人偏肺病毒多表位抗原的构建与表达

Construction and expression of multi-epitope antigen of human metapneumovirus

目的构建含人偏肺病毒(h MPV)B细胞和T细胞表位的多表位抗原,并在原核系统表达,评价其免疫原性和免疫反应性。方法以人偏肺病毒NL/1/00不同蛋白为模板,采用在线生物信息学软件Bepipred、ABCpred、Bcepred、LEPS及LBTOPE预测B细胞表位,以Net MHCpan及Net MHC预测CTL细胞表位,以Net MHCⅡ预测Th细胞表位。综合各软件预测结果,确定候选B细胞及T细胞表位,各表位间加入间隔序列"GPGPG"和"KK",串联为多表位抗原基因mea,与p ET32a(+)连接后转化E.coli BL21(DE3)宿主菌,经含氨苄的LB平板培养基筛选、鉴定获得重组p ET32a(+)-mea,经IPTG诱导表达获得多表位抗原(MEA),以镍层析柱纯化,以Western blot鉴定表达蛋白,以MEA免疫小鼠,以ELISA法检测产生抗体效价,以间接免疫荧光法(IFA)检测抗体特异性。结果共预测筛选人偏肺病毒B细胞表位6个,CTL表位4个,Th细胞表位2个,串联为多表位抗原基因mea,经与p ET32a(+)重组后,转化E.coli BL21(DE3)宿主菌,经IPTG 30℃诱导5 h,上清及沉淀均有目的蛋白表达。上清经镍层析柱纯化后获得重组多表位抗原MEA,浓度为1 mg/ml。Western blot检测显示,表达的MEA可与抗His抗体结合。经MEA免疫的BALB/c小鼠的抗血清经ELISA检测,抗体效价达105以上;经间接免疫荧光检测,抗血清能与感染vero-E6细胞的h MPV结合。结论成功构建了h MPV多表位抗原基因,并在原核系统成功表达,获得多表位抗原MEA。该抗原具有很好的免疫原性和免疫反应性,并可与h MPV病毒发生结合,具有病毒特异性。

Objectives To construct multi-epitope antigen of human metapneumovirus（h MPV） including both B cell epitopes and T cell epitopes, and to analyze its immunogenicity and immunoreactivity. Methods Online software Bepipred, ABCpred,Bcepred, LEPS and LBTOPE were used to predict B cell epitopes, Net MHCpan and Net MHC were used to predict CTL epitopes, Net MHC II was used to predict Th cell epitopes. All these predicted epitopes were assembled in series as well as linker GPGPGP and KK were added between different epitope to generate multi-epitope antigen gene（mea）. Recombinant p ET32a（＋）-mea were constructed and transformed to E.coli BL21（DE3）. Multi-epitope antigen peptide（MEA） were inducible expressed by IPTG. MEA were purified by Ni-NTA agarose and identified by Western blot. Purified MEA was used to immune BALB/c mice, anti-MEA serum titre was tested by ELISA, indirect fluorescent assay（IFA） was used to evaluate antibody specificity. Results Six B cell epitopes, four CTL epitopes and two Th cell epitopes were selected to construct mea. Both supernatant and precipitate of host bacteria after inducing had anticipated protein MEA. After purification of supernatant, 1 mg/ml of MEA were obtained. MEA could react with anti-His antibody using Western blot analysis. BALB/c mice were immunonized with MEA three times, above 105 antibody titer were detected by ELISA. IFA showed that anti-MEA serum could react with h MPV which indicated the serum had h MPV-specificity. Conclusion h MPV mea are developed and expressed successfully.Obtained MEA shows good immunogenicity, immunoreactivity and good h MPV-specificity.