广西巴马小型猪ApoE的表达、真核载体构建及功能验证

发布时间：2018-04-30 02:09

本文选题：广西巴马小型猪 + 阿尔茨海默病　；参考：《广西大学》2013年硕士论文

【摘要】：阿尔茨海默病(Alzheimer's disease, AD)是继心脏病、肿瘤、中风之后,成为人类死亡的第四大死因,严重危害到老年人身体健康。研究发现,载脂蛋白E(apolipoprotein E, ApoE)与AD高度相关。因此,本研究以广西巴马小型猪为研究对象,测定了ApoE在不同组织、不同年龄和大脑不同部位的表达模式；构建ApoE真核表达载体并对其功能进行验证,同时进行转ApoE基因小鼠的生产,为后续构建广西巴马小型猪AD动物模型的研究奠定基础。根据GenBank公布的猪ApoE cDNA序列设计特异引物,利用RT-PCR (reverse transcription PCR, RT-PCR)分析广西巴马小型猪ApoE在心、脾、肝、肾、胰和脑等组织中的表达情况,并测定了ApoE在不同年龄(10,14,24,24月龄)猪脑组织中的表达模式。结果发现ApoE在脑和胰腺中的表达最高；老年(24月龄)猪脑组织中ApoE的表达量高于其它年龄；免疫组化的结果显示,ApoE在前脑皮质的表达量高于海马组织。将ApoE cDNA序列克隆到pMD18-T载体,双酶切并连接构建ApoE真核表达载体pEGFP-C1-ApoE,利用PK15和PC12细胞对载体的功能进行了验证。结果显示,在两种细胞中都检测到绿色荧光,证明真核表达载体pEGFP-C1-ApoE在PK15和PC12细胞中均能正常表达目的蛋白。为了进一步验证重组质粒的功能,将质粒注入小鼠睾丸,自然交配获得57只F1代小鼠,PCR检测获得阳性小鼠2只,转基因阳性率为3.5%。
[Abstract]:Alzheimer's disease (ADD) is the fourth leading cause of death after heart disease, cancer and stroke, which seriously endangers the health of the elderly. Apolipoprotein E(apolipoprotein E (ApoE) was found to be highly correlated with AD. Therefore, the expression patterns of ApoE in different tissues, different ages and different parts of brain were determined in Guangxi Bama miniature pig, and the eukaryotic expression vector of ApoE was constructed and its function was verified. At the same time, the production of transgenic mice with ApoE gene was carried out, which laid a foundation for further study on the construction of AD animal model of Guangxi Bama miniature pig. According to the porcine ApoE cDNA sequence published by GenBank, specific primers were designed to analyze the expression of ApoE in the heart, spleen, liver, kidney, pancreas and brain of Guangxi Bama mini-pig by RT-PCR reverse transcription PCR. The expression pattern of ApoE in the brain tissue of different ages (1014 ~ 24 ~ 24 months) was determined. The results showed that the expression of ApoE was the highest in the brain and pancreas, the expression of ApoE in the brain of the pig was higher than that in the other ages, and the expression of ApoE in the forebrain cortex was higher than that in the hippocampal tissue. The ApoE cDNA sequence was cloned into pMD18-T vector and ligated with two enzymes to construct ApoE eukaryotic expression vector pEGFP-C1-ApoE. The function of the vector was verified by PK15 and PC12 cells. The results showed that green fluorescence was detected in both cells, indicating that the eukaryotic expression vector pEGFP-C1-ApoE could normally express the target protein in both PK15 and PC12 cells. In order to further verify the function of the recombinant plasmid, the plasmid was injected into the testis of mice, and 57 F1 generation mice were obtained by natural mating, and 2 positive mice were obtained by PCR detection. The positive rate of transgenic gene was 3.5%.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16;Q78

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