CN102344914B - Construction and application of recombinant MDV (Marek's Disease Virus) strain for expressing REV env (Reticuloendotheliosis Viruses envelope) - Google Patents

Abstract

The invention relates to construction and application of a recombinant MDV (Marek's Disease Virus) strain for expressing REV env (Reticuloendotheliosis Viruses envelope), and relates to construction and application of recombining a strain into the REV env by taking an MDV Guangdong isolate (GD07) as a vector. The obtained recombinant strain MDV GD07-env is used as a vaccine prepared for producing the strain to immune a one-day chicken, so that marek's disease induced by super-strong strain MDV can be prevented; the protective immune effect of the vaccine is superior to that of MDV CVI (Common Variable Immunodeficiency) 988/Rispens strain vaccine which is most widely applied on the markets at home and abroad; and the recombinant virus can also have a good immune protection effect on the REV virus.

Description

A kind of structure and application of expressing the recombinant mdv strain of REV env

Technical field the present invention relates to structure and the application of the recombinant mdv strain of a kind of REV of expression env, belongs to the veterinary biologics biology field.

Background technology

Tumour is the regular incidence of kind of chicken and commodity egg, although its M ﹠ M is not high, the financial loss that causes is very large, major cause be oncorna virus to the caused immunosuppression of chicken body, and and then cause decline to various vaccine immune responses.In recent years, the incidence of neoplastic disease had again the trend of new line in China chicken group.

The virus that can cause tumour among the chicken group has three kinds, be marek's disease virus (Marek ' s disease virus, MDV), fowl leukosis virus (Avian leucosis virus, ALV) and the sick virus of fowl RE hyperplasia (Reticuloendotheliosis viruses, REV).For a long time, need only one and tumour occurs in the chicken group, just suspecting even asserting is Marek, and has ignored the pathogenic of other two kinds of viruses.The A of ALV, B, C, D hypotype can be in layer induced tumor, the J subgroup of ALV is main induced tumor in meat type kind chicken then.But REV is equal induced tumor in egg type and meat type chicken then.

Change and epidemic according to clinical pathology, although can distinguish some typical Marek or leukemia, but actual the showing of tumour that occurs gradually changed among the chicken group in recent years, was difficult to only come the actually tumour of any virus initiation of differential diagnosis according to pathology.Particularly, in same chicken group and in the same sick chicken, the phenomenon of two kinds of different virus coinfections is very general, and this has more brought difficulty to differential diagnosis.

Fowl RE hyperplasia virus (REV), this is another kind of retrovirus, but the multiple birds of natural infection are extensive in distributed in nature.REV may pass through chicken embryo generation vertical infection, and the chick of vertical infection or early infection short and small syndromes the most easily occurs and exempts to suppress syndromes, simultaneously also can induced tumor.Be difficult to distinguish according to clinical pathology and MDV or ALV tumour.At present, REV infects quite generally among China chicken group, undoubtedly, has a certain proportion of tumour relevant with the REV infection.

In nearly two, three years, even in some chicken groups who has used MDV CV1988/Rispens strain liquid nitrogen seedling (the present invention is called for short again the CV1988 vaccine) immunity, the trend that still has tumor incidence to come back.Because people still impute it to MDV, therefore many people infer the epidemic strain of special ultra-intense virus type whether occurred.But up to now, not yet can experimental results show that this point both at home and abroad.Yet, from the neoplastic disease chicken of this class chicken house, separate by virus, found that the sick chicken of significant proportion exists the coinfection of MDV and REV.In addition, doing simultaneously in the Experimental infection with REV and two kinds of viruses of MDV, also really can bring out the tumor nodule of different cell types.

Because it is quite general on China chicken group that REV infects, have and think, the coinfection of REV is to cause the major reason that tumor incidence raises among the CV1988 vaccine immune chicken group.Owing to causing immunosuppression in the REV of some chickens infection, having caused that also the protective immunity of being brought out by the CV1988 vaccine descends, thereby caused vaccinated flock to still have tumour to occur.

The infection conditions of REV among nearly 5 years In Guangzhou Areas cultivation chicken group is followed the trail of in this laboratory, and the result shows, the infection rate of REV in our province chicken group from 0 to 100% on average is about 20%.Artificial challenge's experiment shows, REV has very strong immunosuppressive action to chick, it had both significantly suppressed the growth of central immune organ thymus gland and the fabricius bursa, also significantly suppress the antibody response of infected chicken after to NDV and AIV vaccine immunity, wherein, to bird flu (Avian influenza virus, AIV) the more remarkable (Sun Shuhong of the restraining effect of vaccine immunity, Cui Zhi is medium, Chinese virusology (English edition), 2006; Li Yanpeng, Cui Zhi is medium, Chinese animal doctor's journal, 2008).Existing comparative experiments shows, with respect to other immunosuppressive virus, and such as MDV, CAV, ARV, ALV etc., REV is to the immunosuppressive action that chicken shows the strongest (Liu Fengxiang, Cui Zhi is medium, Scientia Agricultura Sinica, 2009).Can show very strong immunosuppressive action when REV infects separately, with other viral coinfection the time, this immunosuppressive action also can further be strengthened, and the immunosuppression that shows is even more serious.Take to the immune response of AIV-H5 vaccine as example, the coinfection of two-strain all suppresses the immune response to vaccine significantly.

