【作者】 张军；

【导师】 于海泉；

【作者基本信息】 内蒙古大学 ， 生物工程（专业学位）， 2015， 硕士

【摘要】 动物乳腺生物反应器是基于转基因技术平台,通过构建乳腺特异性表达的表达元件,驱动外源基因在乳腺中特异表达,并在乳汁中获得重组蛋白的技术。CRISPR/Cas9技术具有靶向性强、剪切效率高的特点,在实现基因定点删除和外源基因定点敲入方面显示出极大地优势和应用前景。肿瘤坏死因子α(tumor necrosis factor α, TNF-α)是一种主要由激活的巨噬细胞和单核细胞产生的细胞因子,具有肿瘤杀伤活性、参与免疫调节、促进炎症反应以及增强机体抗病毒等作用。本研究构建了以β-casein基因座为靶标的人源TNF-α基因同源重组载体,同时针对牛β-casein第二外显子区域构建具有特异性切割作用的CRISPR/Cas9载体,采用电转染的方式将两种载体共同导入牛胎儿成纤维细胞,通过G418筛选,挑选阳性克隆,利用PCR法对阳性克隆细胞进行了整合位点的分析,结果表明目的基因成功地整合到β-casein特定位点。为检测外源基因的乳腺特异性表达,本研究同时将同源重组载体和CRISPR/Cas9载体共转染牛乳腺上皮细胞,利用RT-PCR和Western Blot检测所获得转目的基因阳性细胞中外源基因的表达,结果显示在细胞中获得了TNF-α mRNA基因和蛋白的表达。本研究为乳腺特异性表达人TNF-α转基因牛研究奠定了基础。更多还原

【Abstract】 Animal mammary gland bioreactor is a transgenic technology for development of recombinant proteins, in which the foreign gene is expressed under control of mammary gland-specific regulatory elements. CRISPR/Cas9 technology has characters of high targeting efficacy and a robust of sequence-specificity. It has a broad range of applications value in introducing gene knock-out and knock-in in the specific sites. Tumor necrosis factor-α (TNF-α) is a kind of important cytokine, is mainly secreted by activated macrophages and monocytes, has roles of tumor lethal activity, immune regulation, inflammation promotion and antiviral enhancement and so on. In this study, the homologous recombination vector with human TNF-a gene and a CRISPR/Cas9 vector targeting to the second exon of beta-casein gene locus were constructed. Then, the two vectors were co-transfected into the bovine fetal fibroblasts by electroporation. After screened with G418, positive clone cells were examined for the interation site by PCR. The results indicate that the exogenous gene was inserted correctly into the specific site of β-casein. To test the specifically expression of the exogenous gene TNF-α mRNA in mammary gland, the homologous recombination vector and the CRISPR/Cas9 vector were co-transfected into the bovine mammary epithelial cells, RT-PCR and Western Blot were used to detect the expression of mRNA and protein of TNF-α, results showed that bovine mammary epithelial cells expressed mRNA and protein of TNF-α gene. This study is of great importance for further study of producing of TNF-a transgenic cow.更多还原