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The Expression of pEGFP-C3-PENK in NIH3T3 Cell and the Detection of Activity of the Expressed Protein

Author: HuYiLe
Tutor: ZuoWeiDong
School: Zhengzhou University
Course: Human Anatomy and Embryology
Keywords: Analgesic Preproenkephalin gene Protein kinase A Green Fluorescent Protein
CLC: R402
Type: Master's thesis
Year: 2009
Downloads: 11
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Abstract

Objective: The most commonly used method for the treatment of pain opioid analgesics. But there are many side effects, such as causing excessive sedation, respiratory depression, constipation, and addiction. Looking for more convenient and effective, the role lasting economic, security analgesic become the subject of attention. Since the discovery of endogenous opioid peptides since its analgesic effect, side effects, addiction and other advantages growing concern, in the pre-test will be enhanced green fluorescent protein (EGFP) gene eukaryotic expression vector pcDNA3. () to connect to construct the recombinant plasmid pcDNA3.1 ()-EGFP was intrathecal injected directly into the subarachnoid space of the rat, be subarachnoid epithelial cell uptake and expression of a long six weeks, suggesting that if the plasmid carrying the town the pain gene intrathecal injection analgesic effect may play a longer period of time (January) and in vivo expression. Eukaryotic plasmid carrying enkephalin gene expression sites and expression aging for further research, the experimental PENK fluorescent eukaryotic expression vector and the recombinant plasmid pEGFP-C3-PENK and transferred to its expression in NIH3T3 cells enkephalin, observe the location and time of the expression, and the positive clone cells and cells of neuronal cells co-cultured the synthetic enkephalin nerve cell surface receptor binding activation of cell signaling pathways by neurons within The change in the amount of phosphate kinase A activation detection proved synthesized enkephalin biological activity. The proenkephalin gene sequence (NM ₀ 06211) method RT-PCR according to the GenBank human before using Primer5.0 primers designed inner primer 5 'and 3' ends, plus the corresponding restriction sites integrated into the carrier. Organization RNA extracted from human brain. The proenkephalin gene single-stranded cDNA reverse transcription people PCR amplified by reverse transcription product. 2 eukaryotic expression vector Construction of the correct sequencing of the fragment, digested with the corresponding double digestion of pEGFP-C3 vector fragment was ligated to transform E.coli JM109 strain, Kana screening and selection of positive colonies, plasmids were extracted, bis enzyme digestion. Transfection of the cells, and positive clones were screened first determine the lethal dose of G418, Lipofectamine 2000 transfection. 24 hours after transfection with a fluorescence microscope to observe green fluorescent carrier transfection, and containing 600mg / L G418 complete DMEM selection medium screening for ten days, 4 positive clones cellular immune staining and supernatant enkephalin content determination. Green fluorescent cells to be close to completely cell supernatants were collected after confluence, i.e. containing a culture solution of enkephalin, by radioimmunoassay supernatant enkephalin content. The screening of the positive clone cells L-ENK primary antibody cells red immunofluorescence detection combined intracellular green fluorescent protein's expression, observed pEGFP-C3-PENK plasmid in the cell before the synthetic green fluorescence with people brain brown peptide-binding site of the protein and L-ENK secretion. Culture and grouping of primary cultured neurons from newborn rat cortical neuron cells. 10 ^{6 / ml density planting to 250ml of cell culture flasks, at 37 ° C, 5% CO ₂ incubator cultured for one week. Divided into a control; stimulation of prostaglandin (PG); Add the enkephalin PG stimulation; naloxone preincubation enkephalin PG stimulates four groups. 6.Western blotting determination of the amount of PKA phosphorylation in neurons. Each group of the extracted total proteins do neurons protein content curve measured, and the sample according to the amount of total protein determination of the amount of PKA phosphorylation of each group neurons do Western blotting. Results. Extraction of human brain tissue RNA, single-stranded cDNA reverse transcription nested PCR amplification of the reverse transcription product, been around 800bp fragment after double digestion with the large fragment of the corresponding digested vector was constructed recombinant green fluorescent vector plasmid pEGFP-C3-PENK. 2. Immunohistochemical display, turn transfection pEGFP-C3-PENK plasmid of positive cells under a fluorescence microscope displayed green fluorescence, the cells surrounding especially membrane displayed a large number of red fluorescence, prompts in the intracellular synthesis of green fluorescence with human before brain The FQ-binding protein, the human preproenkephalin after enzymatic cleavage L-ENK wear cell membrane distribution within the cell supernatant. NIH3T3 cells transfected clones were determined by radioimmunoassay pEGFP-C3-PENK positive supernatant content of L-ENK 903.53 82.83pg/ml. 4.Western blotting enkephalin the plasmid transfected pEGFP-C3-PENK cells secrete allows neuronal cell PKA activation reduced, adding naloxone could antagonize the role of enkephalin. Conclusion 1. Preproenkephalin gene green fluorescent protein eukaryotic expression vector was transfected into NIH 3T3 cells, and the expression of a long-term (more than one month). 2 new synthetic enkephalin can be suppressed the PKA system activation of nerve cells, the inhibitory effect can be antagonized by naloxone.}

The Expression of pEGFP-C3-PENK in NIH3T3 Cell and the Detection of Activity of the Expressed Protein

Abstract

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