發(fā)布時(shí)間：2018-03-18 23:34

  本文選題：人組織型纖溶酶原激活劑突變體(ht-PA_m)　切入點(diǎn)：轉(zhuǎn)基因動(dòng)物乳腺生物反應(yīng)器　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2005年碩士論文　論文類型：學(xué)位論文

【摘要】：組織型纖溶酶原激活劑(t-PA)是治療血栓栓塞性疾病的重要藥物。本項(xiàng)目的t-PA突變體,活性提高,半衰期延長,已成功獲得表達(dá)。目前t-PA主要來自于哺乳動(dòng)物細(xì)胞大規(guī)模培養(yǎng),成本高昂,價(jià)格昂貴。利用動(dòng)物乳腺生物反應(yīng)器來生產(chǎn)不僅維持生產(chǎn)的成本低,而且產(chǎn)量高,能夠進(jìn)行翻譯后修飾、正確折疊。但位置效應(yīng)的影響是其主要技術(shù)瓶頸之一。細(xì)菌人工染色體容量大,可裝載乳蛋白基因的所有調(diào)控序列,乳蛋白基因座中的一些邊緣序列具有絕緣子的功效,能使乳蛋白基因表達(dá)調(diào)控不受周圍序列的影響,作為轉(zhuǎn)基因載體就有可能克服外源基因的位置效應(yīng)。 本研究主要構(gòu)建了由β-酪蛋白基因調(diào)控序列指導(dǎo)的t-PA突變體BAC轉(zhuǎn)基因載體。采用了λ噬菌體Red同源重組系統(tǒng)介導(dǎo)的同源重組方法對(duì)含有鼠β-酪蛋白基因的RPCI23-440C1 BAC進(jìn)行了快速改構(gòu)。這種方法要明顯優(yōu)于傳統(tǒng)的——利用微生物本身的RecA重組系統(tǒng)進(jìn)行重組的方法。首先通過PCR方法,獲得兩端帶有鼠β-酪蛋白基因同源序列的tPAm-Zeo和tPAm-BGHpA-Zeo同源重組片段,將其電擊轉(zhuǎn)化至已含有編碼Red重組酶質(zhì)粒和BAC的宿主菌。在λRed重組系統(tǒng)的幫助下,通過同源重組片段兩端與β-酪蛋白同源的序列在體內(nèi)與β-酪蛋白基因發(fā)生同源重組,將其置換。經(jīng)Southern blot和測(cè)序鑒定,得到了t-PAm基因定點(diǎn)敲入的tPAm-Zeo/tPAm-BGHpA-Zeo BAC轉(zhuǎn)基因載體。最后通過FLP位點(diǎn)專一性重組,利用Zeocin抗性基因兩側(cè)的FRT位點(diǎn),將抗性基因剔除。Southern blot和序列分析鑒定,獲得了無抗性基因且編碼兩種重組酶質(zhì)粒丟失的t-PAm BAC載體。最后利用上述兩種轉(zhuǎn)基因載體分別從細(xì)胞和動(dòng)物水平初步驗(yàn)證了其有效性。 由此建立了快速高效的BAC重組技術(shù),獲得了轉(zhuǎn)基因所需的BAC載體,為轉(zhuǎn)基因動(dòng)物以及動(dòng)物乳腺生物反應(yīng)器的制備提供了新的方法,這一方法的建立也為利用BAC研究基因調(diào)控和功能提供了新途徑。
[Abstract]:Tissue plasminogen activator (t-PA) is an important drug in the treatment of thromboembolic diseases. The activity of t-PA mutants in this project has been increased, the half-life of t-PA has been prolonged, and t-PA has been successfully expressed. At present, t-PA is mainly derived from the large-scale culture of mammalian cells. Using animal mammary gland bioreactor not only to maintain the cost of production, but also high production, can be modified after translation, Correct folding. But the influence of position effect is one of the main technical bottlenecks. Bacteria artificial chromosome capacity, can carry all the milk protein gene regulatory sequence, milk protein gene in some of the edge sequence has insulator effect, It can make the regulation of lactoprotein gene expression not affected by the peripheral sequence. As a transgenic vector, it is possible to overcome the position effect of exogenous gene. In this study, t-PA mutant BAC transgenic vector guided by 尾 -casein gene regulatory sequence was constructed. RPCI23-440C1 BAC containing mouse 尾 -casein gene was transfected by RPCI23-440C1 BAC mediated by 位 phage Red homologous recombination system. This method is obviously superior to the traditional method of recombination using the RecA recombination system of the microorganism itself. First of all, through the PCR method, TPAm-Zeo and tPAm-BGHpA-Zeo homologous recombination fragments with mouse 尾 -casein gene homologous sequences were obtained and transformed into host bacteria containing recombinant plasmid encoding Red and BAC. With the help of 位 Red recombination system, 尾 -casein gene was homologous recombined with 尾 -casein gene at both ends of homologous recombination fragment in vivo, and was replaced by Southern blot and sequencing. The tPAm-Zeo/tPAm-BGHpA-Zeo BAC transgenic vector with t-PAm gene knockout was obtained. Finally, by the specific recombination of FLP locus, the resistance gene was eliminated by FRT locus on both sides of Zeocin resistance gene and identified by sequence analysis. The t-PAm BAC vector with no resistance gene and encoding two recombinant enzyme plasmids was obtained. Finally, the effectiveness of t-PAm BAC vector was preliminarily verified by the above two transgenic vectors at the cell and animal levels, respectively. Therefore, a rapid and efficient BAC recombination technique was established, and the BAC vector for transgenic animals was obtained, which provided a new method for the preparation of transgenic animals and mammary gland bioreactor. The establishment of this method also provides a new way to study gene regulation and function by using BAC.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉士輝,黃培堂,黃翠芬;組織型纖溶酶原激活劑突變體的構(gòu)建、表達(dá)及特性分析[J];中國科學(xué)(B輯 化學(xué) 生命科學(xué) 地學(xué));1995年04期

2 王恒，

本文編號(hào)：1631860