Korean Journal of Veterinary Research 1999;39(4):786-796.
Production of Theileria sergenti recombinant protein by E coli expression system
Jin-ho Park1, Joon-seok Chae2, Dae-hyuk Kim3, Yong-suk Jang3, Oh-deong Kwon1, Joo-mook Lee1
1College of Veterinary Medicine, Chonbuk National University
2School of Veterinary Medicine
3Faculty of Biological Sciences, Chonbuk National University
Abstract
As an attempt to develop an effective control method against theileriosis, recombinant antigen protein was produced. Thirty-two kDa membrane protein(MP) gene of T sergenti was amplified through RT-PCR from extracted total RNA of T sergenti isolated in Chonbuk, Korea. The amplified 869 bp of Korean T sergenti membrane gene was cloned and the base sequences were analyzed. The amplified gene was cloned into E coli expression vector, pQE32 plasmid vector, and the vector was introduced into E coli strain M15 to produce the recombinant membrane protein. For the induction of T sergenti membrane protein(KTs-MP), the plasmid harboring E coli strain M15 were cultured in the presence of IPTG, and the recombinant protein were purified by $Ni^+$-NTA agarose. Then, to confirm the authenticity of the produced membrane protein, molecular weight of expressed recombinant KTs-MP was analyzed by SDS-PAGE and Western blotting. The molecular weight of expressed recombinant protein was 32 kDa as expected. The recombinant KTs-MP was successfully recognized by anti-His Tag antibody, antisera of T sergenti infected cattle and monoclonal antibody of T sergenti membrane protein. Therefore, we concluded that the authentic 32 kDa membrane protein of T sergenti was produced as immunologically recognizable form.
Key Words: Theileria sergenti, recombinant protein, E coli expression vector


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