Existing result of study has proposed urgent requirement to the REV vaccine, yet up to the present, a kind of REV vaccine product unexpectedly do not occur and come out! Because REV is retrovirus, use living vaccine to exist transfection to advance the potential risk of host cell gene group; And the rate of propagation of REV determined at present can't be by a large amount of cultivation REV to satisfy the need of production of inactivated vaccine.Therefore express the immune protective gene of REV by genetic engineering means, making the recombination engineered vaccine is the good method that addresses this problem.

MDV's is popular from the chicken group, from take weak strain (mMDV) as main, to take strong virus strain (vMDV) as main, so that superpower virus (vvMDV) strain is more and more general over nearly 20 years, special superpower virus strain (vv+MDV) (Witter RL.Increased virulenced of Marek ' s Disease Viurs field isolates.Avian Pathology, 1997) also appearred before 10 years.Scientist utilizes the whole bag of tricks to make up the effect that various MDV candidate strains attempt to improve vaccine, but all unsuccessful.For example; Witter and Kreager etc. have compared the protective immunity effect of the new vaccine candidate strain of 10 strains; but the result is unlike the good (Witter of CVI988/Rispens strain; R.L.and K.S.Kreager.Serotype 1 viruses modified by backpassage or insertional mutagenesis:approaching the threshold of vaccine efficacy in Marek ' s disease.Avian Dis; 2004,48 (4): 768-782.).It seems, in the face of the trend of the pathogenic enhancing of MDV street strain, improve vaccine with ordinary method and as if gone to the end.But according to MDV development law in past more than 40 year, its virulence probably can become stronger at a few years from now on.In recent years, in China, even some chicken groups who has used the CV1988 vaccine immunity, the trend that still has Tumor incidence to rise, although domestic also not have to be correlated be separated to the report of special highly virulent strain, the variation of MDV is being carried out always.

Utilizing the strong malicious pathogenic gene of genetic engineering means (insert, suddenly change or disappearance) inactivation to make virus weakening is a Critical policies that makes up the MDV vaccine.U.S. poultry diease tumor experiment chamber utilizes the clay system to knock out respectively the pp38 of highly virulent strain rMD5, vIL18 and Meq gene (Reddy, S.M., B.Lupiani, I.M.Gimeno, R.F.Silva, L.F.Lee and R.L.Witter.Rescue of apathogenic Marek ' s disease virus with overlapping cosmid DNAs:use of a pp38 mutant to validate the technology for the study of gene function.Proc Natl Acad Sci U S A, 2002,99 (10): 7054～7059.; Cui, X., L.F.Lee, H.D.Hunt, W.M.Reed, B.Lupiani and S.M.Reddy.A Marek ' s disease virus vIL-8 deletion mutant has attenuated virulence and confers protection against challenge with a very virulent plus strain.Avian Dis, 2005,49 (2): 199～206.; Cui, X., L.F.Lee, W.M.Reed, H.J.Kung and S.M.Reddy.Marek ' s disease virus-encoded vIL-8 gene is involved in early cytolytic infection but dispensable for establishment of latency.J Virol, 2004,78 (9): 4753～4760.; Lupiani, B., L.F.Lee, X.Cui, I.Gimeno, A.Anderson, R.W.Morgan, R.F.Silva, R.L.Witter, H.J.Kung and S.M.Reddy.Marek ' s disease virus-encoded Meq gene is involved in transformation of lymphocytes but is dispensable for replication.Proc Natl Acad Sci U S A, 2004,101 (32): 11815～11820.; ), the result shows equal virulence attenuation of or the disappearance that can make MDV of knocking out of said gene, utilizes the said gene gene-deleted strain to come immune chicken as candidate vaccine, all can provide to a certain degree protection to chicken.Wherein, the strain of disappearance Meq gene is the most promising.Up-to-date studies show that: after attacking with virulent and special virulent, Meq genetically deficient strain can provide the better protection than CVI988, this also is the best MDV candidate vaccine strain of the protection effect (Lee that reports at present, L.F., B.Lupiani, R.F.Silva, H.-J.Kung and S.M.Reddy.Recombinant Marek ' s disease virus (MDV) lacking the Meq oncogene confers protection against challenge with avery virulent plus strain of MDV.Vaccine, 2008,26 (15): 1887～1892.; Lee, L.F., K.S.Kreager, J.Arango, A.Paraguassu, B.Beckman, H.Zhang, A.Fadly, B.Lupiani and S.M.Reddy.Comparative evaluaion of vaccine efficacy of recombinant Marek ' s disease virus vaccine lacking Meq oncogene in commercial chickens.Vaccine, 2010,28 (5): 1294～1299).

