title: [Prokaryotic expression and function detection of Mycobacterium tuberculosis Hsp16.3].
abstract: OBJECTIVE
  To construct a prokaryotic expression vector of Mycobacterium tuberculosis (MTB) small heat shock protein Hsp16.3, express and purify the fusion protein of His-Hsp16.3, and identify its function.
  
  
  METHODS
  Hsp16.3 gene was amplified from H37Rv DNA by PCR, and then cloned into prokaryotic expression vector Pet28a. The positive recombinant plasmid Pet28a-Hsp16.3 was selected and identified by double enzyme digestion and sequencing. Then the recombinant plasmid of correct sequence was transformed into E.coli BL21 (DE3), and induced by IPTG. The expressed recombinant protein was analyzed by SDS-PAGE and Western blotting; the fusion protein was purified by Hislink spin protein purification system, and then detected for its concentration; real-time quantitative PCR (qRT-PCR) was used to determine the expressions of IL-10 and IFN-γ in mouse peritoneal macrophages treated by the purified fusion protein of different concentrations. Meanwhile, the blank control group and positive control group were set up and compared.
  
  
  RESULTS
  Double enzyme digestion and sequencing showed that the recombinant plasmid Pet28a-Hsp16.3 was successfully constructed, and SDS-PAGE and Western blotting revealed that the fusion protein was expressed in E.coli BL21(DE3). Moreover, qRT-PCR showed that the purified fusion protein when added to the mouse peritoneal macrophage cells could significantly promote the produce of IFN-γ and restrain the expression of IL-10.
  
  
  CONCLUSIONS
  MTB Hsp16.3 was successfully cloned, expressed and purified. His-Hsp16.3 could significantly promote the produce of IFN-γ and restrain the expression of IL-10.
authors.affiliation: Department of Immunology, Immunology Postgraduate Education Innovation Base of Guizhou Province, Zunyi 563000, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Qin
authors.affiliation: Department of Immunology, Immunology Postgraduate Education Innovation Base of Guizhou Province, Zunyi 563000, China.
authors.forename: Shaoying
authors.initials: S
authors.lastname: Gao
authors.affiliation: Department of Laboratory Medicine, Zunyi Medical College, Zunyi 563000, China.
authors.forename: Jianbo
authors.initials: J
authors.lastname: Yuan
authors.affiliation: Department of Immunology, Immunology Postgraduate Education Innovation Base of Guizhou Province, Zunyi 563000, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Xu
authors.affiliation: Department of Immunology, Immunology Postgraduate Education Innovation Base of Guizhou Province, Zunyi 563000, China.
authors.forename: Junmin
authors.initials: J
authors.lastname: Luo
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D018833:Chaperonins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D007371:Interferon-gamma / Q000235:genetics; D016753:Interleukin-10 / Q000235:genetics; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D001426:Bacterial Proteins; C431133:Hsp16.3 protein, Mycobacterium tuberculosis; D011993:Recombinant Fusion Proteins; D016753:Interleukin-10; D007371:Interferon-gamma; D018833:Chaperonins