CN104962581A - Recombined new castle disease virus vaccine strain for expressing African swine fever virus p72 proteins - Google Patents

Abstract

The invention provides a preparation method of a recombined new castle disease virus vaccine strain which expresses African swine fever virus p72 proteins and a recombined new castle disease virus vaccine strain. The method provided by the invention comprises the following steps: constructing a recombinant transcriptional plasmid which is inserted with a p72 gene of African swine fever virus (ASFV); constructing a transcriptional helper plasmid system; carrying out a contransfection for the transcriptional plasmids and the transcriptional helper plasmids into host cells BHK-21 which can be duplicated in new castle disease attenuate strains; and saving and obtaining the recombined virus stain. The vaccine strain for expressing the African swine fever virus p72 proteins provided by the invention has important preservation and strategy meaning in the aspect of animal epidemic disease prevention and control, and can be applied to the treatment and prevention of African swine fever virus.

Description

A kind of recombinant viral vaccine strain expressing African swine fever virus p72 albumen

Technical field

The invention provides a kind of preparation method and recombinant Newcastle disease virus vaccine strain of expressing the recombinant Newcastle disease virus vaccine strain of African swine fever virus p72 albumen.

Background technology

African swine fever (African Swine Fever, ASF) is a kind of acute, the heating caused by the unique member African swine fever virus (ASFV) of African swine fever virus section, disease [1] that infectivity is very high.Family pig and the general susceptible of aper, presents complicated clinical symptom, causes congested, the hemorrhage and dysfunction of digestive system and respiratory system, is that a kind of high degree in contact spreads through sex intercourse epidemic disease [2-4] in pig of being in.Red river hog usually presents subclinical and does not have the infection of clinical symptom, is the storage host of this disease; In soft ticks, the member (Ornithodoros erraticus, Ornithodoros moubata) of Ornithodoros can as the biology medium of this disease and viral storage host [5,6].This sick pathogenic process is short, and latent period, 5-15 days, had very high M & M, and mortality ratio even can up to 100% [7,8].

As far back as nineteen twenty-one, African swine fever, in Kenya's reported first, causes the death [9] of a pig 100%.After this, this disease is always at Africa, until nineteen fifty-seven, owing to feeding to pig, rubbish on flight and leftover cause this disease to break out [10] Portuguese.Spread in some other country in Europe subsequently, comprise Malta (1978), Italy (1967,1980), France (1964,1967,1977), Holland of Belgium (1985) (1986), afterwards, except Sardinia, this disease is eliminated [5] in succession in these countries.From 2005 to 2010, the whole world has 25 country's reports breaking out of African swine fever, mainly comprises African country and the European countries such as Italy, Russia [11].2007, this disease imported the georgia of Caucasus region into due to the pork of international ship transport infection, and rapid spread is to adjacent country, comprises Armenia, Azerbaijan and Russia [12].At present, China there is no the report of this disease, and China exists the boundary line of more than 4300 kilometer with Russia, simultaneously due to the relation of world commerce, the further reinforcement of trade and contact between non-particularly, exacerbate China face African swine fever introduction risk increase situation.At present, African swine fever there is no effective vaccine can prevent this disease, and this disease of prevention and control mainly relies on quick diagnosis and slaughters.Research abroad for African swine fever vaccine has had many reports, but traditional inactivated vaccine can not infect the effective immunoprotection of generation to African swine fever virus.Nineteen sixty, African swine fever breaks out period in Portugal, the strain weakened that goes down to posterity is utilized to carry out immunoprophylaxis, the pig morbidity of partial immunity is even dead, in addition, because immunization causes occurring a large amount of band poison pig, very unfavorable to putting out of this disease of later stage, and there is virus virulence and return strong risk in the go down to posterity African swine fever attenuated vaccine that weakens of inoculation.

Summary of the invention

The invention provides a kind of method and vaccine strain thereof of expressing the recombinant Newcastle disease virus MG7 vaccine strain of African swine fever virus p72 albumen.

The preparation method of the recombinant viral vaccine strain of expression African swine fever virus p72 albumen of the present invention comprises:

(1) build restructuring and transcribe plasmid, the genome cDNA sequence that plasmid comprises the Newcastle disease attenuated vaccine strain MG7 of the p72 gene wherein inserting African swine fever virus (ASFV) is transcribed in this restructuring, in transcription vector plasmid pBSK, then assembles acquisition restructuring transcribe plasmid pBSK-MG7-ASFVp72.Wherein, Newcastle disease attenuated vaccine strain MG7 is transformed by reverse genetics, its F protein cracking site is replaced with the cracking site of Lasota strain F protein, and sudden change causes weak artificial attenuated IBDVs thus;

(2) helper plasmid system transcribed by structure, and this helper plasmid system comprises: the helper plasmid pBSK-L of the cDNA sequence of the helper plasmid pBSK-P of the cDNA sequence of the helper plasmid pBSK-NP of the cDNA sequence of the nucleoprotein (NP) of described new castle disease virus weakening strain of encoding and the phosphorprotein (P) of described new castle disease virus weakening strain and the large polymerase protein (L) of described new castle disease virus weakening strain.

(3) by the above-mentioned host cell BHK-21 transcribing plasmid and transcribe that newcastle disease weakening strain described in helper plasmid cotransfection can copy, the host cell after transfection is cultivated;

(4) supernatant liquor is gathered in the crops, inoculated into chick embryo allantoic cavity rescue recombinant virus after filtering.

The preparation method of the recombinant viral vaccine strain of expression African swine fever virus p72 albumen of the present invention is: the gene of amplification coding African swine fever virus p72 albumen, add new castle disease virus specific gene initial sum terminator sequence at its two ends, be inserted into manually-injected between gene P, M of new castle disease virus weakening vaccine strain pme Irestriction enzyme site, be built into total length recombinant plasmid, the BHK-21 cell of recombinant plasmid and the process of helper plasmid pBSK-NP, pBSK-P and pBSK-L common transfection poxvirus infection is cultivated, again the cell conditioned medium liquid of transfection is inoculated 9 age in days SPF chick embryo allantoic cavities, save the recombinant virus infection BHK-21 cell obtained.

The gene order of the recombinant viral vaccine strain of embodiments of the invention disclosed expression African swine fever virus p72 albumen is as shown in SEQ ID No.4.

Further, the newcastle disease weakening strain described in recombinant viral vaccine strain of expression African swine fever virus p72 albumen of the present invention is MG7, and its nucleotides sequence is classified as SEQ ID No.5.

The recombinant viral vaccine strain of expression African swine fever virus p72 albumen prepared by method of the present invention or can prepare the application in anti-African swine fever virus medicine in preparation African swine fever virus vaccine.

The recombinant viral vaccine strain of expression African swine fever virus p72 albumen of the present invention or can prepare the application in anti-African swine fever virus medicine in preparation African swine fever virus vaccine.

Peeters in 1999 etc. take the lead in establishing the reverse genetic operating system (reverse genetic system, RGS system) of first Avian pneumo-encephalitis virus (NDV).Research shows, NDV genome inserts external source reporter gene or immunogen gene in different loci, still keeps the Inheritance and expression stability of height after cell or the continuous passage of chicken embryo.Therefore, NDV can as the vaccine carrier of expressing foreign protein.Recombinant Newcastle disease virus (NDV) carrier bacterin has following outstanding advantage: the weak malicious recombiant vaccine of NDV can induce humoral immunization, locally mucosa-immune and the cellullar immunologic response of body simultaneously, produces and protects more comprehensively, effectively; NDV attenuated vaccine is always for poultry epidemic prevention, and restricted concerning having kind Mammals, be the natural weak poison of height, its security is by sufficient proof; NDV can stimulate mammalian organism to produce strong immune response; Weak poison has the chicken embryonic development characteristic of high titre, can high level and jumbo expression alien gene, and low production cost; NDV genetic stability is better, only has a serotype, and restructuring or virulence between strain occur, and to return strong possibility very little; Virus replication completes in endochylema, does not have DNA replication dna intermediate, and its genome can not be integrated with the genomic dna of host cell, so there is no artificial genetically modified risk.NDV is different from adenovirus, and mammalian organism lacks already present immunological memory, and shows as transient infection in vivo, does not affect booster immunization, and research proves that NDV is a kind of for mammiferous excellent carrier.NDV effectively can infect via respiratory tract approach, and can the humoral immunization of inducing mouse mucous membrane and whole body system and cellular immunization [13-17].

In the present invention, p72 albumen is the main capsid protein of African swine fever virus, have antigen neutralization epi-position and T cell identification epi-position, and tool plays a very important role in poisoning intrusion host cell attachment process; In addition, African swine fever virus p72 gene is relatively conservative, and the vaccine for p72 albumen can produce Cross immunogenicity to African swine fever virus different genotype strain.

The exquisite weak newcastle disease vaccine strain of people used in the present invention is the artificial attenuated IBDVs transformed by reverse genetics based on genotype VII newcastle disease virus strain, there is very strong replication, and be beneficial to production, cause weak after still keep its biological characteristics, measured by OIE required standard method, for bird and Mammals, all there is very strong security; In addition, the exquisite weak newcastle disease vaccine strain of people is when expression alien gene, and compare classical lasota strain, the foreign gene of insertion is more easily expressed.That concrete employing is MG7 in an embodiment of the present invention.

Avian pneumo-encephalitis virus builds control African swine fever vaccine as live vector and has its outstanding feature and potential using value.African swine fever is a kind of exotic for a lot of country, and there is no vaccine effectively for this disease at present can utilize, and its prevention and control mainly rely on quick diagnosis and slaughter.African swine fever has very high lethality rate, once breaking out this disease for pig industry will be destructive strike.For development and the development of African swine fever vaccine, inactivated vaccine can not be induced and be produced effective anti-infectious immunoprotection, and attenuated vaccine exists virulence and returns strong risk, and the application of attenuated vaccine can produce the malicious pig of a lot of band, makes the prevention and control more difficult of this disease.Different from traditional classical vaccine development strategy, the backgrounds such as the newcastle disease virus gene group structure function used in the present invention are clear, heredity is relatively stable, and recombinant virus inserts foreign gene at cell or chicken embryo through the still Absorbable organic halogens high expression that repeatedly goes down to posterity, and is a kind of suitable vaccine carrier; Recent decades, Avian pneumo-encephalitis virus as the safety and effectiveness of vaccine carrier by sufficient proof; This type of vaccine immunity can simultaneously elicit humoral immune, local mucosal immunity and cellular immunization, forms more comprehensively immunoprotection; Its immunization ways and immunization route various; Production cost is very cheap.At present, China there is no the generation of African swine fever epidemic disease, and this disease breaking out with popular and Chinese with the economy and trade of the popular country of African swine fever, the further reinforcement of cooperation and people-to-people contacts etc. in surrounding countries such as Russia, exacerbates the risk that African swine fever imports China into.

The present invention causes the vaccine strain of weak Avian pneumo-encephalitis virus for vector construction expression African swine fever virus p72 albumen with genetically engineered; Cross immunogenicity can be produced to African swine fever virus different genotype strain; and the humoral immunization of body, local mucosa-immune and cellullar immunologic response can be induced simultaneously; produce and protect more comprehensively, effectively, in this animal epidemic of prevention and control, there is important deposit and strategic importance.

Accompanying drawing explanation

Fig. 1: the structure schematic diagram of plasmid pBSK-MG7-ASFVp72 is transcribed in restructuring.

Fig. 2: rMG7-ASFVp72 foreign protein p72 expression identification.

Fig. 3: recombinant virus growth curve.

Fig. 4: recombinant virus is to the pathogenic detection of Mammals.

Specific IgG detection by quantitative in serum after Fig. 5: rMG7-ASFVp72 immune mouse.

Fig. 6: recombinant virus immune mouse specific cell immunoreaction.

