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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。
Objective To construct a prokaryotic expression vector for truncated G protein of respiratory syncytial virus (RSV) and to detect the immunogenicity and immunological parameters of the expressed and purified protein. Methods The partial nucleotide sequences of G protein gene were synthesized. The CX3C motif was replaced by the CTL epitope of RSV M protein by overlapping PCR. The prokaryotic expression vectors GCX3C-pET22b and GCTL-pET22b were constructed and transformed into E. coli BL21 (DE3) , IPTG induced expression. After the expression product was identified by SDS-PAGE, the target protein was purified by Ni2 + affinity chromatography. The purified product was identified by SDS-PAGE and Western blot. Immunized Kunming mice were divided into three groups: GCX3C group (100μg GCX3C), GCTL group (100μg GCTL) and negative control group (100μl PBS) The mice were immunized with intramuscular injection of the double posterior muscle and blood was taken via the caudal vein to separate the serum. The level of IgG and the number of eosinophils were detected. The specific CTL response of RSV was identified by in vitro lymphocyte killing assay. Results The recombinant prokaryotic expression vector was confirmed by double enzyme digestion and sequencing. The recombinant protein was successfully transformed. The relative molecular mass of the recombinant protein was about 14 000 and the expression level was about 40% of the total bacterial protein. The recombinant protein was mainly expressed in soluble form. The purity of recombinant protein was above 90%, which could react specifically with anti-RSV serum. The level of specific IgG in serum increased with the increase of immunization times (P <0.01). The geometric mean of GCX3C protein and GCTL protein Degrees were 1 584.89 and 1 995.26, respectively. The number of eosinophils in GCX3C group was significantly higher than that in GCTL group (P <0.01). CTL-specific RSV response was induced by GCTL in mice. Conclusion GCX3C protein and GCTL protein have been successfully expressed in prokaryotic cells. Both proteins have good immunogenicity. GCTL protein can eliminate the eosinophilia induced by CX3C motif and enhance the CTL effect. The expression of G protein gene Transformation achieved the desired effect.