CN101838322A - Malaria recombinant antigen, IgY immune body and malaria detection kit - Google Patents
Malaria recombinant antigen, IgY immune body and malaria detection kit Download PDFInfo
- Publication number
- CN101838322A CN101838322A CN200910080270A CN200910080270A CN101838322A CN 101838322 A CN101838322 A CN 101838322A CN 200910080270 A CN200910080270 A CN 200910080270A CN 200910080270 A CN200910080270 A CN 200910080270A CN 101838322 A CN101838322 A CN 101838322A
- Authority
- CN
- China
- Prior art keywords
- antibody
- malaria
- lys
- asp
- igy
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a malaria recombinant antigen, an IgY immune body and a malaria detection kit. In the invention, the malaria antigen with the sequence as shown in SEQ ID No: 11 is designed; the IgY immune body is obtained from an antigen-immunized chicken; and a malaria infection detection kit containing the IgY immune body is prepared. The sensitivity of the detection kit of the invention is 1000 times higher than that of the similar rapid detection card in the prior art.
Description
Technical field
The present invention relates to a kind of new malaria recombinant antigen.Particularly, the present invention relates to a kind of new malaria antigen, by the IgY antibody of this antigen immune chicken acquisition, and the test kit that contains this IgY detection of antibodies malaria infection.
Background technology
Malaria occupies one of the most serious three big transmissible diseases in the whole world, threatens nearly 4,000,000,000 health of living in the torrid zone and subtropical zone population.Wherein the annual infection of pernicious malaria 3-5 hundred million people seize millions of people's life.Malaria also so has seriously hindered Economic Growth of Developing Countries.Because the singularity of malaria treatment medicine, accurately malaria patients can not only be in time found and treat in diagnosis fast, reduce case fatality rate greatly, also can in time save the life that has infected other communicable disease patient by the early stage malaria of getting rid of.Meanwhile, accurately early diagnosis fast also is the necessary means of malaria being carried out real-time epidemiological surveillance and control.The classical way of current diagnosis malaria still is a blood smear microscope inspection method, this method requires very high to proofer's skills and experience, can't satisfy far away and mostly in Africa and the south American countries live in patient's from far-off regions needs, cause a large amount of patients because of the death that delays the diagnosis and treatment.In addition, the method that is used for malaria early diagnosis malaria antigen is double antibody sandwich method and PCR method.Wherein, receive increasing concern in recent years based on the double antibody sandwich method of monoclonal antibody, they mainly use three class monoclonal antibodies: 1, and anti-HRP-II antibody; 2, anti-pLDH antibody; 3, anti-Aldolase antibody.Up to now, the subject matter that existing these class methods exist is: 1) susceptibility is low: because monoclonal antibody can only be discerned single epi-position and antigen, therefore susceptibility is lower than blood smear microscope inspection method: between 82%-95.2%, minimum only has 57%, especially when worm blood rate was lower than 0.01%, susceptibility obviously dropped to 50%; 2) specificity is low: owing to be subjected to the influence of popular district's worm strain difference and variation, still not ideal enough: generally about 93%, minimum only has 76.9%; 3) cross reaction: the use of monoclonal antibody easily and Rheumatoid factors, polyclonal, heterophile antibody, antinuclear antibody cross reaction is arranged; The false positive rate that detects is higher; 4) transformation period is short: most of commercial quick detection kit require to deposit at 2-30 ℃, but this condition is difficult to assurance when really using in the epidemic-stricken area; The ideal test kit should be able to be preserved 2 years under 40-50 ℃ of high temperature; 5) cost height: especially the research and development of monoclonal antibody and production cost are difficult to reduce significantly again, and these people of developing country of spreading unchecked for malaria still are difficult to accept.Therefore, set up stable, the responsive and cheap diagnostic method of a cover and become the major issue that global malaria control needs to be resolved hurrily.
Chicken yolk antibody---IgY more and more is applied in medical diagnosis on disease, prevention, treatment, and fields such as Food science and veterinary science are considered to produce polyclonal antibody optimum antibody source except that Mammals.
Because therefore bird and Mammals source far away not only can produce immune response at the conservative antigen of many Mammals camber, and can produce antibody at more epi-position.
Prior art disclose IgY antibody also not with Rheumatoid factors, polyclonal and other IgG molecular reaction, avoided the false positive results in the immunoassay and reduced the experiment background.IgY antibody is very high to the tolerance of acid, alkali, heat, proteolytic enzyme, is fit to the environmental quality in malaria epidemic-stricken area.
Method through collecting egg production IgY is not only convenient but also quick, but and set up the IgY purification technique that efficient, economic automatization is controlled at present, be fit to large-scale production and application.These advantages all can become the key factor that improves detection sensitivity, reduces cost, solves above-mentioned malaria diagnosis problem.
The report that uses IgY in the malaria immunodiagnosis is not arranged in the prior art, and the specific IgY antibody that especially uses antigen of the present invention to obtain is used in the malaria immunodiagnosis and is not appeared in the newspapers.
