Construction and identification of recombinant retroviral vector with controllable human preproenkephalin gene / 中华麻醉学杂志

ABSTRACT

Objective To construct a recombinant retroviral vector with human preproenkephalin gene regulated by tetracycline. Methods Human preproenkephalin gene was amplificated by polymerase chain reaction (PCR) and was cloned into retrovirus tetracycline responsive plasmid. Then this recombinant plasmid and regulatory plasmid pRev Tet-On were transferred into packaging cell PT67 respectively. The transfected PT67 / Tet-On and PT 67 / TREhPPE cells were selected by the corresponding antibiotics and identified by RT-PCR. The selected cells were enriched and virus liter was assayed using NIH3T3. Results The restriction endonuclease digestion, PCR analysis and DNA sequencing confirmed that the recombinant RevTRE/hPPE vector was constructed successfully. The virus liter of PT 67/TREhPPE was 3.2 ? 103 CFU?ml-1 and the virus titer of PT67/Tet-On was 2.6?105 CFU?ml-1.Conclusion A recombinant retroviral vector with human preproenkephalin gene regulated by trtracycline and stable virus producing lines have been successfully constructed, providing a good basis for further research on regulated cell therapy of chronic pain.