鸡传染性法氏囊病病毒J1C7株全基因组的克隆与分子系统进化树分析

为了获得鸡传染性法氏囊病病毒J1C7株的全基因组cDNA，确定其分子进化关系。以IBDV J1C7株细胞培养液为材料，用蛋白酶K消化、酚/氯仿抽提法提取IBDV基因组dsRNA，利用特异性引物将基因组A、B节段分两段进行RT-PCR并通过融合PCR方法将具有部分重叠序列的A、B节段的上、下游基因组进行拼接，从而构建出完整的A、B节段基因组，利用pMD-18T载体快速克隆PCR产物。结果表明：获得了3260 bp的A节段全长（Genbank登录号EF646854）和2827 bp的B节段全长（Genbank登录号EF646853）。氨基酸同源性比对结果表明：J1C7株与弱毒疫苗株CEF94（芬兰）、P2（德国）、JD1（中国）、HZ2（中国）的亲缘关系最近（蛋白同源性为VP5：99.3%；VP2：99.3%~100%；VP4：97.9%~99.2%；VP3：96.4%~98.1%；VP1：99.2%~99.9%）。BDV J1C7株属于弱毒疫苗株，且与欧洲和中国的弱毒疫苗株有密切的亲缘关系，在进化树上归为一簇。本研究为构建IBDV的感染性cDNA，了解IBDV基因组的结构与功能打下了基础。

Cloning and Genetic Analysis of Complete cDNA Sequences of Both Segments ofInfectious Bursal Disease Virus J1C7 Strain

In order to obtain the genome-wide cDNA of infectious bursal disease virus J1C7 strain and analyze its phylogenetie relationship. A proteinase K digestion and phenol-chloroform extraction method was used to extract dsRNA genome from IBDV J1C7 strain infected chicken embryo fibroblast （CEF）. The specific primers were applied to amplify the two fragments of A and B segments by RT-PCR respectively. Then the fusion PCR was conducted to ligate the two fragments of A and B segments which possessed the overlapped sequences in order to construct the complete genome. The amplified PCR products were cloned by pMD-18T cloning vector. The results showed that the analysis of sequences identified that they were full-length segment A of 3260 bp （Genbank ID：EF646854） and segment B of 2827 bp （Genbank ID：EF646853） respectively. The amino acid alignment result indicates that IBDV J1C7 strain has the closest relationship with the attenuated IBDV strains （CEF94, P2, JD1, HZ2）. The amino acid identity percent of VP5, VP2, VP4, VP3, VP1 were 99.3%, 99.3%-100%, 97.9%-99.2%, 96.4%-98.1%, 99.2%-99.9% respectively. The IBDV J1C7 strain belongs to the attenuated vaccine strains and the phylogenetic tree analysis indicates IBDV J1C7 strain is more closelyrelated to the European and Chinese attenuated strains than those of vvlBDV and variant strains. This study laid the foundation for the construction of IBDV J1C7 strain ＇ s infectious cDNA. It also provided a reliable guarantee for further study of the relations between its structures and functions.