CN1099460C

CN1099460C - Chicken Marek's disease virus genic mutant strain and its application

Info

Publication number: CN1099460C
Application number: CN97125858A
Authority: CN
Inventors: 崔治中; Lee·L
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 1997-12-26
Filing date: 1997-12-26
Publication date: 2003-01-22
Anticipated expiration: 2017-12-26
Also published as: CN1196390A

Abstract

The present invention relates to a recombined Marek's disease virus low-virulent strain CVI/rpp 38 of chickens, which is obtained from directional point mutation. When a cell disruption product labelled by <35>S-methionine is used for the immune precipitation, the monoclonal antibody H19 can not identify pp 38 in natural CVI988 strain MDV, but can identify an MDVpp 38 protein band from the cell infected with the recombined virus CVI/rpp 38, just like identifying the pp 38 from other high virulent strains of the MDV. The titer of the MDV specific antibody of the chicken inoculated with the CVI/rpp 38 virus is obviously lower than that of the MDV specific antibody of the chicken inoculated with the cloning strain of the original vaccine virus CVI988/Rispens, so as to be used as the screening index in breeding for disease resistance. A low-virulent vaccine strain can be obtained from the high-virulent MDV by the reverse point mutation method.

Description

Marek's disease virus genic mutant strain and application thereof

The technical field of the invention

The present invention relates to a kind of chicken Marek's disease virus (Marek ' s disease virus, MDV) mutant strain and the application of this mutant strain in the chicken breeding for disease resistance thereof, promptly utilize this chicken Marek's disease virus mutation strain inoculation chicken involved in the present invention through after a while, blood sampling also separates chicken serum, utilize the antibody titers of serological method mensuration to chicken Marek's disease virus, determining existing of anti-chicken Marek's disease virus chicken, thereby set up the population of anti-chicken Marek's disease virus.

Background of the present invention

Marek's disease virus is a kind of tumorigenicity simplexvirus, its 38kd phosphorprotein (pp38) is counted as a kind of Marek tumor correlated albumen matter (Ikuta K. always, Nakajima K, Naito M.et al.Int.J.Cancer, 1985,35:257-264.Nakajima K.et al.J.Gen.Virol.1987,68:1379-1389.Calnek B W.Marek ' s disease.In Diseases of Poultry, Calnek B W.et al.Eds, Iowa State University, Press, Ames, 1992,302-334), but it is caused a disease and to cause biological action concrete in the neoplastic process unclear always at MDV.Not long ago the inventor finds, at the pp38 of expressed in insect cells gene product (Cui Zhizhong, the viral journal of Lee LF. China, 1992,7:106-112), can show certain immunosuppressive action (Cui Z and Qin A.Immunodepressive effects of therecombinant 38 kd phosphorylated protein of Marek ' s disease virus.In:Current Research on Marek ' s Diseasea to chick, Silva R F.et al.Eds.Rose Printingcompany, Inc., Tallahassee, Florida, USA, 1996,278-283. among the Cui Zhi, Qin Aijian. journal of animal science and veterinary medicine, 1997,28:71-76).But this system still can not represent the effect of pp38 under natural infection and morbidity state fully, makes up and utilize pp38 mutant MDV then to help addressing this problem.Existing report shows, can discern pp38 monoclonal antibody H19 can with the pathogenic MDV strain of all I types of testing (the Lee L F that reacts, X Liu, and R L Witter.J.Immunol.1983,130:1003-1006.Silva R F, and Lee L F.Virology, 1984,136:307-320), but can not react (Rispens BH, Hvan Vloten, N Mastenbroek et al.Avian diseeases with I type MDV vaccine strain CVI988,1972,16:108-125.Witter R L, R F Silva, and L F Lee.Avian diseases, 1987,31:829-840).We showed that the strong malicious MDV of I type was in causing weak process the past to the pp38 analysis of the molecular structure, a base in the pp38 gene is undergone mutation and caused an amino acid variation (Cui Z, L F Lee, J.L.liu et al.J.Virology, 1991,65:6509-6515); The antigenic determinant of monoclonal antibody H19 identification is constant and compare with strong malicious GA strain, the vaccine virus CVI988 of I type MDV has two base mutations and causes two amino acid whose variation (Endoh D in the pp38 gene, MNikura, K Hirai et al.J.Vet.Med.Sci.1994,56:823-826).And cause losing the strong malicious type antigenic determinant that is discerned for monoclonal antibody H19.

Purpose of the present invention

The present invention attempts to be structured in the recombinant C VI988 strain virus that the orientation point sudden change takes place in the pp38 gene, in the hope of the biologic activity of the pp38 of research MDV under crudeness.Utilize its corresponding biological activity simultaneously in the breeding for disease resistance system, when carrying out other character screening, carry out that with this chicken Marek's disease virus is had the disease resistance seed selection.The present invention yet attempts to adopt orientation point to suddenly change strong poison in marek's disease virus to obtain the seedling low virulent strain.Feature of the present invention

