单纯疱疹病毒Ⅱ型gC2蛋白在CHO细胞中的表达、纯化及其免疫活性

Expression of gC2 of herpes simplex virus type Ⅱ in CHO cells and purification and immune activity of expressed product

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 the extracellular domain fragment of gC2 of herpes simplex virus （HSV gC2） typeⅡ in CHO cells, purify the expressed product and determine its immune activity. Methods The amino acid sequence of HSV type Ⅱ gC2 in GenBank was analyzed by ANTHEWIN software, from which the epitope-rich extracelluar fragments with good hydropathy were screened, optimized by OptimumGene software using prokaryotic cells- and eukaryotic cells- preferred codons, synthesized chemically, amplified by PCR and cloned into eukaryotic expression vector pMCE5. The constructed recombinant plasmid pMCES-gC2 was transfected into CHO cells. The expressed protein gC2 was purified by affinity chromatography, and identified by SDS-PAGE and Western blot. Mice were immunized i. p. with the purified recombinant gC2 protein at weeks 0, 2 and 4, using PBS as control, and determined for serum antibody level by indirect ELISA, for frequencies of splenic lymphocytes secreting IFNγ （Thl） and IL-4 （Th2）. Results Recombinant plasmid pMCE5-gC2 was constructed correctly as proved by PCR, restriction analysis （EeoR Ⅰ /Not Ⅰ ） and sequencing. Thepurified gC2 protein, with a relative molecular mass of about 90 000, reached a purity of about 85% and a concentration of 40 μg/ml, which showed specific binding to mouse monoelonal antibody against His. High serum antibody level was induced in mice after immunization for 3 times, which showed significant difference with that before immunization （P 〈 0. 001 ）. The frequencies of mouse splenic lymphocytes secreting IFNγ and IL-4 after stimulation with specific antigen were （13.46 ± 2. 83 ）/10^6 ceils and （19. 20 ±2. 48）/10^6 cells respectively, both of which were significantly higher than those in control group （P 〈 0. 001 ）. Conclusion Recombinant gC2 protein was expressed in CHO cells, which induced good humoral and cellular immune responses in mice after purification. It laid a foundation of further preparation of HSV subunit vaccine.