African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12200

Full Length Research Paper

High-level expression, purification, polyclonal antibody preparation against recombinant OprD from Pseudomonas aeruginosa

Yong-Mei Han1, Xin-Hua Zhang1, Ya-Gang Chen2, You-Ming LI3, Hao Chen4*and Li-Zheng Fang1
  1Department of General Practice, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, China. 2State Key Laboratory of Infectious Disease Diagnosis and Treatment, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China. 3Department of Gastroenterology, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China. 4The Key Lab of Organ Transplantation of Public Health Ministry, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China.
Email: [email protected]

  •  Accepted: 31 January 2011
  •  Published: 14 February 2011

Abstract

 

OprD is a specific porin which can binds imipenem and carbapenems inPseudomonas aeruginosa. OprD loss plays a central role in mediating carbapenem resistance. Therefore, purification of oprD protein lays a pavement for the study in vivo and in vitro. In our study, the oprD gene was cloned into pQE30 expression vector, in frame with a sequence coding an N-terminal hexahistidine tag to allow purification by Ni2+ column. The recombinant OprD-6His was overproduced in inclusion form in Escherichia coli M15. OprD-6His was purified under denatured conditions using Ni-NTA conjugates. Antiserum against this recombinant OprD-6His protein was prepared in rabbit. Western blot analysis and enzyme linked immunosorbent assay (ELISA) were carried out to identify the reaction abilities and sensitivity of anti-OprD-6His polyclonal antibody to purified OprD-6His. Our results indicated that it was induction of E. coli M15 cells with 0.5 mM of isopropylthio-d-galactoside (IPTG) at 33°C for 4 h that the predicted 48 kDa OprD-6His fusion protein was expressed as the form of inclusion bodies with about 55-65 mg of OprD-6His per liter of culture. Western blot showed that recombinant OprD-6His protein could be identified by self-developed anti-OprD-6His polyclonal antibody. Anti-OprD-6His polyclonal antibody was detectable with 1000 times dilution of the original polyclonal antibody solution. In conclusion, higher level expression of OprD was available in the pQE30 expression system at optimal condition. Self-prepared polyclone antibody can effectively detect difference of porin expression in P. aeruginosa with higher sensitivity and specificity.

 

Key words: Pseudomonas aeroginosa; OprD; polyclonal antibody.

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