j亚群禽白血病病毒ahj11株全基因组序列分析及其感染性克隆的构建-analysis of the whole genome sequence of avian leukosis virus subgroup j ahj 11 strain and construction of its infectious clone.docx

I II 元件构建输送或表达外源基因的质粒载体奠定了基础。 关键词：ALV-J，全基因组，感染性克隆，LTR 启动子 PAGE PAGE IV Abstract Avian Leukosis is a general designation of a variety of avian neoplastic diseases caused by Avian Leukosis Virus,which widespread by vertical and horizontal transmission in chicken flocks around the world. Avian Leukosis can cause fleshless, maldevelopment, immunodepression and death. Because virus can be transmitted through horizaontal and vertical transmission,currently,some poultry took some measures to purify chicken flocks through condemning infected breeding chickens. Eleven pairs of primer were designed and synthesized according to ALV-J HPRS-103 strain and SD07LK1 strain complete genome sequence published in GenBank. Eleven overlapping fragments covering the whole proviral genome of ALV-J AH-J11 strain were amplified by PCR.The cDNAs were cloned into pMD18-T for sequencing. Sequences were aligned and phylogenetic tree were drawn as previously described. The results of sequence analysis demonstrated that the full lenth of the AH-J11 strain was 7844nt,made of three constructed protein(gag、pol and env) and ends of UTR.Gag and pol gene were relatively conservative regions,but env gene ws high variable region. The AH-J11 strain was closed with HPRS-103 strain with 97.4% genome nucleotide.This study results enriched information data of ALV-J genome and provided a valuable basis for further study on biological characteristics and molecular genetic variation of ALV-J. According to provius from in the virul life style, 4 fragmeents of provirus cDNA of avian leukosis virus subgroup J(ALV-J) AH-J11 strain were fused and amplified from 11fragments of the genomic DNA by using fusion PCR,then combined in the right direction and sequences into recombinant plasmid pBlALV-AH-J11,containing the whole genome of AH-J11. pBlALV-AH-J11 was transfected into chicken embryo fibroblast (CEF) cells and the ALV was rescued. The rescued ALV(rALV-J) was identified by indirect immu