The present invention is transfection reagent with LipofectAMINE, the chick embryo fibroblast (CEF) that the vaccine virus CVI988 strain MDV that is not discerned by monoclonal antibody H19 with the reorganization pHA25 plasmid DNA transfection of the PP38 complete genome sequence that contains strong malicious type GA strain infects, with indirect fluorescent antibody test (FA), screen and be cloned into the CVI/rpp38 strain recombinant virus that can be expressed as the strong malicious type pp38 antigenic determinant that monoclonal antibody H19 discerned.With ³⁵The virus infection CEF solute and the monoclonal antibody of S-methionine mark show (Fig. 2) do immunoprecipitation, and the banding pattern of the virus envelope Glycoprotein B of recombinant C VI/rpp38 strain virus and natural vaccine poison CVI988 is identical, and is different from virulent strain.Yet monoclonal antibody H19 can not identify any band from the CEF solute that natural virulent strain infects, but can identify very strong pp38 protein band from the CEF solute of recombinant virus CVI/rpp38 and the infection of natural virulent strain.Recombinant virus CVI/rpp38 is infected CEF make direct virus plaque counting simultaneously and do FA dyeing to show the specificity virus spot that both comparison shows that, recombinant C VI/rpp38 virus quite stable with monoclonal antibody H19.The clone strain of natural CVI988/Rispens is almost completely identical with the duplication characteristic of recombinant virus CVI/rpp38 on CEF.Pcr analysis is done in 132bp fragment iteron on the viral genome show, the banding pattern of the different 132bp repeated fragment copy numbers with containing of natural two strain virus of recombinating is identical.But two kinds of viruses are induced the active significantly different of antibody response at the chicken body.To inoculate 3 age in days SPF7 * 15 respectively with recombinant C VI/rpp38 through the natural CVI988 of equal passage number of times almost is to hybridize chicken (every plumage about 40,000pfu), make indirect fluorescent antibody test and measure antibody titers at took a blood sample respectively in back 10 days, 16 days and 26 days CEF that GA strain MDV is infected of infection MDV.The result shows, the chicken that has infected CVI/rpp38 strain recombinant virus significantly delays the antibody response of MDV, and antibody titers significantly is lower than the chicken that natural vaccine poison CVI988 infects.Inoculate the chicken of natural CVI988 strain, promptly had the part chicken MDV antibody to occur in back 10 days in infection, anti-MDV antibody all appears in all 14 infected chickens in the time of 16 days, and average titer is 1: 86 ± 29; In the time of 28 days, reach 1: 379 ± 158; And the chicken of inoculation CVI/rpp38, there was not one to show the reaction of MDV specific antibody in back 10 days in inoculation, in the time of 20 days, only there are indivedual chickens to produce very low anti-MDV antibody titers, though antibody response has all appearred in all chickens in the time of 28 days, but its average titer only is 1: 65 ± 33, significantly is lower than natural CVI988 strain infected chicken (P＜0.001) (Fig. 3).

Because it is slow and low that the anti-MDV immune response that CVI/rpp38 brings out chicken not only produces more natural CVI988 strain, and individual difference also relatively large (table 1), this just might be the Different Individual of chicken to the antibody horizontal of CVI/rpp38 as a kind of selection markers, be used for breeding for disease resistance to MD.Detailed description 1 virus of the present invention

Weak malicious MDV (the Rispens B H of CVI988/Rispens strain I type, Hvan Vloten, N Mastenbroeket al.Avian diseeases, 1972,16:108-125) the kind poison of preserving for the USDA institute of oncology is gone up cultivation in chick embryo fibroblast (CEF).I type MDV monoclonal antibody specific H19 (Lee LF, X Liu andRL Witter.J Immunol, 1983,130:1003-1006) can discern the I type MDV that all were tested, but only to CVI988 strain virus be negative reaction (Witter RL, R F Silva, and LF Lee.Avian diseases, 1987,31:829-840).Before construction of recombinant virus, the CEF that the CVI988/Rispens strain is infected with monoclonal antibody H19 makes indirect immunofluorescence assay (1FA), on the CEF individual layer of two 60mm plates that contain 6000 virus plaques of having an appointment respectively, and the plaque that last discovery can be discerned by monoclonal antibody H19.The transfection of 2 cells infecteds

In order to be structured in the recombinant C VI988/Rispens strain virus that to express strong malicious type determinant on the pp38 gene, be transfection reagent with LipofectAMINE (Gibco BRL company product) respectively, with the CEF of the pp38 gene clone pHA25 plasmid DNA transfection CVI988/Rispens strain infection that contains strong malicious GA strain MDV.Plasmid pHA25 is at the intermediate product to pp38 gene sequencing process, it comprises from the sub-upstream of start code 580 bases until the MDV of the common 2490bp that crosses transcription termination signal specific sequence (Cui Z, LF Lee, J.L.liu et al.J.Virology, 1991,65:6509-6515).Transfection process is undertaken by the working instructions that LipofectAMINE producer provides in principle.For more optimum transfection conditions, respectively before virus inoculation and inoculation back with the LipofectAMINE and the plasmid DNA transfection CEF individual layer of different concns, and adopt the different transfection time.Treat that cells transfected is the s-generation CEF that is inoculated in the 60mm plate, when cell monolayer is the density of 80-90%, began virus inoculation or beginning transfection in back 18 hours in inoculation usually.About 6000 plaques of virus inoculation amount on the CEF individual layer of every 60mm plate.After the CEF individual layer was washed secondary with the basic culture solution DMEM of serum-free antibiotic-free before the transfection, adding contained the transfection liquid of various dose LipofetAMINE and plasmid DNA again, incubator (37 ℃, 5%CO ₂) in when placing different time and waiting to stop transfection, with the nutrient solution 5mL that keeps that contains calf serum the CEF individual layer is washed secondary earlier, add 4～5ml again and contain calf serum and keep nutrient solution and continue to cultivate 3～5 days.

