一种CPPs介导的基因转化方法的建立及其应用  

Establishment of a CPPs-mediated gene transformation method and its application

作　　者：闫可心 闫宗斌 韩金成 薛书江[1] 于龙政[1] 宋建臣[1] 许应天[1] YAN Kexin;YAN Zongbin;HAN Jincheng;XUE Shujiang;YU Longzheng;SONG Jianchen;XU Ying-tian(Agricultural College,Yanbian University,Yanji Jilin 133002,China)

机构地区：[1]延边大学农学院,吉林延吉133002

出　　处：《中国兽医学报》2021年第11期2275-2279,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(32060807);吉林省教育厅基金资助项目(JJKH20200522KJ);高等学校学科创新引智计划基金资助项目(D20-034)。

摘　　要：为了弥补常规基因转化法存在的缺点,本研究采用琼脂糖凝胶电泳分析法探讨了细胞穿膜肽(cell penetrating peptides,CPPs)与DNA的最适比例,用转化率探讨了CPPs-DNA复合物的制备条件、保存条件和解冻条件等对CPPs基因转化的影响,再用CPPs基因转化法进行了牛瑟氏泰勒虫p33基因的克隆与原核表达,进一步验证CPPs基因转化的可行性。结果表明,CPPs与质粒pMD19-T最适电荷比为16∶1;CPPs-DNA复合物的最适制备条件为室温孵育30 min;将培养液D_(600)约为0.6的大肠杆菌菌液在-20℃冰箱保存3月的转化率为(5.58±0.19)×10^(5) CFU/μg,仍符合常规转化要求。按照所优化的条件成功地克隆与表达了牛瑟氏泰勒虫p33基因。本研究结果将为基因转化提供一种新方法。In order to compensate for the shortcomings of conventional gene transformation methods,the optimal ratio of cell penetrating peptides(CPPs)to DNA was explored by agarose gel electrophoresis,and the effects of preparation conditions,preservation conditions and thawing conditions of CPPs-DNA complex on CPPs gene transformation were determined by conversion rate.Then,the p33 gene of Theileriasergenti was cloned and expressed in prokaryotic cells by CPPs-mediated gene transformation method,which further verified the feasibility of CPPs-mediated gene transformation.The results showed that the optimum charge ratio between CPPs and plasmid pMD19-T was 16∶1.The optimum preparation condition of CPPs-DNA complex was incubation at room temperature for 30 min.The E.coli solution with D_(600) of about 0.6 was stored at-20℃for 3 months,and the conversion rate was(5.58±0.19)×10^(5)CFU/μg,it still met the requirements of conventional transformation.According to the optimized conditions,p33 gene of Theileriasergenti was successfully cloned and expressed.The results of this study will provide a new method for gene transformation.

关 键 词：细胞穿膜肽(CPPs) 基因转化 牛瑟氏泰勒虫 p33基因 

分 类 号：Q74[生物学—分子生物学]