摘要
目的原核表达、纯化结核杆菌热休克蛋白Acr2分子,并分析其免疫反应性。方法采用PCR法扩增结核杆菌Acr2基因,并构建重组表达质粒PET22b-Acr2,转化大肠埃希菌BL21(DE3)菌株,并用IPTG诱导Acr2蛋白表达。以及使用镍离子亲和层析柱纯化该表达产物,并通过Western blot检测其免疫反应性。结果 PCR、双酶切和测序结果均表明PET22b-Acr2重组质粒构建成功,其在大肠埃希菌中主要以包涵体蛋白形式进行表达。复性后的包涵体蛋白经镍离子亲和层析柱纯化后,可获得纯化的Acr2重组蛋白,该重组蛋白分子质量约为18.3ku,与结核分枝杆菌Acr2蛋白的理论预测值相符。经Western blot检测显示该重组蛋白能被结核病人血清特异性识别。结论原核表达质粒PET22b-Acr2被成功构建,以及Acr2重组蛋白被有效进行表达和纯化,从而为进一步研究其生物学功能奠定了基础。
Objective To prokaryotically express and purify the protein Acr2 of Mycobacterium tuberculosis and identify its immunoreactivity. Methods The Acr2 gene was amplified by PCR , and the recombinant plasmids PET22b - Acr2 was constructed and transformed into Escherichia coli BL21 （ DE3 ） cells. Expression of the recombi-nant plasmid was induced with IPTG. Then the expressed products were purified with Ni - NTA affinity chroma-tography column, and Western blotting was performed to determine the immunoreactivity of the recombinant pro-tein Acr2. Results PCR, double enzyme digestion and DNA sequencing confirmed that the recombinant plasmid PET22b - Acr2 was successfully constructed, and the target protein was primarily expressed in inclusion body in E. coli. Recombinant protein Acr2 was obtained by the renatured inclusion body that was purified by Ni - NTA affinity chromatography, with molecular weight of about 18. 3 ku, which was consistent with the theoretical molecular weight of M. tb Acr2 protein. Western blotting verification indicated that the recombinant protein was able to be recognized by specific serum antibodies from pulmonary tuberculosis patients. Conclusion Successful prokaryotic ex-pression and construction of PET22b - Acr2 plasmid as well as expression and purification of the recombinant Acr2 protein may lay a basis for further study on the biological function of Acr2 protein.
出处
《热带病与寄生虫学》
2017年第3期131-135,共5页
Journal of Tropical Diseases and Parasitology
基金
安徽省省级大学生创新训练项目(№:201610367024)
国家级大学生创新训练项目(№:201610367010)
安徽省高校自然科学研究重点项目(№:KJ2015A146)
关键词
结核分枝杆菌
热休克蛋白
Acr2
原核表达
纯化
Mycobacterium tuberculosis,Heat shock protein , Acr2 , Prokaryotic expression, Purification
