Production of Theileria sergenti recombinant protein by E coli expression system

Park, Jin-ho; Chae, Joon-seok; Kim, Dae-hyuk; Jang, Yong-suk; Kwon, Oh-deong; Lee, Joo-mook

Korean Journal of Veterinary Research 1999;39(4):786-796.

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