结核分枝杆菌Rv3432c蛋白的原核表达与生物信息学分析

易海波; 高兴红; 罗果; 徐鹏; 王欢

doi:10.12182/20240360401

结核分枝杆菌Rv3432c蛋白的原核表达与生物信息学分析

Prokaryotic Expression and Bioinformatic Analysis of Rv3432c From Mycobacterium tuberculosis

摘要

摘要:
目的构建结核分枝杆菌（Mycobacterium tuberculosis, M.tb）Rv3432c基因的原核表达质粒并进行体外表达，运用生物学信息学软件分析蛋白Rv3432c特征，探讨抗M.tb新型药物靶点。
方法以灭活的结核分枝杆菌H37Rv基因组DNA为模板，PCR法扩增Rv3432c基因，并与表达载体pET-28a构建原核表达重组质粒。SDS-PAGE分析表达产物并利用亲和层析的方法进行纯化。采用Protparam、Pfam online tool、SOMPA、Protscale、TMHMM、Signalp 6.0、NetPhos3.1、SUMOsp 2.0、SWISS-MODEL等生物信息学软件分析蛋白Rv3432c的生物学特性。
结果 pET-28a-Rv3432c重组质粒测序结果与目的基因完全一致；SDS-PAGE分析表明，该融合蛋白以可溶性蛋白形式存在，相对分子质量约为55×10³，与预期大小相符。蛋白Rv3432c为亲水性蛋白（GRAVY值为－0.079）。蛋白Rv3432c为无跨膜结构域和信号肽的蛋白。Rv3432c二级结构主要由无规则卷曲（39.78%）和α-螺旋（39.57%）构成，结构比较松散。
结论成功构建M.tb蛋白Rv3432c的原核表达质粒，并利用生物信息学分析其结构，为进一步研究Rv3432c在结核病发生发展中的作用以及抗M.tb新型药物靶点奠定了基础。

Abstract:
Objective To express the protein enconded by the Rv3432c gene of Mycobacterium tuberculosis (M.tb) in vitro by prokaryotic expression, to analyze the structure of the Rv3432c protein by using bioinformatics software, and to explore for new drug targets against M.tb.
Methods The Rv3432c gene was amplified by PCR using the genomic DNA of the inactivated M.tb strain H37Rv as the template and a recombinant plasmid was constructed with the expression vector pET-28a. The expression products were analyzed by SDS-PAGE and purified using affinity chromatography. The biological properties of Rv3432c were analyzed with Protparam, the Pfam online tool, SOMPA, Protscale, TMHMM Signalp 6.0, NetPhos3.1, SUMOsp 2.0, and SWISS-MODEL.
Results pET-28a-Rv3432c recombinant plasmid sequencing results were fully consistent with those of the target gene. SDS-PAGE analysis showed that the fusion protein existed in the form of a soluble protein with a relative molecular mass of about 55×10³, which matched the expected size. ProtParam analysis showed that the Rv3432c protein was hydrophilic (showing a GRAVY value of －0.079). Rv3432c was a protein with no transmembrane structural domains or signal peptide. The secondary structure of Rv3432c mainly consisted of random coils (39.78%) and α-helix (39.57%) and was relatively loosely structured.
Conclusion We successfully constructed a prokaryotic expression plasmid of the Rv3432c protein and analyzed its structure using bioinformatics, laying the foundation for further research on the role of Rv3432c in the pathogenesis and progression of tuberculosis as well as the identification of new drug targets against M.tb.

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