猪瘟病毒分离株E_2基因的克隆与酵母表达系统转移载体的构建

Cloning of E_2 gene of CSFV and construction of Pichia pastoris transfer vector

用猪肾细胞 (PK- 15 )繁殖猪瘟病毒 (CSFV)分离株 HL - L Y,根据基因库已发表的 CSFV E2 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产物先克隆到 p MD18- T载体上 ,构建了重组质粒 T- E2 ,再通过双酶切亚克隆到表达载体 p PIC9的多克隆位点 (MCS)上 ,经酶切、PCR鉴定和测序分析 ,表明均已成功获得了 CSFV E2 基因的重组体 p PIC9- E2 。将该测序结果与国内几个毒株 SHIMEN,C,C- V- L Z,GS- L T,GS- L X分别进行序列同源性比较 ,核苷酸同源性分别为 96 .5 1% ,95 .5 3% ,89.12 % ,78.6 6 % ,77.2 3% ;氨基酸同源性分别为 97.32 % ,95 .71% ,89.5 4 % ,83.16 % ,83.4 2 %。表明该毒株与国内标准毒株 SHIMEN株和

The HL- L Y strain of classical swine fever virus(CSFV) was propagated and harvested on pk- 15 .One pair of primers were designed to amplify E2 gene by RT- PCR according to published sequence of CSFV's c DNA.The product of RT- PCR,which is about 1.1kb was cloned into p MD 18- T vector,then the recombinent plasmid which is named T- E2 was cleavaged by double restriction endonuclease Eco R I and Not I to get E2 gene.The fragment was inserted into Eco R I and Not I multiple cloning site(MCS) of p PIC9vector. The recombinantof p PIC9- E2 was identified by corresponding restriction endonuclease and PCR,and was ana- lyzed by sequencing and homology comparison.The result showed that CSFV E2 gene of HL- L Y strain share 96 .5 1% ,95 .5 3% ,89.12 % ,78.6 6 % and 77.2 3% with Shimen strain,C strain,C- V- L Z strain,GS- LT strain and GS- L X strain in nucleotide sequence respectively,and share97.32 % ,95 .71% ,89.5 4% ,83.16 % and83. 4 2 % identity in amino acids sequence respectively,which showed that HL- L Y strain has high homology with domestic standard SHIMEN strain and C strain.