重组腺病毒载体Ad-EGFP-Klotho构建的实验研究  

Experimental study on construction of recombinant adenovirus vector Ad-EGFP-Klotho

作　　者：贾政 刘茜[2] 邢正江[1] 邹弘麟[1] 李亚雄[1] 魏玲 韦杰[1] 李春城[1] 孟凡棣 JIA Zheng;LIU Qian;XING Zhengjiang;ZOU Honglin;LI Yaxiong;WEI Ling;WEI Jie;LI Chuncheng;MENG Fandai(Department of Cardiac Vascular Surgery/Cardiovascular Surgery Research Institute of Yunnan Province,Yan′an Hospital,Kunming Medical University,Kunming,Yunnan 650051,China;Department of Geriatrics,Kunming Medical University Affiliated Yan′an Hospital,Kunming,Yunnan 650051,China;Staff Ward,the 920th Hospital of the Joint Service Support Force of Chinese People′s Liberation Army,Kunming,Yunnan 650032,China)

机构地区：[1]昆明医科大学附属延安医院心脏大血管外科/云南省心血管外科研究所,昆明650051 [2]昆明医科大学附属延安医院老年医学科,昆明650051 [3]中国人民解放军联勤保障部队第920医院干部病房,昆明650032

出　　处：《重庆医学》2019年第23期3970-3973,共4页Chongqing medicine

基　　金：云南省基础研究计划（昆医联合专项）（2018FE001-276）;云南省昆明市卫生科技人才培养千工程科研项目[2018SW（后备）06];昆明医科大学附属延安医院院内项目（yyky016-020）

摘　　要：目的 本研究旨在构建并制备增强型荧光蛋白(EGFP)基因和Klotho基因重组腺病毒载体AdEGFP-Klotho,并将其感染HEK293细胞,为基因治疗提供研究基础。方法 设计含有NheⅠ与NotⅠ双酶切位点的引物,应用PCR方法扩增Klotho基因,将其连接到EGFP标记的pDC316-mCMV穿梭质粒上,构建重组穿梭质粒pDC316-mCMV-EGFP-Klotho,利用Polyfectin脂质体将骨架质粒和重组穿梭质粒共转染HEK293细胞进行同源重组,得到重组腺病毒Ad-EGFP-Klotho,并包装扩增,测定病毒颗粒数及滴度。采用PCR方法对重组腺病毒载体Ad-EGFP-Klotho进行鉴定,并进行Klotho基因测序。结果 经PCR和NheⅠ、NotⅠ双酶切鉴定,重组腺病毒载体Ad-EGFP-Klotho中证实含有Klotho基因,测序结果和设计序列比对一致,重组腺病毒载体Ad-EGFP-Klotho构建成功。滴度为2.0×1010 Tu/mL,成功感染HEK293细胞,感染复数(MOI)=100,感染效率达91.75%,从Ad-EGFP-Klotho重组腺病毒载体中可以检测到3 045bp的条带,表明目的基因已成功整合在重组腺病毒载体Ad-EGFP-Klotho基因组中。结论 应用细胞内同源重组方法成功构建了含有EGFP和Klotho基因的重组腺病毒载体Ad-EGFP-Klotho,制备获得高滴度的病毒,可高效感染HEK293细胞并表达目的蛋白。Objective The aim of this study was to construct and prepare an enhanced fluorescent protein(EGFP)gene and Klotho gene recombinant adenovirus vector Ad-EGFP-Klotho,and infect it into HEK293 cells to provide research basis for gene therapy.Methods The primer containing NheⅠand NotⅠdouble restriction site was designed.The Klotho gene was amplified by PCR and ligated into the EGFP-labbledpDC316-mCMV shuttle plasmid to construct the recombinant shuttle plasmid pDC316-mCMV-EGFP-Klotho.The backbone plasmid and the recombinant shuttle plasmid were co-transfected into HEK293 cells by Polyfectin liposome for homologous recombination to obtain recombinant adenovirus Ad-EGFP-Klotho,which was packaged and amplified,and the number of virus particles and titer were determined.The recombinant adenovirus Ad-EGFP-Klotho was identified by PCR and the Klotho gene was sequenced.Results The recombinant adenovirus Ad-EGFP-Klotho was identified by PCR and NheⅠ+NotⅠdouble enzyme digestion,then the Klotho gene was confirmed.The sequencing result was consistent with the sequence of the designed fragment,and the recombinant adenovirus Ad-EGFP-Klotho was constructed successfully.The virus titer was 2.0×1010 Tu/mL.When the HEK293 cells were successfully infected,the MOI=100,the infection efficiency was 91.75%,and the 3045 bp band was detected from the recombinant adenovirus Ad-EGFP-Klotho,indicating that the target gene was successfully integrated into the viral genome of Ad-EGFP-Klotho.Conclusion The recombinant adenovirus Ad-EGFP-Klotho containing EGFP and Klotho gene was successfully constructed by intracellular homologous recombination method to prepare high titer virus,which can efficiently infect HEK293 cells and express the target protein.

关 键 词：KLOTHO基因 绿色荧光蛋白质类 腺病毒 人 杂合子 

分 类 号：Q782[生物学—分子生物学]