發(fā)布時(shí)間：2018-07-11 12:02

  本文選題：人血清白蛋白 + cDNA��； 參考：《西北農(nóng)林科技大學(xué)》2005年碩士論文

【摘要】：人血清白蛋白Human serum albumin (HSA)是人血漿中最豐富的蛋白質(zhì),在人體內(nèi)起著維持血液滲透壓、運(yùn)輸、營養(yǎng)等作用。轉(zhuǎn)基因動(dòng)物乳腺生物反應(yīng)器是基因制藥的新方法,其生產(chǎn)藥用蛋白具有安全、高效、成本低廉等優(yōu)點(diǎn),因此,通過制備動(dòng)物乳腺生物反應(yīng)器生產(chǎn)人血清白蛋白將會呈現(xiàn)美好的前景。本試驗(yàn)以奶牛β-酪蛋白基因(β-casein,CSN2)5′端和3′端為調(diào)控區(qū)序列,人血清白蛋白HSA　cDNA 為表達(dá)序列,構(gòu)建了含有新霉素抗性基因、綠色熒光蛋白報(bào)告基因的人血清白蛋白乳腺特異性表達(dá)載體,為下一步的轉(zhuǎn)染乳腺細(xì)胞和將來通過體細(xì)胞核移植法制備乳腺生物反應(yīng)器生產(chǎn)人血清白蛋白打下堅(jiān)實(shí)的基礎(chǔ)。相關(guān)的研究結(jié)果如下: 1.根據(jù)Genebank(登陸號V00495)中HSA 的cDNA 序列,設(shè)計(jì)擴(kuò)增引物。采用RT-PCR自人肝臟中擴(kuò)增出HSA 基因的1995　bp 全長cDNA。產(chǎn)物純化后連至pMD-18T 載體,轉(zhuǎn)化大腸桿菌DH5a。篩選陽性克隆菌,酶切鑒定并進(jìn)行序列分析。結(jié)果與GeneBank 中登錄的人、猴子、牛、豬、綿羊和小鼠的血清白蛋白cDNA 序列的同源性分別為99%、92%、82%、82%、81%和75%,而且,突變并沒有引起最大讀碼框的改變,即成功的克隆了HSA 的cDNA 序列。　 2.本試驗(yàn)以肝臟為材料,用Trizol 法提取總RNA,經(jīng)甲醛凝膠變性電泳,能清晰的看到28s rRNA和18s rRNA 以及一條較弱的5s rRNA帶,表明,所提取的RNA完整性好,完全可以用于RT-PCR 反應(yīng)。 3.根據(jù)表達(dá)載體pEGFP-C1 上的多克隆位點(diǎn)以及質(zhì)粒pBL 中的CSN2 啟動(dòng)子序列,重新設(shè)計(jì)引物,在HSA　cDNA 兩端添加限制性內(nèi)切酶NheⅠ和BamHⅠ酶切位點(diǎn),并采用雙酶切后定向克隆的方法,成功的將HSA　cDNA 表達(dá)序列與CSN2 調(diào)控序列融合在一起。測序結(jié)果顯示,兩者正確融合,HSA　cDNA 讀碼框正確無誤,沒有發(fā)生影響表達(dá)的突變。　 4.采用雙酶切定向克隆的方法,利用真核表達(dá)載體pEGFP-C1 構(gòu)建了含有抗性基因、報(bào)告基因的人血清白蛋白非融合型乳腺表達(dá)載體pEGFP-BH。用PCR 方法和酶切方法檢測,表明其中的整個(gè)人血清白蛋白乳腺表達(dá)構(gòu)件插入位點(diǎn)準(zhǔn)確無誤�！� 5.脂質(zhì)體法將pEGFP-BH 轉(zhuǎn)染體外培養(yǎng)的乳腺上皮細(xì)胞,經(jīng)過G418 的初步篩選,通過熒光顯微鏡觀察到報(bào)告基因綠色熒光蛋白的表達(dá)。設(shè)計(jì)特異性的檢測引物,利用PCR
[Abstract]:Human serum albumin (HSA) is the most abundant protein in human plasma, which plays a role in maintaining blood osmotic pressure, transport, nutrition and so on. The mammary gland bioreactor of transgenic animals is a new method of gene pharmacy, which has the advantages of safety, high efficiency and low cost. The preparation of animal mammary gland bioreactor for the production of human serum albumin will present a bright prospect. In this study, 5 '-terminal and 3' -terminal sequences of 尾 -casein (CSN2) gene and HSA cDNA of human serum albumin (HSA) were used as regulatory regions to construct a neomycin resistant gene. The human serum albumin specific expression vector of green fluorescent protein reporter gene provides a solid foundation for the next step to transfect mammary gland cells and prepare mammary gland bioreactor to produce human serum albumin by somatic cell nuclear transfer. The results are as follows: 1. According to the cDNA sequence of HSA in Genebank (accession number V00495), primers were designed and amplified. A total length of 1995 BP cDNA of HSA gene was amplified from human liver by reverse transcription-polymerase chain reaction (RT-PCR). The product was purified into pMD-18T vector and transformed into Escherichia coli DH 5a. Positive clones were screened, digested and sequenced. Results the homology of the cDNA sequence of serum albumin from human, monkey, cow, pig, sheep and mouse in GeneBank was 99%, 92% and 82%, respectively, and 75%, respectively. Moreover, the mutation did not cause the change of the maximum reading frame. The cDNA sequence of HSA was cloned successfully. 2. 2. In this experiment, total RNAs were extracted from liver by Trizol method. By formaldehyde gel denaturation electrophoresis, 28s rRNA, 18s rRNA and a weak 5s rRNA band were clearly observed, which indicated that the extracted RNA had good integrity. It can be used for RT-PCR reaction. According to the polyclonal sites in the expression vector pEGFP-C1 and the CSN2 promoter sequence in plasmid pBL, primers were redesigned to add restriction endonuclease NHE 鈪，

本文編號：2115135