorganization or synthetic antigen show and such as antigen as described below about at least 80% homogeneity: Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1 are arranged, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API and Ay-TTR.Described vaccine further comprises a kind of pharmaceutically suitable carrier.In preferred embodiments, antigen is Ac-TMP, Ac-MEP-1 or Ac-MTP-1.Antigen can derive from such as Necator americanus, ancylostoma caninum, the ancylostome of Ancylostoma ceylonicum and Ancylostoma duodenale kind.

The present invention further provides the method that to give patient's vaccination infectivity resistant disease.Described method comprises the steps: to treat hookworm infection to being enough to improve lymphopoietic level, and the immunity inoculation patient is anti-such as HIV, tuberculosis, malaria, measles, tetanus, diphtheria, the infectious disease of pertussis or poliomyelitis (polio).

The present invention also provide can the immunoprophylaxis ancylostome method.Described method comprises the steps: the patient of chemotherapy hookworm infection with the alleviation hookworm infection, and derives from reorganization or the synthetic antigen (perhaps its antigen fragment) of ancylostome after alleviating hookworm infection to patient's inoculation.In described method, can be eradicated or can be reduced to the effective degree of ancylostome immunity inoculation fully by the treatment hookworm infection.Reorganization or synthetic antigen may show and such as following antigen about at least 80% homogeneity: Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1 be arranged, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API and Ay-TTR.

The present invention also provides and has reduced the method that infects patient's ischemia that ancylostome is arranged.Described method comprises the step of using a kind of compositions to patient, and described compositions comprises a kind of reorganization or synthetic antigen (perhaps its antigenic fragment) and a kind of pharmaceutically suitable carrier that derives from ancylostome.

The present invention also provides and has reduced the method that infects ancylostome size in the patient body that ancylostome is arranged.Described method comprises the step of using a kind of compositions to patient, and described compositions comprises a kind of reorganization or synthetic antigen (perhaps its antigenic fragment) and a kind of pharmaceutically suitable carrier that derives from ancylostome.

The present invention further provides a kind of method that infects ancylostome quantity in the patient body that ancylostome is arranged that reduces.Described method comprises the step of using a kind of compositions to patient, and described compositions comprises a kind of reorganization or synthetic antigen (perhaps its antigenic fragment) and a kind of pharmaceutically suitable carrier that derives from ancylostome.

The present invention also provides following nucleic acid and aminoacid sequence: SEQ ID NO.11, SEQID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ IDNO.16, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ IDNO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37, SEQID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ IDNO.42, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.47, SEQ ID NO.48, SEQ ID NO.49, SEQ ID NO.50, SEQ ID NO.51, SEQ ID NO.52, SEQ ID NO.55, SEQ ID NO.56, SEQ ID NO.57, SEQ ID NO.58, SEQID NO.59, SEQ ID NO.60, SEQ ID NO.61, SEQ ID NO.62, SEQ IDNO.63 and SEQ ID NO.64.

Description of drawings

Figure 1A and B.Na-ASP-1:A, cDNA (SEQ ID NO.1) and B, deduced amino acid (SEQ ID NO.2).GeneBank accession number AF079521.

Fig. 2 A and B.Na-ACE:A, cDNA (SEQ ID NO.3) and B, deduced amino acid (SEQ ID NO.4).GeneBank accession number AF536813.

Fig. 3 A and B.Na-CTL:A, cDNA (SEQ ID NO.5) and B, deduced amino acid (SEQ ID NO.6).

Fig. 4 A and B.Na-APR-1:A, cDNA (SEQ ID NO.7) and B, deduced amino acid (SEQ ID NO.8).

Fig. 5 A and B.Na-APR-2:A, cDNA (SEQ ID NO.9) and B, deduced amino acid (SEQ ID NO.10).

Fig. 6 A and B.Ac-TMP:A, cDNA (SEQ ID NO.11) and B, deduced amino acid (SEQ ID NO.12).

Fig. 7 A and B.Ac-MEP-1:A, cDNA (SEQ ID NO.13) and B, deduced amino acid (SEQ ID NO.14).GeneBank accession number AF273084.

Fig. 8 A and B.Ac-MTP-1:A, cDNA (SEQ ID NO.15) and B, deduced amino acid (SEQ ID NO.16).GeneBank accession number AY036056.

Fig. 9 A and B.Ac-ASP-1:A, cDNA (SEQ ID NO.17) and B, deduced amino acid (SEQ ID NO.18).GeneBank accession number AF132291.

Figure 10 A and B.Ac-ASP-2:A, cDNA (SEQ ID NO.19) and B, deduced amino acid (SEQ ID NO.20).GeneBank accession number AF089728.

Figure 11 A and B.Ac-ASP-3:A, cDNA (SEQ ID NO.21) and B, deduced amino acid (SEQ ID NO.22).

Figure 12 A and B.Ac-ASP-4:A, cDNA (SEQ ID NO.23) and B, deduced amino acid (SEQ ID NO.24).

Figure 13 A and B.Ac-ASP-5:A, cDNA (SEQ ID NO.25) and B, deduced amino acid (SEQ ID NO.26).

Figure 14 A and B.Ac-ASP-6:A, cDNA (SEQ ID NO.27) and B, deduced amino acid (SEQ ID NO.28).

Figure 15 A and B.Ac-TTR:A, cDNA (SEQ ID NO.29) and B are from nucleotide 25-531 deduced amino acid (SEQ ID NO.30).

Figure 16 A and B.Ac-103:A, cDNA (SEQ ID NO.31) and B, aminoacid sequence (SEQ ID NO.32).

Figure 17 A and B.Ac-VWF:A, cDNA (SEQ ID NO.33) and B, aminoacid sequence (SEQ ID NO.34).

Figure 18 A and B.Ac-CTL:A, cDNA (SEQ ID NO.35) and B, aminoacid sequence (SEQ ID NO.36).

Figure 19 A and B.Ac-API-1:A, cDNA (SEQ ID NO.37) and B are from nucleotide 23-706 deduced amino acid (SEQ ID NO.38).

Figure 20 A and B.Ac-MTP-1:A, cDNA (SEQ ID NO.39) and B, aminoacid sequence (SEQ ID NO.40).

Figure 21 A and B.Ac-MTP-2:A, cDNA (SEQ ID NO.41) and B, aminoacid sequence (SEQ ID NO.42).

Figure 22 A and B.Ac-MTP-3:A, cDNA (SEQ ID NO.43) and B, aminoacid sequence (SEQ ID NO.44).

Figure 23 A and B.Ac-FAR-1:A, cDNA (SEQ ID NO.45) and B, aminoacid sequence (SEQ ID NO.46).GeneBank accession number AF529181.

Figure 24 A-C.Ac-KPI-1:A and B, cDNA (SEQ ID NO.47) and C are from nucleotide 12-2291 deduced amino acid (SEQ ID NO.48).

Figure 25 A and B.Ac-APR-1:A, cDNA (SEQ ID NO.49) and B, aminoacid sequence (SEQ ID NO.50).

Figure 26 A and B.Ac-APR-2:A, Partial cDNA Sequence (SEQ ID NO.51) and B, partial amino-acid series (SEQ ID NO.52).

Figure 27 A and B.Ac-AP:A, cDNA (SEQ ID NO.53) and B, aminoacid sequence (SEQ ID NO.54).

Figure 28 A and B.Ay-ASP-1:A, cDNA (SEQ ID NO.55) and B, aminoacid sequence (SEQ ID NO.56).

Figure 29 A and B.Ay-ASP-2:A, cDNA (SEQ ID NO.57) and B, aminoacid sequence (SEQ ID NO.58).

Figure 30 A and B.Ay-MTP-1:A, cDNA (SEQ ID NO.59) and B, aminoacid sequence (SEQ ID NO.60).

Figure 31 A and B.Ay-API-1:A, cDNA (SEQ ID NO.61) and B are from nucleotide 23-703 deduced amino acid (SEQ ID NO.62).

Figure 32 A and B.Ay-TTR:A, Partial cDNA (SEQ ID NO.63) and B, partial amino-acid series (SEQ ID NO.64).

Joseph Pearman (Spearman) rank correlation between Figure 33 A and B. ancylostome quantity and the anti--MTP-1 antibody titer.A) total borer population; B) intermediate value EPG.

Figure 34 A-C. immunity inoculation has the function of the dog body endoantigen-specificity geometric mean IgG1 antibody titer of ancylostoma caninum recombination fusion protein as the time.Geometric mean is calculated as every group of 6 Canis familiaris L.s, except the Ac-AP group, wherein has only a Canis familiaris L. to develop antigen-specific antibody and replys.Arrow shows the immunity inoculation of time control.(A) anti--Ac-APR-1 replys (n=6).(B) anti--Ac-TMP replys (n=6).(C) anti-Anti-Ac-AP replys (n=1).

Figure 35. the male and female sexual maturity ancylostoma caninum ancylostome of reclaiming from the colon of the Canis familiaris L. of immunity inoculation or injection Alumen.

Joseph Pearman rank correlation between Figure 36 A and B. ancylostome quantity and the anti--MTP-1 antibody titer.

Figure 37 A and B.A) association between reducing of anti--TTR IgE antibody and ancylostome quantity; B) association between anti--TTR IgG1 antibody and ancylostome quantity reduce.

Figure 38 A and B. after L3 attacks, the change of HV-4 dog hemoglobin (B) and hematocrit (A).

Figure 39. than the adjuvant matched group, ancylostome size in the group of TTR immunity inoculation (1 and 2mm between) the statistics on significantly reduce.

Figure 40. the CD4+ lymphocyte that the hookworm infection (the worm's ovum positive) after hook worm (Ancylostoma) the L3 antigenic stimulus of using by oneself is individual.

Figure 41. the CD4+ lymphocyte that the post-stimulatory hookworm infection of Pichia sp. (Pichia) (the worm's ovum positive) of the express recombinant Na-ASP-1 that uses by oneself is individual.

The detailed description of the preferred embodiment of the invention

The invention provides and be used to cause the compositions of mammal at the immunne response of ancylostome.This compositions can be used as vaccine and be used for the treatment of and/or prevent hookworm infection.Described vaccine comprises antigen preparation and pharmaceutically suitable carrier of purification, and described antigen derives from ancylostome.Term " derives from " and is meant that antigen originates from the biomolecule of (that is, separating certainly) ancylostome.For example, antigen can be albumen, and the antigen fragment of polypeptide or albumen or polypeptide, described albumen or polypeptide have been formed the part of ancylostome organism.Usually, this antigen be separate from and at least partial purification separate and purification process is known (for example referring to following embodiment part) for those skilled in the art from ancylostome.When manufacturing is used to cause immunne response or during as vaccine, this antigen can be " synthetic ", promptly synthetic (for example the acquisition, under the situation of polypeptide and protein fragments, make) by peptide is synthetic, or " reorganization ", promptly obtain (for example, by producing, described carrier has the DNA of coding for antigens) in containing the host cell of carrier by gene engineering.Those skilled in the art will recognize that multiple so suitable expression system can adopt, include but not limited to that those use escherichia coli, yeast (for example, Pichia sp.), baculovirus/insect cell, the expression system of plant cell and mammalian cell.In a preferred embodiment of the invention, antigen presentation is in yeast or baculovirus/insect cell expression system.

Here provided specific antigen, its aminoacid primary sequence and their examples of nucleic acid of encoding.For the convenience of reference, Table I has been enumerated some exemplary antigens and their corresponding SEQ ID NOS.Yet, those skilled in the art will recognize that can there be or makes up multiple variant in the sequence of enumerating here, these variants also can be used as antigen in implementing process of the present invention.For example, according to aminoacid sequence, can have or make up variant, described variant performance: conserved amino acid replaces; Non-conserved amino acid replaces; For example by carrying out truncate at amino terminal or carboxyl terminal or at intramolecule disappearance aminoacid; Perhaps by (for example adding aminoacid at amino terminal or carboxyl terminal or at intramolecule, be added with the histidine mark that helps Protein Separation), replace residue to change dissolution characteristics, the residue that replacement comprises the proteolytic cleavage site to be to eliminate shearing site and to improve stability, add or remove glycosylation site etc. or for other any reason).This variant can be abiogenous (for example, as between the kind or the result of natural variation between the individuality); Perhaps can be autotelic importing (for example, utilizing the assay device of gene engineering).If antigenic variant shows with the sequence of describing sufficient homogeneity is arranged, then all variants of sequence disclosed herein all should be included in the instruction of the present invention.Preferably, with the homogeneity of disclosed sequence in about scope of 50 to 100%, more preferably in about scope of 75 to 100%, and most preferably in 80 to 100% scope.Homogeneity is with reference to the aminoacid sequence part corresponding to the original antigen sequence, does not promptly comprise other element that can be added, such as the chimeric antigen of following description.

Table I. ancylostome antigen, describe and corresponding SEQ ID NOS.

The source	Antigen	Describe	?????????????SEQ?ID?NOs.
					??CDNA	Open reading frame
			Necator americanus
	??Na-ASP-1	Excretory albumen			??SEQ?ID?NO.1	??SEQ?ID?NO.2
				??Na-ACE			Acetylcholine esterase	??SEQ?ID?NO.3	??SEQ?ID?NO.4
	??Na-CTL	The C-agglutinin			??SEQ?ID?NO.5	??SEQ?ID?NO.6
				??Na-APR-1			Aspartic protease	??SEQ?ID?NO.7	??SEQ?ID?NO.8
	??Na-APR-2	Aspartic protease			??SEQ?ID?NO.9	??SEQ?ID?NO.10
			Ancylostoma caninum (Ancylostom a caninum)
	??Ac-TMP	The met protease inhibitor			??SEQ?ID?NO.11	??SEQ?ID?NO.12
				??Ac-MEP-1			Zinc metalloproteinase	??SEQ?ID?NO.13	??SEQ?ID?NO.14
	??Ac-MTP-1	3,4,3',4'-tetraketo-.beta.-carotene protease			??SEQ?ID?NO.15	??SEQ?ID?NO.16
				??Ac-ASP-1			Excretory albumen	??SEQ?ID?NO.17	??SEQ?ID?NO.18
	??Ac-ASP-2	Excretory albumen			??SEQ?ID?NO.19	??SEQ?ID?NO.20
				??Ac-ASP-3			Excretory albumen	??SEQ?ID?NO.21	??SEQ?ID?NO.22
	??Ac-ASP-4	Excretory albumen			??SEQ?ID?NO.23	??SEQ?ID?NO.24
				??Ac-ASP-5			Excretory albumen	??SEQ?ID?NO.25	??SEQ?ID?NO.26
	??Ac-ASP-6	Excretory albumen			??SEQ?ID?NO.27	??SEQ?ID?NO.28
				??Ac-TTR-1			??transerythrin	??SEQ?ID?NO.29	??SEQ?ID?NO.30
	??Ac-103	Surface protein			??SEQ?ID?NO.31	??SEQ?ID?NO.32
				??Ac-VWF			The surface agglutinin	??SEQ?ID?NO.33	??SEQ?ID?NO.34
	??Ac-CTL	The C-agglutinin			??SEQ?ID?NO.35	??SEQ?ID?NO.36
				??Ac-API			Aspartyl protease inhibitor	??SEQ?ID?NO.37	??SEQ?ID?NO.38
	??Ac-MTP-1	3,4,3',4'-tetraketo-.beta.-carotene protease			??SEQ?ID?NO.39	??SEQ?ID?NO.40
				??Ac-MTP-2			3,4,3',4'-tetraketo-.beta.-carotene protease	??SEQ?ID?NO.41	??SEQ?ID?NO.42
	??Ac-MTP-3	3,4,3',4'-tetraketo-.beta.-carotene protease			??SEQ?ID?NO.43	??SEQ?ID?NO.44
				??Ac-FAR-1			In conjunction with retinol	??SEQ?ID?NO.45	??SEQ?ID?NO.46
	??Ac-KPI-1	Protease inhibitor			??SEQ?ID?NO.47	??SEQ?ID?NO.48
				??Ac-APR-1			Aspartic protease	??SEQ?ID?NO.49	??SEQ?ID?NO.50
	??Ac-APR-2	Pepsinogen			??SEQ?ID?NO.51	??SEQ?ID?NO.52
				??Ac-AP			Anticoagulant	??SEQ?ID?NO.53	??SEQ?ID?NO.54
Ancylostoma ceylonicum
				??Ay-ASP-1			Excretory albumen	??SEQ?ID?NO.55	??SEQ?ID?NO.56
	??Ay-ASP-2	Excretory albumen			??SEQ?ID?NO.57	??SEQ?ID?NO.58
				??Ay-MTP-1			3,4,3',4'-tetraketo-.beta.-carotene protease	??SEQ?ID?NO.59	??SEQ?ID?NO.60
	??Ay-API-1	Aspartyl protease inhibitor			??SEQ?ID?NO.61	??SEQ?ID?NO.62
				??Ay-TTR			Class-transthyretin	??SEQ?ID?NO.63	??SEQ?ID?NO.64

The present invention also comprises chimeric antigen, for example adds the antigen that other sequence is formed by aminoacid sequence described herein, and described other sequence must link to each other with disclosed sequence when separating, and still adds these aminoacid and has given some other advantage.For example, these advantages can be used to separate and purifying protein (for example, histidine mark, GST, maltose-binding protein); Guide albumen to the interior position (for example yeast secreted protein) of specific cell; Improve proteic antigenicity (for example, KHL, hapten).These all chimeric construct bodies all should comprise within the scope of the invention, as long as show the level of homology at least based on the existence of the part construct of sequence disclosed herein.

