重组血红蛋白原核双表达载体的构建与表达

发布时间：2018-08-22 18:14

【摘要】：输液可以应对很多临床上的状况,从整个世界来看,对输液的需求越来越高。但是献血资源是一种短缺的资源,而且伴随着很多副作用。这些都驱使人们去寻找一种全血的替代品用于输液治疗。其中很重要的一种思路就是使用无细胞的重组血红蛋白来做为氧载体,即重组血红蛋白类氧载体。对于重组血红蛋白类氧载体的研究已经有多年的历史,本文在前人的基础上提出一种新的构建重组血红蛋白类氧载体的方式,通过构建白蛋白血红蛋白融合蛋白以及在血红蛋白基因中引入点突变来克服重组血红蛋白类氧载体输入人体后会引起的副作用。实验目的:本实验的目的是构建白蛋白血红蛋白α融合蛋白,从而显著增大该重组血红蛋白的分子量,降低肾毒性。另一方面在血红蛋白基因中引入三个定点突变,降低其与NO结合的能力,从而解决重组血红蛋白类氧载体输入人体内会引起的高血压问题。然后体外表达该重组血红蛋白类氧载体。方法与结果:本实验用一段短的Linker DNA将白蛋白与血红蛋白α基因连接起来,并采用重叠延伸PCR的方法在人血红蛋白基因中引入三个定点突变。选取合适的酶切位点,将白蛋白血红蛋白α基因以及血红蛋白β基因连入原核双表达载体pETDuet1中,随后将构建好的表达载体转化入大肠杆菌表达菌株Origami DE3中,用IPTG诱导目的基因的表达。本实验成功的构建出了一种重组血红蛋白双表达载体,并在特定的大肠杆菌表达菌株中成功的表达出了该重组血红蛋白基因。本实验为以后进一步纯化该重组血红蛋白并用于临床实验奠定了基础。
[Abstract]:Infusion can cope with many clinical conditions, and the need for infusion is increasing throughout the world. But blood donation is a scarce resource with many side effects. All this drives people to look for a whole-blood substitute for infusion therapy. One of the most important ideas is to use cell-free recombinant hemoglobin as oxygen carrier, that is, recombinant hemoglobin oxygen-like carrier. There have been many years of research on the recombinant hemoglobin oxygen-like carrier. This paper proposes a new way to construct the recombinant hemoglobin oxygen-like carrier on the basis of previous studies. By constructing albumin hemoglobin fusion protein and introducing point mutation into hemoglobin gene, the side effects caused by recombinant hemoglobin oxygen-like vector were overcome. Objective: to construct the albumin hemoglobin 伪 fusion protein, and to increase the molecular weight of the recombinant hemoglobin and reduce the nephrotoxicity. On the other hand, three site-directed mutations were introduced into the hemoglobin gene to reduce its ability to bind to no, thus solving the problem of hypertension caused by the introduction of the recombinant hemoglobin oxygen-like vector into human body. Then the recombinant hemoglobin oxygen-like vector was expressed in vitro. Methods and results: in this experiment, a short Linker DNA was used to link albumin with hemoglobin 伪 gene, and three site-directed mutations were introduced into human hemoglobin gene by overlapping extended PCR. The albumin hemoglobin 伪 gene and hemoglobin 尾 gene were linked into the prokaryotic expression vector pETDuet1, and the constructed expression vector was transformed into the E. coli expression strain Origami DE3. The expression of target gene was induced by IPTG. In this experiment, a recombinant hemoglobin double expression vector was successfully constructed, and the recombinant hemoglobin gene was successfully expressed in a specific Escherichia coli expression strain. This study laid a foundation for further purification and clinical application of the recombinant hemoglobin.
【学位授予单位】：江苏师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R3411;Q78

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