Citation

Yang J, Lu L, Xu P, Liu H, Feng E, Wang H, Zhu L. Military Medical Sciences 2015; (12): 354-356,402.

Copyright

(Copyright © 2015)

DOI

10.7644/j.issn.1674-9960.2015.05.008

PMID

unavailable

Abstract

OBJECTIVE To achieve arabinose-controlled expression of HtrA strain and detect the expression of HtrA protein.

METHODS Arabinose promoter with htrA100 was amplified from pACD-htrA vector by PCR and cloned into pGP704 vector.Then, Shigella flexneri 2a strain 301 was transferred with the recombinant plasmid pGD-htrA and an AraC-expression vector.The expressions of HtrA in whole-cell and periplasmic space were detected by Western blotting.

RESULTS The suicide plasmid-mediated homologous recombinant vector and the inducible HtrA expression strain were successfully constructed.Without arabinose,HtrA protein was hardly detected ,but in the presense of arabinose , HtrA protein could be detected in whole-cell lysate and in periplasmic space lysate by Western blot.

CONCLUSION Homologous recombination using suicide plasmid can significantly knock down the expression of HtrA protein.After being induced with arabinose , HtrA protein can be expressed normally.

Language: zh

Keywords

Western blotting; arabinose operon; htrA gene; Shigella flexneri; suicide plasmid-mediated homologous recombination