SPF鸡胚中内源性白血病病毒全基因序列鉴定与分析

Sequence Analysis for the Complete Provial Genome of Endogenous Avian Leukosis Virus Strain SD0501

以国内某3家SPF鸡场的SPF鸡胚成纤维细胞提取的基因组DNA为模板,参照已发表的序列,设计合成了4对检测内源性白血病病毒引物,分别检测gag基因、pol基因、env基因和LTR片段,结果显示4者检出阳性率很高(gag,29/46;pol,27/46;env,24/46;LTR,31/46)。设计合成了8对引物,选取4片段检测均为阳性的样品之一,经PCR成功扩增出了8段连续的、相互部分重叠的目的DNA片段,分别连接入T载体进行克隆测序。用DNAstar软件对测序结果进行拼接,从一个鸡胚得到了内源性白血病病毒前病毒全基因组序列。比较分析发现,该序列env基因与已知的E亚群内源性病毒代表株env基因的核苷酸序列同源性在98.5%以上,全基因组序列同源性在99.1%以上,而与其他亚群代表株同源性相对较低,env基因同源性仅为56.3%～91.5%。

The genomic DNA extracted from chicken embryo fibroblasts （CEF） of SPF chickens from three chicken farms was used as template to amplify the ALV proviral DNA by PCR with four pairs of primers, high positive detection rates of gag - gene （29/46）, pol - gene （27/46）, env - gene （24/46） and LTR frag- ment （31/46） were achieved. Eight continuous and overlapping fragments were amplified from one DNA sample with 8 pairs of primers according to published sequences, then cloned into the TA vector and sequenced. The complete sequence of the whole genome of ALV strain SD0501 was established and analyzed with DNAstar software. Comparisons of SD0501 sequence with that of other representative endogenous avian virus strains demonstrated that the genomes of ALV were relatively conservative, the nucleotide identity of all the strains was over 99.1%, and env - gene was over 98.5%. However, a low identity was demonstrated among the representative strains of different subgroups, especially, the env -gene showed obvious difference, the corresponding identity was as low as 56.3%～91.5%.