3C蛋白酶表达、纯化及其活性抑制的研究

发布时间：2018-05-12 12:41

本文选题：3C蛋白酶 + 表达　；参考：《昆明医学院》2008年硕士论文

【摘要】： 3C蛋白酶是催化小RNA病毒前体蛋白中非结构蛋白裂解的关键蛋白酶,具有高度的酶切特异性,其在病毒的生命周期中,起着举足轻重的作用,抑制其催化功能可有效抑制病毒前体蛋白的切割,阻断病毒复制,是小RNA病毒药物治疗研究的靶点之一。小RNA病毒科是已知最小的动物RNA病毒,共有7个属,是引起人类严重疾病的病原体,所以对3C蛋白酶的研究为对这些疾病的预防和治疗提供了科学依据和理论基础。3C蛋白酶的识别位点为Leu Glu Val Leu Phe Gln↓GlyPro,而切割位点在Gln—G1y之间,称为Q—G位点。鉴于此我们构想通过基因工程的方法得到重组3C蛋白酶,利用其切割的特异性在体外验证重组3C蛋白酶的活性,从而为以后开发新型的基因工程工具酶奠定基础,并且通过动物实验得到3C蛋白酶抗体,应用抗体技术抑制3C蛋白酶活性,进而达到抑制小RNA病毒科病毒复制的目的。我们克隆到人鼻病毒14型的3C蛋白酶的基因编码区,将其克隆到表达载体pBV220上。经转化表达宿主菌BL-21,获得阳性克隆,通过温度诱导实现了重组3C蛋白酶在大肠杆菌中的高效、稳定表达,并经纯化获得纯度达90%以上的3C蛋白酶;另一方面,我们表达、纯化了含3C蛋白酶切点的融合蛋白白细胞介素—11,作为3C蛋白酶的靶蛋白,以检测其活性。结果,我们表达的重组3C蛋白酶在体外能够将融合蛋白白细胞介素-11的硫氧还蛋白融合头切下,表明我们得到的3C蛋白酶在体外具有酶活性。随后,我们将得到的3C蛋白酶免疫豚鼠,得到3C蛋白酶抗体,培养Vero细胞,检验应用3C蛋白酶抗体抑制3C蛋白酶活性,以阻断小RNA病毒科病毒复制的效果,初步实验没有得到明显的阳性结果。我们希望通过这次实验探索可以为临床治疗小RNA病毒科病毒引起的多种疾病提供有效的治疗思路。另外,冠状病毒属病毒的3C样蛋白酶和3C蛋白酶在序列和空间构象上具有一定的同源性,对3C蛋白酶活性的研究和探索有助于我们了解和研究3C样蛋白酶,进而为治疗SARS等由冠状病毒引起的疾病提供了科学依据和理论基础。
[Abstract]:3C protease is the key protease to catalyze the cleavage of non-structural protein of the precursor protein of small RNA virus. It has a high enzyme specificity and plays an important role in the life cycle of the virus. Inhibition of its catalytic function can effectively inhibit the cleavage of virus precursor protein and block virus replication, which is one of the targets of drug therapy for small RNA virus. The small RNA virus family is the smallest known animal RNA virus. It has 7 genera and is the pathogen causing serious diseases in human beings. Therefore, the study of 3C protease provides a scientific and theoretical basis for the prevention and treatment of these diseases. The recognition site of 3C protease is Leu Glu Val Leu Phe Gln GlyPro.And the cleavage site is between Gln-G1y, called Q-G locus. In view of this, we have conceived to obtain recombinant 3C protease by genetic engineering, and to verify the activity of recombinant 3C protease in vitro by using its cleavage specificity, thus laying a foundation for the future development of novel genetic engineering tool enzymes. The antibody of 3C protease was obtained by animal experiment, and the activity of 3C protease was inhibited by antibody technique, and then the replication of small RNA virus was inhibited. We cloned the encoding region of human rhinovirus type 14 3C protease and cloned it into the expression vector pBV220. After transformation and expression of host strain BL-21, positive clones were obtained. The recombinant 3C protease was expressed efficiently and stably in Escherichia coli by temperature induction. After purification, 3C protease with purity of more than 90% was obtained. On the other hand, we expressed 3C protease. Interleukin-11, a fusion protein containing 3C protease, was purified as the target protein of 3C protease for detection of its activity. The results showed that our recombinant 3C protease could cut off the thioredoxin fusion head of the fusion protein interleukin-11 in vitro, which indicated that our recombinant 3C protease had enzyme activity in vitro. After that, we immunized guinea pigs with 3C protease, obtained 3C protease antibody, cultured Vero cells, and tested the effect of using 3C protease antibody to inhibit 3C protease activity in order to block the replication of small RNA virus. No obvious positive results were obtained in the preliminary test. We hope that this experiment can provide an effective way for clinical treatment of various diseases caused by small RNA virus. In addition, 3C like protease and 3C protease of coronavirus have some homology in sequence and spatial conformation. The study of 3C protease activity is helpful for us to understand and study 3C like protease. It provides a scientific basis and theoretical basis for the treatment of diseases caused by coronavirus such as SARS.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

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