重组金黄色葡萄球菌肠毒素B的原核表达、纯化及其免疫原性  

Prokaryotic expression,purification and immunogenicity of recombinant staphylococcal enterotoxin B

作　　者：韩慧 史伯昌 李欣昱 李华斌 田崇瑜 闫芳[3] 石艳春[1] 郑源强[1] 赵忠鹏 HAN Hui;SHI Bo-chang;LI Xin-yu;LI Hua-bin;TIAN Chong-yu;YAN Fang;SHI Yan-chun;ZHENG Yuan-qiang;ZHAO Zhong-peng(Inner Mongolia Key Laboratory of Molecular Biology,Inner Mongolia Medical University,Hohot 010058,InerMongolia Autonomous Region,China;不详)

机构地区：[1]内蒙古医科大学内蒙古自治区分子生物学重点实验室,内蒙古呼和浩特010058 [2]安徽医科大学基础医学院,安徽合肥230032 [3]山西农业大学动物医学学院,山西晋中030800

出　　处：《中国生物制品学杂志》2022年第9期1065-1068,1075,共5页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2019SWAQ05-3-5,2019YFC1200604);国家自然科学基金(81871252,81660272);内蒙古自治区自然科学基金(2017ZD08)。

摘　　要：目的对金黄色葡萄球菌肠毒素B(staphylococcal enterotoxin B,SEB)基因定点突变体进行克隆、表达及纯化,并检测其免疫原性,为研制马抗SEB免疫球蛋白F(ab′)2制品奠定基础。方法对SEB基因序列进行定点突变和修饰以及偏性密码子优化后,构建重组表达质粒pET-22b-SEB,转化E.coli BL21(DE3),IPTG诱导表达重组SEB蛋白,经离子交换层析纯化后,HPLC法检测重组SEB蛋白抗原纯度。Western blot法检测重组SEB蛋白抗原免疫活性;将不同浓度重组SEB蛋白抗原腹腔注射BALB/c小鼠,进行致病力、抗体效价、中和抗体活性检测。结果质粒pET-22b-SEB经双酶切及测序鉴定证明构建正确。纯化的重组SEB蛋白相对分子质量约28300,纯度大于90%,与天然SEB具有一致的免疫活性,且具有良好的安全性;免疫小鼠抗体效价达1∶81920,可有效诱导小鼠产生中和抗体。结论成功对SEB进行了定点突变减毒,经克隆、表达及纯化获得高纯度的重组SEB蛋白抗原,具有良好的免疫原性。Objective To clone,express and purify the site-specific mutant of staphylococcal enterotoxin B(SEB)gene and determine its immunogenicity so as to lay a foundation of development of horse anti-SEB immunoglobulin F(ab′)products.Methods Recombinant expression plasmid pET-22b-SEB was constructed after site-specific mutagenesis,modification and optimization of E.coli-prefered codon of SEB gene sequence,transformed to E.coli BL21(DE3)and induced with IPTG.The expressed recombinant SEB protein was purified by ion-exchange chromatography,and determined for purity by HPLC and for immune activity by Western blot.BALB/c mice were injected i.p.with recombinant SEB protein antigen at various concentrations,and determined for pathogenicity,antibody titer and neutralizing antibody activity.Results Recombinant plasmid pET-22b-SEB was constructed correctly as proved by restriction analysis and sequencing.Purified recombinant SEB protein,with a relative molecular mass of about 28300 and a purity of more than90%,showed immune activity consistent with that of natural SEB and good safety.The antibody titer of immunized mice was 1∶81920,indicating that neutralizing antibody was effectively induced in mice.Conclusion SEB was successfully attenuated by site-specific mutagenesis.The recombinant SEB protein antigen with high purity was obtained by cloning,expression and purification,which showed good immunogenicity.

关 键 词：金黄色葡萄球菌肠毒素B 重组抗原 表达 纯化 免疫原性 

分 类 号：R392-33[医药卫生—免疫学]