The present invention with inventor's MDV virulent strain that certain chicken house was separated to from Guangdong in 2007 as carrier, its gene organization is built on the bacterial artificial chromosome (BAC), utilize the homologous recombination technique of recE/T mediation, at first lack wherein 1 Meq gene, then replace another Meq gene with REV env expression cassette, thereby be built into 2 Meq genetically deficients, can express the MDV attenuated vaccine strain of REV env gene.This recombinant strain will satisfy the dual needs of protection MDV and REV simultaneously, and the characteristic of MDV vaccine early immune meets the requirement that the immunosuppressive virus needs carry out immunity in early days.

The successful development of recombinant mdv-REV vaccine will bring strong weapon for the disaster that control MDV, REV and MDV-REV coinfection cause poultry farming.

Summary of the invention

The present invention is on the basis that makes up MDV GD07 bacterial artificial chromosome (BAC); adopt the efficient recombinant technology of recE/T; by twice restructuring; finally lack in the MDV GD07 genome two and caused the Meq gene of Tumor-assaciated with MDV; and fowl reticuloendotheliosis virus (REV) envelope protein env gene expression frame has been inserted in the position of a Meq therein; make the recombinant virus of final acquisition both lose the original tumorigenesis ability of MDV; keep again good immunogenicity, also the fowl reticuloendotheliosis has been had certain immune protective effect simultaneously.

Be to knock out 1 Meq gene in the MDV GD07 strain virus by the mutating technology that utilizes recE/T mediation, and again by Red/ET mediation technology, substitute another Meq gene with REV env expression cassette.

Embodiment

One, Mareks disease vaccine virus GD07-env strain structure

1.REV the structure of env expression cassette.

(1) entire reading frame (ORF) by primer amplification REV env, and the PCR product advanced in the pBudCE4.1 vector plasmid acquisition recombinant plasmid pBud-env with HindIII enzyme and XbaI enzymic digestion rear clone.

F (env1): 5 '-CGCCAAGCTTGACAACCAAGAAGAATG-3 ' (sequence 1 contains the HindIII site)

R (env1): 5 '-CGTATCTAGACCTAGGGTATCCATCTC-3 ' (sequence 2 contains the XbaI site)

(2) pcr amplification env gene expression frame.Take pBud-env as template, design following primer and carry out PCR:

F (env2) 5 '-atggccatgtggtctctacggcgcaaatctagcaggagtgtgcaactccgGCGCGC GTTGACATTGATTA-3 ' (base sequence that sequence 3 capitalizations represent is corresponding to the position of env gene expression frame 1#-20#bp).

R (env2) 5 '-ttataagtaggattccccgtctcctgttggcgattcccgaagatttgtcaCCCCAA CTTGTTTATTGCAG-3 ' (base sequence that sequence 4 capitalizations represent is corresponding to the position of env gene expression frame 2673#-2692#bp).

This PCR product that primer is amplified had both comprised the homology arm (lowercase represents) that is used for Marek’s disease virus gene group Meq each 50bp of upstream and downstream of homologous recombination, had comprised again P _(cmv)The full sequence of promotor, the while has also comprised the early stage mRNApolyA structure of SV40.

2.Meq knocking out of gene

With reference to the research of having delivered (Li Yan roc marek's disease virus meq gene-deleted strain biological characteristics and immune effect research thereof, doctorate paper, 2010, animal and veterinary institute of the Chinese Academy of Agricultural Sciences), utilize kalamycin resistance gene (Kan ^r) replace a meq gene after, picking colony is inoculated in the LB liquid nutrient medium shake and cultivates, and treats that its OD value reaches at 0.5 o'clock, adds 10% pectinose and induces 1h, and the plasmid flp recombinase in the Host Strains is activated, thus removal Kan ^rGene.After inducing bacterium coated respectively 32 ℃ of incubated overnight on the LB agar plate that contains 30 μ g/mL paraxin or 50 μ g/mL kantlex, screening is only grown at the LB agar plate that contains 30 μ g/mL paraxin, but the bacterium that can not grow at the LB agar plate that contains 50 μ g/mL kantlex is recombinant bacterium.The site (flp recognition target, FRT) that still keeps a flp recombinase identification in this recombinant plasmid in the position at first Meq gene place that is removed.

3.REV the env expression cassette substitutes another Meq gene

(1) utilize the Red/ET homologous recombination that selectable marker gene rpsL-neo expression cassette is replaced another Meq gene.

After knocking out a Meq gene and empirical tests, with reference to this laboratory research and development method in early stage (Chen Ruiai. express structure and the Efficacy evaluation thereof of the restructuring rCVI988-env of REV env gene based on the BAC platform. the doctorate paper, 2010, China Agricultural University), utilizing the rpsL-neo plasmid in the GeneBridge company test kit (Red/ET recombination system Counter-Selection BAC Modification Kit) is pcr template, and the amplification both sides are with on the MDV genome Meq gene, the selectable marker gene rpsL-neo expression cassette of each 50bp size homology arm of downstream.