Embodiment

Set forth the present invention further below in conjunction with embodiment, content wherein should not be construed as limiting this scope of invention.Reagent described in embodiment, except special instruction, is commercialization reagent.

1 materials and methods

1.1 material

1.1.1 its F protein cracking site of strain and plasmid: Avian pneumo-encephalitis virus MG7(replaces with the cracking site of Lasota F protein, exquisite weak strain of behaving)

In the present invention:

MG7 is that contriver laboratory is separated and the Avian pneumo-encephalitis virus of the genotype VII identified, by reverse genetics operation, the F protein cracking site of G7 is replaced with the cracking site of Lasota strain F protein, sudden change causes weak strain called after MG7 thus, this strain is preserved by this laboratory, and its nucleotides sequence is classified as SEQ ID No.5.In addition, the concrete preparation method of this strain is at application number: open in the patent application of 2013104561123, can see specific practice disclosed in this patent;

The recombinant poxvirus of expressing T7 polysaccharase is presented by Harbin veterinary institute, and its preparation is all undertaken by document with titration process, and the virus after titration is kept in-80 DEG C of refrigerators;

MG7 full-length gene group transcribes plasmid pBSK-FL and helper plasmid pBSK-NP, pBSK-P, pBSK-L prepare preservation by applicant laboratory;

African swine fever virus DNA is so kind as to give by Spain's food and agrotechnique research institute animal health research centre, European Union's African swine fever reference laboratory.

1.1.2 cell and serum: clone BHK-21 purchased from American ATCC company.BHK-21 Growth of Cells and maintenance medium are the DMEM (purchased from Gibco) containing 10% foetal calf serum (FBS).

1.1.3 monoclonal antibody: African swine fever virus p72 albumen mouse monoclonal antibody specific is provided by Hua Zhong Agriculture University.

1.1.4 laboratory animal: 4 week age, female Balb/c mouse was purchased from veterinary institute laboratory animal base, Lanzhou.

The structure of the viral genomic cDNA clone of 1.2 expression African swine fever virus p72 genes

With African swine fever virus E70 pnca gene group for template, introduce PmeI restriction enzyme site recognition sequence (GTTTAAAC) by PCR primer (see table 1) at African swine fever virus p72 open reading frame 5 ' end, the Kozak sequence of exogenous gene expression and the transcription termination sequence GE(TTAAGAAAAA of NDV can be increased) and transcriptional initiation sequence GS(ACGGGTAGAA), introduce PmeI restriction enzyme site at 3 ' end; PCR primer is after sequence verification, and first end PmeI enzyme is inserted into after cutting between MG7 weakening strain genomic fragment 1-4883 (PmeI site is at 3171).Then the large fragment containing p72 gene and pBSK-FL(are comprised the plasmid of MG7 weakening strain full-length genome cDNA, preserved by this laboratory) one reinstate NotI and BatZ17I and carry out double digestion, enzyme cut after product connect, by the recombinant full-lenght Genomic cDNA clone called after pBSK-MG7-ASFVp72 be built into.

The construction process of pBSK-FL: build 5 the cDNA cloned sequences covering whole newcastle disease virus gene group, utilize the restriction enzyme site of each intersegmental lap, the whole cDNA clone obtaining 15192nt is assembled, the plasmid called after pBSK-FL built thus at transcription vector plasmid pBSK.

Amplification African swine fever virus p72 gene inserts the PCR primer of MG7 full-length gene group see table 1.

1.3 virus rescue

Cultivate BHK-21 cell by 6 orifice plates, when cell grows to 90%, 1 h before transfection, with the pre-cells infected of poxvirus.According to total length plasmid pBSK-MG7-ASFVp72:NP:P:L: the ratio transfectional cell (liposome and plasmid composite hatch 20min) of liposome=2 μ g:1 μ g:0.5 μ g:0.5 μ g:8 μ l.TPCK 0.5 μ l(0.2 mg/ml is added) after 6 h.Receive cell after cell cultures 72 h, be placed in-80 DEG C of refrigerators frozen.By the time, when chicken embryo hatches suitable (9-11 age in days), cell is taken out room temperature and melt.Cell is blown down with syringe, after the frit with 0.22 μ l, inoculated into chick embryo allantoic cavity (500 μ l).Chicken embryo is placed in 37 DEG C of thermostat containers to cultivate after 3 days receives allantoic fluid, first surveys blood clotting, tentatively determines whether to save successfully.Through saving successful recombinant virus called after rMG7-ASFVp72, its nucleotides sequence is classified as SEQ ID No.4.

1.4 Identification of recombinant baculovirus

RMG7-ASFVp72 and rMG7 vaccine strain is 1 infection BHK-21 cell with MOI respectively, discards nutrient solution after infecting 24 h, and the cell that results infect prepares protein sample.After protein electrophoresis transferring film, spend the night closed with 4 DEG C, after this mouse source African swine fever p72 protein specific monoclonal antibody that 1:500 doubly dilutes is added, incubated at room 1 h, three times are washed afterwards with PBST, add goat anti-mouse IgG (purchased from Sigma) incubated at room 1 h of the HRP mark that corresponding 1:3000 doubly dilutes again, wash 4 times with PBST afterwards, expose in darkroom.

1.5 recombinant virus growth characteristics

RMG7-ASFVp72 and rMG7 vaccine strain is 0.01 infection BHK-21 cell with MOI respectively, and respectively in 12,24,36,48,60,72 h samplings, inoculation SPF chicken embryo measures the chicken embryo median infective dose EID of each time point sample ₅₀.

1.6 rMG7-ASFVp72 are to the safety experiment of mouse

By rMG7-ASFVp72 and rMG7 vaccine strain with 10 ⁸eID ₅₀simultaneously collunarium (30 μ l), intramuscular injection (100 μ l) immunity 8 female Balb/c mouse in 4 week age, and establish PBS control group.After immunity, set time every day weighs Mouse Weight and records incidence, continues two weeks.

The humoral and cellular immune response evaluation of 1.7 recombinant virus immunity Balb/c mouse.

By rMG7-ASFVp72 and rMG7 vaccine strain with 10 ⁸eID ₅₀intramuscular injection 100 μ l immunity 5 female Balb/c mouse in 4 week age, and establishes PBS control group.Continuous immunity four times, each immunization interval two weeks, 4th immunity is after two weeks, often organize and get 3 mouse respectively and take euthanasia, separating mouse spleen lymphocyte African swine fever virus p72 albumen carries out stimulated in vitro, detect the T cell percentage of secretion of gamma-IFN and IL-4 with ELISPOT, and use CCK-8(cell proliferation experiment) cd8 t cell of detection specificity, in this, as the index of evaluation cellular immunization.

1.7.1 enzyme-linked immunosorbent assay (ELISA) evaluates the humoral immunity level of immune mouse.

ELISA experimentation is summarized as follows: using the African swine fever virus p72 albumen of prokaryotic expression as antigen coated Sptting plate, for detecting the content of IgG in rMG7-ASFVp72 and rMG7 and PBS immunity the 3rd time one week and immune the 4th one week rear mice serum, use the mouse IgG of concentration known (Southern Biotech) to make gradient dilution bag by elisa plate production standard curve simultaneously.Linear relationship between the OD value obtained according to typical curve and the standard antigen concentration of bag quilt obtains calculation formula, calculates the content of antigen-specific IgG in serum according to calculation formula.

1.7.2 the cellular immunization evaluation of rMG7-ASFVp72 immune mouse

Wrap in advance by IFN-γ and IL-4 Sptting plate, put 4 DEG C for subsequent use.Mouse carried out four immunity after two weeks, euthanasia is implemented and lymphocyte in separating spleen cell to mouse, stimulate with the p72 albumen of African swine fever virus, set up PMA and Ionomycin(phorbol exters and ionomycin simultaneously, Sigma) stimulating group is total to as positive control, and uncorrelated peptide section stimulating group is as negative control (HIV Gag PROTEIN C D8 epi-position), stimulatory protein(SP) final concentration used is 10 g/ml.Cultivate 48 h in cell culture incubator after, discard cell, wash 3 times with coating buffer, add and detect antibody in 4 DEG C of night incubation.After this with first washing 2 times with PBS, then 4 times are washed with PBST.Add the AEC nitrite ion prepared, hatch 10-60 min in 37 DEG C, detect spot formation situation every 5 min.

CCK-8(cell proliferation experiment) be summarized as follows: after exempting from two weeks four, euthanasia is implemented and lymphocyte in separating spleen cell to mouse, stimulate with the p72 albumen of African swine fever virus, set up PMA and Ionomycin(phorbol exters and ionomycin simultaneously, Sigma) stimulating group is total to as positive control, and uncorrelated peptide section stimulating group is as negative control (HIV Gag PROTEIN C D8 epi-position), stimulatory protein(SP) final concentration used is 10g/ml.Cultivate 72 h in cell culture incubator after, add CCK-8 substrate specificity, then in cell culture incubator, cultivate 4 h, with surveying its OD value under microplate reader 450nm wavelength.

2 test-results

2.1 the recombinant Newcastle disease virus of expressing African swine fever virus p72 albumen builds and virus rescue

On newcastle disease MG7 weakening strain reverse genetic operating system basis, African swine fever virus p72 gene that 5 ' end introduces the gene initial sum terminator sequence of NDV polysaccharase specific recognition is inserted as an independently transcriptional units in MG7 genome PmeI site, be built into the recombinant full-lenght cDNA expressing African swine fever virus p72 albumen and clone pBSK-MG7-ASFVp72, see Fig. 1.

By pBSK-MG7-ASFVp72 and the helper plasmid common transfection BHK-21 cell of expressing NP, P, L albumen, gather in the crops transfectional cell supernatant after 72 h and inoculate 9 age in days SPF chicken embryos.Gather in the crops HA after 72 h and test the chick embryo allantoic liquid be positive.RT-PCR and sequencing result display, p72 gene is correctly inserted into MG7 genome PmeI site.Save the recombinant virus called after rMG7-ASFVp72 obtained.

2.2 recombinant virus rMG7-ASFVp72 express the checking of African swine fever virus p72 albumen

RMG7-ASFVp72 and rMG7 vaccine strain is 1 infection BHK-21 cell with MOI respectively, discards nutrient solution after infecting 24 h, and the cell that results infect prepares protein sample.There is specific band in corresponding position in Western blot result display recombinant virus, and rMG7 does not have object band, see Fig. 2.Show that recombinant virus can correction African swine fever virus p72 albumen.

The growth characteristics of 2.3 recombinant virus rMG7-ASFVp72

The growth tendency of duplicating dynamics curve experiments result display rMG7-ASFVp72 with rMG7 is consistent, see Fig. 3.Show that recombinant virus maintains the high titre growth characteristics similar with parent's strain.

2.4 recombinant virus rMG7-ASFVp72 are to mouse safety evaluation

RMG7-ASFVp72 and rMG7 vaccine strain is with 10 ⁸eID ₅₀simultaneously collunarium 30ul, intramuscular injection 100ul immunity 8 female Balb/c mouse in 4 week age, and establish PBS control group.Observed after immunization is all survived to three groups of mouse, and the mental status is good, and Mice Body weight average increases to some extent, but no significant difference, see Fig. 4, show that recombinant virus is the same with its parent plant, to mouse, there is good security.

2.5 recombinant virus inducing mouses produce humoral and cellular immune response reaction

Recombinant virus rMG7-ASFVp72 can produce special ELISA antibody by inducing mouse.Three exempt from and four to exempt from after one week take a blood sample and all high-caliber IgG antibody can be detected, see Fig. 5 clearly.

