파스튜렐라 (A:3) 외막 단백질 H의 유전자 클론닝·발현 및 면역혈청 생산
Molecular Cloning and Expression of a Gene for Outer Membrane Protein H in Pasteurella multocida (A:3) : Production of Antisera against the OmpH
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초록
Pasteurella. multocida 균은 광범위한 질병을 야기시키는 악성 감염원으로 알려져 있다. 그람 음성균의 동종 및이종간에 의한 감염에 대한 강력한 백신 후보 물질로써OmpH라고 불리는 porin 단백질이 고려되어 왔다. 이OmpH가 이번 연구에서 분리 및 정제되었다. 선도 단백질을 제거한 재조합 OmpH 단백질은 pRSET A 발현벡터를이용하여 40kDa로 발현되었으며, 친화성 크로마토그래피로 정제되었다. OmpH에 대한 면역혈청을 얻기 위해, 한마리의 실험용 쥐에 한번에 50ug 의 단백질을 복강 주사를 통해 2회에 걸쳐 주사했다. 항 OmpH 면역혈청의 역가는 ELISA로 측정되었다. 이번 실험에서 확인된 면역혈청의 증가는 OmpH 단백질이 병원성 파스튜렐라 균이 일으키는 가금 콜레라를 예방하기 위한 백신의 강력한 후보임을 보여주고 있다.
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/OmpHF2 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 SDSPAGE 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 5×10-2 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).
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