Design of primers is as follows:

F (rpsL-neo) 5 '-atggccatgtggtctctacggcgcaaatctagcaggagtgtgcaactccgGGCCTG GTGATGATGGCGGG-3 ' (sequence 5).

R (rpsL-neo) 5 '-ttataagtaggattccccgtctcctgttggcgattcccgaagatttgtcaTCAGAA GAACTCGTCAAGAA-3 ' (sequence 6).

Capitalization in the primer is the sequence of rpsL-neo of increasing, and lowercase is the homologous recombination arm of MDVMeq gene 50bp.

That fetches that rpsL-neo expression casette PCR product 1～2 μ L (approximately 0.1～0.2 μ g) that receives behind the purifying joins that 30 μ L prepare in advance has not only comprised the pRedET plasmid but also comprise in the DH10B competent cell of MDV GD07 (in BAC), carry out electricity and transform (2000V, 100 Ω, 25 μ F).After electric shock is finished, add immediately 1mL and be preheated in advance mixing in 37 ℃ the LB nutrient solution, be drawn in the EP pipe of 1.5mL sterilization, 37 ℃, 220r/min, vibration activation 70min (at this moment, under the mediation of Red α/Red β albumen homologous recombination occurs), getting 100 μ L cell suspensions coats and contains paraxin (Cmr, 30 μ g/mL), tsiklomitsin (Tet, 3 μ g/mL) and kantlex (Kan, 15 μ g/mL) on three kinds of antibiotic solid agar plates, in 30 ℃ of constant temperature biochemical cultivation cases, cultivate 24h, select the positive bacterium colony with these three kinds of resistances, be inoculated in three kinds of antibiotic 5mL LB nutrient solutions of same adding, 30 ℃, 220r/min, about lucifuge shaking culture 16h, take a morsel the streak inoculation of bacterium liquid in containing paraxin (Cmr, 30 μ g/mL), on kantlex (Kan, 15 μ g/mL) and three kinds of antibiotic flat boards of Streptomycin sulphate (Str, 50 μ g/mL), cultivate about 24h in 37 ℃ of constant temperature biochemical cultivation cases, whether can normally must bring into play in order to the expressive function of verifying selectable marker gene rpsL-neo expression cassette; Getting simultaneously 0.5mL bacterium liquid is inoculated into 100mL in 1% ratio and contains simultaneously paraxin (Cmr, 30 μ g/mL), kantlex (Kan, 15 μ g/mL) and tsiklomitsin (Tet, 3 μ g/mL) in three kinds of antibiotic LB nutrient solutions, 37 ℃, 220r/min, middle amount is extracted and the purification of Recombinant plasmid about lucifuge shaking culture 16h, and process gel electrophoresis preliminary observation is that then the clone of macromole DNA is further cooked the PCR evaluation; Qualification result show have paraxin, tsiklomitsin and kalamycin resistance and do not have streptomycin resistance, and can specific amplification be recombinant clone to the bacterial clone of rpsL-neo gene.

(2) utilize the Red/ET homologous recombination that the env expression casette is replaced the rpsL-neo expression cassette.

To identify that the correct recombinant clone streak inoculation that comprises the rpsL-neo gene is to containing paraxin (Cmr, 30 μ g/mL), tsiklomitsin (Tet, 3 μ g/mL) and kantlex (Kan, 15 μ g/mL) on three kinds of antibiotic LB solid agar plates, cultivate about 24h in 30 ℃ of constant incubators, the positive single bacterium colony of picking is prepared into Electroporation-competent cells with it.Getting 1～2 μ L env expression casette PCR recovery product joins in the Electroporation-competent cells of the above-mentioned fresh preparation of 50 μ L, after mixing, place precooling 3～5min on the ice bath, then be transferred in the 0.2cm electricity revolving cup of ice bath precooling, electricity turns parameter and is set to 2000V, 100 Ω, 25 μ F 1mm.Electric shock adds immediately 1mL after finishing and is preheated in advance 37 ℃ and does not contain antibiotic LB nutrient solution, be transferred to immediately behind the mixing in the EP pipe of new 1.5mL sterilization, 37 ℃, about 220r/min shaking culture 70min, 10000r/min, centrifugal 2min, supernatant discarded, do not contain antibiotic LB nutrient solution Eddy diffusion thalline with 100 μ L, coat and contain paraxin (Cmr, 30 μ g/mL) and Streptomycin sulphate (Str, 50 μ g/mL) on two kinds of antibiotic solid agar plates, cultivate about 24h in 37 ℃ of constant temperature biochemical cultivation cases, when treating single bacterium colony length to suitable size, same single bacterium colony is with two tiny a certain amount of thalline of each picking of rifle head, be inoculated into respectively and contain paraxin (Cmr, 30 μ μ g/mL) with Streptomycin sulphate (Str, 50 μ g/mL) two kinds of antibiotic LB nutrient solutions and contain paraxin Cmr, 30 μ g/mL) with kantlex (Kan, 15 μ g/mL) in two kinds of antibiotic LB nutrient solutions, whether checking selectable marker gene rpsL-neo is replaced, and has two kinds of resistances of paraxin and Streptomycin sulphate, but the bacterium mono-clonal that does not possess kalamycin resistance is required clone, and this clone is named as GD07-env.

