猪瘟病毒国内分离株(HL-LY)E_2基因的克隆及序列分析

Cloning and Sequence Analysis of E_2 Gene from Domestic Isolate Strain of CSFV

用猪肾细胞 (pK - 15 )繁殖猪瘟病毒 (CSFV)分离株HL -LY ,根据基因库已发表的CSFVE2 基因序列 ,设计并合成一对引物 ,以CSFV总RNA为模板 ,通过RT -PCR技术扩增出约 1.1kb的片段 ,即E2 基因。将RT -PCR产物克隆到 pMD18-T载体上 ,构建了重组质粒T -E2 ,经酶切、PCR鉴定和序列测定 ,表明均已成功获得了CSFVE2基因的重组体T -E2 。将该测序结果与国内几个毒株SHIMEN ,C ,C -V -LZ ,GS -LT ,GS -LX分别进行序列同源性比较 ,核苷酸同源性分别为 96 .5 9% ,96 .0 0 % ,88.4 9% ,78.2 4 % ,77.82 % ;氨基酸同源性分别为 99.4 6 % ,98.39% ,93.5 4 % ,90 70 % ,90 .4 4 %。

The HL-LY strain of Classical Swine Fever Virus(CSFV) was propagated and harvested on pk-15.One pair of primers were designed to amplify E 2 gene by RT-PCR according to published sequence of CSFV's E 2 gene of gene bank . The product of RT-PCR,which is about 1.1 kb was cloned into pMD18-T vector,then the recombinent plasmid which is named T-E 2 was identified by corresponding restriction endonuclease and PCR and was analyzed with sequencing and homology compariation.The result showed that CSFV E 2 gene of HL-LY strain share 96.59%,96.00%,88.49%,78.24%,77.82% with SHIMEN strain,C strain,C-V-LZ strain , GS-LT strain,GS-LX strain in nucleotide sequence respectively, and share 99.46%,98.39%,93.54%,90.70%,90.44% identity in amino acids sequence respectively,which showed that HL-LY strain has high homology with domestic standard SHIMEN strain and C strain.