人canstatin基因转化新型生物反应器——杜氏盐藻(Dunaliella salina)的初步研究

Preliminary Study on Transformation of Human canstatin Gene to the Novel Bioreactor——Dunaliella salina

采用RT-PCR技术从人的胎盘组织中克隆canstatin基因,定向连接到表达载体pUΩ上,然后与筛选标记bar盒连接得到真核表达载体pU Ω-Can-Bar。采用玻璃珠转化法将该表达载体转化杜氏盐藻(以下简称盐藻),通过草丁膦固体平板筛选得到转化株,进而对转化株进行阳性鉴定。PCR结果显示,在盐藻转化株中均能够扩增出约700 bp特异的条带,而在阴性对照中没有扩增出该条带。Southern blot结果进一步证明人canstatin基因已经整合到盐藻细胞的基因组中。此外,对盐藻转化株的遗传稳定性进行了分析,结果表明canstatin基因能够在转化藻株中稳定遗传。人canstatin转基因盐藻株的成功制备为利用盐藻反应器大规模生产人canstatin蛋白提供了实验依据,为及早实现canstatin蛋白在治疗肿瘤上的临床应用提供了前期工作基础。

The human canstatin cDNA was amplified by RT-PCR and then directionally cloned into pUΩ expression vector. The recombinant pUΩ-Can vector was connected with the screening marker （ bar box）, to construct a eukaryotic expression vector called pUΩ-Can-Bar. This expression vector was introduced into the D. salina by glass beads method. The screening culture of transformants of D. salina was performed in solid media containing 5 μg/ml PPT, and the analyses of transformants were carried out through PCR and Southern blot. PCR results revealed that specific 700 bp products were detected in the different transformants of D. salina but not in negative control. Southern blot analysis further demonstrated that human canstatin gene was integrated into the D. salina genome. Moreover, the results of genetic stability analyses of transformants demonstrated that canstatin gene was stably inherited in the D. salina transformants. The successful preparation of the D. salina transformants will provide the experimentation evidence for producing canstatin protein cosmically by using the D. salina bioreactor and give a better prophase work basis for clinic application of canstatin protein early.