HSV-1 gC糖蛋白真核表达载体的构建及表达  

Construction and expression of the eukaryotic expression vector carrying HSV-1 gC glycoprotein gene

作　　者：党引利[1] 阎岩[1] 张晓晓[1] 李璞媛[1] 于澜[1] 张蕾[1] 张芳琳[1] 徐志凯[1] 吴兴安[1] 

机构地区：[1]第四军医大学基础部微生物学教研室,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2011年第5期494-497,共4页Chinese Journal of Cellular and Molecular Immunology

摘　　要：目的:构建单纯疱疹病毒1型(HSV-1)囊膜糖蛋白C(gC)哺乳动物表达载体pCI-mCMV-gC-1-IRES-DHFR-L22R,实现其在中国仓鼠卵巢细胞(CHO-K1)中的稳定表达。方法:利用基因重组技术构建同时克隆有gC-1基因和中国仓鼠二氢叶酸还原(DHFR-L22R)基因的真核表达载体pCI-mCMV-gC-1-IRES-DHFR-L22R,按LipofectamineTM2000说明书转染野生型CHO-K1细胞,在G418和氨甲喋呤(MTX)双筛选压力下筛选稳定表达细胞株,Slot blot方法测定重组蛋白的表达,并用His-Ni琼脂糖填料进行目的蛋白纯化,纯化后West-ern blot检测。结果:成功构建了真核表达载体pCI-mCMV-gC-1-IRES-DHFR-L22R。经过两轮筛选获得稳定表达HSV-1型gC基因的细胞系,Western blot成功检测到目的蛋白。结论:HSV-1 gC在CHO-K1细胞中成功表达,并具有良好的特异性结合活性,为进一步的实验研究和临床应用提供了基础。AIM： To stably express herpes simplex virus type 1（HSV-1） glycoprotein C（gC） in Chinese hamster ovary cells（CHO-K1）.METHODS： The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed and transfected into CHO-K1 cells by LipofectamineTM 2000.The transfected cells were selected by G418 and methotrexate（MTX）.The expression of HSV-1 gC was analyzed by Slot blot.HSV-1 gC proteins were purified with His-Ni Sepharose and then detected by Western blot.RESULTS： The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed successfully.CHO-K1 cells stably expressing HSV-1 gC proteins were established and confirmed by Western blot.CONCLUSION： The HSV-1 gC proteins have been expressed successfully and have good bioactivity.The results make it possible for further study and clinical use of HSV-1 gC.

关 键 词：HSV GC DHFR 真核表达 CHO 

分 类 号：R392.12[医药卫生—免疫学]