摘要
目的表达和纯化结核分枝杆菌休眠生存调节子蛋白Rv2628,制备并鉴定其兔多克隆抗体。方法以结核分枝杆菌H37Rv株基因组DNA为模板,扩增Rv2628基因,构建重组表达质粒,转化至大肠杆菌表达菌株中,经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,利用镍-次氮基三乙酸(Ni-NTA)层析柱纯化目的蛋白,以纯化Rv2628蛋白免疫新西兰大白兔获得兔多克隆抗体,分别利用Western blot法和间接ELISA验证多克隆抗体的特异性。结果成功构建pET-30a-Rv2628重组载体,IPTG诱导后Rv2628蛋白在大肠杆菌主要以包涵体形式表达,Ni-NTA层析柱纯化获得高纯度Rv2628蛋白。免疫兔后获得兔抗Rv2628多克隆抗体具有良好的抗原结合特性,且抗体效价达1∶1093500。结论成功制备高纯度Rv2628蛋白及高效价兔抗Rv2628多克隆抗体。
Objective To express and purify the dormancy survival regulator Rv2628 of Mycobacterium tuberculosis,so as to prepare and identify its rabbit polyclonal antibody.Methods Using the genome of Mycobacterium tuberculosis H37Rv strain as a template,the Rv2628 gene was amplified to construct a recombinant expression plasmid which was transformed into an Escherichia coli protein expression strain and induced expression by IPTG.The target protein was purified using Ni-NTA chromatography column,and the rabbit polyclonal antibody was obtained by immunizing New Zealand white rabbits with purified Rv2628 protein.The specificity of polyclonal antibodies was verified by Western blot analysis and indirect ELISA,respectively.Results The PET-30A-RV2628 recombinant carrier was successfully constructed.After induction by IPTG,the RV2628 protein was mainly expressed in the form of inclusion.The high-purity Rv2628 protein was obtained by Ni-NTA column purification.Rabbit anti-Rv2628 polyclonal antibody was obtained after immunizing the rabbit.The antibody had good antigen binding properties and the antibody titer reached 1∶1093500.Conclusion The high-purity Rv2628 protein and high-titer rabbit anti-Rv2628 polyclonal antibody were successfully prepared.
作者
汪欣然
马文潇
孙菀蔚
宁星语
黄蓉蓉
刘淼
WANG Xinran;MA Wenxiao;SUN Wanwei;NING Xingyu;HUANG Rongrong;LIU Miao(The Second Clinical Medical College,Anhui Medical University;The First Clinical Medical College,Anhui Medical University;Department of Microbiology and Parasitology,Anhui Provincial Laboratory of Microbiology and Parasitology,Anhui Key Laboratory of Zoonoses,School of Basic Medical Sciences,Anhui Medical University,Hefei 230032,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2023年第1期75-80,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
安徽高校省级科学研究重点项目(KJ2019A0223)
