绵羊肺腺瘤病病毒NM株衣壳蛋白基因的克隆与原核表达  

Cloning and expressing of capsid protein gene of jaagsiekte sheep retrovirus Neimeng strain in E.coli

作　　者：斯日古楞[1] 么宏强[1] 马学恩[1] 王升启[2] 

机构地区：[1]内蒙古农业大学动物科学与医学学院,内蒙古呼和浩特010018 [2]中国军事医学科学院,北京100850

出　　处：《中国预防兽医学报》2007年第8期584-587,595,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金项目(30260083);教育部高等学校博士点专项科研基金项目(20040129001)

摘　　要：为了建立原核高效表达体系,从自然感染绵羊肺腺瘤病病毒内蒙(NM)株的肺肿瘤组织中提取基因组DNA,应用PCR技术扩增编码gag基因衣壳蛋白(Capsid protein,CA)基因,构建CA蛋白的重组表达质粒pGEX-4T-1-CA,经酶切、PCR及DNA测序鉴定后,将阳性质粒转化入Ecoli BL21 CodonPlus中,在IPTG的诱导下表达,表达产物用SDS-PAGE分析鉴定。结果表明,该基因可以在大肠杆菌中以可溶性和包涵体形式高效表达,表达的CA融合蛋白表观分子量约为37 Ku,表达量占全菌蛋白的20%,利用谷胱甘肽Sepharose 4B亲和层析柱纯化可以得到具有较高纯度的表达蛋白,是一种理想的免疫原。本研究为下一步利用重组蛋白研制绵羊肺腺瘤病病毒NM株的单克隆抗体及诊断试制盒等奠定了基础。Capsid protein （CA） gene was amplified by PCR from genome DNA of lung tumour tissues of sheep with naturally affected pulmonary adenomatosis using specific primers designed from the JSRV-NM sequences. The PCR product was cloned into a pGEX-4T-1 vector and the recombinant plasmid was transformed into E.coli BL21 CodonPlus cells. Expression of CA protein was induced by IPTG and analysed by SDS-PAGE. The results showed that the expressed protein was approximately 37 Ku in size and accounted for 20 % of total bacterial protein. The fusion protein existed both in the form of soluble protein and inclusion bodies. High purity CA fusion protein was obtained from the soluble fraction of the expressed protein by purification with glutathione-sepharose 4B, which could used for the development of monoclonal antibody and diagnostic reagents of JSRV-NM strain.

关 键 词：绵羊肺腺瘤病 绵羊肺腺瘤病病毒内蒙毒株 衣壳蛋白 基因克隆 表达 

分 类 号：S852.65[农业科学—基础兽医学] Q786[农业科学—兽医学]