4. the rescue of recombinant virus

Process specifications according to QIAGEN plasmid Maxi kit test kit (QIAGEN company) carries out a large amount of extractions of recombinant plasmid GD07-env.According to transfection reagent Lipfectamine ^TMThe specification sheets of 2000 (Invitrogen companies), get that QIAGEN plasmid Maxi kit test kit extracts through identifying that correct recombinant plasmid GD07-env DNA 1 μ g is in 100 μ LDMEM (Gibco company), get 10 μ L lipfectamine in 100 μ L DMDM, with both mixings, room temperature is supplemented to 1mL with mixture with DMDM after placing 20min, joins the cell tiling to 90% 6 orifice plates, 37 ℃, 5%CO ₂After cultivating 6h in the incubator, liquid in 6 orifice plates is changed to the DMEM substratum that contains 5% calf serum, continue to cultivate several days, behind viral Plaque Formation, with cell dissociation, reach the former generation CEF that grows up to individual layer, passed so continuously for 3～4 generations to enlarge virus quantity, to be amplified when a certain amount of, with 0.25% pancreatin (containing 0.01% EDTA) with cell dissociation, centrifugal collecting cell is with behind frozen storing liquid (DMSO: calf serum: DMEM is 1: 3: the 6) re-suspended cell it frozenly being preserved in liquid nitrogen.This called after Marek ' s disease virus GD07/ Δ 2Meq-env is called for short Marek ' s disease virus GD07-env, or MDV GD07-env).This virus strain was deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center in No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 08 29th, 2011, and preserving number is CGMCC No.5169.

Two, the identification feature of marek's disease virus GD07-env strain

Can be used as this viral distinctive sign with three particular sequences that are positioned at specific site on this viral genome:

(1) stays the FRT sequence of next 34bp in the site of Meq genetically deficient

(5 '-GAAGTACCTATTCTTTCTAGAGAATAGGAACTTC-3 ') (sequence 7);

(2) at the Meq gene by the site that REV-env substitutes, the env gene expression frame is arranged, available following primer is identified:

F (env): 5 '-GCGCGCGTTGACATTGATTA-3 ' (sequence 8),

R (env): 5 '-CCCCAACTTGTTTATTGCAG-3 ' (sequence 9).

This has comprised P to the PCR product that primer amplifies _(cmv)Promotor, env ORF, and polyA structure.PCR product sequence size is: 2692bp.

Three .MDV GD07-env strain virus are to the pathogenic of chicken and protective immunity effect

For study recombinant mdv GD07-env under laboratory condition to the strong malicious capital-1 of marek's disease virus and reticulo endotheliosis's virus immune protective effect; 1 210 of age in days SPF chickens are divided into 7 groups at random; every group 30, raise respectively in 7 SPF animal rearing cages with the positive press filtration air.The 1st group of chicken is with 2000PFU/ dosage intraperitoneal inoculation GD07-env only during 1 age in days, the 2nd group of chicken is with 2000PFU/ dosage intraperitoneal inoculation CVI988/Rispens vaccine strain only, the 3rd group is that malicious control group is attacked in the strong malicious capital-1 of Marek’s disease poison, the 4th group of chicken is with 2000PFU/ dosage intraperitoneal inoculation GD07-env only, the 5th group is that reticulo endotheliosis's virus is attacked malicious control group, and the 6th group of dosage intraperitoneal inoculation GD07-env with 2000PFU/ is used for detecting REV antibody.The 7th group is the blank group.After the immunization 7 days, attack MDV capital-1 strain with 500PFU/ dosage only respectively for the 1st, 2,3 group.After the immunization 30 days, use REV virus SNV strain (professor Cui Zhizhong of Shandong Agricultural University present) with 10 for the 4th, 5 group ⁵TCID50/ dosage is only attacked poison, later on every the 10d sterile blood sampling, carries out the blood disease separation test and adopts indirect immunofluorescence assay to detect with chick embryo fibroblast, is used for determining whether REV virus exists in the blood.Detect the variation of anti-REV antibody in the serum by ELISA standard reagent box (IDEXX company).Attack the growth situation of observing weekly chicken behind the poison.

