| Avian Leukosis virus (ALV) of subgroup J (ALV-J) belong to retroviruses, which could induce tumors, grow retardation and immunosuppression in domestic and wild birds, having caused severe economic losses in breeding farms all over the world. Myelocytomatosis had been the most common neoplasma observed in infected flocks since ALV-J was firstly isolated in England in 1989s; however, this disease showed development in our country in recent years and spreaded its tumorigenesis host range from meat-type chickens to commercial layer chickens and Chinese local flocks, moreover, few cases of hemangioma caused by ALV-J were reported in recent year. The ALV-J infection condition in China thoroughly showed the specificity and difference.One subgroup J Avian Leukosis Virus strain associated with hemangioma designated SCDY1 was isolated from a grandparent breeding farm with suspected diseased chickens of Sichuan province by inoculating the samples into chicken embryo fibroblast cells, RT-PCR and PCR amplification technologies. The genomic DNA extracted from chicken embryo fibroblasts (CEF) infected with SCDY1 was used as the template to amplify the ALV-J proviral genomic cDNA by polymerase-chain-reaction (PCR). Nine pairs of primers were designed according to the published sequences of HPRS-103 to amplify nine fragments which overlapped with each other and covered the whole genome of ALV-J, then cloned into the pMD19-T vector and sequenced. The complete sequence of the whole genome of ALV-J with hemangioma strain SCDYl was finally established and analyzed. The subtype of avian leukosis virus was mainly determined by the gp85 glycoprotein. Primers specific for the gp85 gene were designed using Primer Premier 5.0 based on the gained cDNA sequences of gp85 gene of strain SCDY1, the highly antigenic region of its gp85 gene was amplified and expressed in Escherichia coli Rostta using the pET-32a (+) vector respectively.Sequence analysis showed that the complete proviral genome sequence of SCDY1 was 7489 bp in length. Comparisons of the three major structural genes revealed that the homology of nucleotide sequence of env, pol and gag gene between SCDY1 and other isolates in GenBank were 90.3-94.2%,96.6-97.6%and 94.3-96.5%, respectively, and the homology of amino acid sequence of env, pol and gag gene between SCDY1 and other isolates in GenBank were 85.1-90.7%,97.4-98.7%and 96.2-98.4%, respectively. The gag and pol genes of ALV-J were more conservative than the env gene relatively. Most of non-functional TM and E element of SCDY1 in 3'UTR were absent and a specia1 llbp deletion was observed in U3 region of 3'UTR of SCDY1. Transcriptional regulatory elements analysis in U3 region of SCDY1 genome showed that the special 11bp deletion was found in the upstream of CArG box and was recognized as a transcriptional regulatory element ABI REP1, which was the regulatory factor of human gene Apo-AI. One binding factor of ABI REP1 named as HNF-4 was also found, and it served as an inhibitor modulator and played an important role in the regulation of eukaryotic genes. In addition, prokaryotic expression plasmid was successfully constructed and gp85 protein of ALV-J strain SCDY1 associated with hemangioma was successfully expressed, whose relative molecular mass was about 57KD, as the same as expectation. The recombinant gp85 was expressed mainly in the form of inclusion body. Western blotting analysis showed that the expressed production was of great reactionogenicity. |
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