Ⅱ型单纯疱疹病毒糖蛋白D在CHO细胞中的表达及其免疫学特性

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。

Expression of glycoprotein of herpes simplex virus type 2 in CHO cells and its immulogical characters

Objective To express the glycoprotein D(gD)of herpes simplex virus type 2(HSV-2)in CHO cells and determine its immulogical characters.Methods HSV-2 was cultured in Vero cells,from which total DNA was extracted and used as a template for amplification of gD gene by PCR.The PCR product was inserted into vector pCMV-sport,and the constructed recombinant plasmid pCMV-gD was transfected to in COS-7 and CHO cells for expression.The expressed product was purified by Anti-flag M2-Agarose chromatography,then analyzed for purity by SDS-PAGE and HPLC,for reactogenicity by Western blot,for spectrum curve by full wavelength scanning,and for isoelectric point by isoelectric focusing electrophoresis.BALB / c mice were immunized with 20 and 40 μg of gD respectively,then determined for specific antibody level against HSV-2 gD in sera by ELISA,and for serum neutralizing antibody level by neutralization test.Results Restriction analysis and sequencing proved that recombinant plasmid PCMV-gD was constructed correctly.Both the transiently expressed product in COS-7 cells and stably expressed product in CHO cells were recognized by specific monoclonal antibody against HSV-2 gD,indicating high reactogenicity of expressed gD.The purified gD,with a relative molecular mass of about 55 000,reached a purity of 65.46% and maintained the reactogenicity of natural HSV-2 gD,of which the optimal ultraviolet adsorption wavelength and isoelectric point were 275.50 nm and 8.3 respectively.The specific antibody titers in sera of mice immunized with 20 and 40 μg of gD were 1 ∶ 125 000 and 1 ∶ 16 000,while the neutralizing antibody titers were 1 ∶ 17 and 1 ∶ 16,respectively,indicating that gD induced neutralizing antibody and high antibody titer against HSV-2 gD.Conclusion HSV-2 gD was stably expressed in CHO cells and showed high immunogenicity,which laid a foundation of development of recombinant vaccine.