Whole experimental session, pathology is all dissected, observed in GD07-env immune group, CVI988/Rispens immune group and capital-1 after attacking the death of malicious control group chicken, record mortality ratio and the pathology situation that is caused by MD.MD pathology percentage ratio multiply by 100 by the pathology chicken divided by the summation of survival chicken and dead chicken and gets.Vaccine immunity is renderd a service by protective index (PI) and is determined; the PI method of calculation produce MD pathology percentage ratio and deduct the immune group chicken and produce MD pathology percentage ratio and produce MD pathology percentage ratio and multiply by 100 divided by attacking malicious control group chicken again for attacking malicious control group; in order to increase result's objectivity, experiment repeats once.

Compare with the blank group, attack malicious control group and infect the individual little and big or small inequality of the rear chicken group in capital-1, growth and development state is poor, and feather is slightly random, matt, ataxia, the asymmetry of a limb or two limbs, carrying out property paresis.GD07-env and CVI988/Rispens immune group chicken infect the rear big or small homogeneous in capital-1, and growing way is normal, and wherein the GD07-env group has 6 chickens dead, and the CVI988/Rispens group has 7 chicken deaths.GD07-env group chicken group Individual Size is even, and growing way is normal.Attacking malicious rear 90 days all survival chickens execution and cuing open inspection, carry out visual inspection.Compare with the blank group, attack malicious control group liver, spleen, cardiac tumor obvious, CVI988/Rispens immunity chicken has the slight MDV symptom of extremely indivedual performances outer (without the visible obviously pathology of naked eyes), all the other chickens are all normal, and group there are 3 MDV symptoms outer (without the visible obviously pathology of naked eyes) that the chickens performance is slight in MDV GD07-env immunity postoperative infection capital-1, and all the other chickens are all normal.

Compare with the blank group, REV attacks that malicious control group Common Swift is unusual, and body weight is obviously on the low side, and is individual short and small, and cut open inspection and find, the serious atrophy of thymus gland and the fabricius bursa, the peripheral nerve enlargement, chronic ulcerative inflammation appears in glandular stomach.GD07-env immune group chicken group Individual Size is even, and growing way is normal.Cut open inspection and do not find the pathology of internal organs.

By observing the protection effect of in SPF chicken group, attacking the strong rear generation in malicious capital-1, compared MDV GD07-env and the MDVCVI988/Rispens vaccine protection ratio of (being called for short CVI988).Not immune SPF chicken group shows distinctive death and the damage phenomenon (table 1) of 97% Marek, the SPF chicken group in whole chickens in MDV GD07-env immunoinfective capital-1 group and CVI988/Rispens immunity postoperative infection capital-1 compares with the blank group, the size homogeneous, growing way is normal.Show 11% MD light symptoms with the CVI988/Rispens immune group and compare, MDV GD07-env immune group shows 8.5% MD light symptoms.Therefore, GD07-env and CVI988/Rispens are respectively 91.5% and 89% to the protective index in strong malicious capital-1.

Table 1MDV GD07-env and MDV CVI988 vaccine compare the immunoprotection of SPF chicken

By to 30 SPF chickens at 1 age in days immunity Recombinan t virus MDV GD07-env, after the immunity 30 days, the S/P value of 15 chicken serums is arranged greater than 0.5, present strong positive, the S/P value of only having 15 chicken serums is about 0.5, although we are negative with standard determination, its value is also far above 0.02 of contrast chicken serum.Along with the prolongation of immunity time, the NAT of REV begins to descend in the chicken body.When 45d detects after immunity, although still have 14 only to be judged to be the positive in 30 chickens.

Four, vaccine preparation

1. seed culture of viruses

(1) making the seed culture of viruses that this product uses is the seed culture of viruses MDV GD07-env strain that the present invention researches and develops, and is identified, is preserved by inventor place company.

(2) the every 1ml viral level of viral level answers 〉=10 ⁵PFU.

(3) security is with 20 of 1 age in days SPF chick, and the virus of every intraperitoneal inoculation 20000PFU was observed 70～90, should be without the MD clinical symptom, cut open that inspection changes without naked eyes and pathology change.

(4) pure property seed culture of viruses should be without the pollution of bacterium, mycoplasma and exogenous virus.

(5) behind the virus liquid and anti-MD specific serum balanced mix of specificity with 100～200PFU, in 18～22 ℃ and 30min, inoculating cell, the plaque decrement should be more than 95%.

(6) Virus passages is in 25 generations.

2. vaccine manufacturing and the inspection of semifinished product

(1) produces the preparation of using seed culture of viruses

1) seed culture of viruses that liquid nitrogen is preserved is got in the seed culture of viruses breeding, and inoculated into chick embryo inoblast (CEF) passed for 2～3 generations, and harvested cell adds cryoprotectant commonly used, is sub-packed in the little ampulla and seals.Be kept in-196 ℃ of liquid nitrogen, indicate harvest date, seed culture of viruses algebraically and PFU quantity etc.

2) the every 1ml viral level of seed culture of viruses identifying virus assay answers 〉=10 ⁵PFU.

Pure property seed culture of viruses should be without the pollution of bacterium, mycoplasma and exogenous virus.

