發(fā)布時(shí)間：2018-03-13 01:25

  本文選題：染色質(zhì)隔離子　切入點(diǎn)：hMMP-12　出處：《新疆醫(yī)科大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：(1)構(gòu)建巨噬細(xì)胞特異表達(dá)且?guī)в腥旧|(zhì)隔離子的人基質(zhì)金屬蛋白酶-12(hMMP-12)轉(zhuǎn)基因表達(dá)載體；(2)構(gòu)建hMMP-12轉(zhuǎn)基因兔動(dòng)物模型。方法：(1)通過RT-PCR方法擴(kuò)增hMMP-12基因，產(chǎn)物亞克隆到pGEM-T載體，經(jīng)測(cè)序準(zhǔn)確無誤后命名為pGEM-T-hMMP-12；(2)利用pJC13(backbone：pGEM4Z)，構(gòu)建帶有染色質(zhì)隔離子和清道夫受體A(scavenger receptor A，SR-A)增強(qiáng)子、啟動(dòng)子和人生長激素尾(hGH tail)的亞克隆pJC13-SR；(3)利用pGEM-T-hMMP-12和pJC13-SR重組質(zhì)粒，構(gòu)建巨噬細(xì)胞特異性的轉(zhuǎn)基因表達(dá)載體pJC13-SR-hMMP-12；(4)pJC13-SR-hMMP-12表達(dá)載體線性化，顯微注射家兔受精卵制備轉(zhuǎn)基因兔，并進(jìn)行PCR擴(kuò)增及Southern Blot轉(zhuǎn)基因效果分析鑒定。結(jié)果：(1)通過DNA測(cè)序和限制性核酸內(nèi)切酶酶切鑒定證實(shí)，hMMP-12成功克隆到所需表達(dá)載體中；(2)轉(zhuǎn)基因后獲得仔兔36只，以PCR檢測(cè)結(jié)果計(jì)算，轉(zhuǎn)基因總效率為1.41％～3.35％，整合率為20％～50％；以Southern Blot檢測(cè)結(jié)果計(jì)算，轉(zhuǎn)基因總效率為0.47％～2.23％，整合率為13.33％～16.67％。結(jié)論：轉(zhuǎn)基因表達(dá)載體的成功構(gòu)建，，為hMMP-12轉(zhuǎn)基因兔動(dòng)物模型的構(gòu)建和研究MMP-12在動(dòng)脈粥樣硬化的形成和發(fā)展中的作用機(jī)制奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 郭愛桃,韋立新,石懷銀,李向紅,游聯(lián)璧;基質(zhì)金屬蛋白酶1與冠狀動(dòng)脈粥樣硬化斑塊破裂的關(guān)系[J];中華病理學(xué)雜志;2000年04期

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