논문 상세보기
pp.123-136
흰쥐의 뇌로부터 읽는구조 전체를 포함하는 1.8kbp 크기인 IP_3K를 암호화 하는 IP_3KcDNA 유전자속의 NotⅠ부위 GCGGCCGC를 GCAGCCGC로 site-directed mutagenesis 하여 얻은 변이 IP_3KcDNA를 pSP72·Not2 운반체의 EcoR I 부위에 클로닝하여 증폭시키고, 유전자 재조합 과정을 거쳐서 포유동물의 발현 운반체인 pZIP·NeoSV(X)의 NotⅠ부위에 IP_3KcDNA를 서브클론하였다. 이것을 CCL39 hamster lung fibroblasts 세포에 transfection 하여 발현시키고, anti-IP_3K antibody를 사용하여 Western blot 법으로 효소량과 활성도를 각각 측정한 결과, 효소량은 5배, 효소의 활성도는 무려 16배로서 기대하였던 것보다 훨씬 발현효율이 높았다. 또한 흰쥐의 각 조직속에 IP_3K의 분포와 생화학적인 특성이 논의되었다.
IP_3KcDNA gene(1.8kbp) encoding rat brain IP_3K enzyme contained NotⅠrestric site in open reading frame. The NotⅠsequence, GCGGCCGC, was converted to GCAGCCGC by site-directed mutagenesis. The mutated IP_3KcDNA was digested with EcoRⅠand ligated with EcoR I-restricted pSP72·Not2 vector. The resulting pSP72·Not2 IP_3KcDNA was digested with the NotⅠrestriction enzyme and then subcloned into the Not I-degested pZIP·NeoSV(X) mammalian expression vector. The pZIP·NeoSV(X)-IP_3KcDNA was transfected into CCL39 hamster lung fibroblast cells. The efficiency of the expressed IP_3KcDNA gene was significantly higher than expected generally, not only a mean 5-fold increase in the amount of enzyme, but also 16-fold increase in enzyme activity from transfected CCL39 cells by the method of Western blot using anti-IP_3K antibodies. Both distribution of IP_3K in various rat tissues and biochemical properties were discussed.