Summary of the invention
The present invention, comes out to connect from 9 epi-positions of five malaria major antigens through on-line analysis and software prediction, thereby designs a kind of new artificial recombination antigen in conjunction with the malaria genome database from certified pernicious malaria specificity epitope.Adopt this recombinant antigen immunization laying hen, from immune ovum gallinaceum, extract the specific IgY antibody that obtains to discern a plurality of target site of plasmodium.With this antibody labeling horseradish peroxidase (HRP), finally set up the method that double-antibody sandwich detects malaria again.
One aspect of the present invention provides a kind of malaria recombinant antigen, and it has the aminoacid sequence shown in the SEQ ID No:11.
The present invention provides the polynucleotide of the aminoacid sequence shown in a kind of SEQ of coding ID No:11 on the other hand.Because the degeneracy of codon, described polynucleotide can have multiple base sequence.For example, for (as intestinal bacteria E.coli) expression in host cell, the codon that can use this host's preference is to improve expression efficiency.Preferably, these polynucleotide have the base sequence shown in the SEQ ID No:10.
Another aspect of the invention provides a kind of specific IgY antibody that obtains by antigen immune chicken of the present invention.
Another aspect of the invention provides the purposes of specific IgY antibody of the present invention in the preparation of preparation detection malaria.Because specific IgY of the present invention can effectively be discerned plasmodium, therefore can be used for preparing the reagent of various detection malaria.
Another aspect of the invention provides the test kit that contains specific IgY antibody of the present invention, and preferably, this test kit uses the ELISA method.This test kit can comprise the enzyme-linked reaction plate of specific IgY antibody sandwich of the present invention, the IgY antibody and the Color Appearance System of horseradish peroxidase-labeled.Described test kit can prepare by means commonly known in the art, can comprise that also other can effectively improve sensitivity of ELISA test kit and specific ancillary component in the prior art.
Another aspect of the invention provides a kind of method for preparing specific IgY antibody of the present invention, and described method comprises:
1) the antigen immune chicken of using the present invention to design;
2) take-up step 1) in the ovum that chicken produced after the immunity, and from yolk, extract and obtain IgY antibody.
Laying hen after 3 weeks, gets final product antibody continual generation high specific, the character homogeneous by a spot of antigen immune of the present invention.
Preferably, can be to above-mentioned steps 2) in the IgY antibody that obtains be further purified.
Utilize the recombinant antigen that the present invention designs and the combination of its specific IgY antibody, increased the target site in the malaria diagnosis, this point obviously is better than the susceptibility of the single identification of traditional monoclonal antibody.In addition,, avoided the cross reaction of traditional method, reduced application cost simultaneously significantly because IgY antibody itself has.The present invention can also provide new research and development thinking for the quick diagnosis of other sudden great infectious diseases, obtains remarkable social benefit and economic benefit.
The term explanation: except as otherwise noted, the term that uses among the present invention has implication well known in the art.
Antigen: be meant that a kind of human or animal's body that can stimulate produces antibody or primed lymphocyte, and can with these products in vivo or the material of external generation specific reaction.Antigenic basic capacity is immunogenicity and reactionogenicity.Modal antigen molecule is a macro-molecular protein.
Epi-position: immunocyte is difficult to discern whole antigen molecule usually, and only discerns a specific part on the antigen macromole.Be called epi-position (epitope) or antigenic determinant (antigenic determinant).Thereby epi-position represented an immunocompetence district on the antigen molecule, is responsible for combining with the antigen receptor or the free antibody molecule on immunocyte surface.In fact strict, the specificity of antibody is at epi-position rather than at complete antigen molecule.
Antibody: antibody is a kind of protein complex that immunity system is used for differentiating and suppressing allogenic material (for example bacterium and virus).Every kind of antibody is only discerned specific target antigen.
IgY antibody: IgY is prevalent in birds, Reptilia and batrachians, is equivalent to Mammals IgG on function, but its many biological properties are still undiscovered.Recent many investigators have analyzed genomic constitution and the biological function of IgY, and are confirmed that it is the direct origin of Mammals IgG and IgE.Available data shows that the purposes of IgY is very extensive, various.Wherein most important function is to transmit IgY by parent to provide immune protective efficiency passively to the newborn infant.The animal that produces IgY is oviparity substantially, by yolk IgY is passed to embryo and new born animal.
Description of drawings
Fig. 1: recombinant antigen 12% reductibility SDS-PAGE collection of illustrative plates; M: low molecular weight protein (LMWP) standard; 1: induce preceding thalline; 2: induce the back thalline; 3: the recombinant antigen behind the purifying.
Fig. 2: IgY antibody 12% reductibility SDS-PAGE collection of illustrative plates behind the purifying; M: low molecular weight protein (LMWP) standard; 1 and 2: different IgY antibody purification elution peaks.