Recombinant virus rMG7-ASFVp72 can produce specific C D8 cell immune response by inducing mouse.The result display of Fig. 6 (A), mouse can produce the T lymphocyte of specific secretion IFN-γ for African swine fever virus p72 albumen and IL-4 cytokine after four immunity.Fig. 6 (B) shows, can induce and produce a large amount of specific cd8 t cell under p72 albumen stimulates.

The present invention utilizes reverse genetic operating system, African swine fever virus p72 gene is inserted into Avian pneumo-encephalitis virus MG7 attenuated IBDVs genome, success builds and has saved out the recombinant Newcastle disease virus rMG7-ASFVp72 expressing African swine fever virus p72 albumen, experiment results proved rMG7-ASFVp72 can correction African swine fever virus p72 albumen, and recombinant virus maintains the high titre growth characteristics consistent with MG7 attenuated IBDVs, genomic genetic stability and to mammiferous security.Show the immunity test result of recombinant virus rMG7-ASFVp72, rMG7-ASFVp72 can produce humoral and cellular immune response reaction by inducing mouse.

The recombinant Newcastle disease virus MG7 vaccine strain of the expression African swine fever virus p72 albumen described in this research at home and abroad still belongs to the first time report.RMG7-ASFVp72 has very important strategic reserves meaning for the prevention and control of African swine fever.

[0050] reference involved in the present invention:

[1] ARIAS M, SáNCHEZ-VIZCAíNO J M. African Swine Fever: Trends in emergingviral infections of swine. Iowa City (IA): Iowa State University Press, 2002, 119–124.

[2] ARIAS M, SáNCHEZ C, GONZáLEZ M A, et al. Peste porcina Africana: Cursodigital de enfermedades infecciosas porcinas. [Online], 2002, http:www.sanidadanimal.info.

[3] ARIAS M, MARTíNEZ ESCRIBANO JA, RUEDA A, et al. La peste porcina Africana [J]. Med Vet, 1986, 3: 333–359.

[4] SáNCHEZ-VIZCAíNO J M. African swine fever: Diseases of swine, 8th ed. Iowa State University Press, Ames, 1999, 93–102.

[5] PLOWRIGHT W, THOMSON G R, NESER J A. African swine fever: Infectious diseases of livestock, with special reference to southern Africa, 1st ed. Cape Town, South Africa: Oxford University Press, 1994, 1: 567–599.

[6] SáNCHEZ BOTIJA C. Reservorios del virus de la peste porcina Africana. Investigación del virus de la PPA en los artro′podos mediante la prueba de la hemoadsorción [J]. Bull Office Int Epizoot, 1963, 60: 895–899.

[7] HESS W R. African swine fever: a reassessment [J]. Adv Vet Sci Comp Med, 1982, 25: 39–69.

[8] VINUELA E. African swine fever [J]. Curr Top Microbiol Immunol, 1985, 116: 151–170.

[9] MONTGOMERY R E. On a form of swine fever occurring in British East Africa [J]. J Comp Pathol, 1921, 34: 59–191.

[10] COSTARD S, WIELAND B, GLANVILLE W D, et al. African swine fever: how can global spread be prevented? [J] Phil Trans R Soc B, 2009, 364: 2683–2696.

[11] OIE WAHID. Office International des Epizooties–World Animal Health Information Database (WAHID) Interface. 2010, http://www.oie.int/wahis/public.php?page=disease_timelines.

[12] REBECCA J R, MICHAUD V, HEATH L, et al. African Swine Fever Virus Isolate, Georgia, 2007 [J]. Emerging Infectious Diseases, 2008, 14: 12.

[13] Nakaya T, Cros J, Park M S, Nakaya Y, Zheng H, Sagrera A, Villar E, Garci’a-Sastrea, Palese P. Recombinant Newcastle disease virus as a vaccine vector. Journal of Virology, 2001, 75: 11868-11873.

[14] Huang Z, Elankumaran S, Panda A, Samal S K. Recombinant Newcastle disease virus as a vaccine vector. Poultry Science, 2003, 82: 899-906.

[15] Huang Z, Elankumaran S, Yunus A S, Samal S K. A Recombinant Newcastle disease virus (NDV) expressing VP2 protein of infectious bursal disease virus (IBDV) protects against NDV and IBDV. Journal of General Virology, 2004, 78: 10054-10063.

[16] Swayne D E, Suarez D L, Schultz-Cherry S, Tumpey T M, King D J, Nakaya T, Palese P, Garcia-Sastre A. Recombinant paramyxovirus type 1-avian influenza-H7 virus as a vaccine for protection of chicken against influenza and Newcastle disease. Avian Diseases, 2003, 47: 1047 -1050.

[17] Engel-Herberta I, Werner O, Teifke J P, Mebatsion T, Mettenleiter T C, Ro¨mer-Oberdo¨rfer A. Characterization of a recombinant Newcastle disease virus expressing the green fluorescent protein. Journal of Virological Methods, 2002, 108: 19-28.。

 

<110> Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences

<120> mono-kind expresses the recombinant viral vaccine strain of African swine fever virus p72 albumen

<160> 5

 

<210> 1

<211> 73

<212> DNA

<213> artificial sequence (p72-Up)

<400>

ccaactctgt ttaaacttag aaaaaatacg ggtagaacgc caccatggca tcaggaggag 60

ctttttgtct tat 73

 

<210> 2

<211> 43

<212> DNA

<213> artificial sequence (p72-Low)

<400>

ccgcttgggt ttaaacgtta ggtactgtaa cgcagcacag ctg 43

 

<210> 3

<211> 1941

<212> DNA

<213> p72 gene order

<400>

atggcatcag gaggagcttt ttgtcttatt gctaacgatg ggaaggccga caagattata 60

ttggcccaag acttgcttaa tagcaggatt tctaacatta aaaatgtgaa caaaagttat 120

gggaaacccg accccgaacc cactttgagt caaatcgaag aaacacattt ggttcatttt 180

aatgcgcatt ttaagcctta tgttccagta gggtttgaat acaataaagt acgcccgcat 240

acgggtaccc ccaccttggg aaacaagctt acctttggta ttccccagta cggagacttt 300

ttccatgata tggtgggcca ccatatattg ggtgcatgtc attcgtcctg gcaggatgct 360

ccgattcagg gcacggccca gatgggggcc catggtcagc ttcaaacgtt tcctcgcaac 420

ggatatgact gggacaacca aacaccttta gagggcgccg tttacacgct tgtagatccc 480

tttggaagac ctattgtacc cggcacaaag aatgcgtacc gaaacttggt ttactactgc 540

gaataccccg gagaacgact ttatgaaaac gtaagattcg atgtaaatgg aaattccctg 600

gacgaatata gttcggatgt cacaacgctt gtgcgcaaat tttgcatccc aggggataaa 660

atgactggat ataagcactt ggtcggccag gaggtatcgg tggagggaac tagtggccct 720

ctcctatgca acattcatga tttgcacaag ccgcaccaaa gcaaacctat tcttaccgat 780

gaaaatgata cgcagcgaac gtgcagccat accaacccga aattcctttc acaacatttt 840

cccgagaact ctcacaatat ccaaacagca ggtaaacaag atattactcc tattacggac 900

gcaacgtatc tggacataag acgtaatgtt cattacagct gtaatggacc tcaaacccct 960

aaatactatc agccccctct tgcgctctgg attaagctgc gcttttggtt taacgagaac 1020

gtgaaccttg ctattccctc ggtatccatt cccttcggcg agcgctttat caccataaag 1080

cttgcatcgc aaaaggattt ggtgaatgaa tttcctggac tttttatacg ccagtcgcgt 1140

tttatacctg gacgccccag tagacgcaat atacgcttta aaccatggtt tatcccagga 1200

gtcattaatg aaatctcgct cacggataat gaactttaca tcaataacct gtttgtaacc 1260

cctgaaatac acaacctttt tgtaaaacgc gttcgatttt ccctgatacg tgtccataaa 1320

acgcaggtga cccacaccaa caataaccac cacgatgaaa aactaatgtc tgctcttaaa 1380

tggcccattg aatatatgtt tataggatta aaacctacct ggaacatctc cgatcaaaat 1440

cctcatcaac accgagattg gcacaagttc ggacatgttg ttaacgccat tatgcagcct 1500

actcaccacg cagagataag ctttcaggat agagatacag ctcttccaga cgcatgttca 1560

tctatatcgg atattagccc cgttacgtat ccgatcacat tacctattat taaaaacatt 1620

tccgtaactg ctcatggtat caatcttatc gataagtttc catcaaagtt ctgcagctct 1680

tacataccct tccactacgg aggcaatgca attaaaaccc ccgatgatcc gggtgcgatg 1740

atgattacct ttgctttgaa gccacgggag gaataccaac ccagtggtca tattaacgta 1800

tccagagcaa gagaatttta tattagttgg gacacggatt acgtggggtc tatcactacg 1860

gctgatcttg tggtatcggc atctgctatt aactttcttc ttcttcagaa cggttcagct 1920

gtgctgcgtt acagtaccta a 1941

 

