人前脑啡肽原真核表达载体构建及其在HEK293细胞中的表达

发布时间：2019-02-15 13:04

【摘要】：目的：人前脑啡肽原(hPPE)作为内源性阿片肽的一种,其镇痛作用明确,同时较外源性阿片类镇痛物质相比副作用小,无成瘾性,为镇痛研究提供了物质基础。本课题通过基因工程技术获得人前脑啡肽原基因序列,构建人前脑啡肽原基因重组载体pcDNA3.1(+)/hPPE,在转染试剂脂质体2000作用下实现重组载体在HEK293细胞中的表达,为后期的转移性骨肿瘤模型镇痛研究提供工具。 方法：提取人脑组织总RNA,在反转录酶作用下合成DNA互补链即cDNA,依据Gene Bank中报道的前脑啡肽原基因序列设计上下游引物,以cDNA为模板PCR法合成hPPE基因序列后克隆于测序载体pMD-18T上,测序分析。hPPE基因序列测序分析正确后,限制性内切酶HindⅢ、NotⅠ双酶切pMD-18T/hPPE和真核表达载体pcDNA3.1(+),在T4DNA连接酶作用下连接hPPE基因和真核表达载体pcDNA3.1(+),酶切凝胶电泳鉴定重组载体pcDNA3.1(+)/hPPE后,转化大肠杆菌(JM109),扩增提取重组载体。在转染试剂脂质体2000作用下,重组载体pcDNA3.1(+)/hPPE转染HEK293细胞。于转染后48h RT-PCR检测人前脑啡肽原基因,72h后放射免疫法检测人前脑啡肽原表达。 结果： 1 RT-PCR法成功扩增出人前脑啡肽原基因序列,经凝胶电泳鉴定可扩增出800bp左右基因片段与文献中报道一致；经测序分析与GeneBank中报道的人前脑啡肽原基因序列比对,同源性达100%。 2限制性内切酶HindⅢ.NotⅠ双酶切重组载体pcDNA3.1(+)/hPPE,凝胶电泳鉴定扩增出5400bp基因片段和800bp左右基因片段,与文献中报道的pcDNA3.1(+)、hPPE基因序列一致。 3重组载体pcDNA3.1(+)/hPPE、真核表达载体pcDNA3.1(+)分别转染HEK293细胞,于转染48h后搜集pcDNA3.1(+)/hPPE转染组、阴性对照pcDNA3.1(+)转染组、HEK293细胞组这三组细胞。以B-action为内参,RT-PCR检测这三组细胞中hPPE基因：结果可见pcDNA3.1(+)/hPPE转染组扩增出800bp左右的hPPE基因片段,阴性对照pcDNA3.1(+)转染组、HEK293细胞组未扩增出基因片段。 4重组载体pcDNA3.1(+)/hPPE.真核表达载体pcDNA3.1(+)分别转染HEK293细胞,于转染72h后搜集pcDNA3.1(+)/hPPE转染组、阴性对照pcDNA3.1(+)转染组、HEK293细胞组这三组细胞上清液。放射免疫法检测这三组细胞中人前脑啡肽原表达：结果可见阴性对照pcDNA3.1(+)转染组、HEK293细胞组未检测到人前脑啡肽原表达。pcDNA3.1(+)/hPPE转染组检测到人前脑啡肽原表达,其表达量可用pg级浓度表示。 结论：成功克隆出人前脑啡肽原基因,成功构建了人前脑啡肽原基因重组载体pcDNA3.1(+)/hPPE,并实现了人前脑啡肽原在HEK293细胞中的表达,为后期转移性骨肿瘤痛模型镇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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