摘要
目的克隆结核分枝杆菌(Mycobacterium tuberculosis,MTB)PE_PGRS35基因,构建pET28a-PE_PGRS35重组表达载体,异源表达并纯化MTB H37Rv的PE_PGRS35蛋白,经生物信息学分析后,探讨抗MTB新型靶点。方法PCR扩增PE_PGRS35编码基因,利用重组克隆技术,构建表达载体pET28a-PE_PGRS35,测序鉴定成功后转化至大肠埃希菌(E.coli)BL21(DE3)中,加入异丙基-β-D半乳糖苷(IPTG)进行诱导表达。用Ni柱亲和层析纯化PE_PGRS35蛋白,并对其进行生物信息学分析。结果经测序证明pET28a-PE_PGRS35表达载体构建正确,表达的PE_PGRS35蛋白相对分子质量约53000,主要以包涵体形式存在,纯度达90%。生物信息学分析表明,PE_PGRS35蛋白为酸性不稳定性蛋白,主要二级结构为β-折叠和无规则卷曲,无跨膜区,推测为膜外蛋白,有39个磷酸化位点和2个保守结构域,有Rv1769、PPE8、PPE64、PPE54、PPE24、PPE16、PPE35、PPE6、PPE28、PE2共10个蛋白与PE_PGRS35蛋白相互作用。结论成功获得高纯度的PE_PGRS35蛋白,为进一步开发抗MTB药物新型靶点提供了参考。
Objective To clone PE_PGRS35 gene of Mycobacterium tuberculosis(MTB),construct recombinant vector pET28a-PE_PGRS35,express and purify the PE_PGRS35 protein of MTB H37Rv heterologously,and explore a new target against MTB after bioinformatics analysis.Methods The PE_PGRS35 coding gene was amplified by PCR and used to construct the expression vector pET28a-PE_PGRS35 by recombinant cloning technology,which was transformed to E.coli BL21(DE3)after successful sequencing and induced by using IPTG.The obtained PE_PGRS35 protein was purified by Ni column affinity chromatography and analyzed by bioinformatics.Results The pET28a-PE_PGRS35 prokaryotic expression vector was constructed correctly as identified by sequencing.The PE_PGRS35 protein was mainly expressed in the form of inclusion bodies,with a relative molecular mass of about 53000 and a purity of 90%.Bioinformatics analysis showed that PE_PGRS35 protein was an acid-labile protein,with main secondary structure ofβ-sheet and random coil,and no transmembrane region,which was presumed to be an extramembrane protein with 39 phosphorylation sites and two conserved domains.Total 10 proteins,including Rv1769,PPE8,PPE64,PPE54,PPE24,PPE16,PPE35,PPE6,PPE28 and PE2,interacted with PE_PGRS35 protein.Conclusion PE_PGRS35 protein with high purity was successfully obtained,which provided a reference for the further development of new targets for drugs against MTB.
作者
袁美丽
王楚彤
李敏英
李柏青
许涛
汪洪涛
YUAN Mei-li;WANG Chu-tong;LI Min-ying;LI Bai-qing;XU Tao;WANG Hong-tao(Laboratory Medicine Experimental Center,Laboratory Medicine College,Bengbu Medical College,Bengbu 233000,Anhui Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2023年第1期32-38,共7页
Chinese Journal of Biologicals
基金
安徽省自然科学基金(1908085MH252,2008085QH405)。
作者简介
通信作者:汪洪涛,E⁃mail:hongtaowang@bbmc.edu.cn。
