论文部分内容阅读
目的在CHO细胞中表达单纯疱疹病毒(herpes simplex virus,HSV)Ⅱ型gC2蛋白,纯化后检测其免疫活性。方法利用ANTHEWIN等软件分析GenBank中HSVⅡ型gC2的氨基酸序列,筛选抗原表位富集、且亲水性较好的蛋白胞外区片段,选择真核和原核生物均偏爱的密码子,通过OptimumGene软件对基因进行序列优化,化学合成全新的基因序列HSV2-gC2,PCR扩增后,插入pMCE5载体,构建重组表达质粒pMCE5-gC2,转染至CHO细胞中进行真核表达。表达的重组gC2蛋白经亲和层析纯化后,进行SDS-PAGE及Western blot鉴定。将纯化的重组gC2蛋白分别于第0、2、4周经腹腔免疫小鼠,以免疫PBS作为对照,采用间接ELISA法检测免疫小鼠的血清抗体水平;酶联免疫斑点试验(ELISPOT)检测免疫小鼠脾淋巴细胞分泌IFNγ(代表Th1细胞因子)和IL-4(代表Th2细胞因子)的细胞频率。结果重组表达质粒pMCE5-gC2经菌落PCR、双酶切(EcoRⅠ/NotⅠ)及测序证实构建正确;纯化的重组gC2蛋白相对分子质量约为90 000,纯度约为85%,浓度为40μg/ml,可与小鼠抗His单克隆抗体特异性结合;通过3次免疫,小鼠产生了高水平的血清抗体,与免疫前相比,差异均有统计学意义(P<0.001);免疫小鼠脾细胞经特异性抗原刺激后,分泌IFNγ和IL-4的细胞频率分别为(13.46±2.832)/106和(19.2±2.48)/106个细胞,均显著高于对照组(P<0.001)。结论在CHO细胞中表达了重组gC2蛋白,纯化的蛋白能够激发小鼠产生良好的体液免疫和细胞免疫应答,为进一步研制HSV亚单位疫苗奠定了基础。
Objective To express gC2 protein of herpes simplex virus (HSV) type Ⅱ in CHO cells and detect its immunogenicity after purification. Methods The amino acid sequence of HSV Ⅱ gC2 gene in GenBank was analyzed by ANTHEWIN software and the extracellular region of protein was screened for antigenic epitopes with high hydrophilicity. The codons preferred by both eukaryotic and prokaryotic organisms were selected and optimized by OptimumGene software HSV2-gC2 was chemically synthesized and inserted into pMCE5 vector. The recombinant plasmid pMCE5-gC2 was constructed and transfected into CHO cells for eukaryotic expression. The expressed recombinant gC2 protein was purified by affinity chromatography and identified by SDS-PAGE and Western blot. The purified recombinant gC2 protein was intraperitoneally immunized at 0, 2 and 4 weeks respectively, and the immunized PBS was used as a control. The serum antibody level of the immunized mice was detected by indirect ELISA. The immune response was detected by ELISPOT Mouse splenic lymphocytes secrete cell frequencies of IFNy (representing Thl cytokines) and IL-4 (representing Th2 cytokines). Results The recombinant plasmid pMCE5-gC2 was confirmed by colony PCR, EcoRⅠ / NotⅠand sequencing. The relative molecular mass of purified recombinant gC2 protein was about 90 000 with a purity of about 85% at a concentration of 40 μg / ml. His-His monoclonal antibody could specifically bind to mouse. After three immunizations, the mice produced high-level serum antibodies, which were significantly different from those before immunization (P <0.001) The cell frequencies of IFNγ and IL-4 secreted by specific antigens were (13.46 ± 2.832) / 106 and (19.2 ± 2.48) / 106 cells, respectively, which were significantly higher than that of the control group (P <0.001). Conclusions Recombinant gC2 protein is expressed in CHO cells. The purified protein can stimulate mice to produce good humoral and cellular immune response, which lays a foundation for further development of HSV subunit vaccine.