Those skilled in the art will recognize that in order to cause the parasite that antigen is originated from has enough antigen to reply, and need not use albumen and or whole primary sequences of polypeptide.In some cases, proteic fragment is enough to immunize.Therefore, the present invention also comprises the antigen fragment of sequence disclosed herein, and the application in bacterin preparation.Usually, the about at least 10-13 aminoacid of this segmental length.Those skilled in the art will recognize that suitable sequence is normally hydrophilic and it is surperficial come-at-able usually to be.

Equally, according to nucleotide sequence disclosed herein, those skilled in the art will recognize that can there be or makes up multiple variant in sequence, this variant still can provide antigen or its part that needs of coding.For example, because the redundancy of genetic code, more than one codons can be used to the seed amino acid of encoding.In addition, as mentioned above, can wish the antigen primary sequence is changed, and this change for nucleic acid sequence encoding is necessary.In addition, the multiple variant that those skilled in the art will recognize that nucleotide sequence can be made up be used to relate to clone's strategy purpose (for example, insert carrier for the ease of the sequence of operation, such as importing restriction endonuclease shearing site etc.), (for example be used to modify the purpose of transcribing, import promoter or enhancer sequence etc.), perhaps be used for other any appropriate purpose.The such variant of all of nucleotide sequence disclosed herein all should comprise within the scope of the invention, as long as sequence table reveals and about 50 to 100% the homogeneity of original series, and preferred, show about homogeneity of 75 to 100%, and most preferably, about homogeneity of 80 to 100%.The part nucleotide sequence of the corresponding original series of homogeneity reference, and should not comprise other element, such as, promoter, the sequence in carrier source derives from the restriction site in other source etc.

Antigen of the present invention can come from the ancylostome of any kind of, and example includes but not limited to Necator americanus, ancylostoma caninum, Ancylostoma ceylonicum and Ancylostoma duodenale.

The antigenic example of suitable ancylostome includes but not limited to Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API, and Ay-TTR.

In some embodiments of the present invention, the antigen entity is and activates relevant secretory protein that these proteic examples include but not limited to Na-ASP-1, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ay-ASP-1, and Ay-ASP-2.

In other embodiments of the present invention, antigen part is a protease, and these examples of proteases include but not limited to metalloproteases (for example, Ac-MTP-2, Ac-MTP-3; Cysteine proteinase; Aspartic protease (for example, Ac-APR-1 and Ac-APR-2); And serine protease.

In other embodiments of the present invention, antigen can be agglutinin (for example, Na-CTL, Ac-CTL).

In other embodiments of the present invention, antigen can be protease inhibitor (for example, Ac-API-1, Ay-API-1, Ac-AP, Ac-KPI-1).

In preferred embodiments, the antigen that uses in the embodiment of this invention is Ac-TMP, and its dna encoding sequence is listed among Fig. 6 A (SEQ ID NO.11), and its aminoacid sequence is listed among Fig. 6 B (SEQ ID NO.12).

In another preferred embodiment, the antigen that uses in the embodiment of this invention is Ac-MEP-1, and its dna encoding sequence is listed among Fig. 7 A (SEQ ID NO.13), and its aminoacid sequence is listed among Fig. 7 B (SEQ ID NO.14).

In another preferred embodiment, the antigen that uses in the embodiment of this invention is Ac-MTP-1, and its dna encoding sequence is listed among Fig. 8 A (SEQ ID NO.15), and its aminoacid sequence is listed among Fig. 8 B (SEQ ID NO.16).

Other preferred antigens includes but not limited to Na-CTL (SEQ ID NOS.5-6); Na-APR-1 (SEQ ID NOS.7-8); Na-APR-2 (SEQ ID NOS.9-10); Ac-TMP (SEQID NOS.11-12); Ac-ASP-3 (SEQ ID NOS.21-22); Ac-ASP-4 (SEQ IDNOS.23-24); Ac-ASP-5 (SEQ ID NOS.25-26); Ac-ASP-6 (SEQ ID NOS.27-28); Ac-TTR (SEQ ID NOS.29-30); Ac-103 (SEQ ID NOS.31-32); Ac-VWF (SEQ ID NOS.33-34); Ac-CTL (SEQ ID NOS.35-36); Ac-API-1 (SEQ ID NOS.37-38); Ac-MTP-1 (SEQ ID NOS.39-40); Ac-MTP-2 (SEQID NOS.41-42); Ac-MTP-3 (SEQ ID NOS.43-44); Ac-KPI-1 (SEQ IDNOS.47-48); Ac-APR-1 (49-50); Ac-APR-2 (SEQ ID NOS.51-52); Ay-ASP-1 (SEQ ID NOS.55-56); Ay-ASP-2 (SEQ ID NOS.57-58); Ay-MTP-1 (SEQ ID NOS.59-60); Ay-API-1 (SEQ ID NOS.61-62); Ay-TTR (SEQ ID NOS.63-64).

The invention provides the compositions that is used to cause immunne response, described compositions can be used as the anti-ancylostome of vaccine.Term " initiation immunne response " is meant the synthetic of a kind of antigenic stimulus specific antibody, titre is approximately＞and 1 to about 1 * 10 ⁶Perhaps bigger.Preferably, titre is from about 10,000 to about 1 * 10 ⁶Perhaps bigger, and most preferred, titre is greater than 1 * 10 ⁶, for example pass through ³Mixing of H thymus pyrimidine measured.Term " vaccine " is meant a kind of antigen that causes immunne response, compares with the matched group organism of immunity inoculation (for example having only adjuvant) not to cause that ancylostome quantity reduces about 30% at least in the organism.Preferably, the decline level is about 50%, preferred about 60 to about 70% or higher.

The invention provides the compositions that is used to cause immunne response, can be used as the anti-ancylostome of vaccine.Described compositions comprises ancylostome antigen or its variant of purification basically described here, and pharmaceutically suitable carrier.This preparation of compositions as vaccine is well known to a person skilled in the art.Usually, this compositions can be prepared to liquid solution or suspension, yet such as, tablet, pill, the solid form of powder etc. are also in the scope of considering.The solid form that is fit to be dissolved in or be suspended in the liquid before administration also can be produced.Preparation also can be emulsive form.Active component can with pharmaceutically useful and compatible mixed with excipients with active component together.Suitable excipient is, for example, and water, saline, glucose, glycerol, ethanol or the like, perhaps its compositions.In addition, compositions can contain a spot of auxiliary substance, such as wetting agent or emulsifying agent, and pH buffer agent etc.In addition, compositions can contain other adjuvant.Use the compositions of oral form if desired, can add various thickening agents, flavoring agent, diluent, emulsifying agent, dispersing aid or binding agent etc.Thereby can containing this arbitrarily adding ingredient, compositions of the present invention provides the compositions that is fit to form of medication.The antigenic final quantity of ancylostome can change in the preparation.But, usually, the amount in the preparation approximately from 1% to 99%.

Bacterin preparation of the present invention may further include adjuvant, and suitable example includes but not limited to Seppic, Quil A, Alhydrogel etc.

Preparation of the present invention can contain a kind of single ancylostome antigen.Perhaps, more than one ancylostome antigens can be used in the preparation, that is, preparation can contain " cocktail " antigen.

The present invention also provides initiation at the method for the immunne response of ancylostome and the method for the anti-ancylostome of immunity inoculation mammal.Term causes immunne response and is meant that administration of antigens causes that (the titre scope is 1 to 1 * 10 to specific antibody ⁶, preferred 1 * 10 ³, more preferably 1 * 10 ³To about 1 * 10 ⁶, and most preferably greater than 1 * 10 ⁶) synthetic and/or cell proliferation, for example pass through ³Mixing of H thymus pyrimidine measured.Described method comprises to administration contain the antigenic compositions of ancylostome in pharmaceutically suitable carrier.Bacterin preparation of the present invention can carry out administration by multiple suitable manner arbitrarily, and described mode is known for those skilled in the art, includes but not limited to injection, and oral, intranasal contains antigenic food or the like by picked-up.In preferred embodiments, administering mode is subcutaneous or intramuscular.

The invention provides initiation at the method for the immunne response of ancylostome and the method for the anti-ancylostome of immunity inoculation mammal.In one embodiment, mammal is human.But, those skilled in the art will recognize that other mammal also exists, because also be useful to the anti-ancylostome of this immunoprophylaxis, promptly preparation also is used for purpose for animals.Described example includes but not limited to companion " house pet ", such as Canis familiaris L., and cat etc.; Food source, work and amusement animal, such as domestic animal, horse, cattle, sheep, pig, goat or the like.

One skilled in the art would recognize that usually for the anti-ancylostome of immunity inoculation (perhaps causing immunne response) purpose species (for example, the people), the antigen of use should derive from the ancylostome kind that parasitizes the purpose species.For example, the antigen that derives from Necator americanus usually is preferred for the immune mankind, and the antigen that derives from ancylostoma caninum is preferred for immune dog class.But be not always this situation.For example, the known parasitic mankind of ancylostoma caninum and primary dog class host thereof.In addition, the ancylostome antigen of cross species sometimes may be for causing the nonhost animal, and promptly the parasitic host's who does not originate as antigen usually immunne response height is effective.Even, the mensuration that antigen is suitable for immunostimulation or bacterin preparation depends on the ability that prevents parasite attack and parasitism that it is given, shown as (for example, amass and/or the concentration of hemoglobin is measured) as indicated in the reduction by ancylostome quantity for example or the inhibition ischemia relevant by haematocrit with ancylostome.For example, in order to be used for bacterin preparation, to cause that ancylostome quantity reduces about at least 30% for antigen after the administration, preferably about at least 50%, and most preferably about at least 60% to about 70%.

In one embodiment of the invention, providing can the anti-infection method of immunity inoculation patient.Described method comprises the treatment hookworm infection to being enough to increase lymphopoietic level, then by the anti-described infectious disease of immunity inoculation patient.Described method is based on the fact that provides among the embodiment 10, and described embodiment shows that ancylostome grows and causes that cell is to ancylostome and other possible antigenic stimulus immunological unresponsiveness.Therefore, by chemical treatment hookworm infection patient before the anti-ancylostome of immunity inoculation or any infectious agent, can be improved for replying of immunity inoculation.The example of the infectious disease that the result of the anti-described infectious disease of immunity inoculation is improved includes but not limited to HIV, tuberculosis, malaria and conventional immunization programs for children inoculation (for example, measles, tetanus, diphtheria, pertussis, poliomyelitis or the like).

Can include but not limited to albendazole (albendazole) and other anthelmintic by its example that carries out chemically treated ancylostome medicament.

Specific antigen described herein also can be used for treating other tumor, autoimmune and cardiovascular disease, and the short scorching state of treatment.The antigenic this application of other ancylostome is described in, for example, licenses to people's such as Capello United States Patent (USP) 5,427,937 and licenses in the United States Patent (USP) 5,753,787 of Hawdon.

The present invention also provides following nucleic acid and aminoacid sequence: SEQ ID NO.11, SEQ IDNO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ IDNO.34, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.47, SEQ ID NO.48, SEQID NO.49, SEQ ID NO.50, SEQ ID NO.51, SEQ ID NO.52, SEQ IDNO.55, SEQ ID NO.56, SEQ ID NO.57, SEQ ID NO.58, SEQ ID NO.59, SEQ ID NO.60, SEQ ID NO.61, SEQ ID NO.62, SEQ ID NO.63 and SEQ ID NO.64.Described sequence represent the cDNA sequence with and amino acid sequence coded (open reading frame).Though sequence itself has been required protection; but other is compared the sequence with high-level homogeneity with the sequence of those descriptions and also is taken into account; for example has at least 65% to 100% homogeneity with the sequence that provides; perhaps 75% to 100% homogeneity preferably approximately, the perhaps sequence of about at least 80% to 100% homogeneity most preferably.

Especially, Ac-APR-2 (SEQ ID NOS.51 and 52) and Ay-TTR (SEQ IDNOS.63 and 64) are the partial sequences of the most of antigen sequence of representative.Therefore, the present invention includes complete Ac-APR-2 antigen and complete Ay-TTR antigen.

The antigen of the Ay-TTR family that the Ay-TTR antigen representative that provides in this application exists in a lot of species nematicides is provided in addition.Equally, the Ay-TTR antigen from any nematicide should comprise within the scope of the invention.Especially, come from and include but not limited to Necator americanus, ancylostoma caninum, any Ay-TTR of the kind ancylostome of Ancylostoma ceylonicum and Ancylostoma duodenale is also included within the scope of the present invention.

Embodiment

The molecular cloning of embodiment 1.Ac-TMP and purification

Material and method

The immunoscreening in ripe ancylostoma caninum libraryPreparation resists-ancylostoma caninum secreted product antibody.(Hotez and Cerami, 1983) as mentioned above are from interior ancylostoma caninum (Ancylostomacaninum) ancylostome of reclaiming the maturation period of 100 work of intestinal of postmortem (infecting 6 weeks of back) infection Canis familiaris L..Ripe ancylostome is washed 3 times in aseptic PBS, then at 37 ℃ of (5%CO ₂) be kept at the HEPES that 15ml contains 25mM, among the RPM 1640 of the streptomycin of the ampicillin of 100 units/ml and 100 μ g/ml 24 hours.Collect supernatant, concentrate, and ((pH 7.2) are 4 ℃ of dialysed overnight with the phosphate buffer of 1L with PEG6000.After the dialysis, the product of separating out is 10, centrifugal 10 minutes of 000xg, and reclaim supernatant.

By the subcutaneous injection excretory albumen of the emulsive ancylostome of complete Freund's adjuvant (400 μ g) immune rabbit.Subsequently, the excretory albumen of the emulsive ancylostome of incomplete Freund of usefulness same amount carries out three immunity altogether with the interval immune rabbit in 2 weeks.Final immunity obtained whole blood in back 10 days, and from separation of whole blood serum and be kept at-20 ℃.

CDNA expresses the structure in ZapII (Stratagene, La Jolla CA) library and once reported people such as (, 1996) Capello in the past.According to the description of manufacturer with rabbit anti--the ripe secretory product antibody screening of ancylostoma caninum is to according to estimates 5 * 10 ⁵Plaque.In brief, 5 * 10 ⁴Plaque be coated with and be layered in the LB agar culture plate.Cover plaque by the nitrocellulose membrane that soaks into 10mM IPTG and induce the antigenic expression of ancylostoma caninum.37 ℃ cultivate 4 hours after, mention film, seal with the PBS of 5% defatted milk, then with rabbit antibody (dilution in 1: 500) 24 ℃ of cultivations 1 hour.Film clean three times with the PBS (PBS-Tween) that contains 0.1%Tween-20 and with the goat of horseradish peroxidase anti--rabbit igg (Sigma) continued incubations 1 hour with 1: 1000 thinner ratio at 24 ℃.Clean film once more three times with PBS-Tween, use 3,3 then '-diaminobenzidine (DAB) substrate and hydrogen peroxide colour developing.Possible positive colony marking and separation are used for programmed screening.

Description (Stratagene) according to manufacturer is resected to immune positive colony in the pBluscript phage, utilizes the method (Qiagen) of alkaline bleach liquor cleavage to extract phasmid DNA, and utilizes flanking vector primer (T ₃And T ₇) carry out two strands order-checking.By blast search the existing sequence among nucleotide and deduced amino acid and the GenBank is compared.Utilize ESEE 3.1 softwares to carry out sequence analysis.

