穿梭质粒pCDH1-GFP-HCN 4的构建及HCN 4重组慢病毒的包装  

Construction of the shuttle plasmid pCDH1-GFP-HCN 4 and packaging of HCN 4 recombined lentivirus

作　　者：周亚峰[1] 李红霞[1] 蔡思达[2] 程铖[1] 韩莲花[1] 赵欣[1] 杨向军[1] 

机构地区：[1]苏州大学附属第一医院心内科,江苏苏州215006 [2]南京大学生命科学学院,江苏南京210093

出　　处：《苏州大学学报（医学版）》2012年第3期354-358,437,共6页Suzhou University Journal of Medical Science

基　　金：国家自然科学基金资助项目(81170174;81070139);江苏省自然科学基金(BK2011304);江苏省"科教兴卫"医学重点人才项目(RC2011111)

摘　　要：目的将全长约3.6 kb的HCN 4目的基因片段从pCDNA3-HCN 4质粒卸载下来,建立稳定的HCN 4重组慢病毒。方法用Eco RⅠ和XbaⅠ从pCDNA3-HCN 4质粒切下HCN 4目的片段连接到Puc19载体中,再用EcoRⅠ和HindⅢ从质粒Puc19切下目的片段并连接到pcDNA3.1(A)载体中,再用XbaⅠ单酶从质粒pcDNA3.1(A)双切到pCDH1-MCS1-EF1-copGFP中。用XbaⅠ或者Eco RⅠ鉴定,将阳性克隆质粒送上海生工测序,慢病毒的包装参照SBI的Lentivector Expression System操作手册进行。将构建好的慢病毒包装质粒混合物混合后感染293T细胞及心肌样骨髓基质干细胞。结果 (1)XbaⅠ及Eco RⅠ鉴定表明,PCR产物和克隆扩增产物的电泳结果显示该基因的大小约为3.6 kb;阳性克隆质粒测序结果与GENEBANK目的基因HCN 4序列完全相同;构建的目的基因质粒转染293细胞和原代猪骨髓间充质干细胞后,均发现有强烈的绿色荧光出现。(2)构建的慢病毒包装质粒混合物转染293细胞后,发现有强烈的绿色荧光出现,细胞感染率达90%以上。结论 (1)成功将全长HCN 4目的基因片段从pCDNA3-HCN 4质粒卸载下来,成功构建穿梭质粒pCDH1-MCS1-EF1-copGFP。(2)成功建立稳定的HCN 4重组慢病毒,该病毒载体有很高的转染效率。Objective HCN 4 gene fragment is cloned from plasmid pCDNA3-HCN 4 and inserted into the shuttle plasmid pCDH1-GFP-HCN 4 and constructed HCN 4 recombined lentivirus. Methods HCN 4 gene fragment was cloned from plasmid pCDNA3-HCN 4 and inserted into plasmid Pucl9 by En- zymes Eco R ｜ and Xba I , Then the target gene was cutted and cloned into plasmid pcDNA3.1 （A） by Enzymes Eco R I and Hind m. In the end, HCN 4 gene fragment was cutted into plasmid pCDH1-MCS1 -EFI-copGFP by Enzymes Xba I ~ Double Enzymes Xba I and Eco R I verified approach and analysising the sequence were used to confirm the positive plasmid. HCN 4 recombined lentivirus was constructed ac- cording to operating manual of Lentivector Expression System. pPACKH1- Lentivector Packaging Kit with three plasmids was used to transfect 293 T cells. After 48 hours culturing, GFP （green） was detected bythe fluorescence microscope. Results Confirmed by enzymes Xba I and Eco R I HCN 4 gene fragment was cloned into the plasmid pCDH1-MCS1 -EFI-copGFP and the sequence in the positive plasmid was confirmed by comparison with the published gene bank. The recombined lentivirus was used to transfect 293 T cells and GFP was observed in the transfected 293 ce cieney of infection was above 90 percents. Conclusion cloned from plasmid pCDNA 3-HCN 4 and inserted into The recombined lentivirus was successfully constructed and lls under the fluorescent microscope. The effi- （1） HCN 4 gene fragment was successfully e shuttle plasmid pCDH1-GFP-HCN 4. （2） had high efficiency of infection.

关 键 词：HCN4基因 穿梭质粒 慢病毒 

分 类 号：R318[医药卫生—生物医学工程] R541-7205[医药卫生—基础医学]