狂犬病毒aG株核蛋白和糖蛋白双表达质粒的构建及其在真核细胞中的表达  

Construction of Recombinant Plasmid for Co-expression of Nucleoprotein and Glycoprotein of Rabies Virus aG Strain in Eukaryotic Cells

作　　者：杨卉娟[1] 陈俊英[1] 孙明波[1] 张新文[1] 钱源[1] 段骞[1] 王东宝[1] 姜述德[1] 廖国阳[1] 李卫东[1] 

机构地区：[1]中国医学科学院中国协和医科大学医学生物学研究所IPV课题组,昆明650118

出　　处：《中国生物制品学杂志》2006年第6期564-567,共4页Chinese Journal of Biologicals

基　　金：2003年度云南省科技厅创新人才引培项目(编号2003PY07).

摘　　要：目的构建狂犬病毒aG株核蛋白(NP)和糖蛋白(GP)双表达重组质粒,在中国仓鼠卵巢细胞(CHO-K1)中表达核蛋白和糖蛋白。方法提取狂犬病毒RNA,应用RT-PCR方法扩增NP和GP基因,分别将其克隆到pIRES载体上,获得同时含有NP和GP基因的双顺反子重组质粒pING,并以脂质体介导法转染CHO-K1细胞,G418筛选,用ELISA和IFA检测NP和GP的表达。结果限制性内切酶分析表明重组质粒pING含有NP和GP基因片段,长度分别为1353bp和1575bp。应用ELISA和IFA方法,在转染细胞中均检测到NP和GP的表达。结论重组双表达质粒可在CHO细胞中同时表达NP和GP,为进一步开发重组狂犬病疫苗奠定了基础。Objective To construct a recombinant plasmid for co-expression of nucleoprotein （NP） and glycoprotein （GP） of rabies virus aG strain in CHO-K1 cells. Methods The genes encoding the NP and GP genes of rabies virus aG strain were amplified by RT-PCR and cloned into vector plRES to construct a dicistonic plasmid pING, then transfect to CHO-K1 cells in mediation of liposome. The transfectants were screened with G418 ,and the expressed product was analyzed by ELISA and IFA. Results The restriction map of recombinant plasmid pING showed NP and GP gene fragments at lengths of 1 353 bp and 1 575 bp respectively. ELISA and IFA proved the expression of both NP and GP in the transfected cells. Conclusion The recombinant plasmid pING for co-expression of NP and GP of rabies virus in eukaryotic cells was constructed. It laid a foundation of further development of recombinant rabies vaccine.

关 键 词：狂犬病毒 糖蛋白 核蛋白 双表达 真核细胞 

分 类 号：R373.33[医药卫生—病原生物学]