Inverse transcription polymerase chain reaction (RT-PCR) amplification

RT-PCR is used to the stage of development specificity of identifying that Ac-tmp mRNA transcribes.Ancylostoma caninum (A.Caninum) ovum, L1 and L2 larval stage and L3 infect larval stage and obtain people such as (, 1999) Hawdon as mentioned above.Utilize TRIzol reagent (GIBCO BRL) to separate total RNA from each life cycle stage.Utilize few d (T) primer and the synthesizing single-stranded cDNA of MMLV-RT (GIBCOBRL).Based on the Auele Specific Primer of the Ac-tmp sequence from 60bp to 440bp (TIMP3 '-the Ac-tmp cDNA that is used to increase of 1HR and TIMP5 '-2ER).The PCR response parameter is: 94 ℃ of degeneration 1 minute, and 55 ℃ of annealing 1 minute, 72 ℃ were extended 2 minutes.Carry out 30 circulations altogether.

Purification Ac-TMP natural productThe optimization that is used for the partly preparation property reversed phase chromatography analysis condition of the ripe secretory product fractionated of ancylostoma caninum is carried out in 510HPLC system (Waters), the 490 E multiwavelength detectors that have one and half preparation property flow cells have been installed by described system, wavelength set is 214,280,260 and 254mm, and a 250mm * 4.6I.D.YMC-Pack Protein-RP, 200 , 5 μ m C ₄Post (Waters).Collect from 1260 ripe ancylostomes after 15 hours and contain 25mM HEPES as the ripe ancylostoma caninum secretory product to 37 of parent material ℃, the ampicillin of 100 units/ml is among the 15ml RPMI 1640 of the gentamycin of the streptomycin of 100 μ g/ml and 100 μ g/ml.7, before centrifugal 1 hour of the 500xg, supernatant is concentrated to 0.3 times of volume by ultrafiltration in the miniature enrichment facility (Amicon) of Centricon-3.The parasite secretory protein of about 0.6mg is carried out chromatographic analysis.Eluent A is the water of 0.01% trifluoroacetic acid (TFA), and eluent B is the acetonitrile of 0.01%TFA.With 1ml/ minute flow velocity from 0 to 80%B linear gradient elution 40-minute.Collect 0.5 minute fraction and carry out lyophilization, be used for further purification then and analyze by SDS-PAGE (Laemmli, 1970).In order to carry out SDS-PAGE, the 2XSDS-PAGE sample buffer (4%SDS, 2.5%2-mercaptoethanol, 15% glycerol) that the HPLC of the secretory product of 2 μ l and 10 μ l separates fraction numbering 51 and equal volume mixes, and boils 5 minutes.Sample is with 100V electrophoresis 2 hours on the SDS-PAGE of 4-20% gradient gel.According to the description (BIO-RAD) of manufacturer gel being carried out silver dyes.

Fraction 51 is being installed aforesaid use 250mm. * 3.0 I.D.YMC albumen RP, and 200A carries out RP-HPLC in the 510 HPLC systems of 5 μ m C4 posts, and described fraction contains from partly preparing isolating most ancylostoma caninum secretory protein.Eluent A is the water of 0.01%TFA, and B is the acetonitrile of 0.01%TFA.Linear gradient from 0 to 60%B was with 1ml/ minute flow velocity eluting 30 minutes.Collect 0.5 minute fraction and carry out lyophilization.The main protein peak of collecting from this separation partly carries out amino acid sequence analysis and SDS-PAGE (Laemmli, 1970).Amino acid sequence analysis based on albumen Edman degraded carries out on the accurate 494 type protein sequencing instrument (Applied Biosystems) that 785A detector able to programme and 140C pumping system (by ProSeq, Inc. (Boxford MA) provides) have been installed.Utilize the accurate 610A software of standard to identify the order-checking product.

In order to confirm N-end sequence, at the synthetic degeneracy oligonucleotide primer of both direction corresponding to the part N-terminal peptide sequence of No. 51 fraction corresponding to Ac-TMP.Paired flank degeneracy carrier primer is used to amplified production the DNA that the ripe cDNA library in being structured in ZapII obtains." heat is initial " PCR condition is the 10mM Tris-HCl (pH 8.5) that contains 50mM KCl, the MgCl of 2.0mM ₂, every kind of dNTP of 0.2mM, and the cDNA library of 1 μ l, totally 20 μ l reactants.Reactant then is reduced to 85 ℃ 94 ℃ of heating 5 minutes, lasts 5 minutes, adds the Taq archaeal dna polymerase (GIBCO BRL) of 1 unit then.Carry out 30 following circulations subsequently: 94 ℃ of degeneration 1 minute, 55 ℃ of annealing 1 minute, and 72 ℃ of extensions 2 minutes.PCR product electrophoresis and dye on agarose gel with ethidium bromide.(Qiagen, Valencia CA) carry out gel-purified to the PCR product, and check order with the gel extraction kit of QIAEX II.

The result of embodiment 1

Ac-TMP cDNA. from ripe ancylostome cDNA library by the rabbit antibody cloning Ac-TMP cDNA of immunoscreening at the ripe secretory product of all ancylostoma caninums (Ancylostoma canium).Be separated to two male identical clones.Full-length cDNA is 559 bp (SEQ IDNO.11), the poly A tail of open reading frame (ORF) and the 3 ' end of its 140 aminoacid of coding (SEQ ID NO.12).The molecular weight that the ORF of prediction calculates is that 16,100 dalton and theoretical pI are 7.55.The hydrophobic signal peptide sequence has a signal peptidase restriction enzyme site, and restriction enzyme site is between amino acid/11 6 and 17.And then after the signal peptide, Ac-TMP has a recognition marks N-terminal Cys-X-Cys sequence.The glycosylation site (N-X-T) that the N that derives connects is present in (Fig. 6 B) between amino acid/11 19 and 122.

The GenBank database search shows, the N-end structure territory of the tissue depressant (TIMP-2) of the aminoacid sequence of the prediction of this molecule and people's metalloid protease 2 has 33% homogeneity and 50% similarity.Contain a single structure territory and lack the characteristic C-end structure territory (data not shown) of second vertebrates TIMP from the TIMP of the Ac-TMP of the beautiful latent rhabditida (Caenorhabditiselegans) of spontaneous nematicide and supposition.

The RT-PCR amplification.For the Ac-TMP that identifies that the life cycle phase specificity is expressed, extract mRNAs from the ancylostoma caninums (Ancylostoma canium) of different stages of development, and be cDNA with the reverse transcription of Ac-TMP Auele Specific Primer.RT-PCR produces the specific band of a 380bp, the only amplification from sophisticated cDNA of described band.Not from ovum, observe amplification among the Li-L2 and the cDNA in L3 life cycle stage.The amplification of ancylostoma caninum (A.caninum) genomic DNA shows two bands, shows possible intron or has second relevant Ac-TMP gene (data not shown).

In the secretory product of ancylostoma caninum adult, identify Ac-TMP.In order to confirm that Ac-TMP is discharged by ripe ancylostoma caninum ancylostome, by RP-HPLC with Identification of Fusion Protein purification in the parasite secretory product and from the parasite secretory product.Each main peak all carries out the part (data not shown) of amino acid sequence analysis as big ancylostoma caninum protein science research.Selection is further studied corresponding to the protein peak of " fraction 51 " and is carried out chromatographic analysis again.Silver dyes back fraction 51 and comprises an outstanding band, moves apparent weight molecule amount Mr=16,000.The ORF sequence of Ac-TMP behind the signal peptide enzyme action site of N-terminal peptide sequence of this fraction (20 aminoacid) and prediction is mated fully.Based on the reference area below the curve at the HPLC peak 51 of composing the gross areas with respect to whole secretory products, Ac-TMP is confirmed as comprising total ancylostoma caninum (A.caninum) secretory product of about 6.3%.This just is a kind of maximum albumen that is discharged by ripe ancylostoma caninum (A.caninum) with this Molecular Identification.By confirming the abundance of Ac-TMP in the ancylostome secretory product in range estimation on the SDS-PAGE.Paired degenerate primer based on the sequence of a seven amino acid is used to from ripe ancylostome cDNA library construction PCR product.The DNA sequence of PCR product has confirmed the homogeneity (data not shown) with Ac-TMP cDNA.

This embodiment shows that TMP is the maximum albumen of ancylostome secretion, and described albumen has been cloned and expression and recombiant protein are separated.

The molecular cloning of embodiment 2.Ac-mep-1 and evaluation.

Material and method

ParasiteThe ancylostoma caninum parasite is grown in the beagle body (Schad1982) as mentioned above.From the Linesless charcoal coproculture, separate the infection larva (L3) of phase III and be kept in the BU buffer people such as (, 1995) Hawdon.Ripe ancylostoma caninum (A.caninum) ancylostome is collected in the Canis familiaris L. body of postmortem postoperative infection.These ancylostomes are cleaned in PBS three times, IQF in liquid nitrogen, and be kept at-80 ℃.

Nucleic acidBy conventional method (people such as Ausubel, 1993) from ripe ancylostoma caninum isolation of genomic DNA.Operating process according to manufacturer under the condition that has Trizol reagent (Gibco BRL) is separated ancylostoma caninum RNA by the ripe ancylostome of grinding freezing in advance (80 ℃).According to the description of manufacturer, prepare cDNA from RNA by ProSTAR article one chain RTPCR test kit (Stratagene).

Ancylostoma caninum genome and cDNA libraryMake up ancylostoma caninum (A.caninum) genome dna library according to following operation: in the recommendation buffer of 100 μ l volumes, the ancylostoma caninum of 30 μ g (A.caninum) genomic DNA is by Sau3A restriction endonuclease (NEB) the part degraded of 8 units (37 ℃ 5 minutes).DNA with degraded carries out ethanol precipitation also by the standard method collecting precipitation then.Precipitation drying with producing is dissolved in the water, and is connected in λ-FIXII carrier (Stratagene) according to the description of manufacturer.Use Gigapack Gold packaging extract (Stratagene) the coupled reaction thing is packed and to be increased then.The cDNA library of the ripe body of ancylostoma caninum people such as (, 1996) Capello in the past is structured in λ ZAPII (Stratagene) carrier.

The metalloprotein enzyme cloneThe clone of Ac-mep-1 cDNA starts from the PCR that utilizes degenerate primer and widow-dT to carry out on ripe ancylostome library cDNA.It is opposite with the conserved sequence that contains zinc binding moiety unit that degenerate primer is designed to, described zinc binding moiety unit's observation (GenBank in the BLAST of the zinc metalloprotein enzyme gene of two supposition that conceal rhabditida (C.elegan) from beauty comparison ^TM, accession number T22668 and Q22523).Reaction condition is as follows: the template DNA of 85ng, 1X is thermophilic DNA buffer (Promega), the MgCl of 2.5mM ₂, the dNTP of 0.2mM, every kind of primer of 2 μ M, the taq archaeal dna polymerase (Promega) of 1U, the cumulative volume of 20 μ l.35 following circulations are carried out in reaction: 94 ℃ 1 minute, 55 ℃ of 1 minute and 72 ℃ 1 minute.This PCR produces a fragment, when its clone (pGEM-T, Promega) and 3 ' Ac-mep-1cDNA of order-checking interval scale 458bp (21 residues that comprise poly A tail) (clone MP-1).Utilize the basis of MP-1, on library DNA, identify other Ac-mep-1 (clone MP-2) sequence by PCR with T3 (carrier) and MEP-R1 gene-specific primer as the Auele Specific Primer design.React on the library DNA of serial dilution, the product of the uniqueness that always increases is also cloned.Reaction condition as mentioned above.

In similarly cloning, with T3 and MEP-R2 primer amplification MP-3.5 ' end of Ac-mep-1 is identified in use from 5 of GibcoBRL '-RACE test kit.In brief, article one cDNA chain is created in the reverse transcription reaction thing on the RNA of preparation recently with Ac-mep-1 Auele Specific Primer RACE-R1.Utilize terminal deoxidation transferring enzyme to give this cDNA then at 3 ' terminal addition polymerization cytosine tail and as the template in the PCR reaction that contains anchor primer AAP (GibcoBRL) and gene specific reverse primer MEP-R2.The product that produces dilutes, and is used as the template of the heminested PCR reaction that contains anchor primer UAP (GibcoBRL) and gene-specific primer MEP-R3.PCR product and called after MP-4 that the clone produces.

Identify more 5 ' sequence from the genomic dna cloning (G-MEP) of Ac-mep-1 similar sequence.A plurality of clones are checked order carry out pcr amplification (clone FL-1) as the independent fragment of using suitable primer under these conditions with the full length coding region that confirms Ac-mep-1 cDNA and Ac-mep-1.

Sequence analysisUtilize the MEGALIGN software (3.7.1 version) of DNASTAR company that part A c-mep-1 clone is compared.The BLAST application program of BLAST analysis and utilization American National biotechnology information centre (National Center for Biotechnology Information) that is used for the Ac-mep-1 open reading frame (ORF) of the initial sequence of degenerate primer design and prediction carries out.Utilize Curatools sequence analysis application program (CuragenCorp., New Haven, CT). carry out the sequence analysis of Ac-mep-1.Having the FGENESH gene polling routine (finder utility) (the CGG webserver (genomic.sanger.ac.uk)) of analyzing beautiful latent rhabditida DNA setting is used to carry out predictive genes from genomic dna cloning G-MEP.The evaluation of possibility exon sequence is finished with Wise2 sequence analysis application program (sanger.ac.uk/Software Wise2/) among the GMEP.

The Northern traceCarry out the Northern engram analysis at the isolating total RNA of Trizol (GibcoBRL) from ten ripe ancylostomes.Fractionated RNA on 1.2% formaldehyde gel, and by the standard method trace on Hybond-N film (Amersham).With ³²The P dna fragmentation of initial flagging at random surveys trace, and described dna fragmentation is represented the base pair 780 to 2688 of Ac-mep-1 cDNA.

The RT-PCR of stage of developmentRT-PCR is used for studying the transcribing of Ac-mep-1 in each life cycle stage of ancylostoma caninum.For these reactions, from worm's ovum, L1, the cDNA of unactivated and activatory L3 and ripe ancylostome tests with Acmep-1 Auele Specific Primer MEP-F1 and MEP-R1.If the quality of these cDNA is verified that in the separating reaction that utilizes primer PKA-F and PKA-R these cDNA will be specific (people such as Hawdon, 1995) for ancylostoma caninum (A.caninum) protein kinase A so.These reaction conditions and the term harmonization that defines in 2.4 parts.

Anti--Ac-mep-1 antibodyRepresentative utilizes suitable primer to increase by PCR from 610 amino acid whose cDNA fragments of Ac-MEP-1C end portion from the cDNA λ library of ripe ancylostoma caninum.This fragment is entered into pGEM (Promega) by the T/A clone, its clone is entered the HindIII site of pET28c expression vector (Novagen) by standard method (Sambrook and Russell, 2001).In the culture of BL21 (DE3) PlysS (Stratagene) cell that transforms with the tAc-MEP-1/pET28c construct, induce the bacterioprotein of the Ac-MEP-1 (tAc-MEP-1) of truncate to express by the IPTG that adds 1mM.

Expressed proteins is insoluble.For purification tAc-mep-1, freezing (after freezing, BL21 (DE3) PlysS lysis) inductive cell precipitation, be resuspended in the tris pH 8.0 of the 50mM of 1/10th volumes, among the EDTA of 2 μ M, ultrasonic disruption is not up to there being viscosity, then 12,000xg centrifugal 15 minutes (Sorvall RC5B, GSA rotary head).The precipitation that produces is resuspended in the 1%SDS of 15ml, and in 0.5% the B-mercaptoethanol, ultrasonic disruption boiled 5 minutes, at room temperature cultivated then 2 hours.Remove undissolved fragment by centrifugal action repeatedly.Supernatant is dialysed to remove BME to phosphate buffer (pH 7.4) up hill and dale.According to the operating process of manufacturer without denaturant purifying protein on HisBind (Novagen) nickel resin compatible column.5 male Balb/c mices (6-age in week) are organized with Alumen-sedimentary tAc-MEP-1 of 20 μ g or have only Alumen to carry out the intraperitoneal immunity in contrast.Subsequently with twice of the interval booster immunization mice in 2-week.With final immunity one week of back, collect serum for the third time, pooled serum also is used as the primary antibody that Western blot and immunostaining are analyzed.

