血红蛋白类纳米氧载体HBOCs的研究
为了克服多聚血红蛋白类氧载体存在的肾毒性和高血压的副作用，本实验室提出了重组表达白蛋白-血红蛋白融合蛋白的制备人造血的方法。人白蛋白和血红蛋白融合蛋白的分子量足够大，使得重组四聚体不能够穿过血管壁而造成肾毒性作用；而且白蛋白带有大量负电荷，与毛细血管内皮细胞糖萼所带的负电荷相互排斥，使得重组四聚体蛋白在血管中央流动，不会产生NO的清除作用，从而降低升高血压的可能。
本实验室设计了能表达人白蛋白-血红蛋白融合蛋白了质粒。本研究首先对该质粒pSUMO-ALB-Hb进行了鉴定。接着，将其导入大肠杆菌进行了诱导表达。最后对表达出的人白蛋白-血红蛋白融合蛋白进行了初步的鉴定。这些工作为进一步深入研究人白蛋白-血红蛋白携氧功能打下了基础。
毕业论文关键词：白蛋白，血红蛋白，氧载体，融合蛋白，原核表达
Research on the nanodimension hemoglobin-based oxygen carrier（HBOCs）
Abstract: The blood shortage has become a worldwide problem in current and next several decades. The restrictions of traditional transfusion (e.g. The need for cross-matching of donated blood, the risk of infecting virus and short-term blood) exacerbate the tension of blood supply, so development of blood substitutes bring good news for this. However, the side effects of HBOCs, nephrotoxicity and hypertension, still a problem for the safety of blood transfusion. In order to solve these problems，we promote a way which is to build a plasmid containing ALB-Hemoglobin fusion gene. It can express human albumin and hemoglobin fusion protein which is five bigger than hemoglobin tetramer. So it cannot pass through kidney. On the other hand, one albumin carrys negative charges, which reject with negative charge of blood capillary endothelial cell sugar calyx, making fusion protein flow in centre of vascular. Consequently, it won’t scavenge NO in endothelial cell. We design a plasmid with ALB-Hemoglobin fusion gene. This research is to identify pSUMO-ALB-Hb and then transform it to E.coil. We also identify ALB-Hb fusion protein preliminarily. This work can be the base of our next deep research on the function of HBOCs.
Keywords:Albumin,Hemoglobin,Oxygen carrier,Fusion protein,Prokaryotic expression
目录
摘要 I
Abstract II
目录 III
绪论 1
1.1引言 1
1.2血红蛋白 1
1.3血液替代品 2
1.4血红蛋白类氧载体（HBOCs） 2
1.5本文提出的解决方案 3
实验设备和耗材 4
2.1实验设备和试剂 4
2.1.1仪器和设备 4
2.1.2试剂 5
2.2 培养基和溶液的配制 5
2.2.1 LB（Luria-Bertani）培养基的配制 5
2.2.2 溶液的配制 6
2.2.3 DNA琼脂糖凝胶和蛋白质SDS-PAGE凝胶的制作 8
2.3表达载体的构建 9
2.3.1 pSUMO-Mut-ALB-Hbα和pSUMO-Mut-ALB-Hbβ质粒的构建 9
2.3.2 使用菌株 9
重组质粒的转化 9
3.1 CaCl2感受态细胞的制备 9
3.2 热激法将转化重组质粒 10
3.3质粒的微量提取及其酶切验证 10
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