泡球蚴Em18重组蛋白的诱导表达、纯化和多克隆抗血清的制备和鉴定

发布时间：2019-02-16 06:03

【摘要】：目的：诱导泡球蚴Em18重组蛋白在大肠杆菌中的表达，优化Em18重组蛋白的纯化方法，将纯化的重组蛋白作为抗原，制备泡球蚴Em18抗原的多克隆抗血清，为进一步运用噬菌体表面抗原展示技术，筛选Em18抗原表位奠定基础。方法：将重组表达载体pET41a-Em18转化导入BL21(E．coli)中。经IPTG诱导，对表达产物用SDS-PAGE进行分析，鉴定表达蛋白的大小。采用超声破碎程序破碎重组大肠杆菌细胞，加入蛋白酶抑制剂组合，分别用Glutathione Sepharose-4B亲合层析柱、His-Bind-Ni resin及两者结合方法，改变不同的平衡、洗涤和洗脱缓冲液浓度，纯化rEm18-GST重组蛋白，并筛选和优化了泡球蚴Em18重组蛋白在大肠杆菌中表达和纯化的方法。纯化后的蛋白用SDS-PAGE鉴定。将纯化后的Em18-GST作为抗原免疫BALB／c小鼠。制备鼠源抗Em18多克隆抗体，用ELISA和Western印迹分析方法鉴定其特异性。结果：①SDS-PAGE检测表明，，Em18-GST重组蛋白得到成功表达，在相对分子量为50kDa处有表达条带。②在超声程序为：工作1s，间歇3s，每个循环时程为10min，加入蛋白酶抑制剂组合，可降低目的蛋白的降解，得到高浓度的rEm18-GST可溶性蛋白；③通过对三种纯化方法的对比和对平衡、洗涤和洗脱缓冲液浓度等条件的优化组合，采用单纯His柱，获得回收量高，纯度高的rEm18-GST重组蛋白。④ELISA结果表明，抗Em18抗血清的抗体滴度为1：25600；Western-blot结果表明抗Em18抗血清能与Em18-GST
[Abstract]:Objective: to induce the expression of Em18 recombinant protein in Escherichia coli, optimize the purification method of Em18 recombinant protein, and use the purified recombinant protein as antigen to prepare the polyclonal antiserum of Em18 antigen of hydatid alveolar hydatid. The results laid a foundation for the screening of Em18 epitopes by using phage surface antigen display technique. Methods: the recombinant expression vector pET41a-Em18 was transformed into BL21 (E.coli). After IPTG induction, the expressed product was analyzed by SDS-PAGE, and the size of the expressed protein was identified. Recombinant Escherichia coli cells were broken by ultrasonic fragmentation procedure, and protease inhibitor combination was added to change the balance by Glutathione Sepharose-4B affinity chromatography column, His-Bind-Ni resin and the combination of the two methods, respectively. The rEm18-GST recombinant protein was purified by washing and eluting buffer concentration, and the method of expression and purification of Em18 recombinant protein in E. coli was screened and optimized. The purified protein was identified by SDS-PAGE. Purified Em18-GST was used as antigen to immunize BALB/c mice. Mouse anti-Em18 polyclonal antibodies were prepared and identified by ELISA and Western blot analysis. Results: 1SDS-PAGE analysis showed that the recombinant Em18-GST protein was successfully expressed, and there were bands at the relative molecular weight of 50kDa. 2 in the ultrasound program: 1 s, intermission 3 s, each cycle time was 10 min, and the protease inhibitor combination was added. The degradation of target protein was reduced and high concentration of rEm18-GST soluble protein was obtained. 3 through the comparison of three purification methods and the optimized combination of equilibrium, washing and elution buffer concentration, rEm18-GST recombinant protein with high recovery amount and high purity was obtained by using simple His column. The titer of anti Em18 antiserum was 1: 25600; The results of Western-blot showed that the antiserum against Em18 could be associated with Em18-GST.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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