Western blottingIn transfering buffering liquid (0.037%SDS, pH 8.3 for the glycine of 39mM, the tris alkali of 48mM), shift 18 hours to the charged Immobilon-P pvdf membrane of methanol (Millipore) by the isolating albumen of 10% SDS-PAGE at 30V.With the PBS (sealing buffer) of 5% skimmed milk along with the vibration of gentleness at room temperature (RT) film was sealed 1 hour and and the elementary mouse anti tAc-MEP-1 antibody (1: 1500) that absorbs of escherichia coli at room temperature cultivated 1 hour, described mouse anti tAc-MEP-1 antibody dilution is in sealing buffer.In the sealing buffer, wash three films (each 10 minutes) then, and under RT, follow vibration to cultivate 1 hour with the sealing buffer of horseradish peroxidase-bonded goat anti-mouse IgG secondary antibodies (1: 5000).Finally, flushing membrane also developed the color with Renaissance (NEN Life Science product) chemiluminescence agent in 15 minutes in PBS.

ImmunolocalizationRipe ancylostoma caninum ancylostome carries out paraffin embedding and section by standard method.By incubation under the ancylostome section RT that will take off paraffin 1: 100 dilution factor (with the PBS dilution, pH 7.4) mouse anti tAc-MEP or control serum (the same) in finished the original position immunolocalization of Ac-MEP-1 in 1 hour.Section is washed in PBS three times and 25 ℃ of incubations in 1: 200 dilution goat anti-mouse IgG 1 hour, succeeded by flushing (three times) in PBS.Use Olympus IX-50 inverted fluorescence microscope (U-MWIG light filter) to observe then and cut into slices and take pictures.

The result of embodiment 2

The cDNA structure of Ac-mep-1The clone's strategy that is used to obtain Ac-mep-1 coded sequence completely is as follows: by order-checking degenerate pcr clone MP-1, and the clone MP-2 in PCR source, MP-3 and 5 ' RACE clone MP-4 identify the Ac-mep-1 transcript of about 2.6kb.Though approach RACE product 5 ' end a methionine codon is arranged, for not comprising 58 in-frame aminoacid of terminator, show that MP-4 does not represent 5 ' end of actual Ac-mep-1 before this codon.In addition, we can not obtain to comprise the cDNA clone (passing through PCR) of spliced leader sequence.Therefore, the predictive genes program of the beautiful latent rhabditida DNA of the inspection utilization of the genomic dna cloning G-MEP of Ac-mep-1 similar sequence (98.7% exon homogeneity) and except that identifying by 5 ' RACE different potential transcriptional start site.This prediction extends beyond 158 bp of 5 ' RACE sequence and increases by 91 aminoacid in coding region of deriving.Use this prediction, the whole coding region of Ac-mep-1 be amplified as the single product of 2.7kb and the part sequence verification by its two ends described clone.The total length of Ac-mep-1 transcript turns out to be about 2.8kb by Northern trace (5 ' and 3 ' terminal noncoding region is not amplified) in total length PCR.The amino acid sequence that this transcript is derived 870 the amino acid whose single ORF that encoded, these aminoacid have the glycosylation site that four possible N-connect (prediction pI=5.5, m.w.=98.7kDa).The amino terminal amino acid of Ac-MEP-1 comprises a hydrophobic signal peptide order-checking, has a prediction restriction enzyme site (referring to the figure with AC-MEP-1 sequence) behind residue 22.Identify two identification marking zinc binding moiety units of endopeptidase 24.11 families of characterizing metal protease.

Ac-mep-1 and the similarity of a kind of metalloproteases (Hc-MEP 2b) existence 66% of the nematicide haemonchus contortus (H.Contortus) of relevant trichostrongyle blood feeding and 48% homogeneity.It is similar to metalloproteases (the T25B6.2) (Gen-Bank from the beautiful latent rhabditida (C.elegans) of non-parasitic nematicide too ^TMT28906).14 cysteine residues high conservative between these three molecules.Other two cysteine (only guarding for one) are present among Ac-MEP-1 and the Hc-MEP1b.

Northern trace and chromogenic assay that Ac-mep-1 expressesThe Northern engram analysis shows the single mRNA transcript (not shown) that an about 2.8kb is arranged among the ripe ancylostome mRNA.The development-specific that adopts RT-PCR research Ac-mep-1 to transcribe.In the cDNA of test, might identify parasite only period of maturation but not hookworm ovum, the transcribing of L1 or activation and non-activated L3 larva.On the contrary, the positive control PCR that implements on identical cDNA with the primer that is specific to ancylostoma caninum (Ancylostoma canium) protein kinase A shows the amplification from all template cDNA.Therefore, Ac-mep-1 likes well and ad hoc is expressed in the ripe ancylostome body.

Western blot analysis and the immunolocalization of Ac-mep-1 in the ripe ancylostome sectionBy Western blotting, mouse anti MEP-1 antiserum recognizes about 90 and ripe ancylostoma caninum (Ancylostoma canium) albumen of 100kDa significantly.The immunohistochemical analysis of ripe ancylostome section navigates to Ac-MEP-1 on the Microvillares surface of ancylostome internal organs.With compare with the section of control serum incubation, the internal organs microvillus of antiserum and ripe ancylostome section reacts intensely.Also record the faint dyeing in the ripe ancylostome skin occasionally.Though the function of Ac-MEP-1 is unknown, will shows that along the position on parasite internal organs microvillus surface described enzyme directly contacts with blood stasis, and may therefore have the effect of nutrient digestion.

This embodiment shows that MEP-1 is a kind of important enzyme, and it makes ancylostome digestion blood, is an attractive vaccine target therefore.Reorganization MEP-1 albumen is cloned and is expressed.

The research of embodiment 3.AC-MTP antigen

The Agchylostoma ancylostome larva (L3) of infectiousness phase III discharges the metalloproteases of a kind of zinc-dependence, and the apparent molecular weight of its migration is 50kDa (1995a such as Hawdon).Described enzyme in response to the L3 stage induce feeding and growth the specific release of stimulation (Hawdon etc., 1995b), and play a role in may or casting off a skin in parasite skin and tissue invasion (Hotez etc., 1990).Because it in the tissue invasion in parasite-source and the effect in the decortication, directly may block larval migration at the antienzyme antibody response of Ac-MTP-1 and parasite enters enteral.Ac-MTP-1 is a phase specificity, and is discharged to restart at external feeding by activated ancylostome L3 under similar host's environment.The release of Ac-MTP-I makes this molecule become attractive vaccine target between active period.

Embodiment 3A. separates cDNA from ancylostoma caninum (A.caninum) the L3 expression library of the zinc-metalloproteases (Ac-mtp-1) of coding 3,4,3',4'-tetraketo-.beta.-carotene family.

Material and method

Antiserum: the people who agrees through IRB-studies agreement, and therefrom 5 of the Nanlin county in state Anhui Province occupy the serum that people's collection is used for immunoscreening ancylostoma caninum (A.caninum) L3 expression library.Ancylostoma duodenale is this geographic main ancylostome, and according to the response rate from the larva and the ripe ancylostome of infected patient, the ratio of Ancylostoma duodenale and Necator americanus was greater than 20: 1 (people .1999 such as Yong).Of other documents (people such as Xue, 2000), obtain serum from Anhui resident at the antigenic circulating antibody of all cracking of ancylostoma caninum L3 with high titre.Two residents are the hookworm ovum feminine gender, and the every gram feces of remaining 3 people has the quantitative faecal egg that is lower than 400 worm's ovums.Because the serum that antibody titer that they are higher and lower infection intensity, these individualities are counted as supposing that resistance is arranged and mix them is used for immunoscreening.In university students's body in Shanghai, collect negative control sera.

The screening of expression library: utilize the screening of blended antiserum to be structured in cDNA library (Stratagene, La Jolla, CA) people 1995 such as () Hawdon of ancylostoma caninum (A.caninum) (Baltimore strain) L3 among the X ZapIl according to the description of manufacturer.In brief, by nitrocellulose membrane was induced 5 * 10 in 4 hours in 37 ℃ of IPTG that immerse 10mM ⁴The plaque expressing protein.Behind the incubation, with film incubation in the PBS of 5% defatted milk powder 1 hour.22 ℃ with 1: 100 dilution PHS with the film incubation of sealing in PBS 1 hour, cleaned three times totally 10 minutes with PBS at 22 ℃, and with 1: 1000 the bonded Anti-Human IgG of dilution horseradish peroxidase (Sigma, St.Louis MO) cultivate altogether.With substrate 3,3 '-diaminobenzidine (DAB) and 0.015% hydrogen peroxide develop the color to film.Pave plate and screen once more positive plaque is carried out many wheel plaque purifications by being coated with once more.By excision rescue plasmid (Short and Sorge, 1992) in the body, and utilize the primer that is complementary to side joint carrier sequence that two chains are checked order.The aminoacid sequence of nucleotide and supposition compares with existing sequence in the GeneBank data base by blast search people such as (, 1997) Altschul.

The clone of total length Ac-MTP cDNA: the separative positive colony of institute 5 ' carry out truncate.In order to obtain 5 ' end, utilize gene-specific primer PI and be used to article one cDNA chain amplification 5 ' end from ancylostoma caninum (A.caninum) L3 corresponding to the PCR of the primer of conservative nematicide spliced leader sequence.The every kind of primer that contains 100ng of 20 μ l, the reactant of the cDNA of the Taq polymerase of 1U (Promega, Madison WI) and 1 μ l carries out 30 following circulations then 95 ℃ of degeneration 2 minutes: 94 ℃ 1 minute, 55 ℃ 1 minute, and 72 ℃ 2 minutes.Amplicon carry out gel-purified and by conventional method be cloned into pGEM Easy-T carrier (Promega, Madison, WI) in.

Phase specificity: determine the phase specificity (people such as Hawdon, 1995) that mtp-1 transcribes by RT-PCR.From the feces of infected dogs, separate ancylostoma caninum worm's ovum people such as (, 1994) Nolan by the sucrose floatation, handle, and be coated with and be layered in the nematicide growth medium agar plate people such as (, 1988) Sulston by carry out asepticize (axenize) with NaOCl.At 26 ℃ of incubation 24-30 hours, clean incubation thing (blended L from flat board subsequently with BU buffer (Hawdon and Schad, 1991) ₁/ L ₂) and in dry ice/ethanol bath, carry out quick freezing.Unhatched worm's ovum also carries out quick freezing to make cDNA.Small intestinal from infected dogs in the postmortem process is collected the ripe ancylostome of ancylostoma caninum (A.caninum).At the ancylostoma caninum worm's ovum, blended L ₁/ L ₂L serum-stimulation and that do not stimulate ₃(as described below), and the following RT-PCR that carries out on the ripe ancylostoma caninum sample.At freezing (liquid N in advance ₂) mortar in the sample grind into powder, and (Life Technologies, Gaithersburg MD) separate total RNA to utilize TRIzol reagent according to the description of manufacturer.With the DNAse 1 of 10 units (do not contain RNase, BoehringerMannheim, Indianapolis IN) handles RNA, and extracts once more with TRIzol.Utilize the BeadBeater machine under the condition of TRIzol (BioSpec, Bartlesville OK) separate total worm's ovum RNA with the bead Mechanical Crushing, carry out DNAse as mentioned above and handle and extract once more existing.Every kind of sample in the 50 μ L reactants that contain following component on 37 ℃ of synthetic article one cDNA chains totally 1 hour: the Tris HCl of 50mM, pH 8.3, the KCl of 75mM, the MgCl of 3mM ₂, the DTT of 10mM, the widow of 500ng (dT) primer, total RNA of 1 μ g, and the Moloney murine leukemia of 200U poison reverse transcription (LifeTechnologies).Reactant is 94 ℃ of incubations 5 minutes, and uses dH ₂O adds to 100 μ L.Article one cDNA chain of 1 μ L is used among the PCR, primer be MTP5 '-I (5 '-CTTCTCATGATCAACAAACACTACG) SEQ ID NO.65 and MTP3 '-1 (5 '-AATCTAACTCCAACATCTTCTGGTG) SEQ ID NO.66.Reactant carries out 30 following circulations: 94 ℃ 1 minute, 55 ℃ 1 minute, and 72 ℃ 2 minutes.Separate amplicon and with the ethidium bromide observation of dyeing by agarose gel electrophoresis.

Recombinant Protein Expression and purification: utilize routine techniques to be cloned in expression vector pET28 (Novagen) in in-frame mode total length Ac-mtp-1 cDNA and to be transformed in competence BL-21 escherichia coli (E.coli) cell.Histidine residues (His-labelling) Recombinant Protein Expression in 6 vector encoded that contain coding 5 ' end was induced 3 hours at 37 ℃ by adding 1mM IPTG.1ml expresses the cell of rMTP-1 by precipitating in centrifugal 5 minutes at 5000xg, abandons supernatant, and cell is carried out cracking in the TE (pH 8.0) of 100ml, and the TE of described 100ml contains the lyase of 100 μ g/ml and 0.1% Triton X-100.After 20 minutes, with sample ultrasonic Treatment (power level 2-3, the duty factor of 20-30%), each sample carries out 10 times fragmentation in 5 seconds, no longer is clamminess up to sample on ice at 30 ℃ of incubations.Under reducing condition,, observe dissolved albumen with Coomassie brilliant blue dyeing by electrophoretic separation solubility and the insoluble cell part of 12%SDS-PAGE.For purification rMTP-1, be suspended in the 1.0%SDS of 60ml from the cell precipitation of 2 liters of inductive bacterial culturess, in the 0.5%2-mercaptoethanol, boil 5 minutes, and cool to room temperature.Extract is changed buffer twice, and is applied in the HisBind nickel resin column (Novagen) of 10ml the PBS of 2 liters of 0.1%SDS dialysis 48 hours.Description according to manufacturer is carried out chromatography, except the SDS of interpolation 0.1% in all buffer.

In the process of the domain structure of making great efforts to improve dissolubility and study MTP-1,3 constructs that lack amino His labelled sequence are prepared by PCR.Total length Ac-MTPcDNA (1-1642bp), do not have 5 '-cDNA of propetide (408-1642bp), and the catalyst structure domain of inferring (408-1101bp) is cloned into upstream, Nco I site among the pET28 in in-frame mode, thereby from carrier the His marker coding sequence removed.Express recombinant protein under aforesaid the same terms.Antiserum production obtains anti--rMTP polyclonal antiserum by the rMTP immunity BABL/C mice with purification.The rMTP of the column purification of 20 μ g carries out co-precipitation with Alumen people such as (, 1996) Ghosh and subcutaneous injection.Other Booster the time carries out administration with alum precipitated rMTP (every kind 20 μ g) in the 3rd, 6 and 9 weeks.

Absorption at the mice serum of the bacterial lysate of e. coli bl21 strain to remove the antibody with the bacterioprotein reaction.The inducing cell of 25ml carries out centrifugal, is dissolved in the 2X sample buffer of 25ml (100mM Tris, pH6.8,2% SDS, 2.5% 2 mercapto ethanol), and 12, centrifugal 10 minutes of 000xg.(4cm * 8cm) immersed in the supernatant 20 minutes, then incubation (Tris of 48mM, the glycine of 39mM (glyine), 0.037% SDS, methanol of 20%) totally 30 minutes in transfering buffering liquid with nitrocellulose filter.In containing the PBS of 0.1%Tween-2, clean film 3 times, and at 22 ℃ of incubations with 1: 100 dilution mouse resisting anteserum incubation 1 hour.Repeat twice of incubation with fresh film.In order to confirm the specificity of antibody, absorption is at the mouse resisting anteserum of the absorption of the equal portions of BL21 (DE3) cell lysate of expressing total length rMTP-1 for the second time.The antiserum of absorption is used for Western blot.

External activation L3 also collects the ES product:Be similar under the intravital condition of host as mentioned above activation ancylostoma caninum (A.caninum) L such as (Hawdon people 1999) ₃In brief, use the BU buffer (Hawdon and Schad, 1991) of 1%HCl to purify 30 minutes at 22 ℃ of L3 that collect from coproculture.About 5000 L ₃At 37 ℃, 5%CO ₂Cultivation is at the 0.5ml RPM in the single hole of tissue culture plate, 24 hole ₁₆₄₀In the tissue culture medium (TCM) 24 hours, described culture medium was added HEPES pH 7.0 and the antibiotic (people 1999 such as Hawdon) of 25mM.By comprise 15% (v/v)＜S-methyl-glutathione of 10kD ultrafiltration dog serum and 25mM people 1995 such as () Hawdon thus activation L3 restarts feeding.Non-activated L ₃Incubation is in RPMI under the situation that does not have stimulation.Determine percentage ratio people 1996 such as () Hawdon of the larva of feeding as mentioned above.

