猪瘟病毒Shimen株E2基因的克隆及其逆转录病毒表达载体的构建  

Cloning and Retroviral Expression Vector Construction of E2 Gene of CSFV Shimen Strain

作　　者：刘建玲[1,2] 苏正元[1] 许信刚[1] 李谱华[1] 张彦明[1] 

机构地区：[1]西北农林科技大学动物科技学院 [2]西北大学生命科学学院,陕西西安710069

出　　处：《中国兽医学报》2006年第3期238-239,242,共3页Chinese Journal of Veterinary Science

基　　金：陕西省科技攻关项目(2003k02-G11-01);陕西省教育厅专项科研计划项目(05JK296)

摘　　要：根据已发表的猪瘟病毒(classicalswinefevervirus,CSFV)Shimen株的全基因组序列,设计2对引物P1/P2和B1/B2,在B1和B2的5′端分别加上EcoR和BamH位点,以CSFV-Shimen株脾毒为材料,一步法提取总RNA,并以此为模板采用反转录PCR(RT-PCR)和套式PCR(nPCR),成功地扩增出约1.2kb的E2基因。将PCR产物回收后与pMD18-T载体连接并转化,经PCR和酶切鉴定获得重组质粒E2-T。将E2-T经EcoR和BamH酶切消化、回收目的基因后,与经BamH/EcoR酶切的逆转录病毒载体pBABE-puro连接并转化,经酶切鉴定获得重组质粒pBABE-puro-E2。序列测定表明,目的基因的插入位置、方向和读码框完全正确。Two pairs of primers, P1/P2 and B1/B2, were designed based on the sequence of the upper stream and down stream of CSFV E2 gene. About 1.2 kb fragment was reversely transcribed and amplified from CSFV Shimen strain using the primers and further identified as the E2 gene by gel analysis. The RT-PCR product was purified and cloned into the pMD 18- T Vector. -Their recombinant plasmid E2-T was indicated containing E2 gene by PCR and restriction enzyme digestion. The E2 gene was subsequently subcloned into expression vector pBABE-puro by restriction enzyme BamH 1 /EcoR 1 digestion and linking. The recombinant pBABE-puro-E2 was generated, which could be further expressed in eukaryotic cell.

关 键 词：猪瘟病毒 E2基因 RT—PCR 逆转录病毒表达载体 

分 类 号：S852.65[农业科学—基础兽医学] S858.28[农业科学—兽医学]