Construction Of HPPE Eukaryotic Expression Vector PcDNA3.1(+)/hPPE And Expression Of HPPE In HEK293 Cell

Objective:The analgestic effect of human prepropenkephalin(hPPE) was clear, which was an endogenous opioid peptides.Compared to the exogenous opioid substances,human preproenkephalin had fewer side effects. It provided material basis for analgesia study. The purpose of this study was to obtain human preproenkephalin gene sequence, construct human preproenkephalin gene recombinant vector pcNDA3.1(+)/hPPE through genetic engineering techniques,and express human preproenkephalin in HEK293 cells by lipfectamineTM 2000. Human prepropenkephalin will provide tools for the late mouse model of metastatic bone cancer pian study.Methods:The total RNA was extracted from human brain tissue. The RNA was reversed into the cDNA via reverse transcriptase enzyme.The cDNA was complementary strand of the NDA. A couple of primers of human preproenkephalin were designed according to the Gene Bank. The cDNA was used to the template,and human preproenkephalin gene sequence is synthesized by PCR.Then it was cloned to pMD-18T that was a sequencing vector and sequenced.If human preproenkephalin gene sequence is correct, pMD-18T/hPPE and eukaryotic expression vector pcDNA3.1(+) were digested by restriction internally tangent enzymes HindⅢand NotⅠ.hPPE and pcDNA3.1(+) were linked by T4DNA linked enzyme.First the recombinant vector of pcDNA3.1(+)/hPPE was digested and identified by gel electrophoresis,then it was transformated into JM109 bacteria,last it was amplified and extracted.The recombinant vector pcDNA3.1(+)/hPPE was transfected into HEK293 cells by lipofectamineTM2000 of liposome transfectiom reagent. After 48 hours of transfection, the preproenkephalin gene was detected by RT-PCR. After 72 hours of transfection, the human preproenkephalin protein was detected by radioimmunoassay (RIA).Results:1 Human preproenkephalin gene sequence was successfully amplified by RT-PCR.Then it was observed about 800bp fragment by gel electrophresis.The fragment was consistent with the reported sequence in the literature.According to the sequence analysis,human preproenkephalin gene is homologous with the reported sequence in GeneBank.2 The recombinant vector pcDNA3.1(+)/hPPE was digested by restriction internally tangent enzymes HindⅢand NotⅠ.Then they were observed about 5400bp and 800bp gene fragments by gel electrophoresis.These fragments were consistent with the reported sequence in the literature.3 The recombinant vector pcDNA3.1(+)/hPPE was transfected into HEK293 cells.After 48 hours of transfection,collect the cell of pcDNA3.1(+)/hPPE transfection group,the cell of pcDNA3.1(+) transfection group and HEK293 cell group.β-action was used to the internal, human preproenkephalin gene of these three cell groups were deteced by RT-PCR.It was found that about 800bp human preproenkephalin gene fragment was observed in the cell group of pcDNA3.1(+)/hPPE transfection and human preproenkephalin gene fragment were not observed in the cell group of pcDNA3.1 (+) transfection and in the group of HEK293cells.4 The recombinant vector pcDNA3.1(+)/hPPE and eukaryotic expression vector pcDNA3.1(+) were transfected into HEK293 cells.After 72 hours of transfection,collect the cell supernatant of pcDNA3.1(+)/hPPE transfection group,the cell of pcDNA3.1(+) transfection group and HEK293 cell group. Human preproenkephalin of these three cell groups were deteced by radioimmunoassay.It was found that human preproenkephalin were not observed in the cell group of pcDNA3.1(+) transfection and in the group of HEK293cells,and human preproenkephalin was observed in the cell group of pcDNA3.1(+)/hPPE transfection. PG was used to express concentration of human preproenkephalin.Conclusion:Human preproenkephalin gene was successfully cloned.The recombinant expressing vector pcDNA3.1(+)/hPPE was successfully constructed.Human preproenkephalin was successfully expressed in the HEK293 cells. It may provide a useful tool for the late mouse model of metastatic bone cancer pian study.