Contain activatory and non-activated L ₃Media transfer to separating in the micro-centrifuge tube and centrifugal 5 minutes of 000rpm 14.The supernatant that comes from the same treatment group mixes, and removes L arbitrarily thereby the injection filter by 0.45 μ m filters ₃With the epidermis that comes off, and be kept at-20 ℃.Before electrophoresis, (ultrafiltration MA) is with supernatant concentration for Amicon, Beverley by utilizing Centricon 10 filter elements.Spissated ES cleans with the BU of 1ml, ultrafiltration and lyophilization.

In order to collect ripe ES, 1260 ripe ancylostomes are at 37 ℃, 10%CO ₂Cultivation is at RPMI ₁₆₄₀In the tissue culture medium (TCM) (people 1999 such as Hawdon) 15 hours.By ultrafiltration in Centricon 3 column spinners with 3 times of supernatant concentration.

Western blot: (4%SDS in the lysate of the bacterial cell of expression rMTP-1 fusion rotein and the sample buffer that cryodesiccated ES product is suspended in 2x SDS-PAGE again, the 5%2-mercaptoethanol, 15% glycerol) and at the gradient SDS-PAG of 4-20% (Invitrogen, Carlsbad separates on CA).(Millipore, Bedford MA) go up people such as (, 1979) Towbin to isolating albumen by transferred to PVDF membrane in 1 hour at the 25V electroblotting.At 22 ℃, film is with sealing 1 hour in the dcq buffer liquid (PBS, pH7.4,0.1% Tween 20) of 5% degreasing dry powder.The film of sealing 22 ℃ with 1: 5000 dilution mice rMTP antiserum incubation 1 hour, described mice rMTP antiserum is inhaled in advance at the bacterial lysate of expression total length rMTP.24 ℃ with dcq buffer liquid flushing membrane three times totally 10 minutes, (Boehringer Mannheim, Indianapolis IN) cultivated 1 hour together with 1: 5000 dilution horseradish peroxidase-bonded goat anti-mouse IgG down at 22 ℃ subsequently.(Piscataway NJ) observes band for ECL+, Amersham.Pharmacia Biotech to utilize chemiluminescence detection reagent.

The result of embodiment 3A

The clone of ancylostoma caninum (A.caninum) MTP cDNA utilizes mixed serum screening ancylostoma caninum (A.caninum) the L3 cDNA expression library of collecting from the inland of China patient with high resisting-ancylostome L3 titre.Identify 12 positive colonies, wherein 6 to be confirmed as by dna sequencing be identical.Each clone contains a 3 ' poly A tail, is truncate at 5 ' end still.Be used to primer from the nematicide spliced leader sequence together with gene-specific primer P1 by PCR from ancylostoma caninum (A.caninum) L ₃That cDNA is separated to is 5 ' terminal (people 1995 such as Hawdon; People such as Bektech, 1988).

The full-length cDNA with poly A tail is not that 1703bp (referring to Fig. 8 A, SEQ ID NO.15) and 547 the amino acid whose open reading frames of encoding (referring to Fig. 8 B, SEQ ID NO.16) calculating molecular weight are 61,730, and pI is 8.72.The ATG start codon starts from two nucleotide places in the terminal downstream of spliced leader sequence, at terminal 23 the untranslated nucleotide altogether that produce of 5 of Ac-mtp-1 cDNA '.A TAA terminator is positioned at nucleotide 1666 to 1668, and then one contains 35bp 3 ' UTR (Blumenthal and Steward, 1997) that AATAAA gathers the adenosine signal, and it is at the 12bp place of poly A tail upstream (base 1687-1692).Hydrophobic signal peptide of the predicted representative of amino acid/11 to 16 of the protein sequence of deriving, possible shearing site between the 6th Ala and the 7th 's Gly people such as (, 1995) Nielson.The sequence of inferring contains the glycosylation site (N-X-S/T) that 2 possible N-are connected at Asn39 and Asn159.

The aminoacid sequence that utilizes Ac-MTP-1 prediction is by blast search (people such as Altschul, 1997) GenBank shows with the family member who is called the zinc metalloprotein enzyme of 3,4,3',4'-tetraketo-.beta.-carotene significant homology (Bond and Benyon, 1995), the zinc metalloprotein enzyme of 3,4,3',4'-tetraketo-.beta.-carotene is by the digestible protein enzyme 3,4,3',4'-tetraketo-.beta.-carotene name of Procambarus clarkii crayfish (Astacus astacu).With Ac-MTP-1 deduced amino acid search protein structure data base (people such as Apweiler, 2000) there is characteristic 3,4,3',4'-tetraketo-.beta.-carotene finger printing in demonstration, comprise the zinc binding structural domain of extension and conservative Met corner, described corner is positioned at 37 aminoacid places, downstream.The catalyst structure domain that contains the zinc binding site is followed one with the domain that comes from epidermal growth factor (EGF), from aminoacid 334 to 368.From aminoacid 374 to 484 is with the CUB domain relatively poor homologous domain to be arranged, this name be because its at complement subfraction C1r/C1s, embryo's Hemicentrotus seu Strongylocentrotus albumen UEgf and BExistence among the MP-1.EGF is the same with the CUB domain in the 3,4,3',4'-tetraketo-.beta.-carotene metalloproteases, and believes the mutual work (Bond and Benyon, 1995) that can participate in protein-protein.

It behind the N-signal peptide preceding-peptide domain that one section 119 aminoacid is rich in spiral.The C-end of preceding peptide domain contains 4 basic amino acids (R-E-K-R) from the 132nd amino acids to the 135 amino acids, it is the possible recognition site (Bond and Benyon, 1995) of furin or other trypsin-like processive enzyme.To activate Ac-MTP-I and become the form after a kind of 412 aminoacid processing of supposition at the Proteolytic enzyme in this site, the MW of calculating is 46419, and pI is 8.04.

The RT-PCR of phase specificity analyzes: the phase specificity that Ac-mtp-1 expresses is studied by the qualitative RT-PCR from the cDNA of the ancylostoma caninum of different developmental phases.The Ac-mtp-1 Auele Specific Primer is designed to increase corresponding to the 434bp part of the Ac-mtp-1 cDNA of the nucleotide 985 to 1419 of sufficient sequence.The product of prediction size is from non-activated and activatory L ₃The cDNA amplification, but not from ancylostoma caninum worm's ovum or L ₁/ L ₂The cDNA amplification of mix stages.In sophisticated cDNA, observe a band that intensity is less.Long fragment is from the genomic DNA amplification, shows that described primer crosses over an intron, and confirmation derives from the amplification of cDNA and the genomic DNA of uncontamination from the amplicon of cDNA.The ancylostoma caninum pKAc of contrast primer amplification part constitutive expression people such as (, nineteen ninety-five) Hawdon, its from all DNA samples success amplify product, show to have the template that can increase.

Expression and the immunoblotting of reorganization MTP: reorganization MTP-1 results from the escherichia coli, and by the Ni column chromatography purification, is used for immune BALB/c mouse and produces specific antisera.Absorption is at the antiserum of colibacillary lysate and be used for determining whether Ac-MTP-1 is by ancylostoma caninum (A.caninum) L ₃External excretory.From 10,000 non-activated (not feedings) and activatory (feeding) L ₃The ES product utilization rMTP-1 antiserum that obtains is analyzed by Western blot.The total length of expressing in antiserum identification e. coli bl21 (DE3) cell and the rMTP-1 of processing (that is, not having preceding peptide domain) form still can not discern any band in the only carrier-containing inducing cell lysate.

MW is 47.5 and 44.5 band in the ES product of rMTP antiserum identification 10,000 ancylostoma caninums (A.caninum) L3, described ancylostoma caninum (A.caninum) thus L3 has been activated the external feeding that restarts.Antiserum can not be discerned only 10,000 non-activated L in culture medium ₃ES in or any band in ripe ancylostoma caninum (Ancylostoma canium) ES product or the ancylostome lysate (not shown).The slower band of migration and the rMTP of form processing have similar MW (47.5 couples 46.5) in activatory ES, and this is presented at ancylostoma caninum between external pot-life (A.caninum) L ₃Discharge the MTP-1 of processing.Lower MW band also by mice serum (not shown) identification before the immunity, shows antiserum identification and the incoherent albumen of Ac-MTP-1.In order to confirm that this identification is nonspecific, absorption is at the mouse resisting anteserum of BL21 (DE3) cell of expressing total length MTP-1 and be used to survey Western blot.The antiserum of absorption fails to discern rMTP-1 arbitrarily, but discerns the band of MWr=44.5 in the activatory ES product, shows that it is nonspecific discerning low-molecular-weight band by antiserum.

Though reorganization MTP-1 is lived in the serum of the individuality in non-infection district (Shanghai) and can not be discerned the rMTP-1 (not shown) by the pooled serum identification that is used to screen the library.

Separation and the evaluation of embodiment 3B.MTP-1 cDNA

Thereby the patient's of China infection ancylostome serum separates the cDNA from ancylostoma caninum (A.caninum) L3 expression library as probe and identifies, the zinc metalloprotein enzyme (Ac-mtp-1) of a 3,4,3',4'-tetraketo-.beta.-carotene family of described expression library coding.Be used to be structured in 1 ZapII (Stratagene according to the description of manufacturer from the patient's in Chinese Anhui Province mixed antiserum screening, La Jolla, CA) ancylostoma caninum (A.caninum) (Baltimore strain) L3 cDNA expression library (people such as Hawdon, nineteen ninety-five), be main ancylostome species little province of middle Guoan Ancylostoma duodenale (A.Duodenale).Use have low faecal egg number and for the patient's of the circulating antibody of the antigenic high titre of the full lysate of ancylostoma caninum (A.caninum) L3 serum, this situation shows that these patients may resist the infection of ancylostome.Behind plaque purification, reclaim the clone of six identical truncates.Utilize the nematicide spliced leader sequence to separate 5 ' terminal people such as (, nineteen ninety-five) Hawdon by nest-type PRC from ancylostoma caninum (A.caninum) L3 cDNA together with two kinds of gene-specific primers, and two the independently 5 ' terminal clones that check order.

The result of embodiment 3B

The sequence of amplification is considered to represent whole 5 ' ends of transcript, because after the ATG start codon of prediction is first methionine of spliced leader sequence, the characteristic signal peptide of the aminoacid of preceding 16 suppositions coding secretory protein (people such as Nielson, 1997), and show that with the comparison of metalloid protease this is complete aminoacid sequence.The full-length cDNA with poly A tail is not the 1703bp and one section 547 the amino acid whose open reading frame of encoding, and the molecular weight of calculating is 61,730, and pI is 8.72.The 1st to the 16th a kind of hydrophobic signal peptide of the predicted representative of aminoacid in the protein sequence of deriving, possible shearing site between Ala16 and Gly17 people 1997 such as () Nielson.Described protein sequence contains the glycosylation site (NX-S/T) that two possible N-are connected at Asn39 and Asn159.The aminoacid sequence that utilizes Ac-MTP-1 prediction is by blast search (people such as Altschul, 1997) GenBank shows and is called 3,4,3',4'-tetraketo-.beta.-carotene (Bond and Beynon, the member of zinc metalloprotein enzyme family nineteen ninety-five) has significant homology, the degrading proteinase of described 3,4,3',4'-tetraketo-.beta.-carotene called after Procambarus clarkii Astacus astacus.This family member is characterised in that one section is used for excretory weak point-terminal signal peptide with its targeting, then propetide, an and catalyst structure domain that contains characteristic zinc land and " methionine corner ".Do not resemble 3,4,3',4'-tetraketo-.beta.-carotene, most other family member is contained C-end structure territory, comprises the EGF and the CUB domain (Bond and Beynon, 1995) of different numbers.With Ac-MTP-1 deduced amino acid search protein structure data base (people such as Apweiler, 2000) existence of indicating characteristic 3,4,3',4'-tetraketo-.beta.-carotene fingerprint, zinc land and a conservative methionine corner that is positioned at 37 aminoacid downstreams of comprising an extension.Contain after the catalyst structure domain of zinc binding site be one with the domain that comes from epidermal growth factor (EGF), from the 334th to the 368th amino acids.Be one from the 374th to the 484th amino acids and have domain than low homology with the CUB domain, this name is because its C1r/C1s in the complement subfraction, existing among embryo's Hemicentrotus seu Strongylocentrotus albumen Uegf and the BMP-1 (Bork and Beckman, 1993).

The 3,4,3',4'-tetraketo-.beta.-carotene metalloproteases is synthesized the preferment into inactivation.Remove propetide by processive enzyme and activated described enzyme (Bond and Beynon, nineteen ninety-five).Ac-MTP-1 contains one section 119 amino acid whose N-end structure territories, its prediction be used for trypsin-or 4 aminoacid recognition site (R of woods albumen processive enzyme not ₁₃₂E ₁₃₃K ₁₃₄R ₁₃₅) be positioned at its C-end (Bond and Benyon, 1995).To activate Ac-MTP-1 and become the form after a kind of 412 aminoacid processing of supposition at the Proteolytic enzyme in this site, the MW of calculating is 46419, and pI is 8.04.Propetide is also predicted to contain four amphiphilic alpha-helixs, separates (people such as Kelley, 2000) by the bonding pad (from the 23rd amino acids to the 86 amino acids) of a weak point.

By carrying out the phase specificity expression that qualitative RT-PCR studies Ac-mtp-1 from the cDNA of ancylostoma caninum (A.caninum) a plurality of stages of development.Auele Specific Primer is designed to increase corresponding to the part A c-mtp-1cDNA of the 434bp of complete sequence nucleotide 985-1419.The product of inferring size increase from disactivation and activatory L3 cDNA and obtains, but does not obtain from the cDNA amplification of ancylostoma caninum (A.caninum) worm's ovum or L1/L2 mix stages, and demonstration Ac-mtp-1 mainly is expressed in the L3 stage.Band than weak intensity is observed in ripe cDNA.Though this band is faint, can not draw conclusion, because RT-PCR only is used for qualitative analysis about gene expression amount.Yet utilize Western blot that mouse anti-rMTP serum carries out ripe lysate to fail to discern arbitrary protein (not shown) in ripe ES or the lysate.This is limited to the L3 stage with regard to the expression that shows Ac-MTP-1, and the courier who is present in the maturation period is not translated or may partly be degraded.

Reorganization MTP-1 results from the escherichia coli, and carries out purification by the Ni column chromatography, and is used for immune BALB/c mouse generation specific antisera.Absorption is at the antiserum of escherichia coli lysate and be used to determine whether Ac-MTP-1 is external excretory by ancylostoma caninum (A.caninum) L3.The ES product of collecting from the L3 of 10,000 non-activated (not feedings) and activatory (feeding) (Hawdon and Schad, 1993) utilizes the rMTP-1 antiserum to analyze by Western blot.Antiserum is identified in the total length expressed in e. coli bl21 (DE3) cell and the rMTP-1 of processing (that is, not having preceding peptide domain) form, but can not discern any band in the only carrier-containing inductive cell lysate.Article one, the band than small-molecular weight is observed and the big or small rMTP (promptly lacking presequence) that is similar to processing, and some rMTP that are presented at expression in escherichia coli accepted external shearing at the C-of propetide end.This may be autocatalytic shearing, though the non-specific shearing of being undertaken by bacterialprotease also is possible.Autocatalysis also may have been represented the body physiological activate mechanism of Ac-MTP-1.

It is 47.5 and 44.5 band that rMTP antiserum identification has been activated with Mr in the ES product of 10,000 ancylostoma caninums (Ancylostoma canium) L3 of external feeding again.Antiserum can not be discerned among the ES that does not activate L3, only in culture medium, or any specific band in ripe ancylostoma caninum (A.aninum) ES product or ancylostome pyrolysis product (not shown).A band that slowly moves has and the similar molecular weight of the form processing of rMTP (47.5 pairs 46.5) in activation ES, has shown that ancylostoma caninum (Ancylostoma canium) L3 discharges the MTP-1 that has processed in external activation process.In addition, MTP-1 only restarts the stimulation of feeding and discharges in response to activation L3, therefore, and most probable work in some stage of infectious process (Hawdon etc., 1996).Previously described metalloprotein hydrolysing activity also by specific release, and has similar molecular size (Hawdon etc., 1995) between active period, show that Ac-MTP-1 may be responsible for this activity to small part.

