呼吸道合胞病毒截短G蛋白的原核表达、纯化及其免疫原性

目的构建呼吸道合胞病毒(Respiratory syncytial virus,RSV)截短G蛋白的原核表达载体,并对表达、纯化后的蛋白进行免疫原性及相关免疫指标的检测。方法人工合成G蛋白基因的部分核酸序列,采用重叠PCR法将CX3C模序替换为RSV M蛋白上的CTL表位,构建原核表达载体GCX3C-pET22b和GCTL-pET22b,转化E.coli BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE鉴定后,采用Ni2+亲和层析柱纯化目的蛋白。纯化产物经SDS-PAGE及Western blot鉴定后,免疫昆明小鼠,实验分GCX3C组(100μg GCX3C)、GCTL组(100μg GCTL)、阴性对照组(100μlPBS),分别于0、1、4周经小鼠双后侧肌肉注射免疫,经尾梢静脉取血,分离血清,检测IgG水平及嗜酸性粒细胞的数量,并通过小鼠体外淋巴细胞杀伤试验鉴定RSV特异性CTL应答。结果重组原核表达载体经双酶切及测序鉴定,证明G蛋白基因改造成功;重组蛋白的相对分子质量约14 000,表达量约占菌体总蛋白的40%,主要以可溶性形式表达;纯化的重组蛋白纯度达90%以上,可与抗RSV血清发生特异性反应;小鼠血清中的特异性IgG水平随免疫次数的增加而升高(P<0.01),GCX3C蛋白和GCTL蛋白的几何平均滴度分别为1 584.89和1 995.26;GCX3C组较GCTL组小鼠血清中嗜酸性粒细胞数量明显增加(P<0.01);GCTL蛋白免疫小鼠后可产生RSV特异性的CTL应答效应。结论已成功原核表达了GCX3C蛋白和GCTL蛋白,两种蛋白均有较好的免疫原性,GCTL蛋白可消除由CX3C模序引起的嗜酸性粒细胞增多,并增强了CTL效应,G蛋白基因的改造达到了预期效果。

Prokaryotic expression,purification and immunogenicity of truncated G protein of respiratory syncytial virus

Objective To construct a prokaryotic expression vector for truncated G protein of respiratory syncytial virus(RSV),and determine the immunogenicity of expressed and purified protein.Methods Overlapping PCR was used to substitute the CX3C motif nucleotides in G protein partly synthesized with one of CTL epitope nucleotides from M protein of RSV.Recombinant plasmids GCX3C / pET22b and GCTL / pET22b were constructed and transformed into E.coli BL21(DE3) for expression under induction of IPTG.The expressed product was identified by SDS-PAGE,and purified by nickel ion affinity chromatography.The purified protein was identified by SDS-PAGE and Western blot.Kunming mice were divided into three groups,and immunized with 100 μg GCX3C,100 μg GCTL and 100 μl PBS(negative control) respectively.Each mouse was injected i.m.for 3 times,at weeks 0,1 and 4 respectively.Venous blood samples were collected at weeks 1 ~ 5,once a week,and determined for IgG level and counted for eosinophils.RSV-specific CTL response was determined by in vitro lymphocyte killing test in mice.Results Restriction analysis and sequencing proved that the G gene in recombinant prokaryotic expression vector was successfully modified.Recombinant protein,with a relative molecular mass of about 14 000,contained about 40% of total somatic protein and mainly existed in soluble form.Purified recombinant protein reached a purity of more than 90%,and showed specific reaction with RSV antiserum.The specific IgG level in sera of mice increased with the increasing doses(P < 0.01).The GMTs of IgG of mice in GCX3C and GCTL groups were 1584.89 and 1995.26 respectively,while the eosinophil count of the former was significantly higher than that of the latter(P < 0.01).GCTL induced RSV-specific CTL response in mice.Conclusion GCX3C and GCTL proteins were successfully expressed in prokaryotic cells,both of which showed good immunogenicity.GCTL eliminated the increase of eosinophils caused by CX3C motif and enhanced the CTL response.Expected results was observed after modification of G gene.