Fig. 1 demonstrates the recombinant virus plaque that can discern for monoclonal antibody H19 that produces in indirect fluorescent antibody test (IFA) on the CEF individual layer that the weak malicious CVI988/Rispens strain MDV of the pp38 gene clone pHA25 of strong malicious GA strain MDV plasmid DNA transfection infects.When observing, a large amount of intensive viral plaques are also arranged around it, but the viral plaque that the protovirus of these CVI988/Rispens strains forms in IFA is not discerned by monoclonal antibody H19 all with ordinary light.The screening purifying of 3 recombinant viruses

Because MDV is strict cell associativity and does not produce free virus on CEF, make the purifying of recombinant virus comparatively complicated.After the DNA transfection, inoculate the CEF of virus or at virus inoculation after the CEF of DNA transfection continue to cultivate 5 days, observe and have or not virus plaque to form.No matter have or not obvious plaque to form, all with the CEF individual layer with 0.02% trypsin solution from plate digestion down, the CEF under every plate suspension is inoculated on the fresh CEF individual layer of two 60mm plates, continues to cultivate 3-5 days.When obvious plaque forms, get one and do indirect fluorescent antibody test (IFA) with monoclonal antibody H19 and observe and have or not the positive-virus spot; As finding the positive spot of IFA is arranged, the CEF on another piece plate with under the trypsin solution wash-out, and is inoculated on the 96-well culture plate that contains fresh CEF, infection CEF inoculation one or a few blocks 96 well culture plates in every 60mm plate.Cultivate treated that plaque obviously in 3～4 days after, suction goes nutrient solution to add 50 μ l to contain 0.02% tryptic serum-free DMEM nutrient solution, after hatching 5 minutes under 37 ℃, in every hole, add the nutrient solution that 100 μ l contain 30% calf serum again, after with 8 duct pipettors cell piping and druming being suspended, the about 30 μ l of CEF suspension that respectively get virus infection from each 96 hole are inoculated in two each corresponding apertures of 96-well culture plate that contain the CEF individual layer.After cultivation virus plaque occurred in 3～4 days, take out a plate, after the record of the plaque number scale in every hole, the nutrient solution that inclines is also fixed with acetone-ethanol (6: 4) mixed solution, H19 makes IFA with monoclonal antibody, observes and write down each hole positive-virus spot number under fluorescence rolling micro mirror.

Selection and mark contain the positive spot of 1FA and count the high hole of ratio, from the respective aperture of another piece culture plate, with the trypsin solution digestion and the infection CEF individual layer that suspends.To do to be inoculated in respectively after the continuous doubling dilution in the CEF individual layer in a series of holes of another piece 96-orifice plate from the cells infected in a hole.Cultivate waited plaque to occur in 2～3 days after, get the hole that contains 5～20 plaques, further inoculate and screen.So repeat 3～4 circulations, all select the highest culture hole of the positive spot ratio of IFA to go down to posterity at every turn, and reduce the plaque number of inoculating in every hole gradually.Until the virus of be sure oing the positive hole of IFA is to go down to posterity from the hole of only containing a plaque, and till all plaques all are the IFA positive.Be defined as recombinant virus with this through the plaque purifying.

In order to get rid of the impure to the interference of experimental result subsequently of the original seed culture of viruses of CVI988, the present invention has promptly used the purity of having checked seed culture of viruses with the IFA that screening method is identical subsequently before the transfection test.Point mutation strain virus CHARACTERISTICS IDENTIFICATION is shown also that the banding pattern of the pp38 complex body of mutant strain CVI/rpp38 and Glycoprotein B complex body (mainly reaching less important band) all is different from natural H19 with immunoprecipitation ⁺The banding pattern of highly virulent strain 684-A; Then the clone strain of CVI988/Rispens is identical for generations with it for its Glycoprotein B complex body banding pattern.This has proved that CVI/rpp38 is not the strong poison that pollutes but derives from H19 ^-Weak malicious CVI988/Rispens.In fact, behind heavy dose inoculation SPF chicken with 40,000 plaque forming units, H19 ⁺The also same H19 of CVI/rpp38 mutant strain ^-The same obvious pathology that can not cause chicken of CVI988 clone strain.In addition, the CVI988 strain virus on SPF duplicate and plaque forms all faster than other I type MDV, and the recombinant C VI/rpp38 that this research obtains duplicates with plaque that to form characteristics also just the same with the CVI988 protovirus on CEF, and these show that all the CVI/rpp38 recombinant virus derives from the CVI988/Rispens prime strain.The clone of 4 cell free recombinant viruses and screening

Through the recombinant virus of plaque purifying at CEF after the several generations amplification, the CEF of virus infection is suspended in the SPGA damping fluid, with ultrasonic treatment CEF cell to obtain cell free virus (Calnek B W.et al.Appl.Microbiol.1970,20:723-726), with lysate high speed centrifugation (10,000rpm/min) 10 minutes, get supernatant after 0.45 μ millipore filter filters, be inoculated in different extent of dilution on the CEF individual layer in a series of holes of several piece 96-orifice plate, after cultivating 7 days, observe virus plaque.Select tens usefulness inoculation and hole that have only a plaque of high dilution free virus liquid as far as possible, be IFA by preceding method with monoclonal antibody H19, screening is through the H19 of cell free virus cloneization ⁺The former H19 of recombinant virus and cloning ^-Non-recombinant virus.

