金黄色葡萄球菌FnbpA免疫优势片段的构建及免疫保护性研究  

Study on construction of Staphylococcus aureus FnbpA immunodominant fragment and its protective rate

作　　者：杨汐静 陈晓婷 刘道龙[2] 杨阳[1] 杨光[1] YANG Xijing;CHEN Xiaoting;LIU Daolong;YANG Yang;YANG Guang(Animal Experiment Center,West China Hospital,Sichuan University,Chengdu,Sichuan 610041,China;College of Life Sciences,Heilongjiang Bayi Agricultural University,Daqing,Heilongjiang 163319,China)

机构地区：[1]四川大学华西医院动物实验中心,成都610041 [2]黑龙江八一农垦大学生命学院,黑龙江大庆163319

出　　处：《重庆医学》2020年第5期813-818,共6页Chongqing medicine

摘　　要：目的构建金黄色葡萄球菌FnbpA蛋白免疫优势片段原核表达菌株并研究其重组蛋白免疫原性。方法根据预测确定N区优势片段,通过聚合酶链反应(PCR)法对目的片段进行基因扩增,转入表达载体pET-32a,并导入宿主菌大肠杆菌(E.coli)Rosstta中进行原核表达;将N区优势片段与重复区优势片段通过重叠延伸聚合酶链反应(overlap PCR)融合,原核表达;将蛋白FL纯化,分别免疫Balb/c小鼠,并检测小鼠IgG水平、γ干扰素(IFN-γ)、白细胞介素-4(IL-4)、免疫保护率。结果各重组蛋白在E.coli Rosstta中均获得表达,表达后FnbpA 301-430aa、FL蛋白大小分别约39.3×103,49.1×103;抗体水平检测结果表明重组蛋白FL抗体水平接近全蛋白FnbpA,高于FnbpA 301-430aa;重组FL组淋巴细胞分泌IL-4、IFN-γ、IL-17的能力与FnbpA 301-430aa比较差异有统计学意义(P<0.05);攻毒结果表明,全蛋白FnbpA实验组免疫保护性最好,FL实验组保护性效果优于FnbpA 301-430aa。结论成功构建了金黄色葡萄球菌FnbpA免疫优势截短体蛋白的原核表达菌株,重组蛋白FL具有良好的免疫保护性。Objective To construct the prokaryotic expression strain of FnbpA protein immuno-dominant fragment of Staphylococcus aureus,and to research the its recombinant protein immunogenicity.Methods The dominant segments of the N region was determined by the prediction.The target fragment was amplified by PCR,then transferred into the expression vector pET-32a,and transformed into E.coli Rosstta for conducting the prokaryotic expression.The dominant fragment of N region was fused with dominant fragment of repetition region by overlap PCR,prokaryotic expression was performed.The fusion protein was purified to immunize balb/c mice.The levels of serum IgG,IFN-γand IL-17,and immune protection rate were detected.Results Each recombinant protein was expressed in E.coli Rosstta,and the relative molecular weight of the FnbpA 301-430aa,FL protein was about 39.3×103,49.1×103.The results of the antibody level showed that the recombinant protein FL level was similar to whole protein FnbpA,which was higher than that of FnbpA 301-430aa.The ability of lymphocyte for secreting IL-4,INF-γand IL-17 had statistical difference between the recombinant FL group and FnbpA 301-430aa group(P<0.05).The challenge results showed that the whole protein FnbpA experimental group had the best immunoprotective effect,and the protective effect of the FL experimental group was better than that of the FnbpA 301-430aa group.Conclusion The prokaryotic expression strain of the S.aureus FnbpA immunodominant truncated fusion protein is successfully constructed.The recombinant protein FL has a good immunoprotective effect.

关 键 词：金黄色葡萄球菌 FnbpA 重组蛋白 免疫保护作用 

分 类 号：R392[医药卫生—免疫学]