坦布苏病毒AH-F10株E蛋白的原核表达及荧光定量PCR检测方法的建立

发布时间：2018-11-28 08:38

【摘要】：坦布苏病毒病是由坦布苏病毒(Tembusu virus)引起的,该病能导致蛋鸭的产蛋下降、生长迟缓以及死亡,同时该病毒对肉鸭及蛋鸭都有致病力,鸭子感染后,临床出现高热、沉郁、厌食等症状;蛋鸭、种鸭的产蛋量开始下降而且下降比较迅速直到蛋鸭、种鸭不产蛋;肉鸭出现生长迟缓。坦布苏病毒病的发病率最高可以达100%,死亡率在5%-10%之间。坦布苏病毒的E蛋白含有病毒的抗原决定簇,构成坦布苏病毒的主要抗原表为,而且在病毒的吸附以及病毒侵入宿主细胞的过程中都起着重要的作用。本研究用RT-PCR检测方法来扩增Tembusu的E基因,然后连接到原核表达载体pET-32a(+),转化入大肠杆菌BL21细胞能成功表达重组蛋白His-E。蛋白大小为54kDa,表达的产物以存在与沉淀中,具体是在包涵体中。Western blot结果表明目的蛋白可与坦布苏病毒鸭的阳性血清发生特异性反应,表明表达的重组蛋白具有良好的反应原性。利用包涵体粗纯化的方法得到的高纯度的重组蛋白,用紫外分光吸收法测定其浓度为2.978g/L。对纯化后的目的蛋白进行乳化,乳化的方法是与弗氏佐剂等体积震荡,然后每只BALB/c小鼠注射0.2mg重组蛋白的剂量,进行背部皮下多点注射,每隔两周免疫一次,3免后,可加强免疫,眼眶取血,分离血清。以该血清作为一抗开展IFA实验。结果表明,坦布苏病毒在感染BHK-21细胞36h后,用倒置荧光显微镜可以看到实验孔有特异性的绿色荧光,而对照组细胞无绿色荧光,表明表达纯化的E蛋白具有良好的免疫原性。根据Tembusu virus E基因序列,利用Primer 5.0软件设计1对特异性引物,对E基因进行PCR扩增,然后将纯化回收的产物连接到pMD19-T载体,构建重组质粒pMD-19-E。然后对重组质粒进行PCR、酶切及测序鉴定,将阳性质粒作为模板建立SYBR-Green 1实时荧光定量PCR标准曲线,同时做特异性检测、敏感性检测和重复性检测。经反应条件优化后,建立的坦布苏病毒的SYBR-Green 1荧光定量RT-PCR的标准曲线的线性关系R2均在0.99以上,平均试验间变异系数为0.26%;检测敏感性可达到2×10~1copies/μL。应用该方法对人工感染坦布苏病毒的麻鸭的脑、脾脏、肝脏、胰腺,进行荧光定量RT-PCR检测,结果从36份病料组织中检出35份为阳性,检出率为97%,而常规PCR的检出率为42%。结果表明,成功建立了检测坦布苏病毒的SYBR-Green 1实时荧光定量PCR检测,该方法较常规RT-PCR检测方法更快捷,敏感,准确,适用于坦布苏病毒临床样品的检测。
[Abstract]:Tambusuvirus disease is caused by Tambusuvirus (Tembusu virus), which can cause egg laying down, growth retardation and death in laying ducks, and the virus is virulent to both meat and laying ducks. When ducks are infected, they develop high fever and depression in clinic. Symptoms such as anorexia; Egg production begins to decline and declines rapidly until it does not lay eggs; the meat duck grows slowly. The highest incidence of Tabusu virus can be as high as 100 and the mortality rate is between 5-10%. The E protein of TBV contains the antigenic determinant of the virus, which is the main antigen of TBV, and plays an important role in the adsorption of the virus and the invasion of the host cells by the virus. In this study, the E gene of Tembusu was amplified by RT-PCR, then ligated into prokaryotic expression vector pET-32a (), and transformed into Escherichia coli BL21 cells to express the recombinant protein His-E. successfully. The protein was 54kDa.The expressed product existed and precipitated. The. Western blot results in the inclusion body showed that the target protein could react specifically with the positive serum of Tambusuvirus duck. The results showed that the expressed recombinant protein had good reactivity. The high purity recombinant protein was obtained by the method of crude purification of inclusion body. The concentration of the recombinant protein was 2.978 g / L by UV spectrophotometry. The purified target protein was emulsified by shaking with Freund's adjuvant in volume, then each BALB/c mouse was injected with the dose of 0.2mg recombinant protein. Can enhance immunity, orbital blood, separation of serum. The serum was used as the first antibody to carry out IFA test. The results showed that the specific green fluorescence was observed by inverted fluorescence microscope after infection with BHK-21 cells for 36 hours, but no green fluorescence was found in the control cells. The results showed that the purified E protein had good immunogenicity. According to the sequence of Tembusu virus E gene, a pair of specific primers were designed by using Primer 5.0 software. The E gene was amplified by PCR. Then the purified and recovered product was ligated into pMD19-T vector to construct the recombinant plasmid pMD-19-E.. Then the recombinant plasmid was digested by PCR, and identified by sequencing. The positive plasmid was used as a template to establish the real-time fluorescence quantitative PCR curve of SYBR-Green 1. The specificity, sensitivity and repeatability were also detected. After optimizing the reaction conditions, the linear relationship R2 of the standard RT-PCR curves of SYBR-Green 1 fluorescence quantitative RT-PCR of Tambusuvirus were all above 0.99, the average coefficient of variation between tests was 0.26, and the detection sensitivity could reach 2 脳 10~1copies/ 渭 L. This method was used to detect the brain, spleen, liver and pancreas of duck infected with TBV by fluorescence quantitative RT-PCR. The results showed that 35 of 36 samples were positive and the detection rate was 97%. The positive rate of routine PCR was 42. The results showed that SYBR-Green _ 1 real-time quantitative PCR was successfully established for detection of Tambusuvirus. The method was more rapid, sensitive and accurate than the conventional RT-PCR detection method, and was suitable for clinical detection of Tambusuvirus.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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