Table 2 shows that different transfection conditions forms and H19 the state of CEF, the plaque of MDV ⁺The influence of orientation point mutant strain positive rate.As seen from the table, for the CEF individual layer in the 60mm diameter plate, the optimum dose of LipofectAMINE in 2.5ml transfection liquid is 35 μ l, transfection liquid was advisable 37 ℃ of function cells in 12 hours, increase the concentration of LipofectAMINE or prolong the transfection time, all can the pair cell state and virus plaque be formed with remarkable detrimentally affect.The DNA transfectional cell should be behind virus inoculation carries out in 12 hours, as inoculate virus behind transfectional cell, then can significantly reduce the formation (comparison test A and B) of viral plaque.From table, be also shown in, when transfection liquid is long to the action time of CEF individual layer (24 hours),, also do not have typical viral plaque to form (test A, B, C) even in normal nutrient solution, continue to cultivate about 5 days after removing transfection liquid.But this does not mean the death of virus, as cell is inoculated fresh CEF individual layer after trysinization suspends, can show virus plaque again.The ratio of IFA positive-virus spot is almost at same level (0.2% and 0.1%) in test B and D, but because in test D, transfected cell monolayer directly can show a large amount of viral plaques after continuing to cultivate, and therefore tests the transfection conditions of D and should regard optimum as.5 immunoprecipitations

Press Yoshida S et al. (Virology, 1994, the mode of 200:484-493) having reported is carried out, be summarized as follows: through having infected the CEF individual layer plate of CVI988/Rispens clone strain and mutant strain CVI/rpp38 or MDV highly virulent strain 648-A, when beginning plaque to occur, the nutrient solution that inclines, after not having methionine(Met) DMEM nutrient solution with serum-free and washing once, adding contains ³⁵The nutrient solution of the no methionine(Met) of S-methionine(Met) (50 μ ci/ml) continued to cultivate after 6～8 hours, was adding an amount of molten cell damping fluid (25mM Tris-HCl, pH7.5 on the washed cell monolayer of PBS liquid; 150mM NaCl; 0.1%SDS; 1% sodium deoxycholate; 1%Triton X-100).Will through the cell pyrolysis liquid of mark respectively with monoclonal antibody H19 (Lee L F, X Liu, and R L Witter.J.Immunol.1983,130:1003-1006) and the monoclonal antibody BA4 of MDV Glycoprotein B (Cui Z.et al.Monoclonal antibodiesagainst serotype 1-specific and groupcommon epitopes on theglycoprotein B of Marek ' s disease viruses.In:Current Research on Marek ' sDiseasea, Silva R F.et al.Eds.Rose Printing company, Inc., Tallahassee, Florida, USA, 1996,233-238) and IAN86 (Lee LF, X Liu and RL Witter.J Immuno, 1983,130:1003-1006) reaction, and, in containing 10% polyacrylamide gel of SDS and 2 mercapto ethanol, make electrophoretic separation protein again with staphylococcal protein A-multiple and thing of Sepharose CL-4B suspension (Pharmacia) absorption antibody antigen.

As seen from Figure 2, using ³⁵The solute of the virus infected cell of S-mark is done in the immunoprecipitation, can from the clone strain of the virulent 684-A strain of MDV, CVI988/Rispens and CEF solute that point mutation CVI/rpp38 strain is infected, identify gp100, gp60 and gp49 complex body to the monoclonal antibody BA4 of MDV membrane glycoprotein B and IAN86, and intensity is identical.This shows that CEF that this three strain virus infects is by isotropic substance peer-level mark.

Monoclonal antibody H19 can not identify any band from the clone strain cells infected of CVI988/Rispens, but can be settled out typical pp38 complex body from the CVI/rpp38 strain bar cells infected lysate of point mutation, as to virulent 648-A strain cells infected.

Can find out also that from immunoprecipitation CVI988/Rispens clone strain and CVI/rpp38 can be identical with the Glycoprotein B antigen banding pattern that monoclonal antibody BA4 and IAN86 produce, and are different from highly virulent strain 648-A.And the pp38 complex body banding pattern of CVI/rpp38 and monoclonal antibody H19 generation also is different from MDV virulent strain 648-A.6. chicken body test

To inoculate three age in days SPF7 * 15 respectively with recombinant C VI/rpp38 through the natural CVI988 of equal passage number of times almost is to hybridize chicken (every plumage about 40,000pfu), make indirect fluorescent antibody test and measure antibody titers at took a blood sample respectively in back 11 days, 17 days and 27 days CEF that GA strain MDV is infected of infection MDV.The result shows, the chicken that has infected CVI/rpp38 strain recombinant virus significantly delays the antibody response of MDV, and antibody titers significantly is lower than natural vaccine poison CVI988 infected chicken.Inoculate the chicken of natural CVI988 strain, promptly had the part chicken anti-MDV antibody to occur in back 10 days in infection, all 14 infected chickens all show anti-MDV antibody in the time of 16 days, and average titer is 1: 86 ± 29; In the time of 28 days, reach 1: 379 ± 158; And the chicken of inoculation CVI/rpp38, there was not a chicken to show the reaction of MDV specific antibody in back 10 days in inoculation, in the time of 20 days, only there are indivedual chickens to produce very low anti-MDV antibody titers, though antibody response has all appearred in all chickens in the time of 28 days, but its average titer only is 1: 65 ± 33, significantly is lower than natural CVI988 strain infected chicken (p＜0.001).But inoculated the weight no significant difference of the chicken of different strain virus at body weight and thymus gland, spleen and the fabricius bursa.

