庚型肝炎病毒NS5区优势抗原表位的表达及其抗原性的初步评价  

Expression and antigenicity analysis of hepatitis G virus NS5 gene

作　　者：丛郁[1] 焦宏远[1] 张文英[1] 田瑞光[1] 詹美云[1] 

机构地区：[1]中国预防医学科学院病毒学研究所肝炎研究室,北京100052

出　　处：《中华实验和临床病毒学杂志》2002年第2期150-153,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：国家"九五"攻关项目(96 90 6A 0 3 0 8)

摘　　要：目的　表达庚型肝炎病毒 (HGV)基因组NS5区部分基因重组抗原 ,分析其抗原性。方法　分别亚克隆了HGVNS5a、NS5b以及NS5b与C区嵌和的基因至pThioC表达载体上 ,构建成 3个重组表达质粒 ,分别转化大肠埃希菌JM1 0 9(DE3) ,用IPTG诱导表达重组蛋白。表达产物经纯化后采用Westernblot和间接ELISA方法分析 3个重组蛋白的抗原性。结果　经酶切和序列分析鉴定正确 ,3种表达蛋白均高效表达且相对分子质量与预期大小相符。Westernblot分析和间接ELISA试验表明 ,3种表达蛋白均能与抗 HGV阳性血清发生特异性反应。将应用重组蛋白检测的抗 HGV抗体与混合重组抗原 (包括C区合成肽、NS5a合成肽、NS3区基因重组抗原 )的检测结果进行了比较 ,在混合重组抗原阳性的 2 2份血清中 ,P5a检出率为 68% (1 5 2 2 ) ,P5b检出率为 91 % (2 0 2 2 ) ,Pc 5b检出率为 73 % (1 6 2 2 )。在 70份阴性标本中 ,3种抗原的检出率分别为P5a:7% (5 70 ) ;P5b :1 % (1 70 ) ;Pc 5b :6 % (4 70 )。3个重组抗原单独检出阳性的标本 ,其中有一部分经RT PCR检测亦为阳性。结论　原核表达的NS5区蛋白所检测的抗 HGV抗体不能完全被其他区段的抗原所覆盖 ,是免疫诊断HGV感染所必需的抗原表位之一。Objective To determine the antigenicity of HGV NS5 recombinant proteins expressed in E.coli. Methods HGV NS5a,NS5b and core/NS5b fusion gene were cloned into pThioC vector.Three expression plasmids were transformed into JM109(DE3) competent cells then expressed with induction by IPTG.Western blot and ELISA were used to determine the antigenicity after the three recombinant proteins were purified.Results After identification by restriction enzyme and sequencing,it was confirmed that the expressed was target proteins espected.Purified expression proteins were found strongly immunoreactive among anti HGV positive sera by Western blot and ELISA.Compared with mixed recombinant antigen(including core,NS5a synthetic peptide and NS3 recombinant proteins),in the 22 positive sera detected with mixed antigen,68%(15/22),90%(20/22) and 73%(16/22) were positive by P5a,P5b and Pc 5b antigens;In the 70 negative samples with mixed antigen,7%(5/70),1%(1/70) and 6%(4/70) were positive by P5a,P5b and Pc 5b antigens.The positive alone was found among RT PCR positive specimen using these recombinant antigens.Conclusion NS5 gene expressed in E.coli which couldn't be covered with other regions of antigens was one of the essential epitopes to HGV immunologic diagnosis.

关 键 词：庚型肝炎病毒 病毒基因表达调控 抗原 

分 类 号：R392[医药卫生—免疫学]