The band (molecular weight is 44.5kDa) of a lower molecular weight in the activation ES product also by the mice serum identification (not shown) before immune, shows a kind of albumen that has nothing to do with Ac-MTP-1 of antiserum identification.In order to confirm that this kind identification is nonspecific, absorption is at the mouse resisting anteserum of the Bacillus coli cells of expressing total length MTP-1 and be used to survey Western blot.The antiserum that is adsorbed can not be discerned rMTP-1 arbitrarily, is 44.5 band but discern molecular weight in the activatory ES product, shows that antiserum is nonspecific to the identification of low-molecular-weight band.Recombinant MTP-1 is used to screen the pooled serum identification in library, but can not discern the rMTP-1 (not shown) from the serum of the individuality of living in non-diseased region (Shanghai).

Though the definite function of Ac-MTP-1 is unknown, the phase specificity of expression and the specificity free list between active period are understood the decisive role in the infectiousness process.Therefore, the intervention to function in the Ac-MTP-1 body provides a kind of available strategy of developing the uncinariatic vaccine of control.

This embodiment has proved that MTP-1 is a kind of by the used important enzyme of ancylostome parasite invasion, and described albumen is immunodominant antigen, although because its discerned from being exposed to the serum that ancylostome has the patient of low ancylostome quantity repeatedly.Therefore MTP is a kind of noticeable material standed for that is used for vaccine antigen.

The canine vaccines test that embodiment 3C. carries out with Ac-MTP-1 antigen

Whether Ac-MTP-1 is a kind of effective vaccine for test, the male beagle of 5 purposiveness-breedings in two groups of 8+1 age in week with reorganization (from expression in escherichia coli with separate) preparation of fusion rotein and AS02A adjuvant, or only adjuvant comes immunity inoculation.It has been successfully used to several malaria vaccine clinical researches the compositions of AS02A, is described in other places (Lalvani etc., 1999; Bojang etc., 2001; Kester etc., 2001).Concrete the care of animal and raising condition before be in the news (Hotez etc., 2002a).The recombination fusion protein purification that contains the poly histidine mark is from the 10mM Tris HCl that is dissolved in 6M HCl guanidine, the E.coli inclusion body that has washed among the pH8.0.Dissolved inclusion body (is containing 0.1NaH2PO4 by gel filtration chromatography, the SaphacrylS-300 that pre-equilibration is crossed in the buffer of 10mMTris-HCl and 6M guanidine, 26/60 solvent resistant column [AmershamPharmacia]) at room temperature (2ml/ minute flow velocity) with the 5-10ml batch machining.Method (2001) according to Singh etc. is mixed the selected fraction that contains Ac-MTP-1 (as by going up assay determination at sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), folding again, go up sample then to the Hi-Trap IMAC post (Amersham Pharmacia) of 5ml, described Hi-TrapIMAC post ZnCl ₂Electrically charged and be equilibrated at the sodium phosphate (pH7.2) of 50mM, among the carbamide of 1M and the NaCl of 0.5M.Subsequently with the level pad of 15 times of column volumes flushing post, and with the sodium phosphate (pH7.2) of 50mM, the carbamide of 1M, the NaCl of 0.5M, and the albumen of the ethylenediaminetetraacetic acid of 50mM (EDTA) elution of bound.Eluting contain that proteic sample mixes and with the Tris-HCl (pH8.0) of 10mM, 5% glycerol, the dithiothreitol, DTT of 1mM, and the EDTA of 2mM dialyses.The reorganization Ac-MTP-1 of purification does not demonstrate enzymatic activity (data not shown).

Reorganization Ac-MTP-1 fusion rotein and SBAS2 adjuvant be mixed to be incorporated in and to carry out 4 intramuscular injections in the 1st, 4,43 and 50 days and be applied in 5 Canis familiaris L.s each.Every Canis familiaris L. is accepted the recombination fusion protein of the about 140 μ g of every dosage and the ASO2A of 0.5ml.Give 5 Canis familiaris L. intramuscular injection AS02A according to identical administration flow process.After the immunity, collect blood and separation of serum cold preservation at-20 ℃ by venipuncture weekly.Antigen-specificity dog IgG2 and IgE antibody are by (Hotez etc., indirect enzyme-linked immunosorbent assay 2002a) (ELISA) is measured as described previously.From non-activated L3 and under host's incentive condition the secretory product of activatory L3 immunoblotting as previously mentioned (Zhan etc., 2002) utilize the pooled serum that obtains from the Canis familiaris L. of Ac-MTP-1-immunity inoculation to carry out.Immune the last time back 14 days of the Canis familiaris L. of each research usefulness is with the subcutaneous infection of 500 ancylostoma caninums (Ancylostoma canium) L3.The source of the ancylostome strain that is used to study be described in other places (Hotez etc., 2002c).The affirmation of the ancylostome kind that is used to study is confirmed succeeded by restriction fragment length polymorphism (Hawdon, 1996) by the polymerase chain reaction.After the infection, weekly Canis familiaris L. is got blood by venipuncture and obtain complete blood count (CBC).Before the last and obduction of vaccination flow process, also obtained the chemical characteristic of serum.The quantitative hookworm ovum counting (McMaster technology) of every Canis familiaris L. (PI) beginning in the 12nd day after infection obtained weekly in 3 days.Infect the 5th week of back, put to death Canis familiaris L. by the intravenous injection barbiturate, and in the autopsy process, reclaim ripe ancylostome and counting from the ripe ancylostome of small intestinal and large intestine (Hotez etc., 2002c).Statistical significant difference between the ripe ancylostome quantity utilizes variance analysis experiment (Anovatest) to determine, as the difference of hematologic parameter and quantitative hookworm ovum quantity.Ancylostome quantity and ovum quantity and antibody titer relatively utilize relevant mensuration of Joseph Pearman grade (non-parametric).

Use Coomassie blue stain after the SDS-PAGE of Ac-MTP-1 recombination fusion protein analyzes, show that the albumen of migration has the apparent molecular weight of the 61kDa of proenzyme prediction.Shown equally with having the ternary band that low apparent molecular weight moves, may be corresponding to the processed Ac-MTP-1 of part.After the immunity, each Canis familiaris L. of accepting vaccine has manifested between 1: 40, and 500 and 1: 364, the IgG2 of the high titre between 500 the scope is anti--the Ac-MTP-1-specific antibody; Between 1: 500 and 1: 1, the anti-Ac-MTP-1-specific IgE antibody between 500 scopes was replied.Discern the albumen of ternary tight migration from the serum of vaccinated Canis familiaris L., the predicted molecular weight that it has proenzyme and activates the Ac-MTP-1 that is processed to form in the secretory product of L3 the host does not then have in non-non-activated L3 secretory product.Other band may be also corresponding to other by the excretory associated metal protease of ancylostoma caninum (Ancylostoma canium) L3; At least 3 sequence marks from the closely related expression of ancylostoma caninum (Ancylostoma canium) L3 are found in (ncbi.nim.nih.gov/dbEST/index.html) among the dbEST data base.

Generally speaking, the ripe ancylostome number (154+34 ancylostome) (average+standard deviation) that reclaims from vaccinated Canis familiaris L. is compared the significant difference that does not have on the statistics with the ripe ancylostome number (143+30 ancylostome) that reclaims from the contrast Canis familiaris L..Yet shown in Figure 33 A, reclaiming ripe ancylostome number in vaccinated Canis familiaris L. intestinal with high resistance ancylostoma caninum IgG2 antibody titer has remarkable minimizing on the statistics.Joseph Pearman correlation coefficient between antibody titer and the ripe ancylostome quantity is-0.89 (P=0.04).Reclaim from the Canis familiaris L. ancylostome number (98 ancylostomes) with the highest antibody titer with reclaim the ripe ancylostome number (189 ancylostomes) that oneself has the Canis familiaris L. of minimum antibody titre and compare the minimizing that is equivalent to 50% helminth number.Identical relation is indicated between IgG2 antibody titer and the intermediate value ovum counting (Figure 33 B).

These studies show that Ac-MTP-1 can provide as the antigenic downstream indication of a kind of anti-hookworm vaccine.

Embodiment 4. uses Ac-TMP, Ac-AP, and the canine vaccines that Ac-APR-1 antigen carries out is tested

In order to estimate directly the therapeutic effect that whether ancylostomiasis is had at the antibody of parasite enzyme and enzyme inhibitor, carry out the inoculation test of dog para-immunity, the recombination fusion protein of encoding mature ancylostoma caninum (A.caninum) protease or protease inhibitor is used in described test.Because can only obtain a small amount of albumen from the ancylostome that lives, testing these molecules as candidate vaccine need be in the expression of the recombinant vector in protokaryon or the eucaryon host system, and the recombination fusion protein with purification carries out immunity to dog then.

The material and the method that are used for embodiment 4.

The research of Canis familiaris L. and animal husbandry:After animal protection that scheme is set up by George Washington University and the approval of using committee (IACUC); bought specific feeding; the male beagle in 8 ± 1 ages in week of parasite naivety; identify by ear thorn stricture of vagina, and raise the AALAC (assessment of laboratory animal protection and evaluation association) that authorizes in zooscopy mechanism of George Washington University.Under 70+4 room temperature, Canis familiaris L. is housed in and is used for research in the room, per hour takes a breath 10-15 time, be made up of 100% fresh air, and the dark cycle of 12 hours illumination period and 12 hours replaces repeatedly.Monitor air-flow and timer function every day.Recipe with the dog food #8727 of Teklad authentication is raised Canis familiaris L., if anorexia is then replenished with canned soft diet.Drinking water is a defeated inherent filtration water factory and by the automatic water supply system transmission; Water analysis is undertaken by U.S.Army Corps ofEngineers.Water from the facility automatic system is cultivated wherein antibacterial and fungus every year.Enclosure is by flushing everyday and per two weeks carry out once sterilizing.Canis familiaris L. in the given seminar was allowed to live before the ancylostome larva is attacked together and is movable, but was separated stable breeding after infecting.All Canis familiaris L.s were in quarantine an about week before beginning to carry out vaccine test.Before vaccination, obtain complete blood count (CBC), serum chemistry characteristic, and the blood serum sample before the vaccination.

Vaccine research scheme and sample size: vaccine test is designed to test three kinds of different antigens, every kind and Alumen preparation, and adsorbed onto alum adjuvant contrast.24 Canis familiaris L.s are four groups by random assortment altogether, and every group comprises 6 Canis familiaris L.s.The quantity of dog sample is selected the statistics power with 80% (α=0.05, two-tail) based on detecting the enteral ripe ancylostome number of described immunity inoculation group with respect to the ability that the contrast Canis familiaris L. reduces 40-50%.Described data are from personal 400 ancylostoma caninums (Ancylostoma canium) L of previous recovery ₃Infect the age-average and the standard deviation (Hotez etc., 2002) of ripe ancylostome of Canis familiaris L. of coupling.

Recombinant antigen: 6 Canis familiaris L.s of each group inoculate with reorganization ancylostome protein immunization, and reorganization ancylostome albumen wherein is expressed in the escherichia coli or insect cell line that have baculovirus as fusion rotein.Ac-AP (Cappello etc., 1995; 1996) and Ac-TMP be expressed in the escherichia coli as pET 28 (Novagen) fusion rotein (Cappello etc., 1996) that contains the poly histidine mark.Ac-APR-1 (Harrop etc., 1996) is expressed in the baculovirus pBacPAK6 carrier (Clontech), and described carrier is contained a kind of poly histidine-coded sequence and other restriction endonuclease sites (Brindley etc., 2001) by modification.By nickel affinity chromatography purification of Recombinant Ac-AP and Ac-TMP fusion rotein, carry out the second step purification subsequently then.(Cappello etc., 1995 under the situation of Ac-AP; 1996), recombiant protein is by mono-S (Amersham-Pharmacia) ion exchange chromatography purification, and Ac-TMP (Zhan etc., 2002) is by superdex 75 (Amersham-Pharmacia) gel filtration chromatography purification.Ac-APR-1 (Harrop etc., 1996) utilizes pepstatin agarose purification (Brindley etc., 2001) by the substrate affinity chromatography.The antigen stock protein concentration is analyzed (Pierce Chemicals) or is utilized extinction coefficient to measure by sample in the absorptance at 289nm place by Pierce Micro BCA, and described extinction coefficient are made up of the aminoacid of the described fusion rotein of derivation and are calculated.The protein content of Alumen absorption is measured by Pierce Micro BCA analysis and utilization bovine serum albumin standard in each doses of antigen.Each antigen is measured by the analysis of SDS-PAGE (SDS-PAGE) with respect to polluting escherichia coli or the proteic relative purity of insect cell.

Adjuvant formulation:Reorganization Ac-TMP and Ac-APR fusion rotein such as front description (Ghosh etc., 1996) are carried out alum precipitate with the mixture of aluminum potassium sulfate 12 hydrate and sodium bicarbonate.Described method need precipitate with aluminum salt proteic aqueous solution under alkali condition, centrifugal then and washing (Ghosh and Hotez, 1999).Utilize this method, reorganization Ac-AP fusion rotein does not detect in the alum precipitate thing.Therefore, the administration that begins the Ac-AP of two dosage does not have adjuvant.Yet the Ac-AP of last two doses is adsorbed on the unbodied noncrystalline calcium phosphate gel.

The dog immunity: select 4-dosage immune programme for children (Table II).Every Canis familiaris L. inoculates by subcutaneous immunity in two sites of shoulder by No. 22 pins.The injection volume between 0.5 and 1.0ml between.The administration in 38-days time of every kind of antigenic four dosage.The double injection (elementary immunity) of beginning was used at the 1st day and the 4th day, and last twice immunity inoculation (reinforcement) used at the 34th day and the 38th day.The Alumen of the Canis familiaris L. injection same amount of matched group.

Table II. be used for the antigen amount and the adjuvant of every dog immunity inoculation.

	????Ac-AP	????Ac-TMP	????Ac-APR-1	Alumen
					Dosage 1 (the 1st day)	????100μg	????71μg	????12.5μg-	??-
Adjuvant	Do not have	Alumen	Alumen	Alumen
					Dosage 1 (the 1st day)	????100μg	????71μg	????12.5μg-	??-
Adjuvant	Do not have	Alumen	Alumen	Alumen
					Dosage 1 (the 1st day)	????180μg	????61μg	????95μg-	??-
Adjuvant	Calcium phosphate	Alumen	Alumen	Alumen
					Dosage 1 (the 1st day)	????273μg	????69μg	????86μg-	??-
Adjuvant	Calcium phosphate	Alumen	Alumen	Alumen

The dog TPPA: collect blood and separation of serum and cold preservation weekly in-20 ℃ by venipuncture.Antigen-specificity dog IgG1 antibody is measured by indirect elisa (ELISA).Owing to do not obtain suitable high-quality dog-specific reagent, other IgG subclass does not have determined.Sample serum and enzyme-joint inspection is surveyed the optium concentration of antibody by the test board titration measuring.Best antigen concentration utilizes saturation technique to measure.NUNC Maxisorp F96 authentication dull and stereotyped (Rosklide, Denmark; Lot number 045638) wraps quilt with the antigenic 0.05M carbonate-bicarbonate buffer of every hole 0.1ml (pH9.6).The flat board of sealing is cultivated at 4 ℃ spend the night (ON), then by DYNEX Opsys ^TM(Chantilly is VA) with PBS (pH 7.2) washing 3 times for dull and stereotyped washing machine.This flat board (pH7.2) was handled 1.5 hours down in room temperature (RT) with the 0.15MPBS that contains 0.5%Tween 20 (PBS-Tween 20) of every hole 0.25ml, decant, and blot with napkin.The test sera preparation of various serial dilution degree is in the PBS-Tween 20 of 0.1ml and 4 ℃ of incubated overnight.After the washing, 0.1ml is coupled to alkali phosphatase, and (anti-dog IgG1 TX) is added in each hole with 1: 1000 dilution factor for Bethyl laboratory, Montgomery.After following 1.5 hours of the room temperature, dull and stereotyped with 0.1ml 2.5mM right-nitrophenyl phosphate (Sigma.St.Louis, MO) 10mM diethanolamine (Sigma, St Louis, MO) and 0.5mM magnesium chloride (Sigma, St.Louis washs 10 times with PBS-Tween20 before MO) solution of (pH9.5) is added in each hole.Flat board was cultivated 30 minutes darkling and ((Molecular Devices, Sunnyvale is CA) at 405nm wavelength place reading for SpectraMax 240PC readout instrument Ca) for Molecular Devices, Sunnyvale by having SOFTmax Pro software.The average optical of contrast dog serum is as benchmark.Serum dilution greater than 3 times of said reference is counted as titration end-point at last.The geometrical mean of these terminal points is calculated according to six dogs from each group.