Use that the titre of antagonism MDV specific antibody comparison shows that the anti-MDV specific antibody reaction of recombinant virus infection chicken is subjected to remarkable inhibition behind natural CVI988 strain and the recombinant C VI/rpp38 inoculation SPF chicken, its titre is starkly lower than natural CVI988 virus.And the unique difference between this two strain virus is exactly, in recombinant virus CVI/rpp38, the pp38 gene of its natural CVI988 strain is replaced by the pp38 gene of virulent strain, and the pp38 gene of vaccine strain CVI988 does not contain the antigenic determinant that can be discerned for monoclonal antibody H19.This test has proved that not only strong malicious pp38 can suppress the specific immune response to MDV, and this restraining effect is relevant with the H19 antigenic determinant.

The chicken that has infected CVI/rpp38 strain recombinant virus significantly delays the antibody response of MDV, and antibody titers significantly is lower than natural vaccine poison CVI988 infected chicken, and individual difference also relatively large (table 1) this just can be the Different Individual of chicken to the humoral antibody level of CVI/rpp38 as a kind of screening index, promptly when carrying out other character screening, the breeding for disease resistance process carries out chicken Marek's disease virus is had the seed selection of disease resistance with this, select there being chicken Marek's disease virus that the chicken individuals of disease resistance is arranged, warp forms with continuous screening of quadrat method and cultivation again.7 cultivate the possibility of MDV attenuated vaccine strain

The present invention also is included in and implements sudden change in the other direction on the pp38 gene of strong malicious type MDV, with possible successful attenuated virus, make it to become this method of MDV attenuated vaccine seedling strain safely and effectively, be and utilize the sudden change method to cause the strong poison of weak MDV and develop this potential application method of novel recombinant vaccine.

Anti-MDV antibody titers distributes behind table 1 inoculation CVI/rpp38 and the CVI988/Rispens

Inoculate inoculation in back 17 days virus inoculation strain in back 27 days

1: 12 1: 25 1: 50 1: 100 1: 12 1: 25 1: 50 1: 100 1: 200 1: 400 1: 800CVI988/ 021 11 00012 10 1R,isp,ens,CVI,/rp,p38 10001256000 annotated: after inoculation CVI988/Rispens strain 17 days, all 14 chickens have all produced the anti-MDV antibody of different titers, and after inoculation CVI/rpp38 strain 17 days, in 14 chickens of test, have only one and produce anti-MDV antibody, this chicken may have certain resistibility to the immunosuppressive action of virulence type pp38, thereby its offspring might also have bigger resistibility to MDV.

The influence of table 2 different transfection conditions pair cell state and transfection and recombination efficiency

Test A Test B test C test DInfect back 12 hours transfection liquid LipofectAMINE 45 μ l 40 μ l 60 μ l 35 μ lpHA25 DNA 4 μ g 4 μ g 6 μ g 8 μ g total amount 2.5ml 2.5ml 2.5ml 2.5ml transfection time length 24hrs 24hrs 24hrs 12hrs before the transfection time opening infective virus

normally keep in the nutrient solution continue to cultivate 3 days CEF individual layers not behind not obvious not obvious about 4,000 tryptic digestions of OK bad good MDV spot blind passage to 96 well culture plates that contain fresh CEF individual layer ¹FA ⁺The hole ²2,/96 74/,396 1/192 19/192FA ⁺Spot ³3/30,000 275/120,000 2/60,000 52/60,000 recombinant virus positive rate 0.01% 0.2% 0.003% 0.1% is annotated: 1. in each transfection, the CVI988-CEF of transfection is infected the 96-well culture plate that contains CEF again and cultivated 3 days.When treating that the MDV viral plaque occurs, every 96-orifice plate is inoculated in two 96-orifice plates, and back two blocks of plates must hole-hole correspondence, and wherein one is used for the IFA screening, and another piece remaines in and is used to the FA that detects in the incubator ⁺The respective aperture virus of recombinant virus is protected and is planted.2. FA appears ⁺The hole count of viral plaque/general inspection hole count.3.FA ⁺The about total virus spot of viral plaque number/inspection window number (average every hole viral plaque is counted x general inspection hole count).Depositing of microorganism

Virus among the present invention-marek's disease virus CVI/rpp38 point mutation strain is deposited with China Committee for Culture Collection of Microorganisms common micro-organisms center on December 11st, 1997, and preserving number is: CGMCC NO.0334.

Caption

Fig. 1.

