PRRSV的GP5和Nsp7原核表达及ELISA检测方法的建立

发布时间：2018-04-20 03:20

本文选题：猪繁殖与呼吸综合征 + GP5蛋白　；参考：《吉林农业大学》2015年硕士论文

【摘要】：猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是一种病毒性传染病,它的传播给全球养猪业带来的经济损失是不可估量的。如何有效的预防和治疗该病是各国研究的热点,比较有效的检测方法包括RT-PCR、ELISA检测等,ELISA检测快速、有效的优点使其能够广泛的应用。接种灭活疫苗是我国预防该病的主要方法,但是单一的结构蛋白或单一的非结构蛋白的ELISA检测不能够判断抗体的产生是由灭活疫苗引起还是感染野毒引起的,这就会影响该病的检测结果,如何建立更加准确、有效的ELISA检测方法是目前的当务之急。GP5蛋白是检测PRRSV抗体的主要靶蛋白,Nsp7基因在非结构蛋白中相对保守,不同毒株之间差异较小,它的这个特点可以使其在检测不同毒株刺激机体产生的抗体时保持较高的特异性。接种灭活疫苗和感染PRRSV都可以刺激机体产生针对结构蛋白的抗体,所以只用结构蛋白作检测抗原会影响检测结果的准确性。本研究在单一结构蛋白ELISA检测的基础上增加一种非结构蛋白以达到能准确判断抗体来源的ELISA检测方法,利用PCR技术,从PRRSV分离株中扩增出GP5和Nsp7基因,将目的基因与表达载体pET28a连接,将构建的重组表达载体pET28a-GP5、pET28a-Nsp7转化到表达菌株BL21(DE3)中,测序鉴定。经IPTG诱导表达,表达产物再进行SDS-PAGE分析。结果表明成功表达了GP5蛋白和Nsp7蛋白,表达蛋白经His Trap FF纯化柱纯化,然后进行Western blot分析,证明表达的重组蛋白均具有良好的抗原性。优化ELISA检测的各种条件,成功建立GP5-Nsp7-ELISA检测方法。
[Abstract]:Porcine reproductive and respiratory syndrome (PRRSs) is a viral infectious disease, and the economic loss caused by its spread to the global pig industry is incalculable. How to prevent and treat the disease effectively is a hot topic in many countries. The more effective detection methods include RT-PCR- Elisa and so on. The advantages of rapid and effective Elisa make it widely used. Inoculation with inactivated vaccine is the main method to prevent the disease in China, but the ELISA detection of single structural protein or single non-structural protein can not determine whether the production of antibody is caused by inactivated vaccine or infected wild virus. This will affect the detection results of the disease, how to establish a more accurate, effective ELISA detection method is the most urgent task at present. GP5 protein is the main target protein for detecting PRRSV antibody, Nsp7 gene is relatively conservative in non-structural proteins. The difference between different strains is small, which makes it more specific in detecting antibodies produced by different strains. Inoculation of inactivated vaccine and infection of PRRSV can stimulate the body to produce antibodies against structural proteins, so using structural proteins as antigen will affect the accuracy of detection results. On the basis of ELISA detection of single structural protein, a non-structural protein was added to determine the origin of antibody by ELISA detection method. GP5 and Nsp7 genes were amplified from PRRSV isolate by PCR technique. The target gene was ligated with the expression vector pET28a and the recombinant expression vector pET28a-GP5pET28a-Nsp7 was transformed into the expression strain BL21DDE3 and sequenced. The expression was induced by IPTG, and the expression product was analyzed by SDS-PAGE. The results showed that GP5 protein and Nsp7 protein were successfully expressed. The expressed proteins were purified by His Trap FF column and then analyzed by Western blot. The results showed that the expressed recombinant proteins had good antigenicity. The conditions of ELISA detection were optimized and the GP5-Nsp7-ELISA detection method was established successfully.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

【共引文献】