【摘要】 目的原核表达、纯化呼吸道合胞病毒(respiratory syncytial virus,RSV)表位重组蛋白F1-F/M2,并检测其在小鼠体内的免疫原性,为RSV亚单位疫苗的研制、疫苗所致免疫病理反应机理的研究提供参考。方法利用定点突变技术对原核表达载体pGEX-6p-1进行改造,在其GST标签前制造一个HindⅢ酶切位点;从RSV long株中扩增F1片段,从质粒pET28a-G1-F/M2中扩增F/M2基因片段,并在克隆载体pUC19的辅助下,将F1-F/M2基因连接至改造后的pGEX-6p-1载体中,构建重组表达质粒pGEX-6p-1-F1-F/M2,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经6×His-tag标签蛋白纯化试剂盒纯化后,经尿素浓度梯度透析复性,SDS-PAGE分析其纯度,BCA法检测其浓度,Western blot法分析其反应原性。将纯化的重组蛋白F1-F/M2经铝佐剂乳化后,经腹腔免疫BALB/c小鼠,共3次,间隔10 d,末次免疫后10 d,采用空斑减少试验检测小鼠血清、肺组织匀浆和鼻腔冲洗液中抗RSV中和抗体水平;取小鼠脾脏,分离脾单个淋巴细胞,采用ELISAPOT技术检测重组蛋白诱导产生IFNγ的细胞免疫水平。结果重组表达质粒pGEX-6p-1-F1-F/M2经双酶切和测序证实构建正确;表达的重组蛋白F1-F/M2相对分子质量约为20 800,表达量约占菌体总蛋白的20%,几乎全部以包涵体形式表达;纯化的重组蛋白的纯度约为90%,浓度为0.42 mg/ml,可与小鼠抗RSV血清发生特异性反应;重组蛋白F1-F/M2能诱导小鼠血清中产生较高滴度的中和抗体(1∶289),肺部组织匀浆中略弱(1∶242),鼻腔冲洗液中未检测到中和抗体;重组蛋白F1-F/M2免疫小鼠的脾淋巴细胞经体外抗原刺激能产生特异性IFNγ斑点。结论成功原核表达了表位重组蛋白F1-F/M2,纯化后的重组蛋白能诱导产生较高滴度的中和抗体及CTL应答,有望开发成RSV亚单位疫苗。更多还原

【Abstract】 Objective To express recombinant respiratory syncytial virus(RSV)epitope protein F1-F / M2 in prokaryotic cells,purify the expressed product and determine its immunogenicity in mice,so as to provide a reference for the preparation of RSV subunit vaccine and the study on mechanism of immune pathological reaction caused by the vaccine.Methods Prokaryotic expression vector pGEX-6p-1 was modified by site-directed mutagenesis,to which Hind Ⅲ restriction site was introduced before the GST tag.F1 gene fragment was amplified from RSV long strain,while F1-F / M2 gene fragment from plasmid pET28a-G1-F / M2.F1-F / M2 gene was cloned into the modified vector pGEX-6p-1 with the help of an intermediate vector pUC19.The constructed recombinant plasmid pGEX-6p-1-F1-F / M2 was transformed to E.coli BL21(DE3)and induced by IPTG.The expressed recombinant protein was purified by 6 × His-tag purification kit,refolded by urea concentration density dialysis,then determined for purity by SDS-PAGE,for concentration by BCA method,and for reactogenicity by Western blot.The purified recombinant F1-F / M2 protein was emulsified with aluminum adjuvant,and immunized i.p.to BALB / c mice for 3 times each at an interval of 10 d.Neutralizing antibody levels against RSV in the sera,lung tissue homogenate and nasal washing of mice were determined by plaque reduction assay 10 d after the last immunization.Spleens of mice were collected,from which lymphocytes were isolated and determined for IFNγ level by ELISAPOT assay.Results Restriction analysis and sequencing proved that recombinant plasmid pEGX-6p-1-F1-F / M2 was constructed correctly.The expressed recombinant F1-F / M2 protein,with a relative molecular mass of about 20 800,contained about20% of total somatic protein,almost all of which were in forms of inclusion bodies.The purified recombinant protein reached a purity of about 90% and a concentration of 0.42 mg / ml,and showed specific reaction with mouse antisera against RSV.Recombinant F1-F / M2 protein induced high neutralizing antibody titer(1 ∶ 289)in sera of immunized mice,while low neutralizing antibody titer(1 ∶ 242)in lung tissue homogenate and no neutralizing antibody titer in nasal washing.The splenic lymphocytes of mice immunized with recombinant F1-F / M2 formed specific IFNγ spots after stimulation with antigen in vitro.Conclusion Recombinant RSV epitope protein F1-F / M2 was successfully expressed in prokaryotic cells.Purified F1-F / M2 protein induced high neutralizing antibody titer and CTL response in mice and might be developed as a RSV subunit vaccine.更多还原