摘要
目的 将全长约3.6 kb的HCN 4目的基因片段从pCDNA3-HCN4质粒卸载下来,建立稳定的HCN4重组慢病毒.方法 用EcoR Ⅰ和Xba Ⅰ从pCDNA3-HCN4质粒切下HCN4目的片段连接到Puc19载体中,再用EcoRⅠ和Hind Ⅲ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 R Ⅰ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 R Ⅰ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pCDHI -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.
Abstract
Objective HCN 4 gene fragment is cloned from plasmid pCDNA3-HCN 4 and inserted into the shuttle plasmid pCDHl-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 Enzymes Eco R Ⅰ and Xba Ⅰ , Then the target gene was cutted and cloned into plasmid pcDNA3.1( A) by Enzymes Eco R Ⅰand Hind Ⅲ. In the end, HCN 4 gene fragment was cutted into plasmid pCDHl -MCS1 -EF1 -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 according to operating manual of Lentivector Expression System. pPACKHl - Lentivector Packaging Kit with three plasmids was used to transfect 293 T cells. After48 hours culturing, GFP (green) was detected by the fluorescence microscope. Results Confirmed by enzymes Xba Ⅰ and Eco R Ⅰ HCN 4 gene fragment was cloned into the plasmid pCDHl-MCSl -EFl-copGFP and the sequence in the positive plastnid 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 cells under the fluorescent microscope. The efficiency of infection was above 90 percents. Conclusion ( 1 ) HCN 4 gene fragment was successfully cloned from plasmid pCDNA 3-HCN 4 and inserted into the shuttle plasmid pCDHl-GFP-HCN 4. (2) The recombined lentivirus was successfully constructed and had high efficiency of infection.
基金项目
国家自然科学基金(81170174)
国家自然科学基金(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)
NETL
NSTL
万方数据