Fig. 3: the dynamic curve of IgY antibody in the leghorn chicken ovum.
Fig. 4: the IgY antibody of purifying is to the identification of single epi-position, mixing epi-position and recombinant antigen.
Fig. 5: the immunoblotting collection of illustrative plates of the IgY antibody recognition recombinant antigen of purifying; M: low molecular weight protein (LMWP) standard; 1: IgY antibody can not be discerned recombinant protein before the immunity; 2: immunity back IgY antibody capable is obviously discerned recombinant protein.
Fig. 6: the IgY antibody of purifying is to natural plasmodial immunofluorescence collection of illustrative plates; Fig. 6 A: IgY antibody can not be discerned natural plasmodium polypide before the immunity; Fig. 6 B: immunity back IgY antibody capable is the natural plasmodium polypide of identification obviously.
Fig. 7: the double-antibody sandwich elisa method detects plasmodium falciparum in the whole blood.
Embodiment
Embodiment 1: the preparation of subtertian malaria recombinant antigen
Analyze in conjunction with the malaria genome database from certified pernicious malaria specificity epitope, finally select 9 epi-positions that comprise from RESA, MSA1, CSP, MSA1, five major antigens of MAg-1, its sequence is respectively SEQ ID Nos:1-9, and the antigenic epi-position order of connection of the present invention's design is 9-6-9-8-6-7-7-3-9-6-4-9-1-5-6-2 (numeral is corresponding sequence numbering).The design polynucleotide sequence of corresponding epi-position of encoding according to the e. coli codon preference, according to well known to a person skilled in the art the primer method of superposition, introduce BamH I and Bcl II restriction enzyme site respectively in the front and back end of epi-position encoding sequence, behind pcr amplification, sequence after the amplification is connected in order to the antigenic encoding sequence of recombinating (adds initiator codon ATG, its polynucleotide sequence is SEQ ID No:10, have 1068 bases), and be connected to pET30 (a) carrier multiple clone site, transformed into escherichia coli E.coli BL21 (DE3) bacterial strain.Also can use synthetic above-mentioned polynucleotide sequence SEQ ID No:10 of additive method well known in the art and transformed into escherichia coli.Use IPTG to induce above-mentioned bacterial strains, its can be in E.coli BL21 (DE3) a large amount of soluble-expressions, expression amount reaches 18%.Obtain recombinant antigen through nickel affinity column and molecular sieve purification, its purity of SDS-PAGE electrophoretic analysis the results are shown in Figure 1 more than 95%.This artificial recombination antigen has 354 amino acid (its sequence is SEQ ID No:11), and predicted molecular weight is 39.19kD.
Embodiment 2: the preparation of anti-plasmodium disease (Plasmodium falciparum) recombinant antigen IgY
(1) antigen immune leghorn chicken
Get antigen (0.5mg/ml) and equal-volume freund adjuvant thorough mixing that 200 μ l embodiment 1 obtain, immune Leghorn (Leghorn) (purchasing animal medicine institute) in China Agricultural University.The 0th week was adopted Fu Shi Freund's complete adjuvant initial immunity, the subcutaneous multi-point injection of chicken neck; Adopt freund 's incomplete adjuvant to carry out the multiple spot booster immunization in the 3rd, 5,11 weeks respectively at shank and wing portion muscle.Collect ovum gallinaceum every day, 4 ℃ of preservations are standby behind the mark.
(2) the thick purifying of IgY
Get to be dipped in the 0.1% bromogeramine solution after the fresh leghorn chicken egg that obtains in the step (1) is cleaned and sterilize, use 75% alcohol swab wiping egg shell again.Remove albumen with the yolk separator behind the shell breaking, sterile needle punctures egg membrane as far as possible, collects yolk liquid,, stirs by dilution in 1: 9 with yolk diluent 0.06M NaAcH5.0.Add the sad of final concentration 5% then while stirring, room temperature is placed 20min, and 8,000g/min room temperature high speed centrifugation 20min removes by filter precipitation with qualitative filter paper, obtains the water-soluble extract of degreasant yolk.Add saturated ammonium sulphate solution again to final concentration 40%, room temperature is placed 20min, and 12,000g/min room temperature high speed centrifugation 20min collects supernatant.
(3) the total IgY antibody of purifying
This step and next step all adopt AKTA FPLC chromatographic system (GE Healthcare company) to handle, and at first with the sample of acquisition in the Phenyl FF hydrophobic chromatography post adsorption step (2), the binding buffer liquid of selecting for use is 0.8M (NH
4)
2SO
4, 25mM TrisCl pH 8.5,50mM NaCl, elution buffer are 25mM TrisCl pH 8.5, and 50mM NaCl, flow velocity are 1ml/min, and linear gradient elution is in charge of and is collected the albumen elution peak.After the SDS-PAGE electrophoresis is identified, collect the high elution samples of purity through 30kD ultrafiltration pipe (Millipore company) concentrate (5,000r/min * 30min).PBS difference pre-equilibration HiPrep 16/60Sephacryl S-100HR and HiLoad 16/60Superdex 30prep grade (GEHealthcare company) with 2 times of column volumes, last sample size is 2ml, flow velocity is 1ml/min, use the 0.2M phosphate buffered saline buffer wash-out IgY antibody of 2 times of column volumes again, be in charge of and collect the albumen elution peak, 12%SDS-PAGE identifies that IgY purity is more than 98.5%, and every ovum output the results are shown in Figure 2 greater than 30mg.