Dog hookworm infection and parasite are reclaimed: immunity in the end is after 14 days, and every Canis familiaris L. being studied is used 400 ancylostoma caninums (Ancylostoma canium) L that is included in the capsule ₃Carry out oral infection.The source of the ancylostome strain that is used to study is described in other places (Hotez etc., 2002).The affirmation of the ancylostome kind that is used to study confirms succeeded by restriction fragment length polymorphism (Hawdon, 1996) by the polymerase chain reaction.After the infection, Canis familiaris L. is got blood weekly to obtain complete blood count (CBC) by venipuncture.The serum chemistry characteristic also finishes and obtained before postmortem examination in vaccination.The quantitative hookworm ovum counting of each Canis familiaris L. (McMaster technology) begins after infecting 12 days with day acquisition on every Wendesdays.After infecting for five weeks, Canis familiaris L. is condemned to death by the intravenous injection barbiturate, reclaims sophisticated ancylostome and counting (Hotez etc., 2002) from small intestinal and large intestine when obduction.The sex of every ripe ancylostome is determined by perusal.When implementing 8 Canis familiaris L.s of euthanasia (two Canis familiaris L.s are from each group in four groups) every day, obduction carried out in three day time.Separate the small intestinal of about 1-2cm and place formalin to be used for histopathological analysis in the future.

Statistical method: the percentage ratio that ripe ancylostome quantity reduces or increases with respect to matched group in the group of immunity inoculation is represented by following formula:

The statistical significance of ripe ancylostome quantity variance is utilized the nonparametric test method; Kruskal-Wallis and Dunn method, and Mann-Whitney U test determination.The difference of hematologic parameter between the group and quantitative hookworm ovum counting is tested by ANOVA and is estimated.When the test above two was calculated by identical variable, the level of significance should be adjusted according to the number of test.The sex difference of the ripe ancylostome of reclaiming compares by Wilcoxon-Signed Ranks test the carrying out statistics that is used for two relevant groups.If the P value of being calculated is equal to or less than 0.10 (bilateral) or-0.05 (one-sided), then difference is considered to statistically evident.

The result of embodiment 4.

Sophisticated ancylostoma caninum (Ancylostoma canium) antigen: 3 reorganization ancylostoma caninum (Ancylostoma canium) antigens are selected for the dog immunity inoculation.Wherein two Ac-AP and Ac-TMP only are by the excretory protease inhibitor of adult stage ancylostome.Ac-AP is 91 amino acid whose factor Xa inhibitor anticoagulants (Cappello etc., 1995; 1996), and Ac-TMP is the tissue depressant of the metalloproteases of 140 amino acid whose suppositions, and is the albumen by the excretory abundance of ancylostoma caninum (Ancylostoma canium).The antigen of the 3rd selection is Ac-APR-1, is 422 amino acid whose aspartic acid cathepsins (Harrop etc., 1996).SDS-PAGE carries out Coomassie blue stain after analyzing recombination fusion protein.Positive according to expectation, the apparent molecular weight that recombination fusion protein Ac-APR-1 and Ac-TMP move on SDS-PAGE is respectively 45,000 and 18,000.The predicted molecular weight that is expressed as the Ac-AP of the pET28 fusion rotein with the terminal poly histidine mark of a N-is 12,191Da (Cappello, 1996).By SDS-PAGE, it is 22,000 main band and one about 15 that reorganization Ac-AP fusion rotein is observed to a molecular weight, the fainter band that 000Da place moves.This observed result can be corresponding to the formation of polypeptide oligomer.This phenomenon appears in the purge process of Ac-AP natural product (Cappello etc., 1995) in the past.The inhibition activity of factor Xa, the dna sequence analysis of the pET28 plasmid of coding reorganization Ac-AP fusion rotein, and the amino terminal peptide sequence analysis that undertaken by the Edman edman degradation Edman of 22kDa band has confirmed the homogeneity (data do not show) of gene outcome.

The dog antibody response. the dog immunization program of selection is, elementary immunity is carried out in the administration of twice (the 1st day and the 4th day) subcutaneous dosage during 4 days of beginning, and then twice subcutaneous immune strengthening subsequently began (the 34th day and the 38th day) in back 30 days in elementary immunity and carry out.Ac-TMP and Ac-APR-1 are with the form injection of Alumen-protein precipitation.On the contrary, Ac-AP does not form precipitate with Alumen.Therefore, for initial two doses, the Ac-AP subcutaneous administration does not have adjuvant.Yet,, use the scheme of calcium phosphate gel to show precipitate A c-AP (data do not show) effectively second and days time period of the 30-between the immunity for the third time.Because this reason, calcium phosphate are selected as the Ac-AP immunity administration that adjuvant is used for last two doses.

The geometrical mean of the IgG1 antibody titer of 3 vaccine antigens is presented among Figure 34 A-C.In Canis familiaris L. (Figure 34 A) at the Ac-APR-1 immunity inoculation, after about 6 weeks of elementary immunity, then carry out twice last immune strengthening, antigen-specific IgG 1 increases.In contrast, anti-Ac-TMP IgG1 antibody response is further strengthened (Figure 34 B), and in elementary immunity 2 weeks of back, is beginning to increase for the third time with before the 4th dosage.Anti-Ac-TMP antibody titer increases for the second time after twice last reinforcement, and titre surpasses 1: 10, and 000.There are five not resist former generation immunne response in the Canis familiaris L. of six anti-Ac-AP of immunity inoculation.Shown in Figure 34 C, demonstrate antigen-specific antibody after single dog twice administration in the end that the Ac-AP immunity inoculation is replied and reply.

The number that reclaims the ripe ancylostoma caninum (Ancylostoma canium) that ripe ancylostoma caninum (Ancylostoma canium) ancylostome reclaims from the small intestinal of immunity inoculation Canis familiaris L. from small intestinal is presented at the Table III.The immunity inoculation Canis familiaris L. with respect to only with Alumen separately in the Canis familiaris L. body of injection the minimizing of ancylostome number within 4.5% to 18% scope.Above-mentioned minimizing can not show fully statistical significance between the group (Kruskal-Wallis test, P=0.19).Yet, the probability that the ancylostome decreased number 18% that the small intestinal of the Canis familiaris L. of Ac-APR-1 reclaims (a group maximum minimizing) is arranged from immunity inoculation by the Dunn method less than 0.05, and by the one-sided inspection of Mann-Whitney U less than 0.03.The Canis familiaris L. of the anti-Ac-TMP of immunity inoculation also demonstrates the minimizing (10.8%) of ripe ancylostome quantity, although these are not statistically evident.Do not demonstrate 5 Canis familiaris L.s of anti-Ac-AP antibody response, demonstrate the minimizing of inapparent ancylostome quantity yet.Yet, have the independent Canis familiaris L. of significant anti-Ac-AP antibody response, demonstrate ripe ancylostome quantity and reduce 34.7%.As shown in Table III, data can not provide the Canis familiaris L. of ample evidence proof immunity inoculation and the quantitative hookworm ovum counting between the contrast Canis familiaris L. that significant minimizing is arranged statistically.Similarly, immunity inoculation does not influence the hematologic parameter of Canis familiaris L., comprises hematocrit, hemoglobin, white blood cell count, and eosinophilic granulocyte's (data do not show).Positive according to expectation, the challenge dose that is used for the ancylostome of this research does not cause anemia (data do not show) in the Canis familiaris L. of contrast Alumen injection.

Reclaim sophisticated ancylostoma caninum (Ancylostoma canium) from colon.

Table III. the Canis familiaris L. of immunity inoculation is with respect to the minimizing of ripe ancylostome in the dog small intestine of Alumen-injection

Experimental group	The Canis familiaris L. numbering	Helminth			Reduce %
								Average	????SD	Intermediate value
Contrast	????6	????176	????22	????180
					??Ac-AP	????5	????168	????36	????170	????4.5
??Ac-AP *	????1	????115		????115							????34.7
					??Ac-TMP	????6	????157	????26	????161	????10.8
??Ac-APR-1	????6	????144	????31	????138							????18 **

^*Positive immunne response

^*P＜0.05 (Dunn method)

Otherwise from the ripe ancylostome decreased number that the small intestinal of immunity inoculation Canis familiaris L. reclaims, the ripe ancylostome number that reclaims from colon but increases (Table IV) accordingly.

Table IV. the Canis familiaris L. of immunity inoculation is with respect to the increase of ripe ancylostoma caninum in the colon of the Canis familiaris L. of Alumen-injection

Test

1 group	The Canis familiaris L. numbering	Helminth	Increase %
						Average	Standard deviation	Intermediate value
Contrast	????4	????6							????8	????4
			?Ac-AP	????5	????17	????17	????14	????183
?Ac-AP *	????1	????71								????71	????1083
			?Ac-TMP	????4	????36	????11	????32	????500 **
?Ac-APR-1	????5	????24							????11	????27	????300 **

^*Positive immunne response

^*P＜0.05 (Dunn method)

The increase of the ripe ancylostome number that reclaims from large intestine be statistically significant (Kruskal-Wallis test, P=0.07).The Canis familiaris L. of immunity inoculation Ac-TMP (increasing by 500%) or Ac-APR-1 (300% increases) demonstrates significantly increase (Dunn method, P＜0.05) on the statistics with respect to the Canis familiaris L. with the Alumen injection.With Ac-AP inoculation but do not demonstrate Canis familiaris L. that antigen-specific antibody replys and significantly increase on the statistics not having aspect the ripe ancylostome number that reclaims from colon.Yet one Canis familiaris L. with significant anti--Ac-AP antibody response demonstrates that ripe ancylostome number has 1083% increase in its colon.

In a word, between inoculation and contrast Canis familiaris L., there is not significant difference (data do not show) for the total statistics that makes up ripe ancylostome from small intestinal and large intestine recovery.On the contrary, the significance migration that causes ripe ancylostome to leave small intestinal to the antigenic antibody response of recombinant ancylostome and enter colon.In the Canis familiaris L. of Ac-TMP and Ac-APR-1 inoculation, ripe ancylostome is with respect to the ratio of ripe ancylostome in the colon 43.9 ratios that are reduced to respectively between 6.6 and 7.3 from Alumen-injection Canis familiaris L. in the small intestinal.Show in the independent dog small intestine of anti-Ac-AP antibody response with colon in the ratio of ancylostome quantity be 1.6, showing has almost half ancylostome quantity to transfer in the colon in this Canis familiaris L..

The difference of sex-dependence. migration is not wait away from the small intestinal and the male and female ancylostome number of moving to colon.As shown in figure 35, more general from the female sexual maturity ancylostome of colon recovery than the male ancylostome that is recovered to.For the Canis familiaris L. that inoculates with Ac-APR-1 (P=0.04) and Ac-AP (P=0.06), colonizing in female ancylostome quantity in the colon more is significant statistically.Male ancylostome more may be recovered to from small intestinal than female ancylostome, although difference is not significant on the statistics.It is not to carry out on the ancylostome of the small intestinal that is saved for histopathological analysis that is attached to the 1-2cm section that male and female are differentiated.The average of ancylostome number is between the scope of 5 and 6 helminths in these sections.These a spot of helminths do not have significance contribution (data do not show) to the scoring of male and female dependency difference.

These examples have proved that it is feasible causing with the recombination fusion protein seeded with mammalian that antigenic specificity replys, and described antibody response is relevant to the transfer of ancylostome parasitic in the minimizing of ancylostome quantity in the intestinal or the intestinal.

The canine vaccines test of embodiment 5.Ac-MTP-1 and Ac-TTR

The minimizing of embodiment 5A. antibody titer and ancylostome

Acquisition is tested in the antigen A c-MTP-1 of escherichia coli and the vaccine test of Ac-TTR Canis familiaris L..Antigen is with adjuvant SBAS2 administration.Vaccinated animal demonstrates high-caliber dog IgG2 antigen-specific antibody, and the appropriateness of antigen-specific IgE increases.Subsequently, Canis familiaris L. is attacked by subcutaneous injection L3 ancylostome larva.

Shown in Figure 36 A and B, have certainly high anti--ancylostoma caninum (Ancylostomacanium) IgG2 is anti--the ripe ancylostome number that reclaims in the intestinal of the immunity inoculation Canis familiaris L. of MTP-1 antibody titer has on the statistics and reduce significantly.Joseph Pearman correlation coefficient between antibody titer and the ripe ancylostome quantity is-0.89 (P=0.04).Compare the minimizing that is equivalent to 50% helminth quantity from the ancylostome number that the Canis familiaris L. with the highest antibody titer (98 ancylostomes) is reclaimed with the ripe ancylostome number that reclaims from Canis familiaris L. with minimum antibody titre (189 ancylostomes).Identical relation is indicated between IgG2 antibody titer and the quantitative ovum counting of intermediate value.

Use Coomassie blue stain after the SDS-PAGE of Ac-MTP-1 recombination fusion protein analyzes, show that migration albumen has the apparent molecular weight of the predicted molecular weight 61kDa of proenzyme.Similarly shown ternary band, with lower apparent molecular weight migration, the Ac-MTP-1 that processes corresponding to part probably.After the immunity, each Canis familiaris L. of accepting vaccine manifests the anti-Ac-MTP-1-specific antibody of IgG2 of high titre, and scope is between 1: 40, and 500 and 1: 364, between 500; Anti-Ac-MTP-1-specific IgE antibody is replied scope between 1: 500 and 1: 1, between 500.Discern the albumen of ternary tight migration from the serum of the Canis familiaris L. of immunity inoculation, predicted molecular weight with proenzyme and form processing Ac-MTP-1, described proenzyme and form processing Ac-MTP-1 are in the activatory L3 of host but not in the secretory product of non-activated L3.

About the antigenic use of TTR, can be referring to Figure 37 A and B, a Canis familiaris L. that has high-level IgE and IgG1 antibody at TTR demonstrates ancylostome quantity reduction (6%).

This embodiment has proved that the mammal that inoculates with MTP or TTR has caused protection antibody and replied, and observes the minimizing of ancylostome quantity in having the mammalian body of high antibody titer.

Embodiment 5B. prevents to lose blood owing to immunity inoculation ancylostome antigen and reduces the ancylostome size

Animal is also tested to determine whether prevent to lose blood with the ancylostome antigen inoculation.Show with Ac-TTR inoculation and to have prevented to lose blood (accompanying drawing 38A and B) significantly.Utilize Mann-Whitney test, the two difference of hemoglobin (38B) concentration (P=0.036) between TTR and the adjuvant matched group and hematicrit (38A) concentration (P=0.009) be on the statistics significantly.

In addition, the difference of hemoglobin concentration is interpreted as significantly reducing on the helminth size statistics.Utilization is collected data based on the imaging system of the scanning of reclaiming helminth from the host.Helminth is taken pictures with the digital gamma camera of CoolSnapPro (Media Cybernetics), thereby and utilizes grand ImagePro software to measure the helminth length (mm) that is compared between the definite processing of imaging.Vaccinated group of TTR is with respect to the adjuvant matched group as shown in Figure 39, the helminth size significantly reduce statistically (1 and 2mm between).

This embodiment shows the inoculation with TTR, except reducing ancylostome quantity in the body, equally also reduces and loses blood.

Embodiment 6. chimeric ancylostome antigens

Present embodiment has been studied the two kind different HBcs particulate protective effects of expression corresponding to the peptide antigenic determinant of the aminoacid 291-303 (equally also finding in Ac-ASP-1) of Na-ASP-1.Relative hydrophilic (hydrophobicity and hydrophilic) by research Na-ASP-1 and Ac-ASP-1 speculating acid sequence it is found that two molecules all demonstrate hydrophilic sequence before, and on behalf of the ring of this molecule, the model prediction of described hydrophilic sequence can go out the zone.Peptide can protect mice to avoid challenge infection to the covalency absorption confirmation chimeric molecule of KLH (key hole keyhole limpet hemocyanin).

