摘要
目的异源表达并纯化结核分枝杆菌(Mycobacterium tuberculosis,Mtb)H37Rv的Rv1872c蛋白,探讨抗Mtb药物新型靶点。方法PCR扩增Rv1872c编码基因,构建融合表达载体pET-Rv1872c,并转化至大肠埃希菌(E.coli)BL21(DE3)中,加入异丙基硫代-β-D-半乳糖苷(IPTG)进行诱导表达。用Co2+亲和层析纯化Rv1872c融合蛋白,并对其进行生物信息学分析。结果经SDS-PAGE和Western blot分析,证明Rv1872c融合蛋白在E.coli中完全以包涵体形式表达,其亚基相对分子质量约46200,且在变性条件下,经亲和层析获得高纯度的Rv1872c融合蛋白。生物信息学分析表明,Rv1872c为不稳定的碱性蛋白,主要二级结构元件为α-螺旋和无规则卷曲,无跨膜区,推测为外周膜蛋白,且为潜在的醌依赖型L-乳酸脱氢酶。结论通过原核表达和亲和层析,成功获得了高纯度的Rv1872c融合蛋白,为进一步功能鉴定和开发抗Mtb药物新型靶点提供了参考。
Objective To express and purify the protein Rv1872 c of Mycobacterium tuberculosis(Mtb)H37Rv strain and investigate the new target of anti-tuberculosis drugs.Methods The gene encoding Rv1872c was amplified by PCR and inserted into plasmid pET-28b(+).The constructed fusion expression vector pET-Rv1872c was transformed into competent E.coli BL21(DE3)cells and induced with IPTG.The expressed Rv1872c fusion protein was purified by cobalt ionimmobilized affinity chromatography and analyzed for bioinformatics.Results SDS-PAGE and Western blot showed that Rv1872c fusion protein,with a relative molecular mass of about 46200,was expressed in E.coli in a form of inclusion body.Under denaturing condition,highly purified Rv1872c fusion protein was obtained by affinity chromatography.Bioinformatic analysis indicated Rv1872c as an unstable basic protein of which the major secondary structural elements wereα-helix and random coli.Without transmembrane region,Rv1872c was predicted to be a peripheral membrane protein and a potential quinone-dependent L-lactate dehydrogenase.Conclusion Pure Rv1872c fusion protein was obtained by prokaryotic expression followed by affinity chromatography,which provides a reference for further functional characterization of Rv1872c and the development of new anti-Mtb drug target.
作者
唐王刚
司雨
张娜
连超群
TANG Wang-gang;SI Yu;ZHANG Na;LIAN Chao-qun(Department of Laboratory Medicine,Bengbu Medical College,Bengbu 233030,Anhui Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
2020年第10期1117-1121,1127,共6页
Chinese Journal of Biologicals
基金
安徽省教育厅重点项目(KJ2018A0221)
蚌埠医学院科技发展基金项目(BYKF17117)
国家级大学生创新训练项目(201810367011)。