(4) the anti-multi-epitope antigen specific IgY of purifying antibody
Get the multi-epitope antigen that obtains in the 10mg step (3), be dissolved in (0.1MNaHCO in the coupling buffer
30.5M NaCl), be blended in the pipe that can seal with ready CNBr-activated SepharoseTM 4B matrix.Room temperature is put upside down mixing 1h or 4 ℃ and is spent the night.
With the coupling buffer flush away of 5 times of volumes link coupled matrix not.Seal not link coupled active group.The matrix of coupling is put into 0.1M Tris-HCl buffer, pH 8.0 or 1M thanomin (ethanolamine), pH 8.0, place 2h.Wash three circulations of matrix of coupling with the damping fluid of PH 4.0 and PH 8.0, every kind of damping fluid is with the volume of 5 times of post beds.The matrix that coupling is good is adorned post with constant pressure, after the 0.2M phosphate buffered saline buffer balance, standby.0.2M phosphate buffered saline buffer dilutes the IgY antibody of thick purifying, with sample on the 1ml/min constant flow rate, with 10 times of volumes of affinity column balance liquid balance, uses affinity column elutriant wash-out instead, collects and takes off the peak, the SDS-PAGE electrophoresis is identified.
The activity identification of embodiment 3:IgY antibody
(1) titer determination of IgY antibody in the immune leghorn chicken ovum:
Use the antibody horizontal that immunization laying hen is measured in indirect enzyme-linked immunosorbent experiment (ELISA), dilute recombinant antigen with coating buffer, add elisa plate with the 0.1ug/ hole, 4 ℃ of bags are spent the night.Outwell coating buffer and wash 5 times, blot with thieving paper then with PBST; With the PBS sealing plank that contains 3%BSA, every hole 200ul is hatched ditto washing after 1 hour for 37 ℃; 2 times of gradient dilution IgY antibody to be measured, every hole adds antibody diluent 100ul, hatches ditto washing after 2 hours for 37 ℃; The goat-anti chicken IgG that every hole adds the anti-HRP mark of 100ul II (purchases the company in Promega, G1351), hatches ditto washing after 2 hours for 37 ℃ with dilution in 1: 10000; Every then hole adds 100ul colour developing liquid, and color development at room temperature is after 15 minutes, and every hole adds 50ul 1M sulfuric acid H
2SO
4Termination reaction; Microplate reader is measured OD450nm; With the negative contrast of IgY antibody before the immunity, empty OD450nm ± 3SD is a threshold value according to negative control, judges experimental result.The highest (extent of dilution is 1 to the titre that shows titre yolk antibody behind initial immunity of antibody in the peripheral blood: 1.6x10 reaching in the 5th week
6), be interior 3 times of producing antibody of mammalian body.Antibody horizontal drops to half after keeping for 6 weeks, this moment again booster immunization once after, the antibody in the yolk can be returned to the highest original titre after two weeks, and can keep more than 8 weeks, the results are shown in Figure 3.
Simultaneously with 9 kinds of single epi-positions wrap respectively by after, use the indirect enzyme-linked immunosorbent measuring and show, the IgY that immunity back leghorn chicken obtains can effectively discern each single epi-position, the results are shown in Figure 4.
(2) immunologic function of IgY antibody is measured behind the purifying:
Being employed the IgY antibody recognition power that indirect enzyme-linked immunosorbent measuring (concrete experimental technique the same) shows the thick purifying of specific IgY antibody behind the purifying with the recombinant antigen bag obviously improves, and when 0.002ug/ml, still can obviously discern recombinant antigen, the results are shown in Figure 5.
Simultaneously, confirmed that with indirect immunofluorescence experiment the IgY antibody of purifying can effectively discern the plasmodium falciparum of cultivation.During concrete enforcement, at first worm strain culture evenly is applied on the slide glass, takes out air-dry after putting into 100% acetone and fixing 5 minutes; With PBS doubling dilution IgY antibody to be measured, carefully be added on the blood smear, hatched 30 minutes in 37 ℃ of wet boxes; Blot I anti-after, PBS gives a baby a bath on the third day after its birth time, each 10 minutes; The goat-anti chicken IgG (1: 200) that adds the FITC mark is again hatched the same washing after 30 minutes, fluorescence microscope result in 37 ℃ of wet boxes.The IgY antibody of purifying can obviously be discerned a plurality of sites of the natural polypide of plasmodium falciparum (plasmodium falciparum), and tires and can reach 1: 1, and 600, and, the results are shown in Figure 6 to neutral red cell no cross reaction.
