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
10.3969/j.issn.1002-2694.2025.00.164
- VernacularTitle: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
- Author:
Juncheng HUANG
1
;
Xinwen BO
;
Jing LI
;
Xuke CHEN
;
Jiaxin ZHAO
;
Yanyan ZHANG
;
Xinwei FENG
;
Yan SUN
;
Zhengrong WANG
;
Meng QI
Author Information
1. 塔里木大学动物科学与技术学院/农业农村部环塔里木畜草资源利用重点实验室,阿拉尔 843300;新疆农垦科学院畜牧兽医研究所/省部共建绵羊遗传改良与健康养殖国家重点实验室,石河子 832000
- Publication Type:Journal Article
- Keywords:
Rv3621c gene;
PPE65 protein;
Mycobacterium tuberculosis;
bioinformatics;
prokaryotic expression;
BEAS-2B cells
- From:
Chinese Journal of Zoonoses
2025;41(10):1025-1033
- CountryChina
- Language:Chinese
-
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 C????H????N???O???S??,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.
