大规模PCR制备线性流感DNA疫苗及其免疫效果的研究

发布时间：2018-06-06 06:39

本文选题：耐热DNA聚合酶 + 毕赤酵母　；参考：《湖北大学》2016年博士论文

【摘要】：流感是由A型禽流感病毒引发的烈性禽类传染病,至今这类传染病已经传播到世界各地。疫苗免疫是目前禽流感防控的最有效的一种手段,由于流感病毒的包膜糖蛋血凝素和神经氨酸酶HA很容易因为转变或者漂变而发生改变,从而使得病毒能够逃脱机体的天然免疫和当前疫苗诱发的保护,所以需要我们随时更新新疫苗来应对流感可能的爆发。要想成功的阻止高传染性的禽流感病毒的大范围蔓延,我们需要在短时间内生产出大量的疫苗来接种。然而传统的疫苗因为生产工艺的原因很难做到这一点。所以想要依靠传统的疫苗来应对高致病性禽流感可能潜在的爆发是不可能的。有研究表明,用表达禽流感HA基因的线性合适结构(LEC)DNA免疫小鼠,能有效的保护小鼠免于致死剂量流感病毒的攻击。由于不仅具有良好的保护效果,并且设计,制备方法简单,未来线性DNA疫苗极有可能替代传统疫苗。目前生产线性DNA疫苗最高效可靠的方法是PCR,然而常规PCR的规模及成本成为限制通过PCR大规模制备线性DNA疫苗的主要因素。耐热DNA聚合酶昂贵的价格是抬高PCR成本的主要因素。为了解决这个难题我们试图通过毕赤酵母的分泌表达来降低DNA聚合酶的成本。我们根据毕赤酵母的密码子偏爱性优化合成了高保真DNA聚合酶KOD基因,并将它和DNA双链结合蛋白Sso融合在一起,然后克隆到毕赤酵母表达载体pHBM905A上,转化至毕赤酵母来进行表达。本研究中我们成功的实现了高保真的耐热DNA聚合酶RKOD在毕赤酵母中的分泌表达,表达量高达250mg/l,纯化之后酶活达到了 19,384U/mg。我们简化了耐热DNA聚合酶的纯化步骤,缩短了工艺流程,极大的降低了 DNA聚合酶的生产成本。在此基础上我们探索出了用简易的水浴设备进行大体系PCR的方法,并成功的实现了 10-1OOml体系的PCR,并用该方法大量制备了禽流感HIN1的线性DNA疫苗。并通过小鼠模型检验了其免疫效果。实验结果表明通过本研究中通过大规模PCR的方法制备的线性流感疫苗能够有效地保护小鼠免于流感病毒的攻击。其中经过硫代修饰的PTO-LEC-HA疫苗对小鼠产生了 100%的保护效果。综上所述,我们成功的建立起了一个通过大规模PCR制备线性DNA疫苗的方法。本研究为快速、大规模制备廉价的DNA疫苗以迅速应对新发传染病提供了一种新的策略。
[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.
【学位授予单位】：湖北大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.4

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