Cloning and Characterization of cDNA for Korean Rockfish (Sebastes schlegeli ) Insulin-like Growth Factor-I

어류의 insulin-like growth factor-I (IGF-I)의 생화학적 작용기작을 연구하기 위하여 한국산 조피볼락의 IGF-I cDNA 유전자 cloning을 행하였다. 완전한 cDNA 유전자 염기서열은 PCR과 RACE 방법을 통하여 얻어진 DNA로부터 결과를 얻을수 있었다. 결정된 IGF-I의 염기서열은 flounder, chinook salmon, human IGF-I의 염기서열과 비교한 결과 각각 93.6%, 90.7%, 85.4%의 높은 상동성을 보였다. 생화학적으로 활성이 있는 재조합 IGF-I을 얻기 위하여 IGF-I의 B-C-A-D domain 부분을 PCR로 얻은 뒤 E. coli BL21(DE3)에 넣어 overexpression 시켰다. Ni-NTA colummn을 사용하여 순수한 재조합 단백질을 정제할수 있었다. 정제된 단백질은 SDS-PAGE 상에서 7 kDa의 단일 band를 보여 주었으며 [$^3H$]-thymidine 결합정도를 측정하는 방법으로 활성을 가지고 있음을 확인할수 있었다.

To understand the comprehensive mechanisms of biological function for insulin-like growth factor-I (IGF-I) in vertebrates, we have investigated the cDNA sequence of this gene in the korean rockfish (Sebastes schlegeli). The mature form of korean rockfish IGF-I was found to be comprised of 67 amino acid residues, showing about a 7 kDa molecular weight. In this study, we used the polymerase chain reaction (PCR) to obtain a korean rockfish IGF-I (KR IGF-I) cDNA fragment, and methods of rapid amplification of cDNA ends (RACE) to obtain a full length of the KR IGF-I sequence. The KR IGF-I encoded for a predicted amino acid sequence showed identities of 93.6 %, 90.7 %, and 85.4 % in comparison with flounder, chinook salmon, and human IGF-I, respectively. To obtain recombinant biologically active polypeptides, korean rockfish B-C-A-D domains were amplified using the PCR, then the isolated cDNA was expressed in the E. coli BL21(DE3). The recombinant KR IGF-I protein biological function was measured by stimulation of [$^3H$] thymidine incorporation, suggesting the cDNA codes for the korean rockfish proIGF-I.