Behind the virus liquid and anti-MD specific serum balanced mix of specificity with 100～200PFU, in 18～22 ℃ and 30min, inoculating cell, the plaque decrement should be more than 95%.

3) seed culture of viruses is preserved and is put in-196 ℃ of liquid nitrogen, and preservation period is 3 years.

(2) preparation of seedling venom

1) preparation of chick embryo fibroblast: select SPF chicken embryo, hatching is prepared into CEF with 0.01% trysinization behind 9～10 ages in days.

2) the production seed culture of viruses that liquid nitrogen is preserved is got in the preparation of work seed culture of viruses, is inoculated on the CEF cell bottle that grows up to individual layer by 1/100 volume ratio, cultivates 48～72 hours, treats that cytopathy (CPE) reaches 70% above person, and harvested cell is for subsequent use.

3) inoculation discards cell growth medium, by 1/50 cell proportion, being inoculated among the CEF that grows up to individual layer with poison cell, uses 199 maintenance mediums instead, and 37 ℃ of rotations (12r/h) are cultivated.

4) observed 1～2 every day after cultivation connect poison with observation, when treating that typical MDV pathology appears in 70% above cell, can gather in the crops.

5) results discard nutritive medium, add trysinization liquid (10000ml glass rolling bottle adds 25m1), rotate gently the glass rolling bottle, be that Digestive system fully contacts with cell, at room temperature digest about 10min, the loose shape that draws in the net appears in cell monolayer, approach when breaking away from the bottle wall, discard pancreatin, the nutritive medium that contains 5% bovine serum of adding and Digestive system equivalent stops digestion immediately.Blow and beat gently with suction pipe simultaneously, make cell all break away from bottle wall and dispersion.Collect the cell of dispersion in centrifuge tube, behind the centrifugal 15min of 1500r/min, the collecting cell precipitation.

(3) inspection of semifinished product is from the sedimentation cell of collecting, and steriling test is made in sampling, answers sterile production.

(4) join seedling and packing the cell of collecting is added frozen solution commonly used by 10% of original fluid amount, and in the abundant mixing of cell precipitation, quantitative separating seals in ampulla., slowly put on the liquid nitrogen surface again about 1 hour 2～8 ℃ of precoolings first, put in the liquid nitrogen behind the 30min.

3. inspection after construction

(1) physical behavior is pink cell suspension.

(2) steriling test by " Chinese veterinary pharmacopoeia " (the Chinese veterinary pharmacopoeia council. three ones of People's Republic of China's veterinary drug allusion quotation versions in 2010. Chinese agriculture press, 2011, the present invention is hereinafter to be referred as " Chinese veterinary pharmacopoeia ") requirement test, answer asepsis growth.

(3) the mycoplasma check is tested by the requirement of " Chinese veterinary pharmacopoeia ", should grow without mycoplasma.

(4) behind the virus liquid and anti-MD specific serum balanced mix of diagnostic test with 100～200PFU, in 18～22 ℃ and 30min, inoculating cell, the plaque decrement should be more than 95%.

(5) safety verification is with 20 of 1 age in days SPF chick, and the virus of every intraperitoneal inoculation 20000PFU was observed 70～90, should be without the MD clinical symptom, cut open that inspection changes without naked eyes and pathology change.

(6) every batch of vaccine of plaque counting is got 3 bottles, be diluted to suitable extent of dilution with Hanks liquid, cultivate according to a conventional method and plaque counting, obtain at last the average plaque of 3 bottles of cells of same extent of dilution, calculate every bottle of plaque number that vaccine is contained, appraise and decide as contained plaque number in the every plumage part of this batch vaccine with minimum mean number in 3 bottles of vaccines the extent of dilution, should be not less than 2000PFU.

Positive effect of the present invention

1. realized the recombinant virus triage techniques as vector expression reticuloendotheliosis virus (REV) immunoprotection gene with MDV;

2. from test data analyzer, MDV GD07-env virus is used as vaccine, the chicken Marek's disease that the prevention virulent brings out, and its protective immunity effect is superior to most widely used CVI988/Rispens strain vaccine on the present domestic and international market;

3. from the angle analysis of evolution, parent's poison GD07 of MDV GD07-env virus is the therefrom local street strain that separates of state in 2007, and its antigenicity should than CVI988/Rispens, reach domestic other MDV vaccine strain better;

4. originally researched and solved the problem of fowl reticuloendotheliosis disease without vaccine prevention;

5. because MDV is the carrier that can avoid maternal antibody to infect unique in the existing gal virus carrier, therefore involved in the present invention relate to will for research take MDV as carrier, express other correlated virus vaccine that is subject to antibody interferes with technical scheme be provided.

The microorganism that the present invention relates to: be Marek ' s disease virus GD07/ Δ 2Meq-env, be called for short Marek ' s disease virus GD07-env, or MDV GD07-env.This virus strain was deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center in No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 08 29th, 2011, and preserving number is CGMCC No.5169.

