霍乱弧菌溶血素共调节蛋白(Hcp)的原核表达、纯化及多克隆抗体制备  

Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of Hemolysin Co-Regulatory Protein(Hcp)from Vibrio Cholerae

作　　者：蔡远凤 贾城壹 王光丽 CAI Yuanfeng;JIA Chengyi;WANG Guangli(Department of Clinical Laboratory,Suiyang County Hospital of Traditional Chinese Medicine,Guizhou Suiyang 563359,China;Department of Blood Transfusion,the First People's Hospital of Zunyi,Guizhou Zunyi 563099,China;Department of Medical Laboratory,Affiliated Hospital of Zunyi Medical University,Guizhou Zunyi 563000,China)

机构地区：[1]绥阳县中医医院检验科,贵州绥阳563399 [2]遵义市第一人民医院输血科,贵州遵义563099 [3]遵义医科大学附属医院医学检验科,贵州遵义563000

出　　处：《现代检验医学杂志》2024年第5期189-192,204,共5页Journal of Modern Laboratory Medicine

基　　金：贵州省科技厅基础研究计划项目(NO:黔科合基础-ZK[2021]一般470)。

摘　　要：目的原核表达、纯化霍乱弧菌溶血素共调节蛋白(hemolysin coregulatory protein,Hcp),并制备其多克隆抗体。方法PCR扩增霍乱弧菌Hcp基因并克隆入pET28a载体中构建重组表达载体;将重组载体pET28a-hcp转化E.coil BL21(DE3)中,进行表达条件优化及表达形式鉴定。获取可溶性Hcp蛋白行Ni-NTA柱纯化,纯化的Hcp蛋白免疫BALB/c小鼠以制备多克隆抗体,并用间接酶联免疫吸附试验(ELISA)检测抗体效价,以评估其免疫原性。再以Western blot法分析抗体对霍乱弧菌Hcp蛋白的特异性识别。结果重组载体pET28a-hcp的酶切片段与预期相符,测序结果与GenBank数据库中hcp基因序列一致,成功构建pET28a-hcp重组质粒,重组质粒经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达相对分子量为28kD的目的蛋白;经Ni-NTA柱纯化后获得较纯的Hcp蛋白,免疫小鼠可获得效价为1∶512000的抗Hcp多克隆抗体(anti-Hcp);Western blot鉴定结果显示anti-Hcp具有识别霍乱弧菌Hcp蛋白的特异性。结论成功获得可溶形式表达的Hcp蛋白,免疫小鼠后获得高效价的抗Hcp多克隆抗体,为后续研究Hcp蛋白在非O1/非O139群霍乱弧菌T6SS致病过程中的作用奠定了基础。Objective To explore prokaryotic expression,purification of hemolysin coregulatory protein(Hcp)of Vibrio cholerae,and preparation of its polyclonal antibodies.Methods PCR was used to amplify Vibrio cholerae Hcp gene and clone it into pET28a vector to construct recombinant expression vector.The recombinant vector pET28a-hcp was transformed into E.coil BL21(DE3)for expression condition optimization and expression form identification.The soluble Hcp protein was purified by Ni-NTA column.The purified Hcp protein was used to immunize BALB/c mice to prepare polyclonal antibodies.The antibody titer was detected by indirect enzyme-linked immunosorbent assay(ELISA)to evaluate its immunogenicity.Western blot was used to analyze the specific recognition of antibodies to Hcp protein in Vibrio cholerae.Results The enzyme fragment digested by recombinant vector pET28a-hcp was consistent with the expected,the sequencing results were consistent with the Hcp gene sequence in the GenBank database,and the pET28a-hcp recombinant plasmid was successfully constructed.The recombinant plasmid was induced by isopropyl-β-D-thiogalactopyranoside(IPTG)to express the target protein with a relative molecular weight of 28 kD.The pure Hcp protein was obtained after purification by Ni-NTA column,and then Hcp polyclonal antibody(anti-Hcp)with a titer of 1∶512000 could be obtained from immunized mice.Western blot results showed that anti-Hcp had specificity in recognizing Hcp protein in Vibrio cholerae.Conclusion The soluble expression of Hcp protein is successfully obtained,and high-titer polyclonal antibodies against Hcp are obtained after immunization of mice,which may lay a foundation for subsequent studies on the role of Hcp protein in the pathogenesis of T6SS in non-O1/non-O139 V.cholerae.

关 键 词：非O1/非O139群霍乱弧菌 溶血素共调节蛋白 原核表达 多克隆抗体 

分 类 号：R378.3[医药卫生—病原生物学] R392-33[医药卫生—基础医学]