Embodiment 4: detect the preparation with test kit
(1) horseradish peroxidase (HRP) mark IgY antibody
6mg HRP (RZ＞3.0) is dissolved in the 1.5ml deionized water, stirs slowly to add the freshly prepared 0.1M sodium periodate of 0.3ml NaIO down
4, stirred 40 minutes under the room temperature.With the HRP reaction solution in the 0.001M sodium acetate soln 4 ℃ the dialysis 20 hours.Simultaneously, get total IgY antibody 12mg of above-mentioned purifying, dialysis is 3 hours in 0.01M yellow soda ash.HRP is transferred in the bottle, stirs adding 300 microlitre 0.2M sodium carbonate solutions (pH9.5) down, add antibody at once, transfer pH to 9.5 with 0.2M sodium carbonate solution (pH9.5).Stirred under the room temperature 3 hours, and added the freshly prepared sodium borohydride solution of 150 microlitres, placed 2 hours for 4 ℃, after dialysed overnight in the PBS, it is standby to add final concentration and be 4 ℃ of preservations of Sodium Mercurothiolate of 0.01%.
(2) preparation of enzyme-linked reaction plate: the specific IgY antibody of above-mentioned purifying coating buffer (1.36g Na
2CO
3/ 7.35g NaHCO
3/ H
2O 1000ml, PH 9.2) by plank, every hole adds 100ul, hatches 2 hours for 37 ℃ with the amount bag in 2ug/ hole in the dilution back; It is inferior to give a baby a bath on the third day after its birth with phosphate buffered saline buffer, and control is done; Every hole adds 3% bovine serum albumin of 150ul, and 37 ℃ were sealed 2 hours, and it is inferior to give a baby a bath on the third day after its birth with the 0.1M phosphate buffered saline buffer, 37 ℃ of dryings, and sealing is preserved standby.
(3) enzyme bound substrates: the IgY antibody of above-mentioned horseradish peroxidase-labeled adds antibody protective agent (0.01% Thiomersalate and 1% bovine serum albumin) and 0.1M phosphate buffered saline buffer.
(4) Color Appearance System: be made up of A liquid and B liquid, equal-volume mixes during use, wherein
A liquid: 6.2g citric acid, 25g Na
2HPO
4-12H
2O, H
2O 500ml, 0.03% hydrogen peroxide;
B liquid: 1.05g citric acid, 0.093g EDTA, H
2O 480ml, 140mg TMB, dimethyl sulfoxide (DMSO) 20ml.
Embodiment 5: detect plasmodium falciparum in the whole blood
According to the specific IgY antibody that above-mentioned method bag is purified, bag is 2ug/ml by concentration.While collector's whole blood plasmodium culture, blood smear method counting plasmodium infection rate, adjusting infection rate with fresh normal people's whole blood is 1%, 0.1%, 0.01%, 0.001% and 0.0001%, again with the phosphate buffered saline buffer lysate sample that contains 0.02%Triton X-100, then the sample after the cracking is added in the good elisa plate of above-mentioned sealing, every hole 100ul was hatched 2 hours for 37 ℃.Anti-1: 2000 dilution back of IgY with the HRP mark adds elisa plate again, reads OD450nm and result of determination after hatching, wash, developing the color.When the falciparum infection rate of vitro culture is diluted to 0.0001% with normal people's whole blood, still can detect polypide according to above-mentioned IgY antibody compatibility and concentration.This with prior art in similar rapid detection card compare, sensitivity will exceed 1000 times, the results are shown in Figure 7.