Embodiment

Embodiment 1

Vaccine is made

1. the preparation of seedling venom

(1) preparation of chick embryo fibroblast: select SPF chicken embryo, hatching is prepared into CEF with 0.01% trysinization behind 9～10 ages in days.

(2) the production seed culture of viruses that liquid nitrogen is preserved is got in the preparation of work seed culture of viruses, is inoculated on the CEF cell bottle that grows up to individual layer by 1/100 volume ratio, cultivates 48-72 hour, treats that cytopathy (CPE) reaches 70% above person, and harvested cell is for subsequent use.

(3) inoculation discards cell growth medium, by 1/50 cell proportion, poison cell is inoculated among the CEF that grows up to individual layer, uses 199 maintenance mediums instead, and 37 ℃ are rotated cultivation.

(4) observed 1～2 every day after cultivation connect poison with observation, when treating that typical MDV pathology appears in 70% above cell, can gather in the crops.

(5) results discard nutritive medium, add trysinization liquid (10000ml glass rolling bottle adds 25m1), rotate gently the glass rolling bottle, be that Digestive system fully contacts with cell, at room temperature digest about 10min, the loose shape that draws in the net appears in cell monolayer, approach when breaking away from the bottle wall, discard pancreatin, the nutritive medium that contains 5% bovine serum of adding and Digestive system equivalent stops digestion immediately.Blow and beat gently with suction pipe simultaneously, make cell all break away from bottle wall and dispersion.Collect the cell of dispersion in centrifuge tube, behind the centrifugal 15min of 1500r/min, the collecting cell precipitation.

2. the inspection of semifinished product is from the sedimentation cell of collecting, and steriling test is made in sampling, answers sterile production.

3. join seedling and packing the cell of collecting is added frozen solution by 10% of original fluid amount, and in the abundant mixing of cell precipitation, quantitative separating seals in ampulla., slowly put on the liquid nitrogen surface again about 1 hour 2～8 ℃ of precoolings first, put in the liquid nitrogen behind the 30min.

Embodiment 2

Inspection after construction

1. physical behavior is pink cell suspension.

2. steriling test is tested by the requirement of " Chinese veterinary pharmacopoeia ", answers asepsis growth.

3. the mycoplasma check is tested by the requirement of " Chinese veterinary pharmacopoeia ", should grow without mycoplasma.

4. behind the virus liquid and anti-MD specific serum balanced mix of diagnostic test with 100～200PFU, in 18～22 ℃ and 30min, inoculating cell, the plaque decrement should be more than 95%.。

5. safety verification is with 20 of 1 age in days SPF chick, and the virus of every intraperitoneal inoculation 20000PFU was observed 70～90, should be without the MD clinical symptom, cut open that inspection changes without naked eyes and pathology change.

6. every batch of vaccine of plaque counting is got 3 bottles, be diluted to suitable extent of dilution with Hanks liquid, cultivate according to a conventional method and plaque counting, obtain at last the average plaque of 3 bottles of cells of same extent of dilution, calculate every bottle of plaque number that vaccine is contained, appraise and decide as contained plaque number in the every plumage part of this batch vaccine with minimum mean number in 3 bottles of vaccines the extent of dilution, should be not less than 2000PFU.

Claims (3)

1. recombination chicken marek's disease virus (MDV) strain of expressing fowl reticuloendotheliosis virus (Reticuloendotheliosis virus) envelope protein env gene (REVenv) is characterized in that:

(1) this recombination chicken marek's disease virus is named as MDV GD07-env strain, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center in No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on August 29th, 2011, preserving number is CGMCCNo.5169;

(2) this recombinant virus had both lost the original tumorigenesis ability of MDV, kept good immunogenicity, can express simultaneously fowl reticuloendotheliosis virus Reticuloendotheliosis virus envelope protein env gene, the fowl reticuloendotheliosis is had certain immune protective effect;

(3) can be used as this viral distinctive sign with two particular sequences that are positioned at specific site on this recombinant virus genomes:

1) at original two Meq gene locuss, after knocking out the Meq gene, stay therein this sequence of sequence FRT of next 34bp to be on the position of a Meq gene: 5 '-GAAGTACCTATTCTTTCTAGAGAATAGGAACTTC-3 ';

2) on the position of another Meq gene, contain REV env gene expression frame.

2. a kind of recombination chicken marek's disease virus strain of expressing fowl reticuloendotheliosis virus envelope albumen env gene as claimed in claim 1, the structure that it is characterized in that this recombinant virus is to knock out 1 Meq gene in the MDV GD07 strain virus by the mutating technology that utilizes recE/T mediation, and again by Red/ET mediation technology, substitute another Meq gene with REV env expression cassette.

A kind of recombination chicken marek's disease virus strain of expressing fowl reticuloendotheliosis virus envelope albumen env gene claimed in claim 1 for the preparation of the prevention chicken Marek's disease or/and the purposes in the chicken Marek's disease of chicken reticuloendotheliosis-reticuloendotheliosis living vaccine.