發(fā)布時間：2019-02-15 13:04

【摘要】：目的：人前腦啡肽原(hPPE)作為內(nèi)源性阿片肽的一種,其鎮(zhèn)痛作用明確,同時較外源性阿片類鎮(zhèn)痛物質相比副作用小,無成癮性,為鎮(zhèn)痛研究提供了物質基礎。本課題通過基因工程技術獲得人前腦啡肽原基因序列,構建人前腦啡肽原基因重組載體pcDNA3.1(+)/hPPE,在轉染試劑脂質體2000作用下實現(xiàn)重組載體在HEK293細胞中的表達,為后期的轉移性骨腫瘤模型鎮(zhèn)痛研究提供工具。 方法：提取人腦組織總RNA,在反轉錄酶作用下合成DNA互補鏈即cDNA,依據(jù)Gene Bank中報道的前腦啡肽原基因序列設計上下游引物,以cDNA為模板PCR法合成hPPE基因序列后克隆于測序載體pMD-18T上,測序分析。hPPE基因序列測序分析正確后,限制性內(nèi)切酶HindⅢ、NotⅠ雙酶切pMD-18T/hPPE和真核表達載體pcDNA3.1(+),在T4DNA連接酶作用下連接hPPE基因和真核表達載體pcDNA3.1(+),酶切凝膠電泳鑒定重組載體pcDNA3.1(+)/hPPE后,轉化大腸桿菌(JM109),擴增提取重組載體。在轉染試劑脂質體2000作用下,重組載體pcDNA3.1(+)/hPPE轉染HEK293細胞。于轉染后48h RT-PCR檢測人前腦啡肽原基因,72h后放射免疫法檢測人前腦啡肽原表達。 結果： 1 RT-PCR法成功擴增出人前腦啡肽原基因序列,經(jīng)凝膠電泳鑒定可擴增出800bp左右基因片段與文獻中報道一致；經(jīng)測序分析與GeneBank中報道的人前腦啡肽原基因序列比對,同源性達100%。 2限制性內(nèi)切酶HindⅢ.NotⅠ雙酶切重組載體pcDNA3.1(+)/hPPE,凝膠電泳鑒定擴增出5400bp基因片段和800bp左右基因片段,與文獻中報道的pcDNA3.1(+)、hPPE基因序列一致。 3重組載體pcDNA3.1(+)/hPPE、真核表達載體pcDNA3.1(+)分別轉染HEK293細胞,于轉染48h后搜集pcDNA3.1(+)/hPPE轉染組、陰性對照pcDNA3.1(+)轉染組、HEK293細胞組這三組細胞。以B-action為內(nèi)參,RT-PCR檢測這三組細胞中hPPE基因：結果可見pcDNA3.1(+)/hPPE轉染組擴增出800bp左右的hPPE基因片段,陰性對照pcDNA3.1(+)轉染組、HEK293細胞組未擴增出基因片段。 4重組載體pcDNA3.1(+)/hPPE.真核表達載體pcDNA3.1(+)分別轉染HEK293細胞,于轉染72h后搜集pcDNA3.1(+)/hPPE轉染組、陰性對照pcDNA3.1(+)轉染組、HEK293細胞組這三組細胞上清液。放射免疫法檢測這三組細胞中人前腦啡肽原表達：結果可見陰性對照pcDNA3.1(+)轉染組、HEK293細胞組未檢測到人前腦啡肽原表達。pcDNA3.1(+)/hPPE轉染組檢測到人前腦啡肽原表達,其表達量可用pg級濃度表示。 結論：成功克隆出人前腦啡肽原基因,成功構建了人前腦啡肽原基因重組載體pcDNA3.1(+)/hPPE,并實現(xiàn)了人前腦啡肽原在HEK293細胞中的表達,為后期轉移性骨腫瘤痛模型鎮(zhèn)痛研究提供了有力的工具。
[Abstract]:Aim: as one of endogenous opioid peptides, human proenkephalin (hPPE) has definite analgesic effect, less side effect and no addiction than exogenous opioid analgesia, which provides a material basis for analgesic research. In this study, the human proenkephalin gene sequence was obtained by genetic engineering, and the recombinant vector pcDNA3.1 () / hPPE, was constructed by transfection reagent liposome 2000 to realize the expression of the recombinant vector in HEK293 cells. To provide a tool for anaphase analgesic study of metastatic bone tumor model. Methods: total RNA, was extracted from human brain tissue to synthesize DNA complementary strand by reverse transcriptase. CDNA, designed upstream and downstream primers according to the sequence of proenkephalin gene reported in Gene Bank. The sequence of hPPE gene was synthesized by using cDNA as template PCR method and cloned into the pMD-18T vector. After the sequence analysis of hPPE gene was correct, the restriction endonuclease Hind 鈪，

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