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Sequence Analysis for the Complete Proviral Genome and the Expression of gp85 Gene of Subgroup J Avian Leukosis Virus Associated with Hemangioma

Author: ShiMin
Tutor: HuangYong
School: Sichuan Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Hemangioma type subgroup J avian leukosis The progenitor Breeders isolates Whole genome sequence gp85 protein The prokaryotic expression
CLC: S852.65
Type: Master's thesis
Year: 2011
Downloads: 36
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Abstract


Subgroup J avian leukemia virus (Avian Leukosis Virus Subgroup J, ALV-J) belong to the retrovirus can induce local chickens and wild birds produce tumor growth retardation, immune suppression, supplies poultry industry has brought huge economic losses and serious threat. Subgroup J avian leukemia virus was isolated and identified by the British scholar since 1989 has been the main cause of broilers myeloma leukemia, but in recent years, previous studies have confirmed less prone to tumors of commercial laying hens and local breeders by ALV-J after infection can also be induced by the high incidence of tumors. In addition, previously rare cases of hemangioma caused by ALV-J gradually increased in recent years. This is enough to demonstrate the specificity and complexity of different geographical ALV-J infection. This study vaccination chick embryo fibroblasts (CEF) RT-PCR and PCR technology, for the first time identified a vascular tumor type subgroup J avian leukemia virus isolated from a progenitor species farms sick chicken named CEF genomic DNA infection isolates SCDY1, as amplified proviral genome template SCDY1., paragraph 9 was amplified by PCR according to the published prototype strain HPRS-103 sequence design and synthesis of nine pairs of primers, continuous, some mutual overlapping DNA fragments, and then are respectively connected into a pMD19-T vector for cloning and sequencing. Final completion of ALV-J the isolates SCDY1 former virus genome nucleotide sequence analysis software. Avian leukosis virus subtype is mainly determined by the surface of the virus membrane glycoprotein gp85, gp85 gene sequencing results SCDY1 virus strain genome sequencing, Primer permier 5.0 Design of expression primers used to amplify the gp85 gene and construct prokaryotic expression vector PET-32a () / gp85, expressed in E. coli Rosetta. The results showed that the cDNA genome SCDYl; strains of a total length of 7489 bp. The other ALV-J strain sequence in GeneBank published genome sequence homology comparison, gag and pol genes SCDY1 genomic relatively conservative env gene variability is relatively large. , Gag, pol, and env gene homology at the nucleotide level were 94.3-96.5% ,96.6-97 .6% ,90.3-94 .2%; were at the amino acid level, gag, pol and env homology 96.2-98.4% ,97.4-98 .7% ,85.1-90 .7%. Strains SCDYl E-element and a non-functional TM sequences are almost completely missing, and 11 consecutive nucleotides of the absence of a sequence in the 3'UTR of the U3 region also appeared. U3 region transcriptional regulatory element analysis showed that the 11-bp deletion of the SCDYl: strains U3 region sequence is located regulatory elements CArGbox upstream, similar to the human gene Apo-AI of transcription factor ABI REP1, it can provide binding sites for HNF-4, and combined, and HNF-4 can be used as a repressor, and play an important role in the regulation of eukaryotic genes. In addition, the prokaryotic expression system successfully hemangioma ALV-J strain of the virus gp85 protein, the protein molecular weight of about 57KD, and is expected to match. The expressed protein exists in the form of inclusion bodies in the bacterial protein. Western-blot analysis showed that the expression product has good reactogenicity.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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