金黄色葡萄球菌IsdB表位蛋白与HBc蛋白融合表达

目的:采用大肠杆菌表达HBc-IsdB_50-285融合蛋白,探讨其在原核表达系统中的表达效果。方法:设计并克隆HBc-IsdB_50-285融合基因片段,构建表达质粒pET-28-HBc-IsdB_50-285,并转化入E.coli BL21,异丙基硫代半乳糖苷(IPTG)诱导蛋白表达。SDS-PAGE分析蛋白可溶性及相对分子质量;BCA法测蛋白浓度;采用免疫接种法检测重组蛋白的免疫活性。40只小鼠随机分为rIsdB+adjuvant组、HBc-IsdB_50-285+adjuvant组、HBc-IsdB_50-285组和PBS组,每组10只,间接ELISA法检测小鼠特异性IgG效价值,采用金黄色葡萄球菌Newman株对免疫后小鼠攻毒,检测各组小鼠重组蛋白免疫保护效果。结果:表达质粒pET-28-HBc-IsdB_50-285经测序及酶切鉴定证明构建正确;SDS-PAGE分析,重组蛋白以部分可溶形式存在于培养上清中,相对分子质量约为44 000,亲和纯化后蛋白质水平为1.0 g·L^-1。ELISA检测,使用rIsdB蛋白包被酶标板,HBc-IsdB_50-285+adjuvan组小鼠血清特异性抗体滴度的效价值低于rIsdB+adjuvant组(P<0.01);使用HBc-IsdB_50-285蛋白包被酶标板,HBc-IsdB_50-285+adjuvant组的小鼠血清特异性抗体滴度的效价值高于rIsdB+adjuvant和HBc-IsdB_50-285组。攻毒实验,HBc-IsdB_50-285+adjuvant组小鼠对金黄色葡萄球菌免疫保护率低于rIsdB+adjuvant组,但差异无统计学意义(P>0.05);且其与HBc-IsdB_50-285未添加佐剂组比较差异也无统计学意义(P>0.05);与PBS组比较,HBc-IsdB_50-285+adjuvant组小鼠对金黄色葡萄球免疫保护率升高(P<0.01),HBc-IsdB_50-285组小鼠对金黄色葡萄球菌攻毒保护率亦升高(P<0.05)。结论:HBc-IsdB_50-285原核表达载体构建成功;在大肠杆菌中成功表达具有较好生物活性的HBc-IsdB_50-285融合蛋白。

Fusion expression of Staphylococcus aureus IsdB epitope protein and HBc protein

Objective To construct the E.coli expressiom system of the HBc-IsdB_50-285 fusion protein, and to explore the expression efficacy of the HBc-IsdB_50-285 fusion protein in prokaryotic system.Methods HBc-IsdB_50-285 fusion gene fragment was designed and cloned,and the expression vector pET-28-HBc-IsdB_50-285 was construct and transformed into E.coli BL21,the expression of protein was inducted by IPTG. The solubility of protein and its relative molecular mass were analyzed by SDS-PAGE method; BCA assay was used to detect the level of protein; the immune activity of the recombinant protein was detected by immune inoculation method.40 mice were divided into rIsdB+adjuvant group(n=10), HBc-IsdB_50-285+adjuvant group(n=10), HBc-IsdB_50-285 group(n=10), and PBS group(n=10).The values of special IgG of the mice were tested by ELISA method. The mice were immunized and attacked by Stathylococcus aureus Newman strain,and the immune protection rate of the recombinant protein was detected.Results The expression vector pET-28-HBc-IsdB_50-285 was successfully constructed through identification of sequencing and restriction enzyme digestion. The SDS-PAGE results showed that the recombinant protein existed in a soluble form in the supernatant with relative molecular mass about 44 000.The level of purificated protein was 1.0 g·L^-1.The ELISA results showed that the value of antibody titer of the mice in HBc-IsdB_50-285+adjuvant group was lower than that in rIsdB+adjuvant group when the rIsdB were regarded as coating antigen(P<0.01);the values of antibody titer of the mice in HBc-IsdB_50-285+adjuvant group was higer than those in rIsdB+adjuvant group and HBc-IsdB_50-285 group when the HBc-IsdB_50-285 were regarded as coating antigen(P<0.05);the attack test results showed that the protection rate of Staphylococcus aureus of the mice in HBC-IsdB_50-285+adjuvant group was lower than that in rIsdB+adjuvant group (P>0.05). Compared with HBc-IsdB_50-285 group, the protection rate of Staphylococcus aureus of the mice in HBC-IsdB_50-285+adjuvant group had no significant difference (P>0.05). Compared with PBS group, the protection rates of Staphylococcus aureus of the mice in HBc-IsdB50-285+adjuvant group(P<0.01) and HBc-IsdB_50-285 group(P<0.05) were increased.Conclusion A prokaryotic expression vector of HBC-IsdB_50-285 is constructed, and the HBC-IsdB_50-285 fusion protein with better biological activities is expressed in E.coli BL21 successfully.