Expression, Sequencing And Analysis Of Pp38 And Pp24 Gene Of Serotype Ⅰ Of Marek's Disease Virus

Marek' s Disease Virus(MDV) phosphoprotein 24(pp24) gene was amplified from Md11 strain by Polymerase Chain Reaction(PCR). Then we cloned it into the downstream of GST gene according to the right open reading frame (ORF) in pGEX-6P-l vector. The recombinant was transformed into E. co2i'BL21 strain for expression with the induce of IPTG. SDS-PAGE and Westen-blotting validated the expression of pp24. The expressed specific band was excised from the gel and injected into mice once two weeks for 5 times, then we collected the antiserum from the mice and used it for IFA with Chicken Embryonic Fibroblasts(CEF) infected by MDV Mdll, CVI988 and GA strains respectively. The positive straining was found in the MDV plaques, which shows the in vitro expressed protein of pp24 has some epitopes of MDV.We constructed pcDNAS. 1-pp38 and pcDNAS. 1-pp24 eukaryotic expressing plasmids, which contain pp38 gene and pp24 gene respectively. These two plasmids were transfected into CEF by lipofectamine reagent. 24, 48, 72 hours later after transfection, we testified the expression of pp38 with Mab H19, and pp24 with the antiserurn of pp24 expressed in E. coli. The tests justified the expression of pp24 in prokaryotic and eukaryotic expressing systems.In order to study the correlation of pp38 and pp24, we cloned pp38 gene and pp24 gene into pBudCE4. 1 vector which can express two different genes at the same time. After transfection, we also testified the co-expression of pp38 and pp24 by IFA and by Western-blotting with Anti-GST-pp24 sera.We cloned and sequenced the pp38 and pp24 gene amplified from 814, CVI988(mMDV), GA(vMDV), Md5(vvMDV), Md11(vvMDV), RBlB(vvMDV), 648A(vv+MDV), and other 7 isolated strains from China. Sequence analysis shows pp24 and pp38 gene are very conservative. The homogeneity of pp24 and pp38 is higher than 98. 9% and 99. 4% in nuclear acid and higher than 98. 1% and 99. 0% in amino acid among different strains.There are two mutations in pp38 gene at the sites of 320 (A-G) and 326(A-G). These mutations cause the changes of the amino acid at 107(Q-R) and 109(E-G). The rest mutations in pp38 and pp24 are at random. Sequence analysis also shows the first 195 nuclear acids of pp38 and pp24 are the same except for the 81 site(G/C), but this mutation does not cause the change of amino acid. We regard this as a genetic marker connecting with geography in the evolution of MDV but not related to isolated time and pathotype of different strain of MDV I .In order to investigate the epidemic of MDV, reticuloen-endotheliosis virus (REV) and chicken anemia virus(CAV), we used the special PCR amplified product of pp38 gene as a probe to detect MDV, the cDNA clone of whole genome of SNV strain as a probe to detect REV, the clone of whole genome of Cux-1 strain as a probe to detect CAV. The tissue samples of 792 diseased chickens from 37 flocks of 10 provinces (including Taiwan Province) in China are tested. The results indicate that the checkout ratios of the three viruses are quite high: 84%(MDV), 62. 8% (CAV) and 58. 8%(REV) ;all chickens have very serious dual infection (32.6%) and triple infection(45. 7%); 28 of 37 chicken flocks occur triple infection (75. 6%) ,5 ones occur dual infection of MDV and CAV(13. 5%),3 ones occur dual infection of MDV and REV(8. 1%), only one not occurs MDV, CAV or REV. Expect CAV is negative in the samples from Guangxi province, the other 9 provinces exist MDV, CAV and REV, it shows that the three viruses have existed widely in China.