Sequence Analysis And Construction Of Infectious Clone Of Avian Leucosis Virus Subgroup J Anhui Strain AH-J11

Avian Leukosis is a general designation of a variety of avian neoplastic diseases caused byAvian Leukosis Virus,which widespread by vertical and horizontal transmission in chickenflocks around the world. Avian Leukosis can cause fleshless, maldevelopment,immunodepression and death. Because virus can be transmitted through horizaontal andvertical transmission,currently,some poultry took some measures to purify chicken flocksthrough condemning infected breeding chickens.Eleven pairs of primer were designed and synthesized according to ALV-J HPRS-103strain and SD07LK1strain complete genome sequence published in GenBank. Elevenoverlapping fragments covering the whole proviral genome of ALV-J AH-J11strain wereamplified by PCR.The cDNAs were cloned into pMD18-T for sequencing. Sequences werealigned and phylogenetic tree were drawn as previously described. The results of sequenceanalysis demonstrated that the full lenth of the AH-J11strain was7844nt,made of threeconstructed protein(gag、pol and env) and ends of UTR.Gag and pol gene were relativelyconservative regions,but env gene ws high variable region. The AH-J11strain was closedwith HPRS-103strain with97.4%genome nucleotide.This study results enrichedinformation data of ALV-J genome and provided a valuable basis for further study onbiological characteristics and molecular genetic variation ofALV-J.According to provius from in the virul life style,4fragmeents of provirus cDNA of avianleukosis virus subgroup J(ALV-J) AH-J11strain were fused and amplified from11fragments of the genomic DNA by using fusion PCR,then combined in the rightdirection and sequences into recombinant plasmid pBlALV-AH-J11,containing the wholegenome of AH-J11. pBlALV-AH-J11was transfected into chicken embryo fibroblast (CEF)cells and the ALV was rescued. The rescued ALV(rALV-J) was identified by indirectimmunofluorescence assay,RT-PCR and western-blot,respectively. The results showed thatthe rALV-J was able to well replicate in CEF. This study provides an useful platform forinvestigation of the pathogenesis and molecular biology of ALV-J.To construct the eukaryotic expression vector used the long terminal repeat(LTR) ofavian leukosis virus subgroup J(ALV-J) as a promoter, LTRs were cloned into pBluescriptII SK+vector, subsequently, eGFP gene and NDV NP gene were inserted into5’LTR and3’LTR, resepectively. The final recombinant plasmids were named pSK-LTR-GFP andpSK-LTR-NP. The two recombinant plasmids were transfected into293T cell、BHK-21cell and chicken embryonic fibroblast cell, and the exogenous genes were successfullyexpressed by detecting with indirect immunofluorescence assay and western-blot.Theresults showed that LTR could well initiate the transcription and expression of foreigngenes (eGFP and NP). The flow cytometry results indicated that the expression level ofreporter gene reached peak at48h in293T cell. This study will land foundation fordeveloping a plasmid vector used ALV-J LTR for delivering and/or expressing exogenousgene.