Two kinds of different chimeric molecules of expressing ASP-1 have been made up.ICC-1546 expresses the bolt architecture of ASP-1 aminoacid 291-303 as " encircling out ", and ICC-1564 expresses identical peptide as the N-end structure.Previous research has proved mouse anti L3 antibody recognition ICC-1546, and nonrecognition ICC-1564.

The antigen chimera use as mentioned above aluminium glue as adjuvant by administration.DSM (the dissolved film extract of detergent of ripe ancylostoma caninum (Ancylostoma canium)) is as negative control.The attack of larva is undertaken by the larva in subcutaneous injection L3 stage.

The result shows that the Canis familiaris L. with arbitrary particle immunity inoculation produces high-caliber anti-particle antibody.The direct anti-hepatitis core antigen composition of most of antibody.Yet, in a Canis familiaris L. body, have high-level anti-ASP-1 (with anti--peptide) antibody with the ICC-1546 inoculation.This Canis familiaris L. demonstrates the obvious minimizing (Table V) of ancylostome quantity.

Table V. the comparison of anti-ASP-1 antibody and ancylostome quantity

????ICC?1546	Total ancylostome	Anti-ASP-1 IgG1	????IgG2
				????A1	????139	????1∶800	????0
????A2	????181	????1∶800	????0
				????A3	????170	????1∶200	????0
????A4	????180	????1∶800	????0
				????A5	????118	????1∶1,600	????1∶1,600
The A average	????158
????ICC?1564	Total ancylostome	Total ancylostome	????IgG2
				????B1	????135	????1∶100	????0
????B2	????143	????1∶100	????0
				????B3	????206	????1；200	????0
????B4	????195	????1∶800	????0
				????B5	????217	????1∶400	????0
The B average	????179
Alumen	Total ancylostome	Total ancylostome	Total ancylostome
				????D1	????176	????0	????0
????D2	????150	????0	????0
				????D3	????161	????0	????0
????D4	????241	????0	????0
				????D5	????255	????0	????0
The D average	????191

This embodiment proof can cause ancylostome quantity reduction in the body at the high antibody titer of the relevant specific antigen determinant of ASP-1.

Embodiment 7. antigens are expressed in baculovirus/insect cell and yeast

Ancylostome antigen such as the expression in baculovirus/insect cell and the pichia pastoris phaff (Pichia pastoris), provides maximum possible for obtaining solubility and bioactive recombiant protein at eukaryotic expression system.

A. the expression in pichia pastoris phaff

Antigen Na-ASP-1, Ac-TTR, Ac-API, and Ay-ASP-2 is successfully expressed with the Pichia sp. fermentation system.By separating antigen with being convenient to isolating poly histidine mark.

B. the expression in baculovirus/insect cell system

Antigen Na-CTL, Ac-MEP-1, Ac-ASP-2, and Ac-MTP-1 is successfully expressed in baculovirus/insect cell expression system.Utilization is convenient to isolating poly histidine mark and is separated antigen.

Embodiment 8. Ancylostoma ceylonicums are directly to isogeneic Ay-ASP-1, the clone of the cDNAs of Ay-ASP-2 and Ay-MTP

Directly successfully being cloned after Ancylostoma ceylonicum larva cDNA library construction from hamster parasitic ancylostome Ancylostoma ceylonicum (A.Ceylancium) to isogeneic.

The straight source of the Ancylostoma ceylonicum of ASP-1 gene (orthologue) is by utilizing a 900bp's ³²The Ac-ASP1 cDNA fragment of P-labelling is cloned as probe screening Ancylostoma ceylonicum L3cDNA library.Screen about 500,000 clones and obtain 85 positive colonies.Wherein have 21 clones to be checked order, this wherein has 19 cDNA that coding is identical again.Do not find the straight source gene of other ASP-1.This clone demonstrates with Na-ASP-1 85% homogeneity and 92% similarity.

By utilizing a 600bp ³²The Ac-asp-2 cDNA fragment of P-labelling obtains 30 positive colonies as about 100,000 plaques in probe screening Ancylostoma ceylonicum L3 cDNA library, and 10 quilts wherein check order and find to be equal to the ORF (directly to homologous clone) that Ay-ASP-2 predicts.

By utilizing a 590bp ³²The Ac-MTP cDNA fragment of P-labelling is screened about 500,000 Ancylostoma ceylonicum L3 cDNA libraries as probe, obtain 700 positive colonies, and 8 quilts wherein checks order.7 coding Ay-MTP-1 are arranged again in 8, and an other coding a kind of isotype of inferring (Ay-MTP-2).

This embodiment proof has the similarity of height between the ancylostome antigen from ancylostoma caninum (Ancylostoma canium) and Ancylostoma ceylonicum kind, and the homogeneity (＞80%) of height is arranged between the most of ancylostome antigens of data show.

Embodiment 9. immunolocalizations

The immunolocalization of the vaccine antigen that some are main carries out in ripe ancylostome section.The mensuration of immunolocalization is as follows: Ac-103 is as the ancylostome surface antigen, Ac-FAR-1 and Ac-API are as the fluidic component of pseudocoele, (Ac-API also is a kind of pharyngeal albumen), Ac-CP-1 is as two device gland (amphidial gland) albumen, Ac-TMP in the secreting gland and ASP-3 are as the albumen of two devices and esophagus.In addition, the total protein of this ancylostome ES product navigates to two devices and secreting gland, and the gastral brush shape of ancylostome boundary film.

This embodiment proves that many ancylostome antigens are exposed on the surface of helminth or by the helminth secretion, and therefore to the targeting sensitivity by host's antibody or host immune competent cell.

Embodiment 10. carries out the human research in Brazilian Minas Gerais state

At China and other local existing report, ancylostomiasis of people's hookworm infection and other soil-propagation (for example, ascariasis and trichuriasis) and schistosomicide were compared and were demonstrated unique epidemiologic feature (Gandhi etc., 2001) in the past.The popularity degree of other these helminthic infections and intensity arrive the peak and descend during adult age subsequently between childhood and adolescence, yet the popularity degree of hookworm infection and intensity increased with the age.Many Chinese and Brazil in than Leah Hess village (and supposition other place) middle aged even old resident demonstrate the most serious infection.

The basic immunology mechanism of explaining this observed result is studied.Shown in accompanying drawing 40 and 41, the CD-4+ lymphocyte carries out gate from hookworm infection resident's whole blood, and stimulates with the reorganization Na-ASP-1 (Figure 41) of L3 solubility ancylostome antigen (Figure 40) or Pichia sp.-expression.The generation of the host cell factor is measured by the intracellular cytokine staining technique.Two kinds of antigen all stimulates high-caliber IL-10 and IL-5 but not IL-4.IL-10 is a kind of downward modulation that has, the strong immunomodulator of anti-inflammatory property, and IL-4 produces relevant with the immunity of TH-2 type with antibody.These discoveries show that the individuality of hookworm infection may be reactionless to ancylostome and other possible antigenic stimulus.

In contrast, discover that the individuality of handling with ancylostome produces IL-4.These observed results show that it removes the immunity that ancylostome helps the reconstruct patient from intestinal.This is an important observed result, may not can the patient who is initiatively infected be played therapeutic vaccine because its prompting lacks the processing of the hookworm vaccine of recombinating, and prompting anthelmintic chemotherapy before immunity inoculation may be necessary.

In addition, these observed results show that also hookworm infection may hinder at the successful immunity inoculation such as HIV and malaria cause of disease.Some zones in Subsaharan Africa, ancylostome and HIV and malaria overlap, and participant's ancylostome situation is studied in monitoring before HIV or malaria immunity inoculation, and those had been found that before immunity it may be essential that the participant who is initiatively infected handles.

Although the present invention is described by the mode of preferred embodiment, those skilled in the art will be appreciated that the present invention can be implemented through changing in the spirit and scope of appended claims.Therefore, the present invention should not limited by described embodiment, and the institute that should further be included in the spirit and scope of this description that provides changes and equivalent.

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Soulsby E.Helminths, arthropod in the performing animal and protozoacide (Arthropods and Protoza of Domesticated Animals), the 7th edition, Philadelphia:Lea and Febiger, 1982.

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Claims (97)

1. compositions comprises:

From reorganization or synthetic antigen or its fragment of ancylostome, and,

Pharmaceutically suitable carrier.

2. the compositions of claim 1, wherein said reorganization or synthetic antigen show and are selected from following antigen about at least 80% homogeneity: Na-ASP-1, Na-ACE, Na-CTL are arranged, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API, and Ay-TTR.

3. the compositions of claim 2, wherein said antigen is Ac-TMP.

4. the compositions of claim 2, wherein said antigen is Ac-MEP-1.

5. the compositions of claim 2, wherein said antigen is Ac-MTP-1.

6. the compositions of claim 1, the kind of wherein said ancylostome is selected from Necator americanus (Nector americanus), ancylostoma caninum (Ancylostoma canium), Ancylostoma ceylonicum (Ancylostoma ceylancium), and Ancylostoma duodenale (Ancylostomaduodenale).

7. one kind is caused the interior method to the ancylostome immunne response of mammalian body, and described method comprises the steps:

Give the compositions of described administration effective dose, described compositions comprises from the reorganization of ancylostome or synthetic antigen or its fragment, and

Pharmaceutically suitable carrier.

8. the method for claim 7, wherein said reorganization or synthetic antigen show and are selected from following antigen about at least 80% homogeneity: Na-ASP-1, Na-ACE, Na-CTL are arranged, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API, and Ay-TTR.

9. the method for claim 8, wherein said antigen is Ac-TMP.

10. the method for claim 8, wherein said antigen is Ac-MEP-1.

11. the method for claim 8, wherein said antigen is Ac-MTP-1.

12. the method for claim 7, the kind of wherein said ancylostome is selected from Necator americanus, ancylostoma caninum, Ancylostoma ceylonicum, and Ancylostoma duodenale.

13. the method for the anti-ancylostome of immunity inoculation mammal, described method comprises the steps:

Give the compositions of described administration effective dose, described compositions comprises from the reorganization of ancylostome or synthetic antigen or its fragment, and

Pharmaceutically suitable carrier.

14. the method for claim 13, wherein said reorganization or synthetic antigen show and is selected from following antigen about at least 80% homogeneity: Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API, and Ay-TTR.

15. the method for claim 14, wherein said antigen is Ac-TMP.

16. the method for claim 14, wherein said antigen is Ac-MEP-1.

17. the method for claim 14, wherein said antigen is Ac-MTP-1.

18. the method for claim 13, the kind of wherein said ancylostome is selected from Necator americanus, ancylostoma caninum, Ancylostoma ceylonicum, and Ancylostoma duodenale.

19. a compositions comprises:

From reorganization or synthetic antigen or its fragment of ancylostome, wherein said reorganization or synthetic antigen show and is selected from following antigen about at least 80% homogeneity: Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API, and Ay-TTR, and

Pharmaceutically suitable carrier.

20. the method for claim 19, wherein said antigen is Ac-TMP.

21. the method for claim 19, wherein said antigen is Ac-MEP-1.

22. the method for claim 19, wherein said antigen is Ac-MTP-1.

23. the method for claim 19, the kind of wherein said ancylostome is selected from Necator americanus, ancylostoma caninum, Ancylostoma ceylonicum, and Ancylostoma duodenale.

24. a vaccine comprises:

From reorganization or synthetic antigen or its fragment of ancylostome, wherein said reorganization or synthetic antigen show and is selected from following antigen about at least 80% homogeneity: Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API, and Ay-TTR, and

Pharmaceutically suitable carrier.

25. the method for claim 24, wherein said antigen is Ac-TMP.

26. the method for claim 24, wherein said antigen is Ac-MEP-1.

27. the method for claim 24, wherein said antigen is Ac-MTP-1.

28. the method for claim 24, the kind of wherein said ancylostome is selected from Necator americanus, ancylostoma caninum, Ancylostoma ceylonicum, and Ancylostoma duodenale.

29. a compositions that causes immunne response, described compositions comprises:

From reorganization or synthetic antigen or its fragment of ancylostome, wherein said reorganization or synthetic antigen show and is selected from following antigen about at least 80% homogeneity: Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API, and Ay-TTR, and

Pharmaceutically suitable carrier.

30. the method for claim 29, wherein said antigen protein, polypeptide or its fragment are Ac-TMP.

31. the method for claim 29, wherein said antigen protein, polypeptide or its fragment are Ac-MEP-1.

32. the method for claim 29, wherein said antigen protein, polypeptide or its fragment are Ac-MTP-1.

33. the method for claim 29, the kind of wherein said ancylostome is selected from Necator americanus, ancylostoma caninum, Ancylostoma ceylonicum, and Ancylostoma duodenale.

34. one kind can the anti-infection method of immunity inoculation patient, described method comprises the steps:

A) the treatment hookworm infection is to being enough to increase lymphopoietic degree; And

B) the anti-described infectious disease of the described patient of immunity inoculation.

35. the method for claim 34, wherein said infectious disease is selected from HIV, tuberculosis, malaria, measles, tetanus, diphtheria, pertussis, and poliomyelitis.

36. a method that is used for the ancylostome immunity inoculation, described method comprises the steps:

A) patient of chemotherapy hookworm infection improves hookworm infection; And

B) after improving hookworm infection, use reorganization or synthetic antigen or the described patient of its fragment immunity inoculation from ancylostome.

37. the method for claim 36, wherein said reorganization or synthetic antigen show and is selected from following antigen about at least 80% homogeneity: Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API, and Ay-TTR.

38. one kind is reduced the method that the hookworm infection patient loses blood, described comprising the steps:

Use a kind of compositions for described patient, described compositions comprises

From reorganization or synthetic antigen or its fragment of ancylostome, and,

Pharmaceutically suitable carrier.

39. a method that reduces ancylostome size in the hookworm infection patient body, described method comprises the steps:

Use a kind of compositions for described patient, described compositions comprises

From reorganization or synthetic antigen or its fragment of ancylostome, and

Pharmaceutically suitable carrier.

40. one kind is reduced the method that infects ancylostome quantity in the ancylostome patient body, described method comprises the steps:

Use a kind of compositions for described patient, described compositions comprises

From reorganization or synthetic antigen or its fragment of ancylostome, and

Pharmaceutically suitable carrier.

41.SEQ?ID?NO.11。

42.SEQ?ID?NO.12。

43.SEQ?ID?NO.13。

44.SEQ?ID?NO.14。

45.SEQ?ID?NO.15。

46.SEQ?ID?NO.16。

47.SEQ?ID?NO.5。

48.SEQ?ID?NO.6。

49.SEQ?ID?NO.7。

50.SEQ?ID?NO.8。

51.SEQ?ID?NO.9。

52.SEQ?ID?NO.10。

53.SEQ?ID?NO.11。

54.SEQ?ID?NO.12。

55.SEQ?ID?NO.21。

56.SEQ?ID?NO.22。

57.SEQ?ID?NO.23。

58.SEQ?ID?NO.24。

59.SEQ?ID?NO.25。

60.SEQ?ID?NO.26。

61.SEQ?ID?NO.27。

62.SEQ?ID?NO.28。

63.SEQ?ID?NO.29。

64.SEQ?ID?NO.30。

65.SEQ?ID?NO.31。

66.SEQ?ID?NO.32。

67.SEQ?ID?NO.33。

68.SEQ?ID?NO.34。

69.SEQ?ID?NO.35。

70.SEQ?ID?NO.36。

71.SEQ?ID?NO.37。

72.SEQ?ID?NO.38。

73.SEQ?ID?NO.39。

74.SEQ?ID?NO.40。

75.SEQ?ID?NO.41。

76.SEQ?ID?NO.42。

77.SEQ?ID?NO.43。

78.SEQ?ID?NO.44。

79.SEQ?ID?NO.47。

80.SEQ?ID?NO.48。

81.SEQ?ID?NO.49。

82.SEQ?ID?NO.50。

83.SEQ?ID?NO.51。

84.SEQ?ID?NO.52。

85.SEQ?ID?NO.55。

86.SEQ?ID?NO.56。

87.SEQ?ID?NO.57。

88.SEQ?ID?NO.58。

89.SEQ?ID?NO.59。

90.SEQ?ID?NO.60。

91.SEQ?ID?NO.61。

92.SEQ?ID?NO.62。

93.SEQ?ID?NO.63。

94.SEQ?ID?NO.64。

95. Ac-APR-2 antigen.

96. Ay-TTR antigen from nematicide.

97. the Ay-TTR antigen of claim 96, wherein said nematicide is an ancylostome.

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