<210> 4

<211> 17178

<212> DNA

The gene order of <213> recombinant viral vaccine strain rMG7-ASFVp72

<400>

accaaacaga gaatctgtga ggtacgataa aaggcgaaga agcaatcgag atcgtacggg 60

tagaaggtgt gaatcccgag tgcgaggccg aagctcgaac ctgaggggac cttctaccga 120

tatgtcgtct gttttcgacg agtacgagca gctcctcgct gctcagaccc gccctaacgg 180

aactcatgga gggggagaga aagggagcac tttaaaagtt gaggtcccag tattcaccct 240

aaacagtgat gatccagagg atagatggaa ttttgcggta ttctgtcttc ggattgctgt 300

tagcgaggat gccaacaaac cactcaggca aggtgctctt atatccctct tatgctccca 360

ttctcaggtg atgagaaacc atgttgccct tgcagggaaa caaaatgagg ccacactggc 420

tgttcttgag atcgatggtt ttgctaacag tgtgccccag ttcaacaata ggagtggagt 480

gtctgaggaa agagcacaga gattcatggt aatcgcagga tctctccctc gggcgtgcag 540

caacggtact ccgtttgtca cggctggggt tgaagatgat gcaccagaag atatcactga 600

cactctggaa agaatcctat ctatccaagt tcaggtatgg gtcacagtag caaaggccat 660

gactgcatat gagacagcag atgagtcaga aacaagaaga ataaataagt atatgcagca 720

aggtcgagtt cagaagaagt acatccttca tcctgtatgc aggagtgcaa ttcaactcac 780

aatcagacat tctctggcag tccgtatctt cctagttagt gagctcaaga ggggccgcaa 840

tacagcaggt gggagctcta catattacaa cttggtcggg gatgtagact catacatcag 900

gaacaccggg cttactgcat ttttcctaac actcaaatat ggaatcaata ccaagacgtc 960

agccctcgca ctcagcagcc tcacaggtga tatccaaaaa atgaaacagc tcatgcgttt 1020

atatcggatg aaaggtgaaa atgcaccata catgacattg ttaggtgaca gtgaccagat 1080

gagctttgca cctgctgagt atgcacaact ttattctttt gccatgggca tggcatcagt 1140

cttagataag ggaactggca agtaccaatt cgccagggac tttatgagca catcattctg 1200

gagacttgga gtagagtatg ctcaggccca gggaagtagc attaatgagg acatggctgc 1260

tgagctaaaa ctaaccccgg cagcaaggag aggcctggca gctgctgccc aacgagtatc 1320

cgaagaaatc ggcagcatgg acattcccac tcaacaggcg ggagtcctca ccgggctcag 1380

tgatgaaggc ccccgaactc cacagggcag atcaaacaag ccgcaagggc aaccagatgc 1440

tggggatggg gagacccaat tcttggattt tatgagagca gtggcgaacg gcatgcggga 1500

agcgccaaat cctgtacaga gcaccaccca tccagagcct cccccaaccc ctggggcatc 1560

ccaagacaac gacactgact gggggtactg atcgacaaca cccagcctgc cttcacagga 1620

tcacaccaaa ccctccgacc aaaaccctcc cacactccct gacccacaac cccgcacgac 1680

cacaccaaca aaagctcccc cccaccctct cccccactcc cagccacacg atcccgccca 1740

cccgggacaa cacaggcaca gctcagttcg tcgacaatcc gcccagagcc caaggtatta 1800

gaaaaaaata cgggtagaag agagacatcc agagaccagg acgagtcacc aagttctctg 1860

ttctcccttc tacccagtga attagggtga agatggccac ttttacagat gcggagatag 1920

atgacatatt tgagaccagt gggactgtca ttgacagcat aattacggcc cagggcaaat 1980

cagctgagac cgttggaaga agtgcgatcc cgcagggcaa gaccaaagct ctaagcacag 2040

catgggagaa gcacgggagt atccagccac acgccagtca ggacgcccct gaccaacaag 2100

acagaataga aaaacagcca tccacacctg agcaggcgac tctacacaac aatccgccga 2160

tcgcatccac tgaaccgcct cccactcagg ccgcaagcga gaccagcgac acacagctca 2220

agactggggc aagcaactcc cttctgtcca tgctcgacaa actgagtaat aaatcgtcca 2280

atgctaaaaa gggcccatgg tcgggatccc aagaagggca tcaccaacct ccggcccaac 2340

aacatgggaa ccagccgagc tatggaagca accagggaag accgcagcat caggccaagg 2400

ccgtccctgg aaaccggggc acagacgaga acacagcata tcatggacag cggaaggagt 2460

cacaaccatc agctggtgca acccctcatg cgccccagtc agggcagagc caagacaata 2520

ctcctgcacc tgtggatcgt gtccagctac ctgccgactt tgtgcaggcg atgatgtcta 2580

tgatggaggc attatcacag aaggtaagta aagttgatca tcagctggac ctagtcttga 2640

aacagacatc ctccattcct atgatgcgat ctgaaatcca acagctcaag acatctgttg 2700

cgatcatgga agctaactta ggcatgatga aaattctgga ccctggttgt gctaatgttt 2760

catccttaag tgatctccgg gcagtagccc gatcccatcc agtcctagtt tcaggccctg 2820

gagacccatc tccttacgtg acacaagggg gtgaaatgac gctcaataaa ctctcacaac 2880

cggtgcagca cccctctgaa ttgattaagt ctgccactgc aagcgggcct gacatgggag 2940

tggagaagga cactgtccgc gcattaatca cctcgcgccc gatgcatcca agctcctcgg 3000

ctaagctcct gagcaagcta gatgcagcca ggtcaattga agagatcagg aagatcaaac 3060

gccttgcgct gaatggttga tggccatcac aactcataac aggctcccgt cactttagcg 3120

tcacacggaa tccctcgggg gccctccctc gcaaatctat gcttcaacac cgtttaaact 3180

tagaaaaaat acgggtagaa cgccaccatg gcatcaggag gagctttttg tcttattgct 3240

aacgatggga aggccgacaa gattatattg gcccaagact tgcttaatag caggatttct 3300

aacattaaaa atgtgaacaa aagttatggg aaacccgacc ccgaacccac tttgagtcaa 3360

atcgaagaaa cacatttggt tcattttaat gcgcatttta agccttatgt tccagtaggg 3420

tttgaataca ataaagtacg cccgcatacg ggtaccccca ccttgggaaa caagcttacc 3480

tttggtattc cccagtacgg agactttttc catgatatgg tgggccacca tatattgggt 3540

gcatgtcatt cgtcctggca ggatgctccg attcagggca cggcccagat gggggcccat 3600

ggtcagcttc aaacgtttcc tcgcaacgga tatgactggg acaaccaaac acctttagag 3660

ggcgccgttt acacgcttgt agatcccttt ggaagaccta ttgtacccgg cacaaagaat 3720

gcgtaccgaa acttggttta ctactgcgaa taccccggag aacgacttta tgaaaacgta 3780

agattcgatg taaatggaaa ttccctggac gaatatagtt cggatgtcac aacgcttgtg 3840

cgcaaatttt gcatcccagg ggataaaatg actggatata agcacttggt cggccaggag 3900

gtatcggtgg agggaactag tggccctctc ctatgcaaca ttcatgattt gcacaagccg 3960

caccaaagca aacctattct taccgatgaa aatgatacgc agcgaacgtg cagccatacc 4020

aacccgaaat tcctttcaca acattttccc gagaactctc acaatatcca aacagcaggt 4080

aaacaagata ttactcctat tacggacgca acgtatctgg acataagacg taatgttcat 4140

tacagctgta atggacctca aacccctaaa tactatcagc cccctcttgc gctctggatt 4200

aagctgcgct tttggtttaa cgagaacgtg aaccttgcta ttccctcggt atccattccc 4260

ttcggcgagc gctttatcac cataaagctt gcatcgcaaa aggatttggt gaatgaattt 4320

cctggacttt ttatacgcca gtcgcgtttt atacctggac gccccagtag acgcaatata 4380

cgctttaaac catggtttat cccaggagtc attaatgaaa tctcgctcac ggataatgaa 4440

ctttacatca ataacctgtt tgtaacccct gaaatacaca acctttttgt aaaacgcgtt 4500

cgattttccc tgatacgtgt ccataaaacg caggtgaccc acaccaacaa taaccaccac 4560

gatgaaaaac taatgtctgc tcttaaatgg cccattgaat atatgtttat aggattaaaa 4620

cctacctgga acatctccga tcaaaatcct catcaacacc gagattggca caagttcgga 4680

catgttgtta acgccattat gcagcctact caccacgcag agataagctt tcaggataga 4740

gatacagctc ttccagacgc atgttcatct atatcggata ttagccccgt tacgtatccg 4800

atcacattac ctattattaa aaacatttcc gtaactgctc atggtatcaa tcttatcgat 4860

aagtttccat caaagttctg cagctcttac atacccttcc actacggagg caatgcaatt 4920

aaaacccccg atgatccggg tgcgatgatg attacctttg ctttgaagcc acgggaggaa 4980

taccaaccca gtggtcatat taacgtatcc agagcaagag aattttatat tagttgggac 5040

acggattacg tggggtctat cactacggct gatcttgtgg tatcggcatc tgctattaac 5100

tttcttcttc ttcagaacgg ttcagctgtg ctgcgttaca gtacctaacg tttaaaccaa 5160

aacaacagcc ctctctcacc ccccccaatc cctcgaatga ccgcacaact gcaaccaatc 5220

cagcagcatt agaaattaag aaaaaatacg ggtagaatca aagtgccttg attgcaccaa 5280

aatggactca tccaggacaa ttgggctgta ctttgattct gccctccctt ccagcagcct 5340

gttagcattt ccgattgtct tacaagacac gggagacggg aagaagcaaa tcaccccaca 5400

atacaggatc cagcgtcttg attcgtggac agacagtaag gaagactcgg tattcatcac 5460

cacctacggg ttcatctttc aaattgggaa tgaagaaacc accgtcggtg tgatcaatga 5520

caatcccagg cacgagctac tctcttccgc aatgctctgc ttagggagtg tcccgaacga 5580

cggagatctt gttgagctgg cgagagcctg cctcaccatg gtggtaactt gcaaaaagag 5640

tgcaactaac actgagagaa tagtcttttc agtagtgcag gctcctcggg tgctgcaaag 5700