Sequence table
＜110〉Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
＜120〉malaria recombinant antigen, IgY antibody and malaria detection kit
<130>890040CG
<160>11
<170>PatentIn?version?3.4
<210>1
<211>8
<212>PRT
＜213〉plasmodium falciparum (plasmodium falciparum)
<400>1
Glu?Glu?Asn?Val?Glu?His?Asp?Ala
1 5
<210>2
<211>20
<212>PRT
＜213〉plasmodium falciparum (plasmodium falciparum)
<400>2
Leu?Asp?Asn?Ile?Lys?Asp?Asn?Val?Gly?Lys?Met?Glu?Asp?Tyr?Ile?Lys
1 5 10 15
Lys?Asn?Lys?Lys
20
<210>3
<211>15
<212>PRT
＜213〉plasmodium falciparum (plasmodium falciparum)
<400>3
Lys?Lys?Ile?Ala?Lys?Met?Glu?Lys?Ala?Ser?Ser?Val?Phe?Asn?Val
1 5 10 15
<210>4
<211>19
<212>PRT
＜213〉plasmodium falciparum (plasmodium falciparum)
<400>4
Glu?Asp?Ser?Gly?Ser?Asn?Gly?Lys?Lys?Ile?Thr?Cys?Glu?Cys?Thr?Lys
1 5 10 15
Pro?Asp?Ser
<210>5
<211>19
<212>PRT
＜213〉plasmodium falciparum (plasmodium falciparum)
<400>5
Asp?Gly?Asn?Cys?Glu?Asp?Ile?Pro?Ile?Ile?Val?Asn?Glu?Phe?Ser?Ala
1 5 10 15
Ile?Asp?Leu
<210>6
<211>18
<212>PRT
＜213〉plasmodium falciparum (plasmodium falciparum)
<400>6
Gly?Asn?Ala?Glu?Lys Tyr?Asp?Lys?Met?Asp?Glu?Pro?Gln?His Tyr?Gly
151015
Lys?Ser
<210>7
<211>19
<212>PRT
＜213〉plasmodium falciparum (plasmodium falciparum)
<400>7
Asp?Gln?Pro?Lys?Gln?Tyr?Glu?Gln?His?Leu?Thr?Asp?Tyr?Glu?Lys?Ile
1 5 10 15
Lys?Glu?Gly
<210>8
<211>17
<212>PRT
＜213〉plasmodium falciparum (plasmodium falciparum)
<400>8
Gly?Ile?Ser?Tyr?Tyr?Glu?Lys?Val?Leu?Ala?Lys?Tyr?Lys?Asp?Asp?Leu
1 5 10 15
Glu
<210>9
<211>10
<212>PRT
＜213〉plasmodium falciparum (plasmodium falciparum)
<400>9
Gln?Thr?Asp?Glu Ile?Lys?Asn?Asp?AsnIle
1510
<210>10
<21l>1068
<212>DNA
＜213〉artificial sequence
<400>10
atggaattcg?gatcacagac?cgacgagatc?aagaacgacc?acatccagac?cgatgaaatt 60
aaaaatgata?atattggatc?aggcaacgcc?gagaagtacg?acaagatgga?cgagccgcag 120
cactacggca?agagcggatc?acagaccgac?gagatcaaga?acgaccacat?ccagaccgat 180
gaaattaaaa?atgataatat?tggatcaggc?atcagctact?acgagaaggt?gctggccaag 240
tacaaggacg?acctggaggg?atcaggcaac?gccgagaagt?acgacaagat?ggacgagccg 300
cagcactacg?gcaagagcgg?atcagaccag?ccgaagcagt?acgagcagca?cctgaccgac 360
tacgagaaga?tcaaggaggg?cggatcagac?cagccgaagc?agtacgagca?gcacctgacc 420
gactacgaga?agatcaagga?gggcggatca?aagaagatcg?ccaagatgga?gaaggccagc 480
agcgtgttca?acgtgggccc?cggccccgga?tcaaacaaga?acgacaacaa?gaacgacgga 540
tcacagaccg?acgagatcaa?gaacgaccac?atccagaccg?atgaaattaa?aaatgataat 600
attggatcag?gcaacgccga?gaagtacgac?aagatggacg?agccgcagca?ctacggcaag 660
agcggatcaa?acaagaacga?caacaagaac?gacggatcag?aggacagcgg?cagcaacggc 720
aagaagatca?cctgcgagtg?caccaagccg?gacagcggat?cacagaccga?cgagatcaag 780
aacgaccaca?tccagaccga?tgaaattaaa?aatgataata?ttggatcaga?ggagaacgtg 840
gagcacgacg?ccggatcaga?cggcaactgc?gaggacatcc?cgcacgtgaa?cgagttcagc 900
