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目的在CHO细胞中表达Ⅱ型单纯疱疹病毒(Herpes simplex virus-2,HSV-2)糖蛋白D(Glycoprotein D,gD),并检测其免疫学特性。方法采用Vero细胞培养HSV-2,提取总DNA,以其为模板,PCR扩增gD基因,与pCMV-sport载体连接,构建重组表达质粒pCMV-gD,将质粒pCMV-gD转染COS-7和CHO-K1细胞,并进行表达。亲和胶Anti-flag M2 Agarose纯化表达蛋白,经SDS-PAGE和HPLC分析重组蛋白纯度,Western blot分析蛋白反应原性,全波长扫描分析蛋白的光谱曲线,等电聚焦电泳测定蛋白的等电点。以20、40μg纯化的gD免疫BALB/c小鼠,ELISA法检测小鼠血清中HSV-2 gD特异性抗体水平,中和试验测定小鼠血清中和抗体水平。结果酶切鉴定及DNA测序表明,重组表达质粒pCMV-gD构建正确,在COS-7细胞的瞬时表达产物和CHO细胞中的稳定表达产物均可被HSV-2 gD单抗特异性识别,表明该蛋白具有较好的反应原性。纯化的gD在相对分子质量约5 500处可见目的蛋白条带,纯度为65.46%;保留天然HSV-2 gD的反应原性,最适紫外吸收波长为275.50 nm,等电点为8.3。gD 20μg组和40μg组小鼠血清特异性抗体滴度分别为1∶125 000和1∶16 000,中和抗体滴度分别为1∶17和1∶16,表明gD可诱导中和抗体的产生,也可诱导高滴度的HSV-2gD特异性抗体。结论成功在CHO细胞中稳定表达了HSV-2 gD,表达的HSV-2 gD具有较好的免疫原性,为基因工程疫苗的开发奠定了基础。
Objective To express glycoprotein D (gD) of herpes simplex virus-2 (HSV-2) in CHO cells and test its immunological properties. Methods Vero cells were used to culture HSV-2 and the total DNA was extracted. The gD gene was amplified by PCR and ligated with pCMV-sport vector to construct recombinant plasmid pCMV-gD. The plasmid pCMV-gD was transfected into COS-7 and CHO-K1 cells and expressed. The protein was purified by affinity-flag M2 Agarose affinity chromatography. The purity of the recombinant protein was analyzed by SDS-PAGE and HPLC. The protein reactivity was analyzed by Western blot. The spectral curve was analyzed by full wavelength scanning. The isoelectric focusing . BALB / c mice were immunized with 20 and 40μg of purified gD. The serum levels of HSV-2 gD-specific antibodies were detected by ELISA. The neutralization test was used to determine the serum levels of neutralizing antibodies in mice. Results Restriction endonuclease digestion and DNA sequencing showed that the recombinant plasmid pCMV-gD was correctly constructed, and the transient expression products of COS-7 cells and the stable expression products of CHO cells could be specifically recognized by HSV-2 gD monoclonal antibody, The protein has good reactionality. Purified gD showed a molecular weight of about 5 500 with a purity of 65.46%. The reactivity of native HSV-2 gD was retained. The optimum UV-vis absorption was 275.50 nm and the isoelectric point was 8.3. Serum specific antibody titers of gD 20μg and 40μg mice were 1:125 000 and 1:16 000, respectively, and neutralizing antibody titers were 1:17 and 1:16, respectively, indicating that gD induces the production of neutralizing antibodies , Can also induce high titers of HSV-2gD-specific antibodies. Conclusion HSV-2 gD is successfully expressed in CHO cells. The expressed HSV-2 gD has good immunogenicity, which lays the foundation for the development of genetically engineered vaccine.