大鼠DAF功能区的重组表达纯化和功能鉴定

发布时间：2018-01-14 02:06

本文关键词：大鼠DAF功能区的重组表达纯化和功能鉴定　出处：《第四军医大学》2006年硕士论文　论文类型：学位论文

【摘要】：补体激活造是炎症反应性疾病的病理反应之一。如何能够控制补体的激活是当前科学研究的一个热点。促衰变因子(DAF)属膜补体调节蛋白，，是一种锚定于细胞膜表面的蛋白质。可以促进经典、旁路途径C3、C5转化酶的解离并防止其组装，这对其宿主细胞起到了重要的保护作用。可溶性DAF的表达在过去已经有过非常多的尝试，但多为带有真核细胞分泌信号肽的方式表达。由于DAF有4个短的保守重复序列(SCR)，含有多达8对二硫键，原核核细胞表达都以包涵体告终。本研究拟采用大肠杆菌表达Trx融合蛋白的方式来表达大鼠DAF的4个SCR，以期得到可溶性的表达产物。首先通过PCR的方法，将编码大鼠DAF四个SCR的基因片段亚克隆到Trx融合原核表达载体pET32a(+)。再将构建好的载体转入大肠杆菌表达宿主BL21(DE3)pLysS。通过比较3种不同的诱导温度和IPTG浓度，经Western Blot检测，发现低温诱导确实能够增加可溶性蛋白的量，但效果并不是很明显。通过Ni~(2+)亲和层析分离出了可溶性组分，经Western Blot检测，进一步确认了该可溶性成分确实为带有His标签的融合蛋白。按标准的37℃，1mmol／L IPTG诱导4h的方案，得到了大量包涵体。通过包涵体复性，得到了具有抑制致敏绵羊红细胞发生溶血的蛋白。综上所述，本研究采用的Trx融合蛋白的方式表达DAF的4个SCR
[Abstract]:Complement activation is one of the pathological reactions of inflammatory response diseases. How to control the activation of complement is a hot spot in the current scientific research.
Decay accelerating factor (DAF) is a membrane complement regulatory protein, is a membrane anchored to the cell surface. The protein can promote the classical pathway, C3, dissociation of the C5 convertase and prevent their formation, which is to protect the important role of the host cell. The expression of soluble DAF in the past has been try very much, but is a eukaryotic secretory signal peptide expression. Because DAF has 4 short consensus repeat (SCR), containing up to 8 to two disulfide bonds, the prokaryotic cells have ended in inclusion. This study used Escherichia coli Trx fusion protein expression to 4 SCR expression of DAF in rats, in order to obtain the soluble expression product.
First, the gene fragment encoding four SCR of rat DAF was subcloned into Trx fusion prokaryotic expression vector pET32a (+) by PCR method. Then the constructed vector was transferred into Escherichia coli to express host BL21 (DE3) pLysS..
Through the comparison of 3 different induction temperature and IPTG concentration by Western Blot detection found induced by low temperature can increase the soluble protein content, but the effect is not very obvious. The Ni~ (2+) affinity chromatography to isolate the soluble components by Western Blot detection, further confirmed the soluble components indeed a fusion protein with His tag.
A large number of inclusion bodies were obtained according to the standard 37 degree temperature, 1mmol / L IPTG and 4H induction. A lot of inclusion bodies were obtained by inclusion body renaturation, and a protein that inhibited hemolysis of sensitized sheep erythrocytes was obtained.
To sum up, 4 SCR of DAF were expressed by the Trx fusion protein used in this study.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R363

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