摘要
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 glycoprotein D of herpes simplex virus type 2 (gD2) in the insect cells, and to determine its immunogenicity. Methods: HSV-2 genome was used as the template for amplification of gD2 extracellular domain fragment gene by PCR. The PCR product was inserted into the vector Bacmind, and the constructed recombinant plasmid gD2-Bacmind was transfected into the sf9 cells to package the recombinant baculovirus. The Sf9 cells were infected by recombinant baculovirus seed derived from the forth passage (P4), the titer of P4 recombinant baculovirus was detected by a plaque assay and the expression of recombinant protein gD2 was determined by Western blotting method. The supernatant of infected cells was collected and purified by Ni-NTA affinity chromatography to obtain the target protein gD2, the purified gD2 protein was used to immunize the BALB/c mice in 0, 2, 4 weeks (gD2 group), and PBS was used as negative control (PBS group) ; the titers of gD2 specific IgG in serum were detected by ELISA assay. Results; The PCR analysis and sequencing results proved that gD2-Bacmind was constructed correctly. The titer of recombinant baculovirus was 2.0 ×10^9 pfu · mE^-1, the purified gD2 was about 37 000 with expectation, the percentage of gD2 in total protein was 90%. The average value of Log10 of titer of gD2 specific IgG in serum detected by ELISA assay in gD2 group at the sixth week was 4. 34, and there was significant difference compared with PBS group (P〈0.01). Oonclusion: The gDZ expressed by insect-baculovirus expression vector system has the immunogenicity and can be selected as candidate protein for HSV-2 vaccine.
出处
《吉林大学学报(医学版)》
CAS
CSCD
北大核心
2017年第4期720-724,共5页
Journal of Jilin University:Medicine Edition
基金
吉林省卫计委青年项目资助课题(2015Q042)
吉林省教育厅大学生创新创业训练计划项目资助课题(2016B326)