本文選題：熱休克轉(zhuǎn)錄因子4 + 慢病毒載體　； 參考：《河南大學(xué)》2010年碩士論文

【摘要】： 背景 先天性白內(nèi)障是導(dǎo)致兒童失明的主要原因,大約0.3- 1.5/1000的兒童患有先天性白內(nèi)障,它的主要病理改變?yōu)榫铙w發(fā)育遲緩,晶狀體內(nèi)非透明瘢塊組織形成,染色體異常為主要致病原因。已經(jīng)發(fā)現(xiàn)多種基因的突變體與臨床及動物實(shí)驗(yàn)性先天性白內(nèi)障發(fā)生有關(guān)。例如編碼晶狀體細(xì)胞結(jié)構(gòu)蛋白相關(guān)基因的突變(MIPs, CP49 and alpha-, beta-和gamma- crystallins)和調(diào)控晶狀體發(fā)育相關(guān)的轉(zhuǎn)錄因子的基因突變(Pax6, FoxE3, Six3, Prox1, Sox2/3, Maf, Pitx3, AP-2a和Hsf4)。2002年,BU等對國人一個先天性白內(nèi)障家系進(jìn)行連鎖分析時,將致病的基因定位于16號染色體,確定了Hsf4是先天性白內(nèi)障的候選基因,它的發(fā)生機(jī)制為Hsf4基因的第348位核苷酸T突變?yōu)镃,使Hsf4的DNA結(jié)合域內(nèi)的第115位高度保守的亮氨酸轉(zhuǎn)變?yōu)楦彼?而最終導(dǎo)致白內(nèi)障的發(fā)生。應(yīng)用轉(zhuǎn)基因技術(shù)敲除小鼠的Hsf4基因可以使小鼠在出生后3-5天(P3-5)出現(xiàn)白內(nèi)障,該結(jié)果有力的支持Hsf4是調(diào)控晶狀體早期發(fā)育的重要轉(zhuǎn)錄因子的論點(diǎn)。但是,調(diào)控Hsf4轉(zhuǎn)錄活性的信號通路及Hsf4調(diào)控晶狀體早期發(fā)育的分子機(jī)理還不清楚。因此,研究Hsf4在晶狀體發(fā)育過程中的生物學(xué)調(diào)控作用對揭示突變型Hsf4的致病機(jī)理及早期診斷和治療與Hsf4突變相關(guān)的先天性白內(nèi)障有著重要的意義。為此我們構(gòu)建了Hsf4的真核表達(dá)載體pcDNA3.0/Hsf4b,然而pcDNA3.0/Hsf4b轉(zhuǎn)染MLEC(Hsf4b-/-)晶狀體上皮細(xì)胞的轉(zhuǎn)染效率較低,對探求Hsf4b調(diào)控晶狀體發(fā)育的分子機(jī)制的研究較難進(jìn)行。體外建立一個轉(zhuǎn)染效率高且穩(wěn)定表達(dá)Hsf4b的細(xì)胞株對我們進(jìn)一步研究Hsf4b的生物學(xué)功能有重要的意義。 目的 構(gòu)建Hsf4b的真核表達(dá)載體,便于研究Hsf4b在體外的功能。構(gòu)建Hsf4b的重組慢病毒載體是為了能獲得一株持續(xù)且穩(wěn)定表達(dá)Hsf4b的細(xì)胞株。為以后繼續(xù)研究Hsf4調(diào)控晶狀體發(fā)育的分子機(jī)制奠定基礎(chǔ)。 方法 用人心臟cDNA文庫為模板,應(yīng)用囊括Hsf4b全長的引物進(jìn)行PCR并在Hsf4bcDNA的N-端加入Flag標(biāo)簽。將PCR產(chǎn)物經(jīng)Kpn I和EcoR I酶切后,與該二酶線性化pcDNA3.0質(zhì)粒一起連接獲得重組質(zhì)粒pcDNA-Flag-Hsf4b;做重組質(zhì)粒上的定點(diǎn)突變;為了獲得能持續(xù)且穩(wěn)定表達(dá)Hsf4b的細(xì)胞株,將重組質(zhì)粒pcDNA-Flag-Hsf4b和慢病毒表達(dá)載體Plentiviral用XholI和NdeI酶切后再連接獲得Plentiviral-Flag-Hsf4b質(zhì)粒,與病毒衣殼蛋白PLP1、PLP2、VSV-G一起轉(zhuǎn)染入HEK293T細(xì)胞,用其分泌的含病毒的上清去感染MLEC細(xì)胞,并通過Blascitidin藥物對其進(jìn)行穩(wěn)定篩選,用Western檢測慢病毒表達(dá)載體Plenti/Hsf4b的表達(dá)情況,并檢測相應(yīng)下游蛋白Hsp25的表達(dá)。 結(jié)果 構(gòu)建了Hsf4b的真核表達(dá)載體pcDNA-Flag-Hsf4b,將其轉(zhuǎn)入HEK293T細(xì)胞顯示能表達(dá)相應(yīng)的蛋白,但是轉(zhuǎn)染MLEC細(xì)胞時轉(zhuǎn)染效率非常低,Western檢測不到,我們研究發(fā)現(xiàn)其主要原因是重組質(zhì)粒被轉(zhuǎn)染入細(xì)胞后其表達(dá)的相應(yīng)蛋白又結(jié)合于重組質(zhì)粒的CMV上抑制Hsf4b相關(guān)蛋白的進(jìn)一步表達(dá)。將構(gòu)建好的Hsf4b真核表達(dá)載體作CMV上的定點(diǎn)突變,使這種抑制作用解除,最后獲得了pcDNA-Flag-Hsf4b載體的突變體。測序結(jié)果及Western結(jié)果也證明了突變成功,插入片斷在MLEC細(xì)胞中得到了表達(dá)。 為了使Hsf4b在MLEC細(xì)胞中穩(wěn)定且持續(xù)表達(dá),構(gòu)建了Hsf4b的慢病毒表達(dá)載體并成功篩選出了一株能穩(wěn)定表達(dá)Hsf4b的細(xì)胞株和一株P(guān)lentiviral(空載體)細(xì)胞株。Western(蛋白質(zhì)免疫印跡)檢測其下游小分子熱休克蛋白Hsp25的表達(dá),結(jié)果顯示Hsf4b促進(jìn)Hsp25的表達(dá)。 結(jié)論 利用基因工程技術(shù),將Hsf4b的cDNA克隆到真核表達(dá)載體pcDNA3.0,構(gòu)建了Hsf4b真核表達(dá)質(zhì)粒pcDNA-Flag-Hsf4b,并在人胚胎腎細(xì)胞系293T中成功表達(dá)出蛋白。對質(zhì)粒pcDNA-Flag-Hsf4b上CMV啟動子成功定點(diǎn)突變,獲重組質(zhì)粒CMVm-Flag- Hsf4b。構(gòu)建了Hsf4b慢病毒表達(dá)載體Plenti-Flag-Hsf4b,并成功轉(zhuǎn)染至MLEC細(xì)胞中。獲得一株穩(wěn)定表達(dá)Hsf4b蛋白的細(xì)胞株MLEC/Plenti-Hsf4b。MLEC/Plenti-Hsf4b細(xì)胞株中小分子熱休克蛋白Hsp25的表達(dá)升高。
[Abstract]:Background


