本文選題：3C蛋白酶 + 表達(dá)��； 參考：《昆明醫(yī)學(xué)院》2008年碩士論文

【摘要】： 3C蛋白酶是催化小RNA病毒前體蛋白中非結(jié)構(gòu)蛋白裂解的關(guān)鍵蛋白酶,具有高度的酶切特異性,其在病毒的生命周期中,起著舉足輕重的作用,抑制其催化功能可有效抑制病毒前體蛋白的切割,阻斷病毒復(fù)制,是小RNA病毒藥物治療研究的靶點(diǎn)之一。小RNA病毒科是已知最小的動(dòng)物RNA病毒,共有7個(gè)屬,是引起人類嚴(yán)重疾病的病原體,所以對(duì)3C蛋白酶的研究為對(duì)這些疾病的預(yù)防和治療提供了科學(xué)依據(jù)和理論基礎(chǔ)。3C蛋白酶的識(shí)別位點(diǎn)為L(zhǎng)eu Glu Val Leu Phe Gln↓GlyPro,而切割位點(diǎn)在Gln—G1y之間,稱為Q—G位點(diǎn)。鑒于此我們構(gòu)想通過(guò)基因工程的方法得到重組3C蛋白酶,利用其切割的特異性在體外驗(yàn)證重組3C蛋白酶的活性,從而為以后開(kāi)發(fā)新型的基因工程工具酶奠定基礎(chǔ),并且通過(guò)動(dòng)物實(shí)驗(yàn)得到3C蛋白酶抗體,應(yīng)用抗體技術(shù)抑制3C蛋白酶活性,進(jìn)而達(dá)到抑制小RNA病毒科病毒復(fù)制的目的。 我們克隆到人鼻病毒14型的3C蛋白酶的基因編碼區(qū),將其克隆到表達(dá)載體pBV220上。經(jīng)轉(zhuǎn)化表達(dá)宿主菌BL-21,獲得陽(yáng)性克隆,通過(guò)溫度誘導(dǎo)實(shí)現(xiàn)了重組3C蛋白酶在大腸桿菌中的高效、穩(wěn)定表達(dá),并經(jīng)純化獲得純度達(dá)90%以上的3C蛋白酶;另一方面,我們表達(dá)、純化了含3C蛋白酶切點(diǎn)的融合蛋白白細(xì)胞介素—11,作為3C蛋白酶的靶蛋白,以檢測(cè)其活性。結(jié)果,我們表達(dá)的重組3C蛋白酶在體外能夠?qū)⑷诤系鞍装准?xì)胞介素-11的硫氧還蛋白融合頭切下,表明我們得到的3C蛋白酶在體外具有酶活性。隨后,我們將得到的3C蛋白酶免疫豚鼠,得到3C蛋白酶抗體,培養(yǎng)Vero細(xì)胞,檢驗(yàn)應(yīng)用3C蛋白酶抗體抑制3C蛋白酶活性,以阻斷小RNA病毒科病毒復(fù)制的效果,初步實(shí)驗(yàn)沒(méi)有得到明顯的陽(yáng)性結(jié)果。我們希望通過(guò)這次實(shí)驗(yàn)探索可以為臨床治療小RNA病毒科病毒引起的多種疾病提供有效的治療思路。另外,冠狀病毒屬病毒的3C樣蛋白酶和3C蛋白酶在序列和空間構(gòu)象上具有一定的同源性,對(duì)3C蛋白酶活性的研究和探索有助于我們了解和研究3C樣蛋白酶,進(jìn)而為治療SARS等由冠狀病毒引起的疾病提供了科學(xué)依據(jù)和理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R346

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