Molecular Cloning and Expression of a Gene for Outer Membrane Protein H in Pasteurella multocida (A:3) : Production of Antisera against the OmpH

파스튜렐라 (A:3)외막 단백질 H의 유전자 클론닝$\cdot$발현 및 면역혈청 생산

  • Kim Younghwan (Department of Cenetic Engineering, Sungkyunkwan University) ;
  • Hwang Heon (Department of Bio-Mechatronic Engineering, Sungkyunkwan University) ;
  • Lee Sukchan (Department of Cenetic Engineering, Sungkyunkwan University) ;
  • Park Eun-Seok (Department of Bio-Mechatronic Engineering, Sungkyunkwan University) ;
  • Yoo Sun-Dong (Department of Bio-Mechatronic Engineering, Sungkyunkwan University) ;
  • Lee Jeongmin (Department of Cenetic Engineering, Sungkyunkwan University) ;
  • Yang Joo-Sung (Department of Cenetic Engineering, Sungkyunkwan University) ;
  • Kwon MooSik (Department of Cenetic Engineering, Sungkyunkwan University)
  • 김영환 (성균관대학교 유전공학과) ;
  • 황헌 (성균관대학교 바이오 메카트로닉스과) ;
  • 이석찬 (성균관대학교 유전공학과) ;
  • 박은석 (성균관대학교 약학과) ;
  • 유선동 (성균관대학교 약학과) ;
  • 이정민 (성균관대학교 유전공학과) ;
  • 양주성 (성균관대학교 유전공학과) ;
  • 권무식 (성균관대학교 유전공학과)
  • Published : 2005.12.01

Abstract

Pasteurella multocida is known to cause widespread infections in husbandry. To induce homologous and heterologous immunity against the infections, outer membrane proteins (OMPs) in the envelope of P. multocida are thought to be attractive vaccine candidates. Outer membrane protein H is considered as the major component of OMPs. In this study, a gene for OmpH was isolated from pathogenic P. multocida serogroup A. The gene was composed of 1,047 nucleotides coding 348 amino acids with signal peptide of 20 amino acids. The amino acid composition showed about 80 to 98 per cent sequence homologies among other 10 strains of P. multocida serogroup A, reported so far. A recombinant ompH, from which signal peptide was truncated, was generated using pRSET A to name 'pRSET A/OmpH-F2'. The pRSET A/OmpH-F2 was well expressed in E. coli BL21(DE3). The truncated OmpH was purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. Its molecular weight was registered to be 40 kDa on SDS-PAGE gel. In order to generate immunesera against the OmpH, 50 ug of the protein was intraperitoneally injected into mice three times. The anti-OmpH immuneserum recognized about $5{\times}10^{-2}$ng quantity of the purified OmpH. It can be used for an effective vaccine production to prevent fowl cholera caused by pathogenic P. multocida (Serogroup A).

Keywords

Pasteurella multocida serogroup A;OmpH;outer membrane protein H;fowl cholera

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