丁型肝炎病毒抗原的原核表达及抗原性分析

Expression of the hepatitis Delta antigen in prokaryotic cell and evaluation of its application as an EIA diagnostic reagent

目的利用含T7启动子和His纯化标签pRSETB质粒构建丁型肝炎病毒抗原(HDAg)重组表达质粒(pRSETB-HDAg),转化宿主菌,表达并纯化,获得生物活性高抗原性强的基因工程重组抗原。方法将中国河南株HDAg基因片段插入pRSETB表达质粒,转化BL21宿主菌,经IPTG诱导表达。表达产物经Chelating亲和层析纯化后,采用EIA方法分析HDAg的抗原性。结果重组HDAg稀释1000倍(10ng/ml)仍与抗体有较强的反应。经与美国HDAg和华美公司生产的HDV诊断试剂同时检测26份HDV阳性参比血清,三者结果比较,除美国抗原漏检1份,检出率为96·15%(25/26份)外,病毒CDC和华美试剂均无漏检,检出率为100%,三者检测结果具有很高的符合率。结论成功构建了丁型肝炎病毒抗原重组表达质粒(pRSETB-HDAg),在原核细胞获得稳定高效表达,EIA检测证明HDAg抗原性好,可用于组装HDV诊断试剂,用于临床丁型肝炎患者的诊断和我国丁型肝炎病毒感染流行病学的调查。

Objective To construct the pRSETB-HDAg recombinant expression plasmid and to obtain soluble hepatitis D virus antigen with high biological and antigentic activity. Methods HDAg gene fragment was inserted into fusion expression pRSET B vector that includes T7 promoter and a polyhistidine tag. The recombinant plasmid was transformed into host bacterium BL21 after induction with IPTG. The expression supernatant was purified by chelating affinity chromatography and the recombinant HDAg antigenic activity was detected by EIA. Results EIA detection using the recombinant HDAg showed strong positive reaction with hepatitis D patients srea. The positive rates of the EIA, compared with HDAg from USA and Hua Mei EIA kit in detecting 26 ceases of anti- HDV positive reference sera,were 100% ,96.15% and 100% ,respectively. Conclusion Recombinant plasmid for HDAg with good antigenicity was successfully constructed and could be used as hepatitis D antibody detection reagent.