结核分枝杆菌Rv1787基因编码蛋白PPE25的原核表达及生物信息学分析

Prokaryotic expression and protein bioinformatic analysis of the Mycobacterium tuberculosis Rv1787 gene encoding the protein PPE25

目的构建结核分枝杆菌Rv1787基因的原核表达质粒进行体外表达,运用生物学信息分析方法分析Rv1787基因编码蛋白PPE25的生物学特征。 方法以结核分枝杆菌H37Rv基因组DNA为模板,PCR法扩增Rv1787基因,并与表达载体pET-32a构建重组质粒。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。利用生物信息学软件ProtParam分析PPE25蛋白的理化性质,TMPRED和SignalP4.1Service预测蛋白的跨膜区和信号肽,NPS@SOPMA和Swissmodel分别预测蛋白的二级结构和三级结构,Bepipred 1.0bserver和DNAStar综合预测蛋白的B细胞抗原表位,综合运用SYFPEITHI、BIMAS、NetCTL、RANKPEP、NetMHCⅡPAN 4.0server预测蛋白的CTL细胞表位,综合运用SYFPEITHI、RANKPEP、NetMHCⅡpan 3.2server预测蛋白的Th细胞表位。 结果pET-32a-Rv1787重组质粒测序结果与目的基因完全一致;SDS-PAGE分析表明,该融合蛋白以包涵体形式存在,相对分子质量为56×10^3,与预期大小相符。生物信息学分析结果显示,PPE25蛋白为疏水性蛋白,含多个跨膜结构域,无信号肽。二级结构主要由α-螺旋和无规则卷曲构成,结构比较松散。综合多种表位预测软件分析结果,筛选出3个优势B细胞抗原表位,7个CTL优势抗原表位和9个Th优势抗原表位。 结论成功构建结核分枝杆菌PPE25蛋白编码基因Rv1787重组原核表达质粒,并利用生物信息学筛选出多个优势抗原表位,为进一步研究PPE25蛋白在结核病发生发展中的作用奠定了基础。

Objective To express the Mycobacterium tuberculosis Rv1787gene encoding the protein PPE25 in vivo by constructing aprokaryotic expression plasmid and to analyze its biological characteristics using bioinformatics. Methods The Rv1787gene was amplified from the genomic DNA of M.tuberculosis strain H37Rv using PCR,and the recombinant plasmid was constructed with the expression vector pET-32a.The induced recombinant PPE25protein was purified using affinity chromatography and confirmed using SDS-PAGE.The physicochemical characteristics of PPE25were analyzed using ProtParam,the transmembrane region and signal peptides were predicted using TMPRED and SignalP4.1 Service,the secondary and tertiary structure were predicted using NPS@SOPMA and Swissmodel,B-cell epitopes were predicted using Bepipred 1.0bserver and DNAStar,TCL-cell epitopes were predicted using SYFPEITHI,BIMAS, NetCTL,RANKPEP,and NetMHC II PAN 4.0server,and Th-cell epitopes were predicted using SYFPEITHI, RANKPEP,and NetMHC II pan 3.2server. Results The results indicated that the recombinant plasmid was cloned and stably expressed,and the DNA sequences of the recombinant plasmid were completely consistent with the sequences in NCBI's GenBank according to BLAST alignment.The recombinant PPE25protein was detected using SDS-PAGE with a molecular weight of 56×10^3,which was consistent with expectations,and it existed in the form of an inclusion body. PPE25was a hydrophobic protein consisting of 365amino acids and including 6inside-out and 5out-inside transmembrane domains and no signal peptides.The secondary structure of PPE25mainly consisted ofα-helixes and random coils,and it was loosely structured.Eight B-cell epitopes in the PPE25protein were predicted using Bepipred 1.0bserver at amino acids 8-10,18-22,169-176,204-206,319-326,254-268,319-32,and 360-365,and five B-cell epitopes were predicted using the software DNAStar.Amino acids 8-13,170-175,and 360-364were screened as the predominant B-cell epitopes.Based on analysis with various pieces of software to predict epitopes,7CTL epitopes and 9Th epitopes were predicted to be predominant epitopes. Conclusion The recombinant prokaryotic expression plasmid Rv1787encoding the PPE25protein was successfully constructed,and several dominant epitopes were screened bioinformatically. These findings provide a basis for further research on PPE25.