ctgtatggtt gtggcaaata ggtactcatc agtgaatgca gtgaagcatg tgaaggcgcc 5760

agaaaagatc cctgggagcg gaaccctaga gtataaagtg aattttgtct ctttgaccgt 5820

ggtgccaaga agggatgtct acaggatccc aactgcagta ttgaaagtgt ctggctcaag 5880

cctgtacaat cttgcgctca atgtcactat tgatgtggac gtggatccga agagcccgtt 5940

agtcaaatcc ctttccaagt ccgatagcgg atactatgcg aatctttttc tgcatatcgg 6000

gcttatgtcc actgtagata agaagggaaa gaaagtgaca tttgacaaga tagaggaaaa 6060

gataaggaga ctcaatctat ccgtcgggct cagtgatgtg ctcggaccct ctgtgcttgt 6120

gaaggcgaga ggtgcacgga ctaagctact tgctcctttt ttctctagca gtgggacagc 6180

ctgctatcct atagcaaatg cctctcccca ggttgccaag atactctgga gccaaactgc 6240

gcacctgcgg agtgtgaaag tcatcattca agccggcact cagcgtgctg tcgcagtgac 6300

cgctgatcat gaggtaacct ccactaagat agagaggagg catgccattg ctaaatacaa 6360

tcctttcagg aaataagttg catccctaag actgcagttc acctgctttc ccgaatcacc 6420

attacaccag acaatgatcc atctcgactg cttatagtta gttcacctgt ctagcaaatt 6480

agaaaaaaca cgggtagaag agtctggatc ccgaccggca cactcaggac gcaacatggg 6540

ctccaaacct tctaccagga tcccagcacc tctgatgctg gtcacccgga ttatgctgat 6600

attgggctgt attcgttcga caagctctct tgacggcagg cctcttgcag ctgcaggaat 6660

tgtagtaaca ggagataagg cagtcaatgt atacacctcg tctcagacag ggtcaatcat 6720

agtcaagttg ctcccgaata tgcccaggga taaggaggcg tgtgcgaaag ccccattaga 6780

ggcatataac agaacactga ctactttgct cactcctctt ggcgactcca tccgcaagat 6840

tcaagggtct gtgtccacgt ctggaggaag gaggcaaaaa cgctttatag gtgctgttat 6900

tggcagtgta gctcttgggg ttgcaacagc ggcacagata acagcagctg cggccctaat 6960

acaagccaac aagaatgctg ccaacatcct tcggcttaag gagagcattg ctgcaaccaa 7020

tgaagctgtg catgaagtca ccgacggatt atcacaacta tcagtggcag ttgggaagat 7080

gcagcagttt gtcaatgacc agtttaataa tacggcgcga gaattggact gtataaaaat 7140

cacacaacag gttggtgtag aactcaacct atacctaact gaattgacta cagtattcgg 7200

gccacagatc acctctcctg cattaactca gctgaccatc caggcacttt ataatttagc 7260

tggtggcaat atggattact tattaactaa gttaggtata gggaacaatc aactcagctc 7320

attaattggt agcggcctga tcactggtta ccctatactg tatgactcac agactcaact 7380

cttgggcata caagtgaatt tgccctcagt cgggaactta aataatatgc gtgccaccta 7440

tttggagacc ttatctgtaa gtacaaccaa aggatatgcc tcagcacttg tcccgaaagt 7500

agtgacacaa gtcggttctg tgatagaaga gcttgacacc tcatactgta tagagtccga 7560

tctggattta tattgtacta gaatagtgac attccccatg tccccaggta tttattcctg 7620

tttgagcggc aacacatcag cttgcatgta ttcaaagact gaaggcgcac tcactacgcc 7680

gtatatggcc cttaaaggct cagttattgc caattgtaag ataacaacat gtagatgtac 7740

agaccctcct ggtatcatat cgcaaaatta tggagaagct gtatccctga tagatagaca 7800

ttcgtgcaat gtcttatcat tagacgggat aactctgagg ctcagcggag aatttgatgc 7860

aacttatcaa aagaacatct caatactaga ttctcaagtc atcgtgacag gcaatcttga 7920

tatatcaact gaacttggaa acgtcaacaa ttcaatcagc aatgccttgg ataagttggc 7980

agaaagcaac agtaagatag aaaaagtcaa tgtcagatta accagcacat ctgctctcat 8040

tacctatatt gttctaactg tcatttctct atttttcggt gcacttagtc tgggtttagc 8100

gtgttacctg atgtacaaac agaaggcaca acaaaagacc ttgctatggc ttgggaataa 8160

tgcccttgat cagatgagag ccactacaag agcatgaatg cagataagag gtggatatat 8220

acccaacagc agcctgtgtg tcaattccga taatctgtca agtagaagac ttaagaaaaa 8280

actactggga acaagcaacc aaagagcaat acacgggtag aacggtcaga ggagccaccc 8340

ttcaatcgga aattaggctt cacaacatcc gttctaccac atcaccatca acaagagtca 8400

atcatggacc gcgcggttaa cagagtcgtg ctggagaatg aggaaagaga agcaaagaac 8460

acatggcgcc tggttttccg gatcgcagtc ttacttttaa tggtaatgac tctagctatc 8520

tccgcagctg ccctggcata cagtatggag gccagtacgc cgcacgacct tgcaggcata 8580

tcgactgtga tctctaagac agaagataag gttacgtctt tactcagttc gagtcaagat 8640

gtgatagata ggatatacaa gcaggtggct cttgaatccc cgctggcgct actaaacact 8700

gaatctacaa ttatgaatgc aataacctct ctttcttatc aaattaacgg ggctgcgaac 8760

aatagcggat gtggggcgcc tgttcatgac ccagattata tcggggggat aggcaaagaa 8820

ctcatagtgg acgacatcag tgatgtcaca tcattttatc cttctgcata tcaagaacac 8880

ctgaatttca tcccggcgcc tactacagga tccggttgca ctcggatacc ctcatttgac 8940

atgagcacca cccattattg ttatactcac aatgtaatac tatccggttg cagagatcac 9000

tcacactcac atcaatactt agcgcttggt gtgcttcgga catctgcaac agggagggta 9060

ttcttctcta ctctgcgctc catcaattta gatgacaccc aaaatcggaa gtcctgcagt 9120

gtgagtgcaa cccctttagg ttgtgatatg ctgtgctcta aggtcacagg gactgaagag 9180

gaggattaca agtcagctgc cccaacatca atggtgcacg gaaggctagg gtttgacggt 9240

caataccatg agaaggactt agacaccacg gtcttattta aggattgggt ggcaaattac 9300

ccaggagtgg gaggagggtc ttttattgac gaccgtgtat ggttcccagt ttacggaggg 9360

ctcaaaccca attcacccag tgacactgca caagaaggga aatatgtaat atacaagcgc 9420

cataacaaca catgccccga tggacaagat taccaaattc ggatggctaa atcttcatat 9480

aaacccgggc gatttggtgg aaagcgcgta cagcaagcca tattatccat caaagtgtca 9540

acatccttgg gtaaggaccc ggtgctgact attccaccta atacaatcac actcatggga 9600

gccgaaggca gaatcctcac agtagggaca tctcacttct tgtaccaacg agggtcttca 9660

tatttctccc ctgccttatt atatcccatg acagtaaata acaaaacggc tacactccat 9720

agtccttata cgtttaatgc tttcactcgg ccaggtagcg tcccttgcca ggcatcagca 9780

agatgcccca actcatgcat cactggggtc tatactgatc catatccctt aatcttccat 9840

aggaatcata ctctacgagg ggtctttggg acgatgcttg atgatgaaca agcgagactt 9900

aaccccgtat ctgcagtatt cgacaacata tcccgcagtc gtgtcacccg ggtgagttca 9960

agcagcacca aggcagcata cacgacatcg acatgtttta aagttgtcaa gaccaataaa 10020

gcttattgtc ttagtattgc agagatatcc aataccctat tcggggaatt taggatcgtt 10080

cccttattag ttgagatcct caaggatgat agagtttaag aagctagact tggccgattg 10140

agccaatcat aggatggttg ggaagacgac accacatcaa tcatctccca caatgcttag 10200

agtcaatctg aatattaaca taagccagga tcccatgttg ttgggcagcc acaatcagac 10260

aatactgaca tgatcattct gagtcctgcc cactatcacc ttattaagaa aaaatacaaa 10320

aagcattgag atataagggg aaacaaccaa caagagggaa cacgggtagg acatggcggg 10380

ctccggtccc gaaagggcag agcaccagat catcctacca gagtcacatc tatcctctcc 10440

attggtcaag cacaaattgc tatactactg gaaattgact gggctaccgc ttcctgatga 10500

atgcgacttt gatcatctca ttatcagcag gcaatggaag agaatactgg agtcggccac 10560

tcctgacaca gagagaatga taaaactcgg gcgggcagtg caccagactc tcaaccacaa 10620

ctccaagata accggagtgc tccatcccag gtgtttagaa gaactggcta gtattgaggt 10680

cccagattca actaacaaat tccggaagat tgaaaagaag atccagattc acaacacaag 10740

gtatggagac ctgttcacaa agctgtgcac gcatgttgag aagaaattgc taggatcatc 10800

ccggtctaat aatgtcccac gatcagagga attcagtagc atccgtacag atccggcatt 10860

ctggtttcac tcaaaatggt ccagagctaa gttcgcgtgg ctccatataa aacaagtcca 10920

aaggcatctg attgtagcag caaggacaag gtctgcagtc aacaagttag taacattaag 10980

tcataagata ggccacgtct ttgttactcc tgagcttgtc attgtgacac atacagacga 11040

gaacaagttc acatgcctca cccaggaact tgtattgatg tatgcggata tgatggaagg 11100

cagggacatg gtcaatataa tatcttctac agcagcacat ctcagaaacc tatccgagaa 11160

aattgacgat attctgcggt tagtagatgc cctggcaaag gacttaggta atcaagtcta 11220

tgacgttgta gcattaatgg agggattcgc atacggtgct gttcagctgc ttgagccatc 11280

aggtacattt gcaggagatt tctttgcatt taacctacag gagctcaaag acactttaat 11340

cgaacttctc ccaaataata tagcggaatc agtaactcac gctattgccg ctgtattctc 11400

cggtttagaa cagaatcaag cagctgagat gttgtgcttg ctacgtttgt ggggtcatcc 11460