gccatcgacc?tgggatcagg?caacgccgag?aagtacgaca?agatggacga?gccgcagcac 960
tacggcaaga?gcggatcact?ggacaacatc?aaggacaacg?tgggcaagat?ggaggactac 1020
atcaagaaga?acaagaaggg?ccccggcccc?ggatccgcta?gctaataa 1068
<210>11
<211>354
<212>PRT
＜213〉artificial sequence
<400>11
Met?Glu?Phe?Gly?Ser?Gln?Thr?Asp?Glu?Ile?Lys?Asn?Asp?His?Ile?Gln
1 5 10 15
Thr?Asp?Glu?Ile?Lys?Asn?Asp?Asn?Ile?Gly?Ser?Gly?Asn?Ala?Glu?Lys
20 25 30
Tyr?Asp?Lys?Met?Asp?Glu?Pro?Gln?His?Tyr?Gly?Lys?Ser?Gly?Ser?Gln
35 40 45
Thr?Asp?Glu?Ile?Lys?Asn?Asp?His?Ile?Gln?Thr?Asp?Glu?Ile?Lys?Asn
50 55 60
Asp?Asn?Ile?Gly?Ser?Gly?Ile?Ser?Tyr?Tyr?Glu?Lys?Val?Leu?Ala?Lys
65 70 75 80
Tyr?Lys?Asp?Asp?Leu?Glu?Gly?Ser?Gly?Asn?Ala?Glu?Lys?Tyr?Asp?Lys
85 90 95
Met?Asp?Glu?Pro?Gln?His?Tyr?Gly?Lys?Ser?Gly?Ser?Asp?Gln?Pro?Lys
100 105 110
Gln?Tyr?Glu?Gln?His?Leu?Thr?Asp?Tyr?Glu?Lys?Ile?Lys?Glu?Gly?Gly
115 120 125
Ser?Asp?Gln?Pro?Lys?Gln?Tyr?Glu?Gln?His?Leu?Thr?Asp?Tyr?Glu?Lys
130 135 140
Ile?Lys?Glu?Gly?Gly?Ser?Lys?Lys?Ile?Ala?Lys?Met?Glu?Lys?Ala?Ser
145 150 155 160
Ser?Val?Phe?Asn?Val?Gly?Pro?Gly?Pro?Gly?Ser?Asn?Lys?Asn?Asp?Asn
165 170 175
Lys?Asn?Asp?Gly?Ser?Gln?Thr?Asp?Glu?Ile?Lys?Asn?Asp?His?Ile?Gln
180 185 190
Thr?Asp?Glu?Ile?Lys?Asn?Asp?Asn?Ile?Gly?Ser?Gly?Asn?Ala?Glu?Lys
195 200 205
Tyr?Asp?Lys?Met?Asp?Glu?Pro?Gln?His?Tyr?Gly?Lys?Ser?Gly?Ser?Asn
210 215 220
Lys?Asn?Asp?Asn?Lys?Asn?Asp?Gly?Ser?Glu?Asp?Ser?Gly?Ser?Asn?Gly
225 230 235 240
Lys?Lys?Ile?Thr?Cys?Glu?Cys?Thr?Lys?Pro?Asp?Ser?Gly?Ser?Gln?Thr
245 250 255
Asp?Glu?Ile?Lys?Asn?Asp?His?Ile?Gln?Thr?Asp?Glu?Ile?Lys?Asn?Asp
260 265 270
Asn?Ile?Gly?Ser?Glu?Glu?Asn?Val?Glu?His?Asp?Ala?Gly?Ser?Asp?Gly
275 280 285
Asn?Cys?Glu?Asp?Ile?Pro?His?Val?Asn?Glu?Phe?Ser?Ala?Ile?Asp?Leu
290 295 300
Gly?Ser?Gly?Asn?Ala?Glu?Lys?Tyr?Asp?Lys?Met?Asp?Glu?Pro?Gln?His
305 310 315 320
Tyr?Gly?Lys Ser?Gly?Ser?Leu?Asp?AsnIle?Lys?Asp?Asn?Val?Gly?Lys
325 330 335
Met?Glu?Asp?Tyr?Ile?Lys?Lys?Asn?Lys?Lys?Gly?Pro?Gly?Pro?Gly?Ser
340 345 350
Ala?Ser
Claims (8)
1. malaria recombinant antigen, it has the aminoacid sequence shown in the SEQ ID No:11.
2. antigenic according to claim 1 polynucleotide of coding.
3. polynucleotide according to claim 2, it has the base sequence shown in the SEQ ID No:10.
4. one kind is passed through the IgY antibody of antigen immune chicken acquisition according to claim 1.
5. the antibody described in claim 4 detects purposes in the preparation of malaria in preparation.
6. contain test kit just like the antibody described in the claim 4.
7. method for preparing as antibody as described in the claim 4, described method comprises:
1) uses antigen immune chicken according to claim 1;
2) take-up step 1) in the ovum that chicken produced after the immunity, and from yolk, extract and obtain IgY antibody.
