带有染色质隔离子的hMMP-12转基因表达载体的构建

发布时间：2018-03-13 01:25

本文选题：染色质隔离子　切入点：hMMP-12　出处：《新疆医科大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的：(1)构建巨噬细胞特异表达且带有染色质隔离子的人基质金属蛋白酶-12(hMMP-12)转基因表达载体；(2)构建hMMP-12转基因兔动物模型。方法：(1)通过RT-PCR方法扩增hMMP-12基因，产物亚克隆到pGEM-T载体，经测序准确无误后命名为pGEM-T-hMMP-12；(2)利用pJC13(backbone：pGEM4Z)，构建带有染色质隔离子和清道夫受体A(scavenger receptor A，SR-A)增强子、启动子和人生长激素尾(hGH tail)的亚克隆pJC13-SR；(3)利用pGEM-T-hMMP-12和pJC13-SR重组质粒，构建巨噬细胞特异性的转基因表达载体pJC13-SR-hMMP-12；(4)pJC13-SR-hMMP-12表达载体线性化，显微注射家兔受精卵制备转基因兔，并进行PCR扩增及Southern Blot转基因效果分析鉴定。结果：(1)通过DNA测序和限制性核酸内切酶酶切鉴定证实，hMMP-12成功克隆到所需表达载体中；(2)转基因后获得仔兔36只，以PCR检测结果计算，转基因总效率为1.41％～3.35％，整合率为20％～50％；以Southern Blot检测结果计算，转基因总效率为0.47％～2.23％，整合率为13.33％～16.67％。结论：转基因表达载体的成功构建，，为hMMP-12转基因兔动物模型的构建和研究MMP-12在动脉粥样硬化的形成和发展中的作用机制奠定了基础。
[Abstract]:Objective to construct hMMP-12 transgenic rabbit model by constructing macrophage specific expression vector of human matrix metalloproteinase-12hMMP-12 with chromatin isolator. Methods the hMMP-12 gene was amplified by RT-PCR and the product was subcloned into pGEM-T vector. Using pJC13 backbone: pGEM4ZN, we constructed the enhancer with chromatin isolon and scavenger receptor Ascavenger receptor (SR-A3), the promoter and human growth hormone htail GH (htail GH) subclone pJC13-SRA3) using pGEM-T-hMMP-12 and pJC13-SR recombinant plasmids. Macrophage specific expression vector pJC13-SR-hMMP-12 was constructed to linearize the expression vector pJC13-SR-hMMP-12. The transgenic rabbits were prepared by microinjection of rabbit fertilized eggs. PCR amplification and analysis of Southern Blot transgenic effect were performed. Results: 1) DNA sequencing and restriction endonuclease digestion confirmed that hMMP-12 was successfully cloned into the required expression vector, and 36 rabbits were obtained. The results of PCR analysis were used to calculate the results of PCR detection. The total efficiency of transgenic gene was 1.41 ~ 3.35 and the integration rate was 20 / 50. Based on the results of Southern Blot test, the total efficiency of transgenic gene was 0.472.23, and the integration rate was 13.33 ~ 16.67.Conclusion: the successful construction of transgenic expression vector, It provides a basis for the construction of hMMP-12 transgenic rabbit model and the study of the role of MMP-12 in the formation and development of atherosclerosis.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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