tttgcttgag tctcgtagtg cagcaagagc agtcaggagc cagatgtgtg caccgaagat 11520

ggtagacttc gatatgatcc tccaggtact atctttcttt aaaggaacaa tcatcaatgg 11580

atacagaaag aagaattcag gtgtgtggcc gcgtgtcaaa gtagatacaa tatacgggaa 11640

tatcattggg cagctgtatg ctgattcagc agagatctca catgatgtca tgttgaagga 11700

gtacaagagt ttatctgctt ttgaatttga gccatgtata gactatgacc ctgttaccaa 11760

tctaagcatg ttcctaaaag acaaggcaat cgcacatcct agtgataatt ggctcgcctc 11820

atttaggcgg aacctactct ctgaggacca gaagaaacag ataaaagagg caacttcaac 11880

taaccgcctc ttgatagagt tcttagaatc aaatgatttt gatccatata aagaaatgga 11940

atacctgaca accctcgagt acctaagagg tgacagtgtg gcagtatcgt actcactcaa 12000

agagaaagag gtaaaagtga atgggcggat ttttgctaag ttaacaaaga aactaaggaa 12060

ctgccaggta atggcagaag gaattctagc tgaccagatt gcacctttct ttcagggaaa 12120

tggggtcatt caagatagca tatccttgac aaagagtatg ttagcgatga gtcaactgtc 12180

ctttaacagc aataagaaac gtatcactga ctgcaaagag agggtttcct cgaaccgcaa 12240

tcatgatcag aagagcaaga atcgtagaag agttgccact tttatcacga ctgacctaca 12300

aaagtattgt cttaactgga gatatcagac agtcaaacta ttcgcccatg ctatcaatca 12360

gctgatgggc ctacctcatt tcttcgagtg gattcatctt aggctgatgg acactacaat 12420

gtttgtaggg gatcctttca atcctccaag tgacccgact gactgtgatc tatcaagagt 12480

cccaaacgat gacatatata ttgtcagtgc tagagggggc attgagggac tctgccagaa 12540

gctatggacg atgatctcaa ttgctgcaat ccaacttgct gcagcaagat ctcattgtcg 12600

agttgcctgc atggtacaag gtgacaatca agtaatggct gtaacgagag aggtaagatc 12660

agatgattcc ccggatatgg tgttgacgca gttgcatcaa gctagtgata atttcttcaa 12720

ggaattgatt catgtcaatc atttgattgg ccataatctg aaggatcgtg aaaccattag 12780

atcagacaca ttcttcatat acagcaaacg aatattcaag gatggagcaa tactcagtca 12840

ggtcctcaaa aattcatcta aattggtgct aatatcaggc gaccttagcg aaaacactgt 12900

aatgtcctgt gccaacattg catccactgt agcacgacta tgtgagaatg ggcttccaaa 12960

ggatttctgt tactatttga actacctaat gagttgtgtg cagacatact ttgattcgga 13020

gttctctatt acccacagct cgcaatcaga ttccaaccag tcctggatcg aggatatctc 13080

tttcgtacac tcatacgtgt taacccctgc ccagctgggg ggactgagca accttcaata 13140

ctcaaggctc tacacaagga atattggtga cccagggacc actgctttcg cagaggtcaa 13200

gcgactggaa gcagtggggt tgctgagtcc cagcatcatg actaacatct taaccaggcc 13260

acctggcaat ggagactggg ccagcctatg caacgaccca tactctttta attttgaaac 13320

tgttgcaagc ccaaatattg tcctcaagaa acatacacag aaagtcctat ttgagacatg 13380

ttcaaacccc ttattatccg gggtacatac agaggacaat gaggcagagg agaaagcatt 13440

ggctgaattc ttactcaatc aagaagtgat tcacccacgt gtcgcacatg ctatcatgga 13500

agcaagctct gtgggtagga gaaagcaaat tcaagggctt gttgacacaa cgaacactgt 13560

gattaagatt gcactgacta ggaggcccct cggtatcaaa aggctgatgc ggataatcaa 13620

ttactcgagc atgcatgcaa tgttgttcag agatgatatt ttcttatcca atagatccaa 13680

ccacccatta gtttcttcca atatgtgctc gctgacgcta gcagattatg cccgaaacag 13740

aagctggtca cctctgacag ggggcaggaa aatactgggt gtatccaacc ctgataccat 13800

agaacttgtg gagggagaga ttctcagcgt cagtggaggg tgcacaaaat gtgacagcgg 13860

agatgagcag tttacttggt tccatcttcc aagcaatata gagctgactg atgacaccag 13920

aaaaaatccc ccgatgagag tgccatatct tgggtcgaag actcaagaga ggagagctgc 13980

ctcgcttgcg aaaatagccc acatgtcacc acatgtgaaa gcagcactaa gggcatcatc 14040

cgtgttaatc tgggcttatg gggacaacga agtgaactgg actgctgctc ttaatattgc 14100

aaggtctcga tgcaacataa gctcagagta ccttcggcta ttgtcacccc tgcccacggc 14160

tgggaatctc caacatagat tggatgatgg cataacccag atgacattta cccctgcatc 14220

tctctacaga gtgtcgcctt acgttcacat atccaatgat tctcaaaggc tattcaccga 14280

agaaggggtc aaagagggaa acgtggttta ccaacaaatt atgctcttgg gtttatccct 14340

aattgaatca ctcttcccaa tgacagcaac cagaacatat gatgagatca cattacacct 14400

ccacagtaaa tttagctgct gtatccgaga agcgcctgtt gcggttccct tcgagctcct 14460

cgggctggca ccggaattaa gaatggtaac ctcaaataag ttcatgtatg atcctagccc 14520

tatatcagag agagatttcg cgagacttga cttagctatc ttcaagagtt acgagcttaa 14580

tttggaatca tattccacgc tggagctaat gaacattctt tcaatatcta gcgggaagtt 14640

gattggccaa tctgtggttt cttatgatga agacacctct ataaagaatg atgctataat 14700

agtgtatgac aacacacgaa attggattag tgaggcgcag aactcagatg tggtccgctt 14760

gtttgagtat gcagcactcg aagtgcttct tgactgtgct tatcaactct actatctgag 14820

ggtaaggggt ctaaacaaca tcgtcctata catgaatgac ttatataaga acatgccagg 14880

gatcctactc tccaatattg cggccacgat atcccacccc atcattcact caaggttgaa 14940

tgcagtaggc ctaattaacc atgacgggtc acaccagctc gcagatatag acttcgtcga 15000

ggtgtctgca aaattgttag tctcttgcac tcgacgcgtg gtctcaggct tatatgcagg 15060

gaataagtac gatctgctgt tcccatctgt cttagatgat aacctgaatg agaagatgct 15120

ccaactgatt tcccggttat gctgtctgta cacagtgctc tttgctacaa caagagaaat 15180

cccaaaaata aggggcctat cagcagaaga gaaatgctca atactcactg agtatctact 15240

gtcagatgct gtaaaaccat tgcttaggcc cgaacaagtg aattctatca tgtctcccaa 15300

cataatcacg ttcccagcca atctatatta catgtctagg aagagcctta atttgatcag 15360

agaacgagag gacagagata ctatcttgtc attgttgttc cctcaggaac cactgcttga 15420

gcttcgccca gtacgggaca ttggtgctcg agtgaaagac ccgtttaccc ggcaacccgc 15480

atcattcata caagagctag atctgagtgc cccagcaagg tacgacgcat ttacactgag 15540

taaggcctgc ttcgagcata cattaccgaa cccaaaggag gattacctag tacggtactt 15600

gttcagagga atagggactg cttcatcttc ttggtataag gcatctcatc ttctatccgt 15660

acctgaggtc aggtgtgcaa gacatgggaa ctccttatac ttagcggaag gaagcggagc 15720

catcatgagt cttcttgaat tgcatatacc acatgagact atctattaca atacactttt 15780

ctcgaatgag atgaaccctc cacagcgaca tttcggacct acaccaacac agtttctaaa 15840

ctcggtcgtt tataggaatc tacaagcgga agtgccatgt aaagatggat atgtccagga 15900

gttctgccca ttatggagag agaatgcaga agaaagtgac ctgacctcag ataaagcagt 15960

tggatatatc acatccgtgg taccctacag gtctgtatca ttactacatt gtgacattga 16020

gattcctcca gggtccaatc aaagcttatt agatcaactg gctactaatt tatccctgat 16080

tgccatgcat tctgtgaggg agggcggggt agtgatcatc aaagtactgt atgcaatggg 16140

gtactacttc catttactca tgaatttatt cactccatgt tccacaaaag gatatatact 16200

ctccaatggc tatgcctgta gaggggatat ggagtgttac ctgatattcg ttatgggcta 16260

cttgggcggg cccaccttcg tgcacgaagt ggtaaggatg gcaaaaactc taatacaacg 16320

acacggtaca ctcctatcta aatcagatga aattacattg actaagctat ttacctcaca 16380

gcagcgtcat gtaacagata tcctatccag tcctttaccg aagctaatga agctcttgag 16440

agaaaatatt gatgccgcac taattgaagc tgggggacag cccgtccgtc cattctgtgc 16500

ggaaagtttg gtgagcacac taacagatat gactcagaca gctcagatca ttgccagcca 16560

cattgacaca gtcattcggt ctgtaattta catggaggct gagggtgacc tcgccgacac 16620

agtgttctta tttactcctt acaatctatc cacagacggt aaaaggagaa catcacttaa 16680

gcagtgcacc aaacagatct tggaagtcac aatactgggt ctcagagcca aagatatcaa 16740

taaagtaggt gatgtaatca gtttagtact cagaggtgcg gtttctctag aggacctcat 16800

cccattaagg acatacctga agcgcagtac ctgcactaaa tacctgaaag cggtcctagg 16860

tattactaaa ctcaaagaaa tgttcacaga tacctcatta ctgtacttga ctcgtgctca 16920

acaaaaattc tacatgaaaa ccataggtaa tgctgccaag ggatattact gtaataatga 16980

ctcttaaagg caatcgtacg ccaatcagtt atcttcctaa ctgatgactc cctcattgac 17040

tcaattatac cagattagaa aaaagttaaa ttccgactct ttggaactcg tattcggatt 17100

cagttagtta actttaagca agagtgcgca aagtcatccc taattatagt gatgtcattc 17160

accaaatctc tgtttggt 17178

 

 

 

