Molecular Identification Of Chongqing Strain Of Avain Leukosis Virus And Its P27 Gene Expression In Escherichia.coli

Avian Leukosis is a general designation of a variety of avian neoplastic diseases caused by Avian Leukosis Virus which belong to Retroviridae.Infection with ALV is widespread with the vertical and horizontal transmission in the poultry flocks in the world.This disease causes emaciation,undergrowth,maldevelopment and evokes nonspecific resistance decline and immunosuppression.In recent years,some poultry raising developed countries took some measures to purify poultry through condemning sicken chicken and decreasing vertical transmission.In some parts of our country,there are some reports about the morbility of ALV and the trend of popularity. Among these cases,Subgroup J is the most frequent,followed by Subgroup A.Suspicious cases also exist in Chongqing.The purpose of this study was to do the research of isolation and molecular identification of ALV Chongqing strain,cloning the gp85 gene and prokaryotic expression of p27 gene.This study was to establish the foundation to futher study on molecular bionomics of ALV Chongqing strain and development of ALV antigen detection kit.Some 165 age in days layers from a chicken house in Chongqing were identified that they were infected with ALV by ALV-p27 Antigen Test kit.This study used layers' sickness material to carry on the viral isolation.Viral suspension prepared from sickness material(liver,tumor and so on)inoculated in chick embryo fibroblast,harvested the cells after cultured for 7 days.ELISA test result was positive.In this way,we isolated an exogenous ALV strain,named ALV CQ01 strain.Next,this study used the RT-PCR method to identify the isolated ALV strain.Viral RNA was extracted from suspension,then we used the RNA to synthesize cDNA by reverse transcription as the template.At first,in order to determine that this strain belonged to Subgroup A-E or Subgroup J,we applied two pairs of specific primers(PU1/PU2 and H5/H7,refer to Subgroup A-E and Subgroup J, respectively)to the detection according to the difference of gp85 nucleotide sequence homology. The amplification showed that the strain belong to the Subgroup A-E.In addition,according to the report,endogenous ALV Subgroup E often coexit in ALV Subgroup A infect chicken,other two pairs of specific primers(refer to Subgroup and Subgroup E,respectively)were designed on the basis of the gp85 gene difference of the two subgroups.The RT-PCR result indicated that exogenous subgroup A(CQ01 strain)and endogenous subgroup E were coexiting in the sickness material.Then the two segments of gp85 gene was cloned into plasmid pMD 18-T Vector and sequenced.Nucleotide sequence analysis showed that the homology between cloned gp85 gene of CQ01 strain and MQNCSU strain was 95.1%,while the homology of the Subgroup E gp85 gene compared with ev-3 strain was 99.2%.Phyletic cladogram analysis indicated that CQ01 strain and other five strains(MQNCSU,RAV-1,B53,RAV 0-A1 and ADSL 6803A)didn't in an evolution branch.The Subgroup E and other strains(ev-1,ev-3,ev-C1,ev-H and SD0507)in an evolution branch.The ALV capsid protein p27,primary group specific antigen of ALV,is a kind of highly conservative non-glycosylated protein.Amino acid sequence of p27 protein shares an about 90% identity between exogenous ALV Subgroup A,B,C,D and J.p27 protein is main component in virion,also it's the ideal target protein for the antigen detection.This study cloned and expressed ALV p27 gene in prokaryotic cell.In this study,p27 gene(total length is 720bp)was amplified by RT-PCR with the p27 specific primers.Then the gene was cloned into plasmid pMD 18-T Vector and sequenced after identified by enzyme digestion.Nucleotide sequence analysis indicated that the homology between cloned p27 gene and other ALV subgroups(A,B,C,D,E,J)was over 94.5% and amino acid sequence was over 97.5%.This result testified that the cloned p27 gene was representative.The whole coding region of p27 gene was subcloned into prokaryotic expression vector pET-32a(+).Then the recombinant plasmid was transformed into E.coli BL21(DE3).The 45KD fusion protein was obtained through the detection of SDS-PAGE after induction by IPTG.In addition,target protein could be induced at optimize condition as following:E.coIi BL21 were incubated in LB containing 200μg/mL ampicillin at 37℃until the OD₆₀₀value of the bacteria was 1.2,IPTG was added at 1mM final concentration,and the bacteria were incubated at 25℃under shaking culture(250 rpm)for 5h,the yield of protein reached the highest.The expressed protein showed favourable reactionogenicity identified by Western-blot.