| Objective:To construct a recombinant lentiviral vector containing human preproenkephalin (hPPE), transfect it into human embryonic kidney cells (HEK293) to obtain a cell line with efficient and stable expression of the target gene, and identify the hPPE gene expression, for the purpose of providing experimental materials for the follow-up studies on HEK293cells infected with human preproenkephalin gene used in biological analgesic research of rat model of cancer pains, and laying a foundation for the future experimental study of endogenous enkephalins in the treatment of chronic pains and cancer pains.Methods:The self-constructed recombinant plasmid pcDNA3.1/hPPE saved by our group was digested with restriction endonucleases Hind III and Not I, and hPPE gene fragment was obtained. Based on hPPE gene sequence described in GenBank database, both the upstream and downstream primers containing EcoR I restriction sites were designed. The target gene was amplified by PCR technology. The expression vector (pLV-UbC-GFP-3FLAG) was digested with EcoR I. The digestion product was recovered by agarose gel electrophoresis and a linear vector was obtained. The amplified gene fragment was ligated to the linear expression vector by using the gene recombinant technique. It was then transformed into E. coli competent cells. The transformant was identified, and the positive clones were inoculated. They were saved and sent to sequencing. Those with correct sequencing results were inoculated and the target gene-containing expression plasmid was extracted. The target gene-containing expression vector (pLV-UbC-hPPE-GFP), pCD packaging plasmid and pLTR-G membrane protein expression plasmid were co-transfected into HEK293cells, followed by lentivirus packaging, amplification and purification, and the recombinant lentiviral vector was then transfected into HEK293cells. The hPPE expression in HEK293cells was detected by Western blot.Results:1. Human preproenkephalin gene sequence was successfully amplified by PCR. The size of DNA product was838bp, with a clear and bright band visible in agarose gel electrophoresis. The purified target gene fragment was ligated to the linear expression vector, followed by transformation and colony PCR identification. The results of positive cloning sequencing were completely consistent with the Blastn results of hPPE sequence from GenBank database, indicating that the recombinant vector was successfully constructed.2. The expression vector of recombinant target gene and empty vector were co-transfected respectively with the pCD packaging plasmid and pLTR-G membrane protein expression plasmid into HEK cells. The next day after transfection, all cells were healthy and the densities were close to60%-80%. After packaging and purification of lentivirus, according to the virus titer formula, the title of lentivirus was3.39x108TU/ml.3. HEK293cells infected with UbC-hPPE-GFP-L.V. showed no GFP fluorescence under the fluorescence microscope. HEK293cells infected with the empty virus of Ubc-GFP-L. V. showed strong fluorescence. The infection rate was100%expressed as the positive rate of green fluorescent protein in the cells observed by fluorescence microscopy.4. The transfected HEK293cells samples and the fifth generation HEK293cells samples were detected by Western Blot. A positive band was visible at the place slightly larger than55KDa, whose size was consistent with the fusion protein hPPE-GFP-Flag (29KDa+28KDa+2KDa=59KDa), thereby it was concluded that hPPE was stably expressed in HEK293cells.Conclusion:A recombinant hPPE-containing lentiviral vector was successfully constructed, and the target gene hPPE was able to be stably expressed in HEK293cells, laying a foundation for further studies on enkephalin in the treatment of pains. |
