人呼吸道合胞病毒蛋白F1片段在大肠杆菌中的表达及纯化

目的在大肠杆菌中表达人呼吸道合胞病毒(Respiratory syncytial virus,RSV)F1蛋白主要抗原表位集中的137～523 aa片段,并对其进行纯化,检测其免疫原性。方法采用RT-PCR扩增RSV F1基因片段,与pThioHisA载体连接,构建重组表达质粒pThioHisA-RSV F1,转化大肠杆菌Top10,IPTG诱导表达,表达产物经SDS-PAGE分析;表达的融合蛋白经稀释复性、离子交换及亲和层析纯化后,进行SDS-PAGE及Western blot分析;将纯化的融合蛋白RSVF1经皮下多点注射分别免疫ICR小鼠,实验分为3组:RSV F1无佐剂组(RSV F1,50μg/只)、RSV F1佐剂组(50μg RSV F1+5μg nOMV佐剂)和阴性对照组(PBS,100μl/只),于第3周加强免疫1次,第4周尾静脉采血,分离血清,采用间接ELISA法检测其免疫原性。结果经双酶切鉴定及DNA测序证明重组表达质粒pThioHisA-RSV F1构建正确;表达的融合蛋白相对分子质量约56 000,主要以包涵体形式表达,表达量约占细胞总蛋白的36%;纯化的融合蛋白纯度可达95%,并可与RSV-F1多克隆抗体发生特异性抗原抗体反应;与阴性对照组相比,RSV F1免疫的小鼠血清特异性IgG水平明显提高(P<0.05)。结论已成功地在大肠杆菌中高效表达了RSV F1片段,纯化的融合蛋白在小鼠体内具有良好的免疫原性,为进一步研究RSV F蛋白亚单位疫苗奠定了基础。

Expression of F1 fragment of human respiratory syncytial virus protein in E.coli and purification of expressed product

Objective To express the 137 ~ 523 aa of F1 protein of respiratory syncytial virus(RSV),in which major antigenic epitopes were centralized,in E.coli,purify the expressed product and determine its immunogenicity.Methods Human RSV F1 gene fragment was amplified from viral RNA by RT-PCR and inserted into vector pThioHisA.The constructed recombinant plasmid pThioHisA-RSV F1 was transformed to E.coli Top10 which was induced with IPTG.The expressed product was identified by SDS-PAGE,refolded by dilution and purified by ion exchange and affinity chromatography,then analyzed by SDS-PAGE and Western blot.ICR mice were divided into three groups,and immunized with 50 μg purified fusion protein RSV F1,50 μg RSV F1 + 5 μg nOMV adjuvant and 100 μl PBS(negative control) respectively,by subcutaneous injection in several sites and boosted at week 3.Venous blood samples were collected at week 4,from which sera were separated and determined for immunogenicity by indirect ELISA.Results Restriction enzymes analysis and DNA sequencing proved that recombinant plasmid pThioHisA-RSV F1 was constructed correctly.The expressed fusion protein,with a relative molecular mass of about 56 000,mainly existed in a form of inclusion body and contained about 36% of total somatic protein.The purified fusion protein reached a purity of 95% and showed specific reaction with polyclonal antibody against RSV F1.The serum specific IgG levels of mice immunized with RSV F1 were significantly higher than that in negative control group(P < 0.05).Conclusion RSV F1 was successfully expressed in E.coli,and the purified fusion protein showed good immunogenicity in mice,which laid a foundation of further study on RSV F protein subunit vaccine.