中国人兽共患病学报2025，Vol.41Issue(10)：1025-1033,9.DOI:10.3969/j.issn.1002-2694.2025.00.164
结核分枝杆菌Rv3621c基因编码蛋白PPE65克隆表达及其对BEAS-2B细胞增殖和TGF-β表达水平的影响
Cloning and expression of PPE65 encoded by the Mycobacterium tuberculosis Rv3621c gene in Escherichia coli,and its effects on proliferation and TGF-β expression of BEAS-2B cells
摘要
Abstract
This study was aimed at preliminarily investigating the molecular biological functions of the PPE65 protein from Myco-bacterium tuberculosis,and providing foundational data for tuberculosis prevention and control.The basic biological properties of the PPE65 gene-encoded protein were predicted with bioinformatics tools.Sequence information for the Mycobacterium tuberculosis Rv3621c gene and PPE65 protein was retrieved from the NCBI database.The Rv3621c gene was amplified through PCR with the H37Rv genome as a template,then cloned into the pET22b(+)expression vector.The recombinant pET22b(+)-PPE65 plasmid was transformed into Escherichia coli BL21(DE3)competent cells for IPTG-induced expression.Solubility analysis,purification,and identification of the recombinant PPE65 protein were performed.BEAS-2B cells were treated with various concentrations of PPE65 protein for 24 h,and cell proliferation was assessed with CCK-8 assays.PPE65 was found to be composed of 413 amino acids and to have a molecular formula of C₁₈₁₉H₂₈₁₀N₄₈₂O₅₅₆S₁₁,a relative molecular mass of 40 679.88,a theoretical isoelectric point of 4.60,an ali-phatic index of 81.94,and an average hydrophilicity value of 0.319,thus indicating a stable hydrophobic protein lacking signal pep-tides or transmembrane domains.Secondary structure analysis revealed 53.03%α-helix(Hh),2.66%β-sheet(Ee),and 44.31%ran-dom coil(Cc).Bioinformatics predictions identified 38 B-cell epitopes and 22 CTL/Th-cell epitopes.The full-length PPE65 gene(1 308 bp)was confirmed through double restriction enzyme digestion and sequencing,thereby validating the correct construction of the pET22b(+)-PPE65 recombinant plasmid.SDS-PAGE analysis demonstrated that the recombinant protein was found in inclusion bodies,and a single band at 43.7 kDa was observed after purification.Western blotting revealed specific binding to mouse-derived His monoclonal antibodies,thereby confirming successful expression of the PPE65 protein.BEAS-2B cells treated with a PPE65 protein concentration gradient(2.5-20 µg/mL)exhibited a dose-dependent increase in cell number.Compared with those in the PBS control group,TGF-β relative expression levels were significantly higher in all treatment groups(t2.5=4.893,P<0.001,t5.0=4.640,P<0.05,t10=7.535,P<0.05,t20=16.44,P<0.000 1).This study elucidated the structural characteristics of the PPE65 protein,successfully obtained the recombinant protein through prokaryotic expression and purification,and demonstrated its ability to promote BEAS-2B cell proliferation.The underlying mechanism might involve suppression of TGF-β/S mad signaling pathway activation.These findings provide a theoretical basis for understanding the role and regulatory mechanisms of PPE65 during M.tuberculosis infection.关键词
Rv3621c基因/PPE65蛋白/结核分枝杆菌/生物信息学/原核表达/BEAS-2B细胞Key words
Rv3621c gene/PPE65 protein/Mycobacterium tuberculosis/bioinformatics/prokaryotic expression/BEAS-2B cells分类
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黄俊程,齐萌,薄新文,李静,陈旭珂,赵家欣,张艳艳,冯昕炜,孙艳,王正荣..结核分枝杆菌Rv3621c基因编码蛋白PPE65克隆表达及其对BEAS-2B细胞增殖和TGF-β表达水平的影响[J].中国人兽共患病学报,2025,41(10):1025-1033,9.基金项目
新疆生产建设兵团重点领域科技攻关计划项目(No.2020AB025) Xinjiang Production and Construction Corps Key Areas of Science and Technology Research Project(No.2020AB025) （No.2020AB025）
