摘要
目的利用原核系统获得重组结核分枝杆菌Lsr2蛋白,制备抗Lsr2多克隆抗体。方法以临床标准株H37Rv DNA为模板,合成引物PCR扩增Lsr2核酸序列,插入原核表达载体pET28a,转化大肠杆菌Rosetta(DE3),经IPTG诱导表达,柱上复性纯化后,重组蛋白免疫新西兰大白兔,制备并纯化抗Lsr2多克隆抗体,采用间接ELISA和Western-blotting实验对抗体进行验证。结果成功构建pET28a-Lsr2表达质粒,重组蛋白Lsr2经SDS-PAGE电泳鉴定分子量为14 kD。亲和层析及柱上复性后,纯化的重组蛋白纯度在95%左右。制备的抗Lsr2多克隆抗体效价在1:5.5×10~6以上,并能特异性识别纯化的重组蛋白和结核分枝杆菌菌体蛋白。结论成功在原核系统内表达并纯化了重组结核分枝杆菌Lsr2蛋白,制备了高效价的抗Lsr2多克隆抗体,为进一步研究Lsr2蛋白的功能,筛选其下游相互作用蛋白以及相关分子机制研究奠定了基础。
Objective To obtain recombinant Mycobacterium tuberculosis(M. tuberculosis) protein Lsr2 in prokaryotic expression system, and to prepare its polyclonal antibody. Methods Primers of Lrs2 sequence were synthesized according to the sequence of the M. tuberculosis H37 Rv genome. Lrs2 was amplified by PCR and cloned into prokaryotic expression vector pET-28 a. The recombinant plasmid pET-28 a-Lrs2 was transformed into Escherichia coli Rosetta(DE3). The expression of the recombinant protein was induced with isopropyl β-D-thiogalactoside(IPTG), then renatured and purified by metal chelate chromatography. New Zealand rabbits were immunized to prepare polyclonal antibody against Lrs2. The antibody was identified by indirect enzyme-linked immunosorbent assay(ELISA) and Western blotting assay. Results pET-28 a-Lrs2 was constructed successfully. Recombinant Lrs2 protein had a relative molecular mass of about 14 kD according to sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). After affinity purification and on-column refolding, the purity of the recombinant protein reached about 95%. The titer of the prepared polyclonal antibody was more than 1∶5.5×10^6, and the antibody could recognize the purified recombinant Lrs2 protein or Lrs2 protein in M. tuberculosis H37 Rv lysate. Conclusion The recombinant M. tuberculosis protein Lrs2 is successfully expressed and purified in prokaryotic expression system, and anti-Lrs2 polyclonal antibody with high specificity is obtained, which lays a foundation for further studying the function of Lrs2, screening its interaction proteins and investigating its related molecular mechanisms.
作者
孙卫国
侯江厚
李改平
杨栗坤
孙雯娜
张灵霞
SUN Wei-guo;HOU Jiang-hou;LI Gai-ping;YANG Li-kun;SUN Wen-na;ZHANG Ling-xia(Army Tuberculosis Prevention and Control Key Laboratory,Beijing Key Laboratory of New Techniques of Tuberculosis Diagnosis and Treatment,Institute for Tuberculosis Research,the Eighth Medical Center of General Hospital of PLA,Beijing 100091,China;Maternal and Child Health Care Hospital of Kunming City,Kunming,Yunnan 650013,China;Department of Respiratory Medicine,the 309th Hospital of Chinese PLA,Beijing 100091,China)
出处
《实用预防医学》
CAS
2019年第4期400-403,共4页
Practical Preventive Medicine
基金
国家传染病重大项目(2013ZX-10003-006)
解放军第309医院面上课题(No.2016MS-001)
关键词
结核分枝杆菌
Lsr2蛋白
原核表达
多克隆抗体
Mycobacterium tuberculosis
Lrs2 protein
prokaryotic expression
polyclonal antibody