Be the viral plaque that IFA male MDV point mutation strain CVI/rpp38 forms with what monoclonal antibody H19 screened on the CEF individual layer.At the ordinary light microscopically, be also shown in many viral plaques around it, but under UV-light, all be the IFA negative reaction.

Fig. 2.

³⁵The immunoprecipitation of S-methionine mark thing.The lysate of the CEF that the clone strain (C) of the virulent 648-A strain (A) of MDV, mutant strain CVI/rpp38 (B) and CVI988/Rispens infects shows different banding patterns with the monoclonal antibody BA4 of anti-MDV Glycoprotein B and the pp38 monoclonal antibody H19 of anti-I type MDV respectively.

Fig. 3.

Behind 3 ages in days inoculation mutant strain CVI/rpp38 (solid line) and natural CVI988/Rispens strain (pecked line) MDV-CEF, the fluorescence antibody titre of anti-MDV is dynamic in the chicken serum

The encoding sequence of the pp38 gene ORF of the strong malicious GA strain of sequence catalogue 1 marek's disease virus

(base 1-870 290 amino acid of encoding are with latter two termination of being separated by coding, totally 879 bases)

According to Cui et al.Journal of Virology, 1991,65 (12): the aminoacid sequence of the pp38 of the strong malicious GA strain of 6509-6515.2 marek's disease virus (totally 290 amino acid whose polypeptide)

According to Cui et al.Journal of Virology, 1991,65 (12): the encoding sequence of the pp38 gene ORF of the exquisite weak malicious R2 strain of 6509-6515.3 marek's disease virus people

According to Cui et al.Journal of Virology, 1991,65 (12): the aminoacid sequence (totally 290 amino acid whose polypeptide) of the pp38 of the exquisite weak malicious R2 strain of 6509-6515.4 marek's disease virus people

According to Cui et al.Journal of Virology, 1991,65 (12): the encoding sequence of the pp38 gene ORF of the weak malicious CVI988/Rispens strain of 6509-6515.5 Marek's disease virus vaccine

According to Endoh et al.Journal of Veterinary Medical Science, 1994,56 (5): 823

-826。The aminoacid sequence of the pp38 of the weak malicious CVI988/Rispens strain of 6 Marek's disease virus vaccines

(totally 290 amino acid whose polypeptide)

The point mutation strain CVI/rpp38 strain of the weak malicious CVI988/Rispens strain of the Marek's disease virus vaccine among-826.7 the present invention

Encoding sequence 8 the present invention of pp38 gene ORF in the point mutation strain CVI/rpp38 strain of the weak malicious CVI988/Rispens strain of Marek's disease virus vaccine

pp38 ( 290 ) 9 CVI/rpp38pp38DNA10 CVI/rpp38pp381 1 ATGGAATTCGAAGCAGAACACGAAGGGCTGACGGCGTCTTGGGTCGCCCCCGCTCCCCAG 61 GGTGGAAAAGGGGCGGAGGGCCGCGCAGGGGTCGCCGACGAGGCAGGGCATGGGAAAACA121 GAAGCGGAATGCGCCGAGGACGGCGAGAAATGCGGGGACGCCGAGATGAGCGCTTTGGAT181 CGGGTCCAGAGGGACCGGTGGAGATTCAGTTCTCCGCCCCCTCACTCTGGAGTCACGGGG241 AAGGGGGCTATTCCAATAAAGGGTGATGGGAAGGCGATAGAATGCCAGGAGCTAACCGGA301 GAGGGAGAGTGGCTGTCACAGTGGGAGGAGCTACCGCCTGAGCCCCGGAGGTCAGGGAAT361 GAACATCTTGACGAAAGTCGGTATGCGAAACAAACCGAAAGGGGTAGCTCTACGGGGAAA421 GAAGAGGGAGATGGTATGAAGCAGATGGGGGAGCTTGCCCAGCAGTGCGAAGGAGGAACA481 TATGCGGACTTGCTTGTCGAAGCAGAGCAAGCTGTTGTACATTCCGTTCGCGCATTAATG541 CTGGCCGAAAGACAAAACCCAAATATATTGGGGGAGCATTTGAATAAAAAACGGGTTCTT601 GTACAACGACCCCGTACTATTCTATCCGTGGAGTCAGAGAATGCAACAATGCGTTCTTAT661 ATGCTGGTTACATTGATCTGTTCTGCAAAATCATTATTACTAGGATCGTGCATGTCATTT721 TTCGCTGGTATGTTAGTCGGTAGAACGGCAGACGTAAAAACACCATTATGGGATACTGTA781 TGTTTGTTAATGGCTTTCTGTGCAGGCATTGTCGTTGGGGGAGTGGATTCTGGGGAGGTG841 GAATCTGGAGAAACAAAATCTGAATCAAATTAASequence 21 MEFEAEHEGLTASWVAPAPQGGKGAEGRAGVADEAGHGKTEAECAEDGEK 51 CGDAEMSALDRVQRDRWRFSSPPPHSGVTGKGAIPIKGDGKAIECQELTG101 EGEWLSQWEELPPEPRRSGNEHLDESRYAKQTERGSSTGKEEGDGMKQMG151 ELAQQCEGGTYADLLVEAEQAVVHSVRALMLAERQNPNILGEHLNKKRVL201 VQRPRTILSVESENATMRSYMLVTLICSAKSLLLGSCMSFFAGMLVGRTA251 DVKTPLWDTVCLLMAFCAGIVVGGVDSGEVESGETKSESN sequences 3

