[Construction of a secreting prokaryotic shuttle expressing plasmid pBCG-sP-HSP65 and its expression in Mycobacterial smegmatis].
To construct the secretive prokaryotic shuttle expression plasmid pBCG-SP-HSP65, the signal peptide sequence of antigen 85B amplified from Bacillus Calmette-guérin (BCG) genome by PCR and the whole HSP65 DNA sequence of human M. tuberculosis obtained from the plasmid pCMV-MTHSP65 by PCR were cloned into the plasmid pBCG-2100 under the control of the promoter of Heat Shock Protein 70 (HSP70) from human M. tuberculosis. Recombinants were electroporated into Mycobacterial smegmatis and induced by heating. Results of the induced expression were detected by SDS-PAGE and the biological activity of the expressed protein was tested by Western-blot analysis. Results showed pBCG-SP-MTHSP65 was constructed successfully and confirmed by restriction endonuclease analysis, PCR detection and DNA sequencing analysis. After it was electroporated into Mycobacterial smegmatis and induced by heating, the percentage of expressed 65kD protein in Mycobacterial smegmatis detected by SDS-PAGE was 20% in total bacterial protein. But the percentage of expressed 65kD protein in recombibinant Mycobacterial smegmatis was up to 34.46% in total bacterial protein and 68.56% in the total protein of cell lysate supernants, Which demonstrated the recombinant HSP65 gene could express in recombinant with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive proteins could specially combine with antibody against human M. tuberculosis HSP65. Orally, pBCG-SP-HSP65 was successfully constructed; HSP65 gene could express in Mycobacterial smegmatis with high efficiency via it. And the expressed proteins possess the biological activity. So it provids experimental evidence for the application of the recombinant Mycobacterial smegmatis and the development of the vaccine against tuberculosis.
Dai
Wu-Xing
WX
Department of Biochemistry and Molecular Biology of Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China. daiwuxing2002@sohu.com
Chen
Zhi-Hao
ZH
Gao
Hong
H
Huang
Hai-Lang
HL
Liang
Liang
L
Cheng
Ji-Zhong
JZ
Huangfu
Yong-Mu
YM
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D018834:Chaperonin 60; D018833:Chaperonins / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020102:Mycobacterium smegmatis / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
D001426:Bacterial Proteins; D018834:Chaperonin 60; D011993:Recombinant Fusion Proteins; C074119:heat-shock protein 65, Mycobacterium; D018833:Chaperonins