Cloning And Analysis Of The Complete Genome Of Duck Hepatitis Vims Type C GX Strain And Prokaryotic Expression Of The Structural Proteins

Duck hepatitis virus (DHV) causes a highly contagious disease in young ducks oftenassociated with liver necrosis, and high mortality. There are three genotypes of DHV,which are named type A DHV (DHV-A), type B DHV (DHV-B) and type C DHV (DHV-C),respectively. Both DHV-A and DHV-C have been found in the main-land of China, butDHV-B is only reported in Taiwan. DHV-C is a newly reported duck hepatitis virus inChina, which has led to serious economic loses to Chinese duck industry. So far, we haveknown less about the molecular biology about DHV-C. Here, we determined the full-lengthgenome of DHV-C GX strain and analyzed the bioinformatics of the strain.（1）To determine the complete sequences of GX strain, a total of six fragmentscovering the complete genome were PCR amplified with LA Taq DNA polymeraseaccording to the manufacturer’s protocol and using specific primers at several positionsalong the template RNA based on the published DHV-C sequence. The results showed thatThe length of the entire genomes GX strain was7800bp (including the poly (A) tail) andcontained a single open reading frame (ORF) containing6756nucleotides which encoded2251aa. The5’UTR and3’UTR of GX strain were562bp and366bp in length,respectively. The poly (A) tail of GX strain consisted of26As at least.（2）The results of sequence alignment analysis of GX strain and the referencedDHV sequences showed that GX strain shared higher homology with the reported DHV-Cstrains isolated in Korea, which was94.6-99.1%. VP1gene showed high variability amongGX strain and the referenced DHV strains, the sequence similarity between GX strain andDHV-A was only76.4%-78.5%. There are not only many base mutations, but also twoglycine insertions at the145amino acid. Besides, the uncoding regions of GX strain alsohave many base mutations; the most important is that the length of5’UTR and3’UTR ofGX strain was different from DHV-A strains.5’UTR of GX strain is62bp shorter that thatof DHV-A, but3’UTR of GX strain is34bp longer than that of DHV-A.（3）According to the complete genomic sequence of Duck hepatitis virusⅠ(DHV-Ⅰ)ZJ-V Strain, we designed three pairs of specific primer to amplify VP0, VP3and VP1gene.Then the sequences of VP0, VP3and VP1were inserted into pGEX-4T-1vector, pET-32a(+) vector, pET-32a (+) vector, respectively. And the three recombinant plasmids(pGEX-VP0，pET-VP3and pET-VP1) were transfected into E.coli BL21(DE3)plysS cells,E.coli Rosetta cells, E.coli BL21(DE3) cells,respectively. The expression products wereanalyzed with SDS-PAGE and Western-blot. Our results showed that VP0, VP3and VP1genes were successfully cloned, and their length were768bp,711bp and714bp,respectively. The SDS-PAGE and Western-blot results showed that VP0, VP3and VP1protein were successfully expressed in E.coli BL21(DE3)plysS cells, E.coli Rosetta cells,E.coli BL21(DE3) cells,respectively. And their molecular weight was about55ku,47ku and47ku, respectively. Among of them, VP3and VP1could react with the positive serumagainst DHV-I. Besides; we also prepared the specific polyclonal antibodies against VP0,VP3and VP1, respectively, by immunized rabbits with the three purified recombinantproteins. The antibody titres of VP0, VP3and VP1detected by with ELISA were1:25600，1:25600and1:25600, respectively.Summary, these results laid well foundation for further exploring the genomicvariation, the replication and translation mechanism of DHV or developing new vaccinesagainst DHV.