8. method as claimed in claim 7, it further comprises step 2) the purifying antibody step that obtains.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910080270A CN101838322A (en) | 2009-03-18 | 2009-03-18 | Malaria recombinant antigen, IgY immune body and malaria detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910080270A CN101838322A (en) | 2009-03-18 | 2009-03-18 | Malaria recombinant antigen, IgY immune body and malaria detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101838322A true CN101838322A (en) | 2010-09-22 |
Family
ID=42742022
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910080270A Pending CN101838322A (en) | 2009-03-18 | 2009-03-18 | Malaria recombinant antigen, IgY immune body and malaria detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101838322A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI460189B (en) * | 2012-08-01 | 2014-11-11 | Univ Nat Taiwan Normal | AN EXTRACTION METHOD FOR γ-LIVETIN (IGY) FROM YOLK |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1354187A (en) * | 2001-12-02 | 2002-06-19 | 重庆和润实业(集团)有限公司 | Hen egg yolk antibody for resisting pyloric helicobacterium cytotoxin, its preparation and application |
| CN1417329A (en) * | 2001-10-31 | 2003-05-14 | 中国人民解放军军事医学科学院生物工程研究所 | Polyepitope malaria vaccine and its encoding gene |
| CN1438245A (en) * | 2003-02-01 | 2003-08-27 | 郭占军 | Infectious causative-agent related egg-yolk antibody preparation and use thereof |
| CN1867588A (en) * | 2003-08-12 | 2006-11-22 | 阿维迪斯公司 | Product comprising a c4bp core protein and a monomeric antigen, and its use |
| CN101298615A (en) * | 2004-10-26 | 2008-11-05 | 中国医学科学院基础医学研究所 | Novel anti-plasmodium falciparum epitope and vaccine containing the same |
-
2009
- 2009-03-18 CN CN200910080270A patent/CN101838322A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1417329A (en) * | 2001-10-31 | 2003-05-14 | 中国人民解放军军事医学科学院生物工程研究所 | Polyepitope malaria vaccine and its encoding gene |
| CN1354187A (en) * | 2001-12-02 | 2002-06-19 | 重庆和润实业(集团)有限公司 | Hen egg yolk antibody for resisting pyloric helicobacterium cytotoxin, its preparation and application |
| CN1438245A (en) * | 2003-02-01 | 2003-08-27 | 郭占军 | Infectious causative-agent related egg-yolk antibody preparation and use thereof |
| CN1867588A (en) * | 2003-08-12 | 2006-11-22 | 阿维迪斯公司 | Product comprising a c4bp core protein and a monomeric antigen, and its use |
| CN101298615A (en) * | 2004-10-26 | 2008-11-05 | 中国医学科学院基础医学研究所 | Novel anti-plasmodium falciparum epitope and vaccine containing the same |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI460189B (en) * | 2012-08-01 | 2014-11-11 | Univ Nat Taiwan Normal | AN EXTRACTION METHOD FOR γ-LIVETIN (IGY) FROM YOLK |
| US9605052B2 (en) | 2012-08-01 | 2017-03-28 | National Taiwan Normal University | Method for extracting IgY (γ-livetin) from egg yolk |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1360678B (en) | Method for early detection of flaviviruses using NS1 glycoprotein and application thereof | |
| CN105461805B (en) | Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus and its application | |
| CN102659923B (en) | Epitope minimal motif peptides of human zona pellucida protein-4 and extended short peptides and application thereof | |
| CN101818129B (en) | Hybridoma cell line for anti-African swine fever virus monoclonal antibody and monoclonal antibody secreted thereby | |
| CN101831407B (en) | Hybridoma cell line of monoclonal antibody against African swine fever virus and secreted monoclonal antibody thereof | |
| CN101833005B (en) | Competitive ELISA (Enzyme-Linked Immuno Sorbent Assay) kit for detecting antibody of African swine fever virus and application thereof | |
| CN101825633B (en) | Competitive ELISA kit for detecting african swine fever virus antibody and purposes thereof | |
| CN101294147B (en) | Monoclone antibody of swine fever virus resistant wild strain E2 protein, preparation method and application thereof | |
| CN102236017A (en) | Indirect ELISA kit for detecting African swine fever virus antibody and application thereof | |
| Blouin et al. | Antibodies to Anaplasma marginale major surface proteins 1a and 1b inhibit infectivity for cultured tick cells | |
| CN107058239A (en) | A kind of swine fever virus resistant E2 protein monoclonal antibodies cell line and its application | |
| CN102236019A (en) | Indirect ELISA kit for detecting African swine fever virus | |
| CN102108355B (en) | Multi-epitope artificial antigen of plasmodium falciparum and application thereof | |
| CN106336458A (en) | Vibrio parahaemolyticus colloidal gold-yolk antibody rapid detection test paper and preparation method thereof | |
| CN105348386B (en) | The monoclonal antibody cocktail and detection kit of the O-shaped virus of resistant to foot and mouth disease | |
| CN105906717A (en) | Preparation method and application of Brucella multi-epitope fusion protein vaccine | |
| CN102851264A (en) | An apyrase encoding gene of Aedes albopictus, and preparation method and application of protein thereof | |
| CN101838322A (en) | Malaria recombinant antigen, IgY immune body and malaria detection kit | |
| CN105296435B (en) | The monoclonal antibody specific and application of hybridoma cell strain and its O-shaped (O/GX/09-7) virus of the resistant to foot and mouth disease of secretion | |
| CN103725697A (en) | Chemically synthesized staphylococcus aureus surface protein FnBPA gene fragment and expression and application thereof | |
| CN106226520A (en) | Antigen of mycobacterium tuberculosis albumen Rv0865 and the application of B cell epitope peptide thereof | |
| CN102360008A (en) | Kit based on duck plague virus gG segmented recombinant protein and its application | |
| CN102690351A (en) | Preparation method of plasmodium vivax aldolase protein monoclonal antibody | |
| CN109900903A (en) | A kind of porcine pseudorabies virus gE blocks ELISA antibody assay kit and its application | |
| CN104965087B (en) | The method and its target proteinses of a kind of efficient detection Infection of Toxoplasma Gondii acute infection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20100922 |