<210> 5

<211> 15192

<212> DNA

<213> MG7 nucleotide sequence

<400>

accaaacaga gaatctgtga ggtacgataa aaggcgaaga agcaatcgag atcgtacggg 60

tagaaggtgt gaatcccgag tgcgaggccg aagctcgaac ctgaggggac cttctaccga 120

tatgtcgtct gttttcgacg agtacgagca gctcctcgct gctcagaccc gccctaacgg 180

aactcatgga gggggagaga aagggagcac tttaaaagtt gaggtcccag tattcaccct 240

aaacagtgat gatccagagg atagatggaa ttttgcggta ttctgtcttc ggattgctgt 300

tagcgaggat gccaacaaac cactcaggca aggtgctctt atatccctct tatgctccca 360

ttctcaggtg atgagaaacc atgttgccct tgcagggaaa caaaatgagg ccacactggc 420

tgttcttgag atcgatggtt ttgctaacag tgtgccccag ttcaacaata ggagtggagt 480

gtctgaggaa agagcacaga gattcatggt aatcgcagga tctctccctc gggcgtgcag 540

caacggtact ccgtttgtca cggctggggt tgaagatgat gcaccagaag atatcactga 600

cactctggaa agaatcctat ctatccaagt tcaggtatgg gtcacagtag caaaggccat 660

gactgcatat gagacagcag atgagtcaga aacaagaaga ataaataagt atatgcagca 720

aggtcgagtt cagaagaagt acatccttca tcctgtatgc aggagtgcaa ttcaactcac 780

aatcagacat tctctggcag tccgtatctt cctagttagt gagctcaaga ggggccgcaa 840

tacagcaggt gggagctcta catattacaa cttggtcggg gatgtagact catacatcag 900

gaacaccggg cttactgcat ttttcctaac actcaaatat ggaatcaata ccaagacgtc 960

agccctcgca ctcagcagcc tcacaggtga tatccaaaaa atgaaacagc tcatgcgttt 1020

atatcggatg aaaggtgaaa atgcaccata catgacattg ttaggtgaca gtgaccagat 1080

gagctttgca cctgctgagt atgcacaact ttattctttt gccatgggca tggcatcagt 1140

cttagataag ggaactggca agtaccaatt cgccagggac tttatgagca catcattctg 1200

gagacttgga gtagagtatg ctcaggccca gggaagtagc attaatgagg acatggctgc 1260

tgagctaaaa ctaaccccgg cagcaaggag aggcctggca gctgctgccc aacgagtatc 1320

cgaagaaatc ggcagcatgg acattcccac tcaacaggcg ggagtcctca ccgggctcag 1380

tgatgaaggc ccccgaactc cacagggcag atcaaacaag ccgcaagggc aaccagatgc 1440

tggggatggg gagacccaat tcttggattt tatgagagca gtggcgaacg gcatgcggga 1500

agcgccaaat cctgtacaga gcaccaccca tccagagcct cccccaaccc ctggggcatc 1560

ccaagacaac gacactgact gggggtactg atcgacaaca cccagcctgc cttcacagga 1620

tcacaccaaa ccctccgacc aaaaccctcc cacactccct gacccacaac cccgcacgac 1680

cacaccaaca aaagctcccc cccaccctct cccccactcc cagccacacg atcccgccca 1740

cccgggacaa cacaggcaca gctcagttcg tcgacaatcc gcccagagcc caaggtatta 1800

gaaaaaaata cgggtagaag agagacatcc agagaccagg acgagtcacc aagttctctg 1860

ttctcccttc tacccagtga attagggtga agatggccac ttttacagat gcggagatag 1920

atgacatatt tgagaccagt gggactgtca ttgacagcat aattacggcc cagggcaaat 1980

cagctgagac cgttggaaga agtgcgatcc cgcagggcaa gaccaaagct ctaagcacag 2040

catgggagaa gcacgggagt atccagccac acgccagtca ggacgcccct gaccaacaag 2100

acagaataga aaaacagcca tccacacctg agcaggcgac tctacacaac aatccgccga 2160

tcgcatccac tgaaccgcct cccactcagg ccgcaagcga gaccagcgac acacagctca 2220

agactggggc aagcaactcc cttctgtcca tgctcgacaa actgagtaat aaatcgtcca 2280

atgctaaaaa gggcccatgg tcgggatccc aagaagggca tcaccaacct ccggcccaac 2340

aacatgggaa ccagccgagc tatggaagca accagggaag accgcagcat caggccaagg 2400

ccgtccctgg aaaccggggc acagacgaga acacagcata tcatggacag cggaaggagt 2460

cacaaccatc agctggtgca acccctcatg cgccccagtc agggcagagc caagacaata 2520

ctcctgcacc tgtggatcgt gtccagctac ctgccgactt tgtgcaggcg atgatgtcta 2580

tgatggaggc attatcacag aaggtaagta aagttgatca tcagctggac ctagtcttga 2640

aacagacatc ctccattcct atgatgcgat ctgaaatcca acagctcaag acatctgttg 2700

cgatcatgga agctaactta ggcatgatga aaattctgga ccctggttgt gctaatgttt 2760

catccttaag tgatctccgg gcagtagccc gatcccatcc agtcctagtt tcaggccctg 2820

gagacccatc tccttacgtg acacaagggg gtgaaatgac gctcaataaa ctctcacaac 2880

cggtgcagca cccctctgaa ttgattaagt ctgccactgc aagcgggcct gacatgggag 2940

tggagaagga cactgtccgc gcattaatca cctcgcgccc gatgcatcca agctcctcgg 3000

ctaagctcct gagcaagcta gatgcagcca ggtcaattga agagatcagg aagatcaaac 3060

gccttgcgct gaatggttga tggccatcac aactcataac aggctcccgt cactttagcg 3120

tcacacggaa tccctcgggg gccctccctc gcaaatctat gcttcaacac ccaaaacaac 3180

agccctctct cacccccccc aatccctcga atgaccgcac aactgcaacc aatccagcag 3240

cattagaaat taagaaaaaa tacgggtaga atcaaagtgc cttgattgca ccaaaatgga 3300

ctcatccagg acaattgggc tgtactttga ttctgccctc ccttccagca gcctgttagc 3360

atttccgatt gtcttacaag acacgggaga cgggaagaag caaatcaccc cacaatacag 3420

gatccagcgt cttgattcgt ggacagacag taaggaagac tcggtattca tcaccaccta 3480

cgggttcatc tttcaaattg ggaatgaaga aaccaccgtc ggtgtgatca atgacaatcc 3540

caggcacgag ctactctctt ccgcaatgct ctgcttaggg agtgtcccga acgacggaga 3600

tcttgttgag ctggcgagag cctgcctcac catggtggta acttgcaaaa agagtgcaac 3660

taacactgag agaatagtct tttcagtagt gcaggctcct cgggtgctgc aaagctgtat 3720

ggttgtggca aataggtact catcagtgaa tgcagtgaag catgtgaagg cgccagaaaa 3780

gatccctggg agcggaaccc tagagtataa agtgaatttt gtctctttga ccgtggtgcc 3840

aagaagggat gtctacagga tcccaactgc agtattgaaa gtgtctggct caagcctgta 3900

caatcttgcg ctcaatgtca ctattgatgt ggacgtggat ccgaagagcc cgttagtcaa 3960

atccctttcc aagtccgata gcggatacta tgcgaatctt tttctgcata tcgggcttat 4020

gtccactgta gataagaagg gaaagaaagt gacatttgac aagatagagg aaaagataag 4080

gagactcaat ctatccgtcg ggctcagtga tgtgctcgga ccctctgtgc ttgtgaaggc 4140

gagaggtgca cggactaagc tacttgctcc ttttttctct agcagtggga cagcctgcta 4200

tcctatagca aatgcctctc cccaggttgc caagatactc tggagccaaa ctgcgcacct 4260

gcggagtgtg aaagtcatca ttcaagccgg cactcagcgt gctgtcgcag tgaccgctga 4320

tcatgaggta acctccacta agatagagag gaggcatgcc attgctaaat acaatccttt 4380

caggaaataa gttgcatccc taagactgca gttcacctgc tttcccgaat caccattaca 4440

ccagacaatg atccatctcg actgcttata gttagttcac ctgtctagca aattagaaaa 4500

aacacgggta gaagagtctg gatcccgacc ggcacactca ggacgcaaca tgggctccaa 4560

accttctacc aggatcccag cacctctgat gctggtcacc cggattatgc tgatattggg 4620

ctgtattcgt tcgacaagct ctcttgacgg caggcctctt gcagctgcag gaattgtagt 4680

aacaggagat aaggcagtca atgtatacac ctcgtctcag acagggtcaa tcatagtcaa 4740

gttgctcccg aatatgccca gggataagga ggcgtgtgcg aaagccccat tagaggcata 4800

taacagaaca ctgactactt tgctcactcc tcttggcgac tccatccgca agattcaagg 4860

gtctgtgtcc acgtctggag gaaggaggca aaaacgcttt ataggtgctg ttattggcag 4920

tgtagctctt ggggttgcaa cagcggcaca gataacagca gctgcggccc taatacaagc 4980

caacaagaat gctgccaaca tccttcggct taaggagagc attgctgcaa ccaatgaagc 5040

tgtgcatgaa gtcaccgacg gattatcaca actatcagtg gcagttggga agatgcagca 5100

gtttgtcaat gaccagttta ataatacggc gcgagaattg gactgtataa aaatcacaca 5160

acaggttggt gtagaactca acctatacct aactgaattg actacagtat tcgggccaca 5220

gatcacctct cctgcattaa ctcagctgac catccaggca ctttataatt tagctggtgg 5280

caatatggat tacttattaa ctaagttagg tatagggaac aatcaactca gctcattaat 5340

tggtagcggc ctgatcactg gttaccctat actgtatgac tcacagactc aactcttggg 5400

catacaagtg aatttgccct cagtcgggaa cttaaataat atgcgtgcca cctatttgga 5460

gaccttatct gtaagtacaa ccaaaggata tgcctcagca cttgtcccga aagtagtgac 5520

acaagtcggt tctgtgatag aagagcttga cacctcatac tgtatagagt ccgatctgga 5580

tttatattgt actagaatag tgacattccc catgtcccca ggtatttatt cctgtttgag 5640

cggcaacaca tcagcttgca tgtattcaaa gactgaaggc gcactcacta cgccgtatat 5700

ggcccttaaa ggctcagtta ttgccaattg taagataaca acatgtagat gtacagaccc 5760

tcctggtatc atatcgcaaa attatggaga agctgtatcc ctgatagata gacattcgtg 5820

caatgtctta tcattagacg ggataactct gaggctcagc ggagaatttg atgcaactta 5880

tcaaaagaac atctcaatac tagattctca agtcatcgtg acaggcaatc ttgatatatc 5940

aactgaactt ggaaacgtca acaattcaat cagcaatgcc ttggataagt tggcagaaag 6000

caacagtaag atagaaaaag tcaatgtcag attaaccagc acatctgctc tcattaccta 6060

tattgttcta actgtcattt ctctattttt cggtgcactt agtctgggtt tagcgtgtta 6120

cctgatgtac aaacagaagg cacaacaaaa gaccttgcta tggcttggga ataatgccct 6180

tgatcagatg agagccacta caagagcatg aatgcagata agaggtggat atatacccaa 6240

cagcagcctg tgtgtcaatt ccgataatct gtcaagtaga agacttaaga aaaaactact 6300

gggaacaagc aaccaaagag caatacacgg gtagaacggt cagaggagcc acccttcaat 6360

cggaaattag gcttcacaac atccgttcta ccacatcacc atcaacaaga gtcaatcatg 6420

gaccgcgcgg ttaacagagt cgtgctggag aatgaggaaa gagaagcaaa gaacacatgg 6480

cgcctggttt tccggatcgc agtcttactt ttaatggtaa tgactctagc tatctccgca 6540

gctgccctgg catacagtat ggaggccagt acgccgcacg accttgcagg catatcgact 6600

gtgatctcta agacagaaga taaggttacg tctttactca gttcgagtca agatgtgata 6660

gataggatat acaagcaggt ggctcttgaa tccccgctgg cgctactaaa cactgaatct 6720

acaattatga atgcaataac ctctctttct tatcaaatta acggggctgc gaacaatagc 6780

ggatgtgggg cgcctgttca tgacccagat tatatcgggg ggataggcaa agaactcata 6840

gtggacgaca tcagtgatgt cacatcattt tatccttctg catatcaaga acacctgaat 6900

ttcatcccgg cgcctactac aggatccggt tgcactcgga taccctcatt tgacatgagc 6960

accacccatt attgttatac tcacaatgta atactatccg gttgcagaga tcactcacac 7020

tcacatcaat acttagcgct tggtgtgctt cggacatctg caacagggag ggtattcttc 7080

tctactctgc gctccatcaa tttagatgac acccaaaatc ggaagtcctg cagtgtgagt 7140

gcaacccctt taggttgtga tatgctgtgc tctaaggtca cagggactga agaggaggat 7200

tacaagtcag ctgccccaac atcaatggtg cacggaaggc tagggtttga cggtcaatac 7260

catgagaagg acttagacac cacggtctta tttaaggatt gggtggcaaa ttacccagga 7320

gtgggaggag ggtcttttat tgacgaccgt gtatggttcc cagtttacgg agggctcaaa 7380

cccaattcac ccagtgacac tgcacaagaa gggaaatatg taatatacaa gcgccataac 7440

aacacatgcc ccgatggaca agattaccaa attcggatgg ctaaatcttc atataaaccc 7500

gggcgatttg gtggaaagcg cgtacagcaa gccatattat ccatcaaagt gtcaacatcc 7560

ttgggtaagg acccggtgct gactattcca cctaatacaa tcacactcat gggagccgaa 7620

ggcagaatcc tcacagtagg gacatctcac ttcttgtacc aacgagggtc ttcatatttc 7680

tcccctgcct tattatatcc catgacagta aataacaaaa cggctacact ccatagtcct 7740

tatacgttta atgctttcac tcggccaggt agcgtccctt gccaggcatc agcaagatgc 7800

cccaactcat gcatcactgg ggtctatact gatccatatc ccttaatctt ccataggaat 7860

catactctac gaggggtctt tgggacgatg cttgatgatg aacaagcgag acttaacccc 7920

gtatctgcag tattcgacaa catatcccgc agtcgtgtca cccgggtgag ttcaagcagc 7980

accaaggcag catacacgac atcgacatgt tttaaagttg tcaagaccaa taaagcttat 8040

tgtcttagta ttgcagagat atccaatacc ctattcgggg aatttaggat cgttccctta 8100

ttagttgaga tcctcaagga tgatagagtt taagaagcta gacttggccg attgagccaa 8160

tcataggatg gttgggaaga cgacaccaca tcaatcatct cccacaatgc ttagagtcaa 8220

tctgaatatt aacataagcc aggatcccat gttgttgggc agccacaatc agacaatact 8280

gacatgatca ttctgagtcc tgcccactat caccttatta agaaaaaata caaaaagcat 8340

tgagatataa ggggaaacaa ccaacaagag ggaacacggg taggacatgg cgggctccgg 8400

tcccgaaagg gcagagcacc agatcatcct accagagtca catctatcct ctccattggt 8460

caagcacaaa ttgctatact actggaaatt gactgggcta ccgcttcctg atgaatgcga 8520

ctttgatcat ctcattatca gcaggcaatg gaagagaata ctggagtcgg ccactcctga 8580

cacagagaga atgataaaac tcgggcgggc agtgcaccag actctcaacc acaactccaa 8640

gataaccgga gtgctccatc ccaggtgttt agaagaactg gctagtattg aggtcccaga 8700

ttcaactaac aaattccgga agattgaaaa gaagatccag attcacaaca caaggtatgg 8760

agacctgttc acaaagctgt gcacgcatgt tgagaagaaa ttgctaggat catcccggtc 8820

taataatgtc ccacgatcag aggaattcag tagcatccgt acagatccgg cattctggtt 8880

tcactcaaaa tggtccagag ctaagttcgc gtggctccat ataaaacaag tccaaaggca 8940

tctgattgta gcagcaagga caaggtctgc agtcaacaag ttagtaacat taagtcataa 9000

gataggccac gtctttgtta ctcctgagct tgtcattgtg acacatacag acgagaacaa 9060

gttcacatgc ctcacccagg aacttgtatt gatgtatgcg gatatgatgg aaggcaggga 9120

