The Expression Of Pegfp-c3-penk In Nih3t3 Cell And The Detection Of Activity Of The Expressed Protein

Objective:Currently the most popular method for the treatment of pain is the opiod analgesics.But it has many side effects,such as over-calmness,breath-inhibition, constipation and addiction.Finding a more effective,convenient,long-lasting, low-cost and safe way to cure pain becomes a topic that attracts more and more attention.Since the discovery of endogenous opioid peptides,it has gained wide acceptance due to its effectiveness in pain relief,less side effects and non-addictive nature.In our studies,we linked the enhanced eGFP gene with the eukaryotic expression pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1(+)-eGFP, which was injected directly into the cavitas subarachnoidealis of the rats.The plasmid was injected intrathecally into the cavitas subarachnoidealis of the rats and was absorbed by the endothelial cell of cavitas subarachnoidealis and was expressed as long as six weeks,indicating that the plasmid carrying the analgesia gene and being injected intrathecally may have analgesic effects and can be expressed for more than a month.Furthermore,to study where and how long the EK gene was expressed in brain,we also linked PENK with FL eukaryotic expression vector to construct recombinant plasmid that is pEGFP-C3-PENK,and transfected it into the NIH3T3 cell line.We identified the position and time the plasmid was expressed.At the same time,co-culturing the positive clone cells and the nerve cells made the expressed EK combine with the surface receptor of the nerve cell to activate the cell signal pathway.Last,the bioactivity of expressed EK was identified through the detection of activation of phosphokinase A in the nerve cell.Methods1.PT-PCRPrimers were designed according to Genbank's report on the human preproenkephalin gene sequence(NM₀06211) by Primer 5.0 program.The restriction endonuclease sites were incorporated into 5'and 3'primers allowing the insertion of the PCR products into retroviral vector.After the transformation,the total RNA was isolated and first strand of PENK cDNA synthesis reactions were performed using cDNA kit according to the manufacturer's recommendation.2.The construction of eukaryotic expression vectorThe right sequencing fragment was cut with bi-enzyme,and was linked with the large fragment of the corresponding peGFP-C3 vector,and was transformed into the strain of E.coli JM109,which was screened by Kana.The positive clones were selected and identified by bi- enzyme cutting.3.The transfection and positive clone screen of the cellFirstly the lethal dose of G418 was determined.Then transfection was performed by using bangosome2000.After 24 hours the green FL carrier was observed with FL microscope and selected with DMEM selective medium which contains 600mg/L G418 for 10 days.4.Positive clone cells immunohistochemistry and the detection of supernatant EKThe supernatant of the cells was collected when the green FL cell was close to the converge,which contains the expressed EK.The amount of EK in supernatant was determined through radio-immunity.Then the red FL immunohistochemistry was performed by using the L-ENK as the first antibody on the screened masculine clone cells.Combined with the expression of the green FL protein in cells,the position of expressed green FL in cell and human proenkephalin binding protein and the secretion of L-ENK were identified. 5.The cultivation and subgroup of the origin generation nerve cellCortical neuron cells were isolated from the neonate rat and were cultured in the cell culture bottle with a volume of 250ml in the density of 106/ml in the incubator with the temperature of 37℃and 5%CO2.Four groups were as following:control group,PG stimulating group,PG stimulating and EK group,PG stimulating after allylnoroxymorphone preincubate group.6.Detection of the amount of phosphorylated PKA in each group by Western blotting.The total protein was isolated from each group and the amount was determined with the protein level curve.Then the amount of phosphorylated PKA in each group was detected with Western blotting according to the total protein.Results1.A total RNA was extracted from the human brain using Trizol Reagent.The human proenkephalin cDNA were obtained from the total RNA by RT-PCR.The PCR product was about 800bp and purified from agarose gel.After the digestion,the PCR product was linked with the corresponding large vector fragments to construct green FL vector plasmid of pEGFP-C3-PENK.2.According to the cell immunohistochemistry,the masculine cell which was transfected with pEGFP-C3-PENK plasmid showed green FL in the FL microscope. Red FL appeared in the surroundings of the cell especially the cellular membrane, indicating that the cells synthesized green FL and human proenkephalin binding protein,and after the enzymaticlysis of human proenkephalin the cells produces L-ENK which transfers through the cellular membrane to the cell supernatant.3.Through radio-immunity detection,the L-ENK amount in the supernatant of pEGFP-C3-PENK masculine clone cells of NIH3T3 is 903.53+82.83pg/ml.4.Western blotting shows that the EK secreted from the cells transfected with pEGFP-C3-PENK plasmid reduced the amount of activate PKA in nerve cells and the addition of allylnoroxymorphone reversed the action of EK. Conclusions1.The eukaryotic expression vector of green FL protein of human preproenkephalin was transfected into NIH3T3 cell and was expressed stably at least one month.2.The newly synthesized EK inhibited the activation of PKA system in the nerve cell and the inhibition can be reversed by allylnoroxymorphone.