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.
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