本文選題：丙型肝炎病毒　切入點(diǎn)：Core蛋白　出處：《西北大學(xué)》2006年碩士論文

【摘要】：丙型肝炎病毒(Hepatitis C virus,HCV)感染是導(dǎo)致慢性肝炎、肝硬化和肝癌的重要原因之一。目前尚無(wú)有效的疫苗及治療藥物,對(duì)HCV感染的早期診斷是控制HCV傳播的重要手段。 HCV感染的臨床診斷途徑有:檢測(cè)血清HCV RNA、HCV抗體及HCV抗原。目前PCR檢測(cè)HCV RNA是HCV診斷最靈敏、特異的方法,但其操作復(fù)雜,易造成污染,且價(jià)格昂貴,不適于大規(guī)模的篩查應(yīng)用。HCV患者血清中HCV抗原水平很低,常規(guī)免疫學(xué)方法難以獲得陽(yáng)性結(jié)果。故HCV診斷最常用的方法是利用HCV特異性蛋白質(zhì)抗原來(lái)篩查血清中HCV抗體。 HCV核心蛋白(Core蛋白)高度保守且有很強(qiáng)的免疫原性,可誘發(fā)高水平的特異性體液免疫及細(xì)胞免疫,是HCV診斷試劑中必不可少的組分之一。近年來(lái)Core蛋白先后在大腸桿菌中獲得了表達(dá),但是大腸桿菌不具有真核生物的基因表達(dá)調(diào)控機(jī)制和蛋白質(zhì)的加工修飾能力,表達(dá)的蛋白往往形成的包涵體,需要經(jīng)過(guò)變性、復(fù)性等處理才能應(yīng)用。與大腸桿菌相比,畢赤酵母是單細(xì)胞真核生物,既具有生長(zhǎng)快的特點(diǎn),又能有效克服大腸桿菌系統(tǒng)缺乏蛋白翻譯后加工、修飾的不足,表達(dá)外源蛋白可分泌到胞外,便于產(chǎn)品的分離純化。因此本研究在大腸桿菌系統(tǒng)中表達(dá)Core蛋白并構(gòu)建其畢赤酵母表達(dá)系統(tǒng),初步對(duì)兩個(gè)系統(tǒng)表達(dá)的Core蛋白進(jìn)行對(duì)比。 將克隆有Core基因的原核表達(dá)載體pBVIL1-Core轉(zhuǎn)化大腸桿菌HB101,溫度誘導(dǎo)表達(dá)Core蛋白,進(jìn)行SDS-PAGE及Western-Blot分析;同時(shí)通過(guò)PCR方法擴(kuò)增Core基因,克隆入表達(dá)載體pPICZaA,篩選陽(yáng)性克隆pPICZaA-Core。將經(jīng)過(guò)酶切鑒定和測(cè)序正確的陽(yáng)性克隆電轉(zhuǎn)入畢赤酵母GS115中,挑取酵母轉(zhuǎn)化單菌落,接種于BMGY培養(yǎng)基,30℃、250r/min培養(yǎng)至OD_(600)=2.0時(shí)室溫1,500r/min離心5min,用BMMY培養(yǎng)基重懸菌體使OD_(600)=1.0,重新放入搖床進(jìn)行誘導(dǎo)。每24h補(bǔ)加甲醇至終濃度為1.0%,連續(xù)誘導(dǎo)72h,誘導(dǎo)后上清進(jìn)行Western-Blot分析。 結(jié)果顯示pBVIL1-Core的表達(dá)產(chǎn)物經(jīng)SDS-PAGE分析出現(xiàn)一條約31KD的帶,與預(yù)期融合蛋白的分子量相符,表達(dá)蛋白存在于包涵體中且表達(dá)量占菌體總蛋白的20%,Western-blot顯示誘導(dǎo)后菌體在相應(yīng)位置出現(xiàn)特異性雜交帶,表明
[Abstract]:Hepatitis C virus (HCV) infection is one of the important causes of chronic hepatitis, liver cirrhosis and liver cancer.At present, there are no effective vaccines and therapeutic drugs. The early diagnosis of HCV infection is an important means to control the spread of HCV.The methods of clinical diagnosis of HCV infection were as follows: detection of serum HCV RNA antibody and HCV antigen.At present, PCR detection of HCV RNA is the most sensitive and specific method for the diagnosis of HCV, but its operation is complex, easy to cause pollution, and expensive, so it is not suitable for large-scale screening application. The level of HCV antigen in serum of patients with HCV is very low.It is difficult to obtain positive results by routine immunological methods.Therefore, the most commonly used method for the diagnosis of HCV is to screen HCV antibody in serum by HCV specific protein.HCV core protein is highly conserved and has strong immunogenicity, which can induce high level of specific humoral immunity and cellular immunity. It is an essential component of HCV diagnostic reagents.In recent years, Core protein has been expressed in E. coli, but E. coli does not have eukaryotic gene expression regulation mechanism and protein processing modification ability, the expressed protein often forms inclusion body, which needs to be denatured.Renaturation and other treatment can be applied.Compared with Escherichia coli, Pichia pastoris is a single cell eukaryote, which has the characteristics of fast growth and can effectively overcome the deficiency of protein translation and modification in Escherichia coli system. The expression of exogenous protein can be secreted out of the cell.It is easy to separate and purify the product.In this study, Core protein was expressed in Escherichia coli and Pichia pastoris expression system was constructed, and the two Core proteins were compared.The prokaryotic expression vector pBVIL1-Core with Core gene was transformed into Escherichia coli HB101, and the Core protein was expressed by temperature induction. The Core gene was amplified by PCR method and cloned into the expression vector pPICZaA to screen the positive clone pPICZaA-Core.The positive clones identified by enzyme digestion and sequenced were transferred into Pichia pastoris GS115.At room temperature, 1500r/min was centrifuged for 5 mins at room temperature when inoculated on BMGY medium at 30 鈩，

本文編號(hào)：1722430