1 ATGGAATTCGAAGCAGAACACGAAGGGCTGACGGCGTCTTGGGTCGCCCCCGCTCCCCAG 61 GGTGGAAAAGGGGCGGAGGGCCGCGCAGGGGTCGCCGACGAGGCAGGGCATGGGAAAACA 121 GAAGCGGAATGCGCCGAGGACGGCGAGAAATGCGGGGACGCCGAGATGAGCGCTTTGGAT 181 CGGGTCCAGAGGGACCGGTGGAGATTCAGTTCTCCGCCCCCTCACTCTGGAGTCACGGGG 241 AAGGGGGCTATTCCAATAAAGGGTGATGGGAAGGCGATAGAATGCCAGGAGCTAACCGGA 301 GAGGGAGAGTGGCTGTCACAGTGGGGGGAGCTACCGCCTGAGCCCCGGAGGTCAGGGAAT 361 GAACATCTTGACGAAAGTCGGTATGCGAAACAAACCGAAAGGGGTAGCTCTACGGGGAAA 421 GAAGAGGGAGATGGTATGAAGCAGATGGGGGAGCTTGCCCAGCAGTGCGAAGGAGGAACA 481 TATGCGGACTTGCTTGTCGAAGCAGAGCAAGCTGTTGTACATTCCGTTCGCGCATTAATG 541 CTGGCCGAAAGACAAAACCCAAATATATTGGGGGAGCATTTGAATAAAAAACGGGTTCTT 601 GTACAACGACCCCGTACTATTCTATCCGTGGAGTCAGAGAATGCAACAATGCGTTCTTAT 661 ATGCTGGTTACATTGATCTGTTCTGCAAAATCATTATTACTAGGATCGTGCATGTCATTT 721 TTCGCTGGTATGTTAGTCGGTAGAACGGCAGACGTAAAAACACCATTATGGGATACTGTA 781 TGTTTGTTAATGGCTTTCTGTGCAGGCATTGTCGTTGGGGGAGTGGATTCTGGGGAGGTG 841 GAATCTGGAGAAACAAAATCTGAATCAAAT TAA4 1 MEFEAEHEGLTASWVAPAPQGGKGAEGRAGVADEAGHGKTEAECAEDGEK 51 CGDAEMSALDRVQRDRWRFSSPPPHSGVTGKGAIPIKGDGKAIECQELTG 101 EGEWLSQWGELPPEPRRSGNEHLDESRYAKQTERGSSTGKEEGDGMKQMG 151 ELAQQCEGGTYADLLVEAEQAVVHSVRALMLAERQNPNILGEHLNKKRVL 201 VQRPRTILSVESENATMRSYMLVTLICSAKSLLLGSCMSFFAGMLVGRTA 251 DVKTPLWDTVCLLMAFCAGIVVGGVDSGEVESGETKSESN5 1 ATGGAATTCGAAGCAGAACACGAAGGGCTGACGGCGTCTTGGGTCGCCCCCGCTCCCCAG 61 GGTGGAAAAGGGGCGGAGGGCCGCGCAGGGGTCGCCGACGAGGCAGGGCATGGGAAAACA 121 GAAGCGGAATGCGCCGAGGACGGCGAGAAATGCGGGGACGCCGAGATGAGCGCTTTGGAT 181 CGGGTCCAGAGGGACCGGTGGAGATTCAGTTCTCCGCCCCCTCACTCTGGAGTCACGGGG 241 AAGGGGGCTATTCCAATAAAGGGTGATGGGAAGGCGATAGAATGCCAGGAGCTAACCGGA 301 GAGGGAGAGTGGCTGTCACGGTGGGGGGAGCTACCGCCTGAGCCCCGGAGGTCAGGGAAT 361 GAACATCTTGACGAAAGTCGGTATGCGAAACAAACCGAAAGGGGTAGCTCTACGGGGAAA 421 GAAGAGGGAGATGGTATGAAGCAGATGGGGGAGCTTGCCCAGCAGTGCGAAGGAGGAACA 481 TATGCGGACTTGCTTGTCGAAGCAGAGCAAGCTGTTGTACATTCCGTTCGCGCATTAATG 541 CTGGCCGAAAGACAAAACCCAAATATATTGGGGGAGCATTTGAATAAAAAACGGGTTCTT 601 GTACAACGACCCCGTACTATTCTATCCGTGGAGTCAGAGAATGCAACAATGCGTTCTTAT 661 ATGCTGGTTACATTGATCTGTTCTGCAAAATCATTATTACTAGGATCGTGCATGTCATTT 721 TTCGCTGGTATGTTAGTCGGTAGAACGGCAGACGTAAAAACACCATTATGGGATACTGTA 781 TGTTTGTTAATGGCTTTCTGTGCAGGCATTGTCGTTGGGGGAGTGGATTCTGGGGAGGTG 841 GAATCTGGTGAAACAAAATCTGAATCAAATTAA6 1 MEFEAEHEGLTASWVAPAPQGGKGAEGRAGVADEAGHGKTEAECAEDGEK 51 CGDAEMSALDRVQRDRWRFSSPPPHSGVTGKGAIPIKGDGKAIECQELTG 101 EGEWLSRWGELPPEPRRSGNEHLDESRYAKQTERGSSTGKEEGDGMKQMG 151 ELAQQCEGGTYADLLVEAEQAVVHSVRALMLAERQNPNILGEHLNKKRVL 201 VQRPRTILSVESENATMRSYMLVTLICSAKSLLLGSCMSFFAGMLVGRTA 251 DVKTPLWDTVCLLMAFCAGIVVGGVDSGEVESGETKSESN7 1 ATGGAATTCGAAGCAGAACACGAAGGGCTGACGGCGTCTTGGGTCGCCCCCGCTCCCCAG 61 GGTGGAAAAGGGGCGGAGGGCCGCGCAGGGGTCGCCGACGAGGCAGGGCATGGGAAAACA121 GAAGCGGAATGCGCCGAGGACGGCGAGAAATGCGGGGACGCCGAGATGAGCGCTTTGGAT181 CGGGTCCAGAGGGACCGGTGGAGATTCAGTTCTCCGCCCCCTCACTCTGGAGTCACGGGG241 AAGGGGGCTATTCCAATAAAGGGTGATGGGAAGGCGATAGAATGCCAGGAGCTAACCGGA301 GAGGGAGAGTGGCTGTCACAGTGGGAGGAGCTACCGCCTGAGCCCCGGAGGTCAGGGAAT361 GAACATCTTGACGAAAGTCGGTATGCGAAACAAACCGAAAGGGGTAGCTCTACGGGGAAA421 GAAGAGGGAGATGGTATGAAGCAGATGGGGGAGCTTGCCCAGCAGTGCGAAGGAGGAACA481 TATGCGGACTTGCTTGTCGAAGCAGAGCAAGCTGTTGTACATTCCGTTCGCGCATTAATG541 CTGGCCGAAAGACAAAACCCAAATATATTGGGGGAGCATTTGAATAAAAAACGGGTTCTT601 GTACAACGACCCCGTACTATTCTATCCGTGGAGTCAGAGAATGCAACAATGCGTTCTTAT661 ATGCTGGTTACATTGATCTGTTCTGCAAAATCATTATTACTAGGATCGTGCATGTCATTT721 TTCGCTGGTATGTTAGTCGGTAGAACGGCAGACGTAAAAACACCATTATGGGATACTGTA781 TGTTTGTTAATGGCTTTCTGTGCAGGCATTGTCGTTGGGGGAGTGGATTCTGGGGAGGTG841 GAATCTGGTGAAACAAAATCTGAATCAAATTAASequence 81 MEFEAEHEGLTASWVAPAPQGGKGAEGRAGVADEAGHGKTEAECAEDGEK 51 CGDAEMSALDRVQRDRWRFSSPPPHSGVTGKGAIPIKGDGKAIECQELTG101 EGEWLSQWEELPPEPRRSGNEHLDESRYAKQTERGSSTGKEEGDGMKQMG151 ELAQQCEGGTYADLLVEAEQAVVHSVRALMLAERQNPNILGEHLNKKRVL201 VQRPRTILSVESENATMRSYMLVTLICSAKSLLLGSCMSFFAGMLVGRTA251 DVKTPLWDTVCLLMAFCAGIVVGGVDSGEVESGETKSESN sequences 9

