重组GBR蛋白免疫动物的实验研究

Construction of expression vector containing S. mutans GBR gene and confirmation of the immunogenicity of the recombinant GBR protein

目的:构建变形链球菌葡糖基转移酶葡聚糖结合区段(GBR)基因的表达载体,探讨重组GBR蛋白的免疫原性。方法:通过PCR、T-A克隆等技术,构建GBR基因表达载体pTriEx-4-GBR,对所构建的表达载体进行DNA序列测定和酶切图谱分析;IPTG诱导pTriEx-4-GBR中目的基因在大肠杆菌JM109(DE3)表达,纯化、浓缩重组GBR蛋白;Balb/c小鼠随机分为2组:实验组用重组GBR蛋白经皮下注射免疫,对照组用生理盐水代替重组GBR蛋白;ELISA法检测小鼠血清抗GBR特异性抗体;用统计软件PEMS3.0对数据进行处理。结果:载有GBR目的基因的表达质粒pTriEx-4-GBR序列及读码框架正确;诱导表达获得的重组GBR蛋白浓度和纯度均较高;用该重组蛋白对小鼠作皮下接种免疫后,诱导产生血清抗GBR特异IgG显著应答(P<0.005)。结论:表达载体pTriEx-4-GBR构建成功,所获得的重组GBR蛋白能诱导小鼠特异免疫应答,为进行进一步相关实验研究奠定了基础。

PURPOSE： The purpose of this study is to construct the expressing plasmid vector containing the GBR gene of Streptococcus mutans and study the immunogenicity of recombinant GBR protein. METHODS： The GBR gene was cloned into the expression vector pTriEx-4 through gene cloning techniques by PCR,T-A clone and etc,the recombinant plasmid pTriEx-4-GBR was analyzed by DNA sequencing and endonuclearase digestion mapping. The recombinant GBR （rGBR） protein expression was induced with IPTG in E. coli JM109（DE3） which was transformed with plasmid pTriEx-4- GBR and then the rGBR protein was purified by affinity chromatography. Balb/c mice were randomly divided into two groups：mice of experimental group were inoculated subcutaneously with the rGBR protein and the rGBR protein was replaced for NS in the control group, then the anti-serum was evaluated by ELISA.The data were analysed by statistical software PEMS3.0. RESULTS： The DNA sequence and the reading frame of GBR gene in the reconstructed vector pTriEx-4-GBR was in corresponding with the initial design and the rGBR protein was purified. The level of specific anti- rGBR serum IgG in the experimental group was significantly higher than that in the control group （P 〈0.005）. CONCLUSION： The results showed that the expressing plasmid carrying the GBR gene was constructed successfully and the purified recombinant GBR protein can elicit specific murine system immune response, which is necessary for further experiments including construction of bivalent anti-caries DNA vaccine and studies both in vitro and in vivo. Supported by National Natural Science Foundation of China （Grant No.30371323,30171010）.