發(fā)布時(shí)間：2018-06-06 06:39

  本文選題：耐熱DNA聚合酶 + 畢赤酵母��； 參考：《湖北大學(xué)》2016年博士論文

【摘要】：流感是由A型禽流感病毒引發(fā)的烈性禽類傳染病,至今這類傳染病已經(jīng)傳播到世界各地。疫苗免疫是目前禽流感防控的最有效的一種手段,由于流感病毒的包膜糖蛋血凝素和神經(jīng)氨酸酶HA很容易因?yàn)檗D(zhuǎn)變或者漂變而發(fā)生改變,從而使得病毒能夠逃脫機(jī)體的天然免疫和當(dāng)前疫苗誘發(fā)的保護(hù),所以需要我們隨時(shí)更新新疫苗來應(yīng)對(duì)流感可能的爆發(fā)。要想成功的阻止高傳染性的禽流感病毒的大范圍蔓延,我們需要在短時(shí)間內(nèi)生產(chǎn)出大量的疫苗來接種。然而傳統(tǒng)的疫苗因?yàn)樯a(chǎn)工藝的原因很難做到這一點(diǎn)。所以想要依靠傳統(tǒng)的疫苗來應(yīng)對(duì)高致病性禽流感可能潛在的爆發(fā)是不可能的。有研究表明,用表達(dá)禽流感HA基因的線性合適結(jié)構(gòu)(LEC)DNA免疫小鼠,能有效的保護(hù)小鼠免于致死劑量流感病毒的攻擊。由于不僅具有良好的保護(hù)效果,并且設(shè)計(jì),制備方法簡(jiǎn)單,未來線性DNA疫苗極有可能替代傳統(tǒng)疫苗。目前生產(chǎn)線性DNA疫苗最高效可靠的方法是PCR,然而常規(guī)PCR的規(guī)模及成本成為限制通過PCR大規(guī)模制備線性DNA疫苗的主要因素。耐熱DNA聚合酶昂貴的價(jià)格是抬高PCR成本的主要因素。為了解決這個(gè)難題我們?cè)噲D通過畢赤酵母的分泌表達(dá)來降低DNA聚合酶的成本。我們根據(jù)畢赤酵母的密碼子偏愛性優(yōu)化合成了高保真DNA聚合酶KOD基因,并將它和DNA雙鏈結(jié)合蛋白Sso融合在一起,然后克隆到畢赤酵母表達(dá)載體pHBM905A上,轉(zhuǎn)化至畢赤酵母來進(jìn)行表達(dá)。本研究中我們成功的實(shí)現(xiàn)了高保真的耐熱DNA聚合酶RKOD在畢赤酵母中的分泌表達(dá),表達(dá)量高達(dá)250mg/l,純化之后酶活達(dá)到了 19,384U/mg。我們簡(jiǎn)化了耐熱DNA聚合酶的純化步驟,縮短了工藝流程,極大的降低了 DNA聚合酶的生產(chǎn)成本。在此基礎(chǔ)上我們探索出了用簡(jiǎn)易的水浴設(shè)備進(jìn)行大體系PCR的方法,并成功的實(shí)現(xiàn)了 10-1OOml體系的PCR,并用該方法大量制備了禽流感HIN1的線性DNA疫苗。并通過小鼠模型檢驗(yàn)了其免疫效果。實(shí)驗(yàn)結(jié)果表明通過本研究中通過大規(guī)模PCR的方法制備的線性流感疫苗能夠有效地保護(hù)小鼠免于流感病毒的攻擊。其中經(jīng)過硫代修飾的PTO-LEC-HA疫苗對(duì)小鼠產(chǎn)生了 100%的保護(hù)效果。綜上所述,我們成功的建立起了一個(gè)通過大規(guī)模PCR制備線性DNA疫苗的方法。本研究為快速、大規(guī)模制備廉價(jià)的DNA疫苗以迅速應(yīng)對(duì)新發(fā)傳染病提供了一種新的策略。
[Abstract]:Influenza is a severe avian infectious disease caused by avian influenza A virus, which has spread all over the world. Vaccine immunization is one of the most effective methods to prevent and control avian influenza at present, because the encapsulated glycosylated egg hemagglutinin and neuraminidase HA of influenza virus are easily changed by transformation or bleaching. This allows the virus to escape innate immunity and current vaccine-induced protection, so we need to update new vaccines to respond to possible outbreaks of influenza. To successfully stop the spread of the highly infectious avian influenza virus, we need to produce a large number of vaccines in a short time. Traditional vaccines, however, are difficult to do because of the manufacturing process. So it is impossible to rely on traditional vaccines for possible potential outbreaks of highly pathogenic avian influenza. Some studies have shown that mice immunized with LEC-DNA, a linear and appropriate structure expressing HA gene of avian influenza, can effectively protect mice from lethal dose of influenza virus. Because of its good protective effect and simple design and preparation, the linear DNA vaccine is likely to replace the traditional vaccine in the future. At present, the most effective and reliable method to produce linear DNA vaccine is PCR.However, the scale and cost of conventional PCR are the main factors restricting the large-scale production of linear DNA vaccine through PCR. The high price of heat resistant DNA polymerase is the main factor that increases the cost of PCR. In order to solve this problem, we try to reduce the cost of DNA polymerase by secretory expression of Pichia pastoris. According to the codon preference of Pichia pastoris, we synthesized the high-fidelity DNA polymerase KOD gene, fused it with DNA double-stranded binding protein Sso, and cloned it into Pichia pastoris expression vector pHBM905A. Transformed to Pichia pastoris for expression. In this study, we successfully realized the secreting expression of high fidelity heat-resistant DNA polymerase RKOD in Pichia pastoris, and the expression amount was as high as 250 mg / l. After purification, the enzyme activity reached 19384 U / mg. We simplify the purification process of heat-resistant DNA polymerase, shorten the process and greatly reduce the production cost of DNA polymerase. On the basis of this, we explored the method of using simple water bath equipment to carry out large-scale PCR, and successfully realized the 10-1OOml system. By using this method, we prepared a large number of linear DNA vaccine of avian influenza HIN1. The immune effect was tested by mouse model. The results show that the linear influenza vaccine prepared by large-scale PCR in this study can effectively protect mice from influenza virus attack. The thiothioate modified PTO-LEC-HA vaccine had a 100% protective effect on mice. In conclusion, we have successfully established a method for preparing linear DNA vaccine by large-scale PCR. This study provides a new strategy for rapid and large-scale preparation of cheap DNA vaccines for rapid response to emerging infectious diseases.
【學(xué)位授予單位】：湖北大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.4

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本文編號(hào)：1985618