In 2002 , it has been found that Hsf4 is a candidate gene for congenital cataract . It has been found that Hsf4 is a candidate gene for congenital cataract . It has been found that Hsf4 is a candidate gene for congenital cataract .


Purpose


The eukaryotic expression vector of HSV4b was constructed to study the function of Hs4b in vitro . The recombinant lentivirus vector was constructed in order to obtain a cell line which was continuously and stably expressing Hs4b . The molecular mechanism of Hsf4 was used to regulate the development of Hsf4 .


method


The recombinant plasmid pcDNA - Flag - HSV4b was digested by KpnI and EcoR I . The recombinant plasmid pcDNA - Flag - HSV4b and lentivirus expression vector were digested with XholI and NdeI .


Results


The eukaryotic expression vector pcDNA - Flag - Hs4b was constructed , and the recombinant plasmid pcDNA - Flag - Hs4b was transfected into the cells . However , the transfection efficiency was very low when transfected into MLEC . The results showed that the recombinant plasmid was transfected into the cells . The results showed that the recombinant plasmid was successfully transfected into the vector . The results of the sequencing and the Western blot showed that the mutation was successful and the inserted fragment was expressed in MLEC .


In order to stably and persistently express HSV4b in MLEC cells , a cell strain expressing Hs4b was constructed and a cell line stably expressing Hs4b was constructed . Western blot was used to detect the expression of Hsp25 in the downstream small molecular heat shock protein .


Conclusion


The recombinant plasmid pcDNA - Flag - HSV4b was constructed and the recombinant plasmid CMVm - Flag - HSV4b was successfully transfected into MLEC .
【學(xué)位授予單位】：河南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R776.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 林燕瑜;李青;楊佩菲;;熱休克轉(zhuǎn)錄因子4與先天性白內(nèi)障[J];福建醫(yī)藥雜志;2007年04期

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本文編號：2034275