吉林农业大学学报2025，Vol.47Issue(1)：161-168,8.DOI:10.13327/j.jjlau.2021.1598
结核分枝杆菌Rv2529基因的原核表达及功能初步分析
Prokaryotic Expression and Function of Rv2529 Gene in Mycobacte-rium tuberculosis
摘要
Abstract
The Myc tag sequence was amplified using specific primers and inserted into the pMV261 plasmid by seamless cloning to construct pMV261-Myc recombinant plasmid.Using the genome of the standard strain of Mycobacterium tuberculosis H37Rv as a template,Rv2529 gene fragment was amplified by PCR,and recombinant plasmid was connected to electrotransform E.coli DH5α compe-tent.The correctly identified positive plasmids were extracted and transformed into Mycobacterium smegmatis to induce protein expression.The target protein was detected and analyzed by SDS-PAGE gel electrophoresis and Western Blot.The recombinant strains of Ms_Rv2529 and Ms_pMV261 were inoculated in 7H9 medium and cultured in a constant temperature shaker,and OD600 value was de-tected every 3 h to draw a growth curve.The recombinant strains were inoculated into RAW264.7,and the invasion rate and intracellular survival rate of the recombinant strains were calculated.Electro-phorese after digesting the positive plasmid by HindⅢ and Eco RⅠ,two bands of 4 488 and 1 428 bp were obtained,respectively,which were consistent with the expected size.The Rv2529 gene fragment was amplified by PCR and compared with the NCBI database after sequencing,and the results were consistent.The molecular weight of the expressed protein detected by SDS-PAGE was 50 kDa,a spe-cific positive signal was detected by Western Blot,and the band size was consistent with SDS-PAGE.The growth rate of the Ms_Rv2529 and Ms_pMV261 recombinant strains was not significantly differ-ent.The invasion rate and survival rate of the Ms_Rv2529 recombinant strain were significantly stron-ger than those of the Ms_pMV261 recombinant strain.This experiment successfully constructed a positive recombinant plasmid of the unknown gene Rv2529 of Mycobacterium tuberculosis,and ex-pressed the Rv2529 protein in Mycobacterium smegmatis.The expression of the Rv2529 protein in M.smegmatis enhances its ability to invade RAW and its anti-kill ability,indicating that the Rv2529 protein is related to the virulence of Mycobacterium tuberculosis,which will lay a foundation for fur-ther exploring its function and role in Mycobacterium tuberculosis.关键词
结核分枝杆菌/Rv2529基因/原核表达/RAW264.7细胞Key words
Mycobacterium tuberculosis/Rv2529 gene/prokaryotic expression/RAW264.7 cell分类
农业科技引用本文复制引用
吴虞,秦守涛,丛薇,张蕊,李东,徐金彪,时坤,李健明,曾范利..结核分枝杆菌Rv2529基因的原核表达及功能初步分析[J].吉林农业大学学报,2025,47(1):161-168,8.基金项目
吉林省科技发展计划重点研发项目(20210204038YY) （20210204038YY）
