摘要
目的克隆Rv1040c结核分枝杆菌脯氨酸-谷氨酸8(PE8)基因、构建pET28a-PE8重组载体和表达纯化PE8蛋白,制备抗PE8多克隆抗体。方法利用重组克隆技术,将PE8基因克隆至原核表达载体pET28a,测序鉴定后,转化至大肠杆菌BL21(DE3),用0.5 mmol/L异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达、稀释复性与纯化。用纯化PE8蛋白免疫新西兰大白兔,制备多克隆抗体,间接ELISA和Western blot法进行效价及特异性鉴定。结果成功构建pET28a-PE8原核表达载体,ITPG诱导后PE8蛋白在大肠杆菌主要以包涵体的形式表达,复性后纯化PE8蛋白纯度达90%,纯化多克隆抗体效价为1∶430080以上,能与PE8蛋白发生特异性反应。结论大肠杆菌表达的PE8重组蛋白免疫新西兰大白兔获得高效价的多克隆抗体。
Objective To clone proline-glutamate 8(PE8)gene segment from Mycobacterium tuberculosis(H37Rv),construct the recombinant plasmid pET28a-PE8,express recombinant PE8 protein,and prepare its polyclonal antibody.Methods Using a standard homologous recombination cloning technology,we cloned the PE8 gene into the prokaryotic vector pET28a.After sequence confirmation,it was transformed into E.coli BL21(DE3)and treated with 0.5 mmol/L isopropyl-beta-D-thiogalactopyranoside(IPTG)to induce protein expression.We purified and renatured the recombinant PE8 protein,and immunized New Zealand rabbits to prepare the polyclonal antibody.Antibody titer was determined by indirect ELISA and the specificity was evaluated by Western blot analysis.Results The recombinant plasmid pET28a-PE8 was successfully constructed,and the PE8 protein was primarily expressed in an inclusion body in E.coli.After renaturation and purification,a purity of about 90%of the recombinant protein was achieved.The titer of the polyclonal antibody was higher than 1∶430080.The polyclonal antibody could specifically recognize the recombinant PE8 protein.Conclusion We have successfully expressed and purified recombinant PE8 protein,which can be further utilized to generate PE8 polyclonal antibody with acceptable titer and specificity.
作者
许涛
张丽
钱中清
李柏青
汪洪涛
XU Tao;ZHANG Li;QIAN Zhongqing;Li Baiqing;WANG Hongtao(Department of Clinical Laboratory,School of Laboratory Medicine,Bengbu Medical College,Bengbu 233030,China;Research Center of Laboratory Medicine,Bengbu Medical College,Bengbu 233030,China;Department of Immunology,Anhui Key Laboratory of Infection and Immunity,Bengbu Medical College,Bengbu 233030,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2020年第6期549-554,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
卫生部重大传染病专项(2012ZX10003006-004)
安徽省自然科学基金(1908085MH252,2008085QH405)
安徽省高校自然科学研究重点项目(KJ2018A0233)
2018年国家级大学生创新创业项目(201810367001)。