* GA strain #319 CAG-TGG-GAG #327R2 strain CAG-TGG-GGGCVI988/Rispens CGG-TGG-GGGCVI/rpp38 CAG-TGG-GAG sequence 10

* GA strain #105S-Q-W-E-E #109R2 strain S-Q-W-G-ECVI988/Rispens S-R-W-G-ECVI/rpp38 S-Q-W-E-E

Claims

The 1 one kinds of still non-existent marek's disease virus of nature (Marek ' s disease virus---MDV) weak poison, it is characterized in that: it is to import sudden change to obtain the MDV low virulent strain by recombinant DNA technology in genome, called after CVI/rpp38, this virus strain has been sent the China Committee for Culture Collection of Microorganisms common micro-organisms center that is hidden in, preserving number: CGMCC NO.0334 on December 11st, 1997.
2 MDV according to claim 1 is characterized in that: it have with vaccine a little less than the identical virus envelope protein B of malicious CVI988 banding pattern, MDV virulent strain virus do not have with vaccine a little less than the identical virus envelope protein B of malicious CVI988 banding pattern.
3 MDV according to claim 1 is characterized in that: it contain can be discerned for monoclonal antibody H14 with the same antigenic determinant of strong malicious type pp38 antigen.
4 MDV according to claim 1 is characterized in that: 319-327 is identical with the strong malicious GA strain of MDV in its genome sequence, promptly be all CAG-TGG-GAG, and low virulent strain CVI988/Rispens is CGG-TGG-GGG.
5 MDV according to claim 1 is characterized in that: 105-109 is identical with the strong malicious GA strain of MDV in its aminoacid sequence, promptly be all S-Q-W-E-E, and low virulent strain CVI988/Rispens is S-R-W-G-E.
6 MDV according to claim 1 is characterized in that: behind its infected chicken, the antibody response of MDV is significantly delayed, and antibody titers significantly is lower than natural vaccine virus CVI988 infected chicken.