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目的:在昆虫细胞中表达2型单纯疱疹病毒(HSV-2)糖蛋白D(gD2)胞外区基因,检测其免疫原性。方法:以HSV-2病毒基因组为模板,PCR扩增得到gD2片段,构建至杆状病毒质粒Bacmind中,转染sf9细胞,包装重组杆状病毒,收集后连续感染sf9细胞,收获第4代重组杆状病毒(P4),通过Western blotting法鉴定蛋白表达情况,采用空斑实验检测重组病毒滴度,将P4代重组病毒作为种子大量感染sf9细胞,上清经镍柱亲和层析进行纯化,将纯化后的蛋白在第0、2和4周免疫BALB/c小鼠(gD2组),以PBS作为阴性对照(PBS组),ELISA检测小鼠血清中gD2特异性IgG滴度。结果:PCR鉴定和DNA测序,重组表达质粒gD2-Bacmind构建正确,空斑实验检测重组病毒滴度为2.0×109 pfu·mL-1,纯化的gD2重组蛋白在相对分子质量37 000处出现目标条带,纯度达90%以上。第6周gD2组小鼠免疫血清中gD2特异性IgG抗体滴度Log10平均值达到4.34,与PBS组比较差异有统计学意义(P<0.01)。结论:利用昆虫-杆状病毒表达系统表达的gD2胞外区蛋白具有良好的免疫原性,可作为HSV-2疫苗的候选。
OBJECTIVE: To express the extracellular domain of glycoprotein D (gD2) of herpes simplex virus type 2 (HSV-2) in insect cells and test its immunogenicity. Methods: The fragment of gD2 was amplified by PCR from the HSV-2 virus genome. The recombinant plasmid was subcloned into Bacmind baculovirus and transfected into sf9 cells. The recombinant baculovirus was packaged and then infected into sf9 cells continuously to obtain the fourth generation recombinant Baculovirus (P4) was used to identify the protein expression by Western blotting. The plaque assay was used to detect the titer of the recombinant virus, and the P4 generation recombinant virus was used as a seed to infect sf9 cells. The supernatant was purified by nickel affinity chromatography. The purified protein was immunized BALB / c mice (gD2 group) at week 0, 2 and 4, and PBS as negative control group (PBS group). The serum gD2-specific IgG titer was detected by ELISA. Results: The recombinant plasmid gD2-Bacmind was constructed correctly by PCR and DNA sequencing. The titer of the recombinant virus was 2.0 × 109 pfu · mL-1 by plaque assay, and the target fragment of purified gD2 recombinant protein appeared at the relative molecular mass of 37 000 With purity of 90% or more. At the 6th week, the average Log10 value of gD2-specific IgG antibody in gD2 mice serum reached 4.34, which was significantly different from PBS group (P <0.01). CONCLUSION: The gD2 extracellular region expressed by the insect-baculovirus expression system has good immunogenicity and can be used as a candidate for HSV-2 vaccine.