人血清白蛋白基因的克隆及乳腺特异性表达载体的构建

发布时间：2018-07-11 12:02

  本文选题：人血清白蛋白 + cDNA　； 参考：《西北农林科技大学》2005年硕士论文

【摘要】：人血清白蛋白Human serum albumin (HSA)是人血浆中最丰富的蛋白质,在人体内起着维持血液渗透压、运输、营养等作用。转基因动物乳腺生物反应器是基因制药的新方法,其生产药用蛋白具有安全、高效、成本低廉等优点,因此,通过制备动物乳腺生物反应器生产人血清白蛋白将会呈现美好的前景。本试验以奶牛β-酪蛋白基因(β-casein,CSN2)5′端和3′端为调控区序列,人血清白蛋白HSA　cDNA 为表达序列,构建了含有新霉素抗性基因、绿色荧光蛋白报告基因的人血清白蛋白乳腺特异性表达载体,为下一步的转染乳腺细胞和将来通过体细胞核移植法制备乳腺生物反应器生产人血清白蛋白打下坚实的基础。相关的研究结果如下: 1.根据Genebank(登陆号V00495)中HSA 的cDNA 序列,设计扩增引物。采用RT-PCR自人肝脏中扩增出HSA 基因的1995　bp 全长cDNA。产物纯化后连至pMD-18T 载体,转化大肠杆菌DH5a。筛选阳性克隆菌,酶切鉴定并进行序列分析。结果与GeneBank 中登录的人、猴子、牛、猪、绵羊和小鼠的血清白蛋白cDNA 序列的同源性分别为99%、92%、82%、82%、81%和75%,而且,突变并没有引起最大读码框的改变,即成功的克隆了HSA 的cDNA 序列。　 2.本试验以肝脏为材料,用Trizol 法提取总RNA,经甲醛凝胶变性电泳,能清晰的看到28s rRNA和18s rRNA 以及一条较弱的5s rRNA带,表明,所提取的RNA完整性好,完全可以用于RT-PCR 反应。 3.根据表达载体pEGFP-C1 上的多克隆位点以及质粒pBL 中的CSN2 启动子序列,重新设计引物,在HSA　cDNA 两端添加限制性内切酶NheⅠ和BamHⅠ酶切位点,并采用双酶切后定向克隆的方法,成功的将HSA　cDNA 表达序列与CSN2 调控序列融合在一起。测序结果显示,两者正确融合,HSA　cDNA 读码框正确无误,没有发生影响表达的突变。　 4.采用双酶切定向克隆的方法,利用真核表达载体pEGFP-C1 构建了含有抗性基因、报告基因的人血清白蛋白非融合型乳腺表达载体pEGFP-BH。用PCR 方法和酶切方法检测,表明其中的整个人血清白蛋白乳腺表达构件插入位点准确无误。　 5.脂质体法将pEGFP-BH 转染体外培养的乳腺上皮细胞,经过G418 的初步筛选,通过荧光显微镜观察到报告基因绿色荧光蛋白的表达。设计特异性的检测引物,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，

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