狂犬病病毒CTN-1株G蛋白的原核表达及基本功能评价  

Prokaryotic expression and basic functional evaluation of G protein of rabies virus CTN-1 strain

作　　者：巩蔚[1] 严丽蔚 朱文兵[1] 张雪梅[1] 徐婧雯[1] 吴忠香[1] 卢孔杰[1] 孙明[1] 董少忠[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室,云南昆明650118

出　　处：《中国生物制品学杂志》2016年第10期1032-1036,共5页Chinese Journal of Biologicals

基　　金：云南省科技厅重点新产品研发计划(2015BC007)

摘　　要：目的原核表达狂犬病病毒(rabies virus,RV)CTN-1株G蛋白,并评价其基本功能。方法采用RT-PCR法扩增RV CTN-1株G蛋白基因,插入原核表达载体p GEX-5X-1,构建重组表达质粒p GEX-5X-1-CTN,转化入Rosetta(DE3)p Lys S,IPTG诱导表达。表达的重组蛋白经亲和层析纯化后,采用间接ELISA法检测其与阳性血清的结合性能及亲和力。结果重组表达质粒经双酶切及测序证实构建正确;纯化的重组蛋白RV-G相对分子质量约85 000,纯度可达85%,能与阳性血清特异性结合,且具有较好的区分性,与阳性血清的亲和力常数为1.2×1010 M-1。结论成功原核表达、纯化获得了纯度较高、特异性较好、亲和力较强、灵敏度较高的RV CTN-1株G蛋白,为研究G蛋白的抗原表位、建立针对RV G蛋白的ELISA抗体检测方法及其中和抗体的分选检测奠定了基础。Objective To express the G protein of rabies virus（RV） CTN-1 strain in prokaryotic cells and evaluate its basic function. Methods The gene encoding G protein of RV CTN-1 strain was amplified by RT-PCR and inserted into prokaryotic expression vector p GEX-5X-1. The constructed recombinant plasmid p GEX-5X-1-CTN was transformed to E. coli Rosetta（DE3） p Lys S for expression under induction of IPTG. The expressed protein was purified by affinity chromatography, and determined for binding capacity and affinity by indirect ELISA. Results Restriction analysis and sequencing proved that recombinant plasmid p GEX-5X-1-CTN was constructed correctly. The purified recombinant protein RV-G, with a relative molecular mass of about 85 000, reached a purity of 85%, showed specific binding to positive serum and good distinguishability, of which the affinity constant to positive serum was 1. 2 × 10^10M^-1. Conclusion The G protein of RV CTN-1 strain, with high purity, specificity, affinity and sensitivity, were expressed in prokaryotic cells and purified, which laid a foundation of study on the antigenic epitope of G protein and development of ELISA method for antibody against G protein as well as sorting of neutralizing antibody to the protein.

关 键 词：狂犬病病毒 CTN-1株 G蛋白 原核细胞 基因表达 亲和力 酶联免疫吸附测定 

分 类 号：R373.9[医药卫生—病原生物学] Q786[医药卫生—基础医学]