摘要
目的 克隆、表达和纯化结核分枝杆菌脯氨酸-脯氨酸-谷氨酸15(PPE15)蛋白,制备并鉴定PPE15蛋白兔多克隆抗体。方法 通过PCR从结核分枝杆菌H37Rv株基因组扩增PPE15基因,利用同源重组克隆技术构建原核表达组氨酸标签PPE15蛋白的pET28a-PPE15载体。将pET28a-PPE15转化至大肠杆菌BL21(DE3)中,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导PPE15蛋白表达,通过SDS-PAGE鉴定PPE15蛋白表达。用亲和层析法Ni-NTA柱纯化PPE15蛋白,用复性纯化后的PPE15蛋白免疫新西兰大白兔,制备和纯化多克隆抗体。Western blot法鉴定PPE15蛋白与人血清和多克隆抗体结合特异性,间接ELISA检测多克隆抗体效价。结果 成功构建pET28a-PPE15重组表达载体,PPE15蛋白主要以包涵体形式存在,纯化后在相对分子质量(M;)38 000位置处有单一条带。纯化后PPE15蛋白与结核病患者和多克隆抗体发生特异性反应,多克隆抗体效价达1∶1 300 480以上。结论 成功表达和纯化重组PPE15蛋白,并制备出高效价兔源性多克隆抗体,为进一步研究PPE15蛋白的功能提供了实验基础。
Objective To clone, express and purify PPE15 recombinant protein from Mycobacterium tuberculosis(H37Rv), as well as prepare and characterize its rabbit polyclonal antibody. Methods By The PPE15 gene was amplified from the genome of Mycobacterium tuberculosis H37Rv by PCR, and the His-tagged prokaryotic PPE15 prokaryotic expression plasmid pET28a-PPE15 was constructed by homologous recombination cloning technique, and transformed into E. coli BL21(DE3). PPE15 expression was induced by isopropyl-β-D-thiogalactopyranoside(IPTG). Recombinant PPE15 was identified by SDS-PAGE, and further purified by affinity chromatography with a Ni-NTA column. The renaturation purified PPE15 protein was used to immunize New-Zealand rabbit to prepare polyclonal antibodies. The antibody specificity was analyzed by Western blot analysis, and antibody titer was determined by indirect ELISA. Results Recombinant prokaryotic PPE15 protein was successfully expressed and purified with a molecular weight of 38 kDa. The purified PPE15 protein exhibited positive reaction with the serum of TB patients and the PPE15 protein, the titer of the polyclonal antibodies reaches more than 1∶1 300 480. ConclusionThe recombinant protein PPE15 was successfully expressed and purified, and high titer rabbit-derived polyclonal antibody was prepared which provided an experimental basis for further functional studies of PPE15 protein.
作者
许涛
李敏英
王楚彤
常先友
钱中清
李柏青
汪洪涛
XU Tao;LI Minying;WANG Chutong;CHANG Xianyou;QIAN Zhongqing;LI Baiqing;WANG Hongtao(Department of Clinical Laboratory,School of Laboratory Medicine,Bengbu 233030;Depart me nt of Immunology,Anhui Province Key Laboratory of Immunology in Chronic Diseases,Bengbu 233030;Research Center of Laboratory,Bengbu 233030;Department of Infectious Diseases,The Fifth People's Hospital of Bengbu City,Bengbu 233030,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2022年第1期78-83,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
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)
蚌埠医学院“512人才培育计划”项目(by51201309)。