catggtcaat ataatatctt ctacagcagc acatctcaga aacctatccg agaaaattga 9180

cgatattctg cggttagtag atgccctggc aaaggactta ggtaatcaag tctatgacgt 9240

tgtagcatta atggagggat tcgcatacgg tgctgttcag ctgcttgagc catcaggtac 9300

atttgcagga gatttctttg catttaacct acaggagctc aaagacactt taatcgaact 9360

tctcccaaat aatatagcgg aatcagtaac tcacgctatt gccgctgtat tctccggttt 9420

agaacagaat caagcagctg agatgttgtg cttgctacgt ttgtggggtc atcctttgct 9480

tgagtctcgt agtgcagcaa gagcagtcag gagccagatg tgtgcaccga agatggtaga 9540

cttcgatatg atcctccagg tactatcttt ctttaaagga acaatcatca atggatacag 9600

aaagaagaat tcaggtgtgt ggccgcgtgt caaagtagat acaatatacg ggaatatcat 9660

tgggcagctg tatgctgatt cagcagagat ctcacatgat gtcatgttga aggagtacaa 9720

gagtttatct gcttttgaat ttgagccatg tatagactat gaccctgtta ccaatctaag 9780

catgttccta aaagacaagg caatcgcaca tcctagtgat aattggctcg cctcatttag 9840

gcggaaccta ctctctgagg accagaagaa acagataaaa gaggcaactt caactaaccg 9900

cctcttgata gagttcttag aatcaaatga ttttgatcca tataaagaaa tggaatacct 9960

gacaaccctc gagtacctaa gaggtgacag tgtggcagta tcgtactcac tcaaagagaa 10020

agaggtaaaa gtgaatgggc ggatttttgc taagttaaca aagaaactaa ggaactgcca 10080

ggtaatggca gaaggaattc tagctgacca gattgcacct ttctttcagg gaaatggggt 10140

cattcaagat agcatatcct tgacaaagag tatgttagcg atgagtcaac tgtcctttaa 10200

cagcaataag aaacgtatca ctgactgcaa agagagggtt tcctcgaacc gcaatcatga 10260

tcagaagagc aagaatcgta gaagagttgc cacttttatc acgactgacc tacaaaagta 10320

ttgtcttaac tggagatatc agacagtcaa actattcgcc catgctatca atcagctgat 10380

gggcctacct catttcttcg agtggattca tcttaggctg atggacacta caatgtttgt 10440

aggggatcct ttcaatcctc caagtgaccc gactgactgt gatctatcaa gagtcccaaa 10500

cgatgacata tatattgtca gtgctagagg gggcattgag ggactctgcc agaagctatg 10560

gacgatgatc tcaattgctg caatccaact tgctgcagca agatctcatt gtcgagttgc 10620

ctgcatggta caaggtgaca atcaagtaat ggctgtaacg agagaggtaa gatcagatga 10680

ttccccggat atggtgttga cgcagttgca tcaagctagt gataatttct tcaaggaatt 10740

gattcatgtc aatcatttga ttggccataa tctgaaggat cgtgaaacca ttagatcaga 10800

cacattcttc atatacagca aacgaatatt caaggatgga gcaatactca gtcaggtcct 10860

caaaaattca tctaaattgg tgctaatatc aggcgacctt agcgaaaaca ctgtaatgtc 10920

ctgtgccaac attgcatcca ctgtagcacg actatgtgag aatgggcttc caaaggattt 10980

ctgttactat ttgaactacc taatgagttg tgtgcagaca tactttgatt cggagttctc 11040

tattacccac agctcgcaat cagattccaa ccagtcctgg atcgaggata tctctttcgt 11100

acactcatac gtgttaaccc ctgcccagct ggggggactg agcaaccttc aatactcaag 11160

gctctacaca aggaatattg gtgacccagg gaccactgct ttcgcagagg tcaagcgact 11220

ggaagcagtg gggttgctga gtcccagcat catgactaac atcttaacca ggccacctgg 11280

caatggagac tgggccagcc tatgcaacga cccatactct tttaattttg aaactgttgc 11340

aagcccaaat attgtcctca agaaacatac acagaaagtc ctatttgaga catgttcaaa 11400

ccccttatta tccggggtac atacagagga caatgaggca gaggagaaag cattggctga 11460

attcttactc aatcaagaag tgattcaccc acgtgtcgca catgctatca tggaagcaag 11520

ctctgtgggt aggagaaagc aaattcaagg gcttgttgac acaacgaaca ctgtgattaa 11580

gattgcactg actaggaggc ccctcggtat caaaaggctg atgcggataa tcaattactc 11640

gagcatgcat gcaatgttgt tcagagatga tattttctta tccaatagat ccaaccaccc 11700

attagtttct tccaatatgt gctcgctgac gctagcagat tatgcccgaa acagaagctg 11760

gtcacctctg acagggggca ggaaaatact gggtgtatcc aaccctgata ccatagaact 11820

tgtggaggga gagattctca gcgtcagtgg agggtgcaca aaatgtgaca gcggagatga 11880

gcagtttact tggttccatc ttccaagcaa tatagagctg actgatgaca ccagaaaaaa 11940

tcccccgatg agagtgccat atcttgggtc gaagactcaa gagaggagag ctgcctcgct 12000

tgcgaaaata gcccacatgt caccacatgt gaaagcagca ctaagggcat catccgtgtt 12060

aatctgggct tatggggaca acgaagtgaa ctggactgct gctcttaata ttgcaaggtc 12120

tcgatgcaac ataagctcag agtaccttcg gctattgtca cccctgccca cggctgggaa 12180

tctccaacat agattggatg atggcataac ccagatgaca tttacccctg catctctcta 12240

cagagtgtcg ccttacgttc acatatccaa tgattctcaa aggctattca ccgaagaagg 12300

ggtcaaagag ggaaacgtgg tttaccaaca aattatgctc ttgggtttat ccctaattga 12360

atcactcttc ccaatgacag caaccagaac atatgatgag atcacattac acctccacag 12420

taaatttagc tgctgtatcc gagaagcgcc tgttgcggtt cccttcgagc tcctcgggct 12480

ggcaccggaa ttaagaatgg taacctcaaa taagttcatg tatgatccta gccctatatc 12540

agagagagat ttcgcgagac ttgacttagc tatcttcaag agttacgagc ttaatttgga 12600

atcatattcc acgctggagc taatgaacat tctttcaata tctagcggga agttgattgg 12660

ccaatctgtg gtttcttatg atgaagacac ctctataaag aatgatgcta taatagtgta 12720

tgacaacaca cgaaattgga ttagtgaggc gcagaactca gatgtggtcc gcttgtttga 12780

gtatgcagca ctcgaagtgc ttcttgactg tgcttatcaa ctctactatc tgagggtaag 12840

gggtctaaac aacatcgtcc tatacatgaa tgacttatat aagaacatgc cagggatcct 12900

actctccaat attgcggcca cgatatccca ccccatcatt cactcaaggt tgaatgcagt 12960

aggcctaatt aaccatgacg ggtcacacca gctcgcagat atagacttcg tcgaggtgtc 13020

tgcaaaattg ttagtctctt gcactcgacg cgtggtctca ggcttatatg cagggaataa 13080

gtacgatctg ctgttcccat ctgtcttaga tgataacctg aatgagaaga tgctccaact 13140

gatttcccgg ttatgctgtc tgtacacagt gctctttgct acaacaagag aaatcccaaa 13200

aataaggggc ctatcagcag aagagaaatg ctcaatactc actgagtatc tactgtcaga 13260

tgctgtaaaa ccattgctta ggcccgaaca agtgaattct atcatgtctc ccaacataat 13320

cacgttccca gccaatctat attacatgtc taggaagagc cttaatttga tcagagaacg 13380

agaggacaga gatactatct tgtcattgtt gttccctcag gaaccactgc ttgagcttcg 13440

cccagtacgg gacattggtg ctcgagtgaa agacccgttt acccggcaac ccgcatcatt 13500

catacaagag ctagatctga gtgccccagc aaggtacgac gcatttacac tgagtaaggc 13560

ctgcttcgag catacattac cgaacccaaa ggaggattac ctagtacggt acttgttcag 13620

aggaataggg actgcttcat cttcttggta taaggcatct catcttctat ccgtacctga 13680

ggtcaggtgt gcaagacatg ggaactcctt atacttagcg gaaggaagcg gagccatcat 13740

gagtcttctt gaattgcata taccacatga gactatctat tacaatacac ttttctcgaa 13800

tgagatgaac cctccacagc gacatttcgg acctacacca acacagtttc taaactcggt 13860

cgtttatagg aatctacaag cggaagtgcc atgtaaagat ggatatgtcc aggagttctg 13920

cccattatgg agagagaatg cagaagaaag tgacctgacc tcagataaag cagttggata 13980

tatcacatcc gtggtaccct acaggtctgt atcattacta cattgtgaca ttgagattcc 14040

tccagggtcc aatcaaagct tattagatca actggctact aatttatccc tgattgccat 14100

gcattctgtg agggagggcg gggtagtgat catcaaagta ctgtatgcaa tggggtacta 14160

cttccattta ctcatgaatt tattcactcc atgttccaca aaaggatata tactctccaa 14220

tggctatgcc tgtagagggg atatggagtg ttacctgata ttcgttatgg gctacttggg 14280

cgggcccacc ttcgtgcacg aagtggtaag gatggcaaaa actctaatac aacgacacgg 14340

tacactccta tctaaatcag atgaaattac attgactaag ctatttacct cacagcagcg 14400

tcatgtaaca gatatcctat ccagtccttt accgaagcta atgaagctct tgagagaaaa 14460

tattgatgcc gcactaattg aagctggggg acagcccgtc cgtccattct gtgcggaaag 14520

tttggtgagc acactaacag atatgactca gacagctcag atcattgcca gccacattga 14580

cacagtcatt cggtctgtaa tttacatgga ggctgagggt gacctcgccg acacagtgtt 14640

cttatttact ccttacaatc tatccacaga cggtaaaagg agaacatcac ttaagcagtg 14700

caccaaacag atcttggaag tcacaatact gggtctcaga gccaaagata tcaataaagt 14760

aggtgatgta atcagtttag tactcagagg tgcggtttct ctagaggacc tcatcccatt 14820

aaggacatac ctgaagcgca gtacctgcac taaatacctg aaagcggtcc taggtattac 14880

taaactcaaa gaaatgttca cagatacctc attactgtac ttgactcgtg ctcaacaaaa 14940

attctacatg aaaaccatag gtaatgctgc caagggatat tactgtaata atgactctta 15000

aaggcaatcg tacgccaatc agttatcttc ctaactgatg actccctcat tgactcaatt 15060

ataccagatt agaaaaaagt taaattccga ctctttggaa ctcgtattcg gattcagtta 15120

gttaacttta agcaagagtg cgcaaagtca tccctaatta tagtgatgtc attcaccaaa 15180

tctctgtttg gt 15192

Claims (8)

1. express the preparation method of the recombinant viral vaccine strain of African swine fever virus p72 albumen, it is characterized in that the method comprises:

(1) build restructuring and transcribe plasmid, this transcribes the genome cDNA sequence that plasmid comprises the described Newcastle disease attenuated vaccine strain of the p72 gene wherein inserting African swine fever virus (ASFV), then in transcription vector plasmid pBSK, assemble acquisition restructuring transcribe plasmid pBSK-MG7-ASFVp72, wherein: Newcastle disease attenuated vaccine strain MG7 is transformed by reverse genetics, its F protein cracking site is replaced with the cracking site of Lasota strain F protein, sudden change causes weak artificial attenuated IBDVs thus;

(2) helper plasmid system transcribed by structure, and this helper plasmid system comprises the helper plasmid pBSK-L of the cDNA sequence of the helper plasmid pBSK-P of the cDNA sequence of the helper plasmid pBSK-NP of the cDNA sequence of the nucleoprotein (NP) of described new castle disease virus weakening strain of encoding and the phosphorprotein (P) of described new castle disease virus weakening strain and the large polymerase protein (L) of described new castle disease virus weakening strain;

(3) by the above-mentioned host cell BHK-21 transcribing plasmid and transcribe that newcastle disease weakening strain described in helper plasmid cotransfection can copy, the host cell after transfection is cultivated;

(4) supernatant liquor is gathered in the crops, inoculated into chick embryo allantoic cavity rescue recombinant virus after filtering.

2. the preparation method of the recombinant viral vaccine strain of expression African swine fever virus p72 albumen according to claim 1, it is characterized in that the gene of amplification coding African swine fever virus p72 albumen, add new castle disease virus specific gene initial sum terminator sequence at its two ends, be inserted into manually-injected between gene P, M of new castle disease virus weakening vaccine strain pme Irestriction enzyme site, be built into total length recombinant plasmid, the BHK-21 cell of recombinant plasmid and the process of helper plasmid pBSK-NP, pBSK-P and pBSK-L common transfection poxvirus infection is cultivated, again the cell conditioned medium liquid of transfection is inoculated 9 age in days SPF chick embryo allantoic cavities, save the recombinant virus infection BHK-21 cell obtained.

3. express a recombinant viral vaccine strain for African swine fever virus p72 albumen, it is characterized in that its gene order is as shown in SEQ ID No.4.

4. the recombinant viral vaccine strain of expression African swine fever virus p72 albumen according to claim 4, is characterized in that described newcastle disease weakening strain is MG7.

5. the application of recombinant viral vaccine strain in preparation African swine fever virus vaccine of expression African swine fever virus p72 albumen prepared by the method described in claim 1 or 2.

6. the application of recombinant viral vaccine strain in the anti-African swine fever virus medicine of preparation of expression African swine fever virus p72 albumen prepared by the method described in claim 1 or 2.

7. the application of recombinant viral vaccine strain in preparation African swine fever virus vaccine of the expression African swine fever virus p72 albumen described in claim 3 or 4.

8. the application of recombinant viral vaccine strain in the anti-African swine fever virus medicine of preparation of the expression African swine fever virus p72 albumen described in claim 3 or 4.