CAJ | 학술논문

建立了一种单细胞海水绿藻--杜氏盐藻(Dunaliella salina Teod.)的外源基因稳定表达系统.通过电激法将携带乙肝病毒表面抗原基因(HBsAg)和氯霉素乙酰转移酶基因(CAT)的质粒转入盐藻细胞内,CAT基因为筛选基因.PCR和Southern杂交结果显示,HBsAg基因已经整合到盐藻基因组中.Northern杂交结果表明,转化成功细胞内的该基因已转录成mRNA.HBsAgELISA和Western杂交检测证明,HBsAg蛋白在转化的盐藻细胞内稳定地表达.同时,PCR和Southern杂交显示,CAT基因也已整合到盐藻基因组中.且CATELISA检测证明,CAT蛋白在转化体中也已稳定地表达.进一步对转化盐藻进行无氯霉素筛选培养,60代后,HBsAg基因依然稳定地存在并表达.本实验第一次报道了外源基因在杜氏盐藻细胞内的稳定表达.
건립료일충단세포해수록조--두씨염조(Dunaliella salina Teod.)적외원기인은정표체계통.통과전격법장휴대을간병독표면항원기인(HBsAg)화록매소을선전이매기인(CAT)적질립전입염조세포내,CAT기인위사선기인.PCR화Southern잡교결과현시,HBsAg기인이경정합도염조기인조중.Northern잡교결과표명,전화성공세포내적해기인이전록성mRNA.HBsAgELISA화Western잡교검측증명,HBsAg단백재전화적염조세포내은정지표체.동시,PCR화Southern잡교현시,CAT기인야이정합도염조기인조중.차CATELISA검측증명,CAT단백재전화체중야이은정지표체.진일보대전화염조진행무록매소사선배양,60대후,HBsAg기인의연은정지존재병표체.본실험제일차보도료외원기인재두씨염조세포내적은정표체.
A stable transformation system for the expression of foreign genes in the unicellular green marine alga (Dunaliella salina Teod.) was established. Using electroporation, the alga was transformed with a plasmid containing the hepatitis B surface antigen (HBsAg) gene and the chloramphenicol acetyltransferase(CAT) gene as a selectable gene. PCR and Southern blotting analysis indicated that the HBsAggene was integrated into the D. Salina genome. Northern dotting analysis showed that the HBsAggene was expressed at the mRNA level. The stable expression of HBsAg protein in transformants was confirmed by HBsAg enzyme-linked immunosorbent assay (HBsAg ElISA) and Western blotting analysis. Aiso, PCR and Southern blotting analyses showed that the CA Tgene was integrated into the D. Salina genome, and CAT ELISA indicated that CAT protein was stably expressed in the cells. The introduced HBsAg DNA and HBsAg protein expression were stably maintained for at least 60 generations in media devoid of chloramphenicol.This is the first report of the stable expression of foreign genes in D. Salina.