日本血吸虫重组蛋白rSjGST的表达纯化及单克隆抗体的制备和特性鉴定

发布时间：2018-05-09 16:24

本文选题：日本血吸虫 + SjGST蛋白　；参考：《安徽医科大学》2011年硕士论文

【摘要】：目的获取高纯度的日本血吸虫重组GST蛋白( rSjGST) ,并制备出该蛋白的单克隆抗体,并对其相关的生物学特性进行鉴定,建立日本血吸虫病患者GST蛋白水平检测方法。方法将含有重组质粒pET28a-SjGST的工程菌BL21进行培养并进行IPTG诱导表达后,破碎大肠杆菌菌体并利用His-tag亲和层析和高效液相色谱(HPLC)纯化出rSjGST蛋白,纯化出的目的蛋白使用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)以及免疫印迹技术进行鉴定。之后采用B淋巴细胞杂交瘤技术,以高纯度的rSjGST蛋白免疫Balb/c雌性小鼠,常规聚乙二醇介导融合,以间接ELISA方法筛选阳性克隆,有限稀释法亚克隆筛选单克隆细胞株,并使用秋水仙素对其核型进行鉴定,使用ISO-2鼠源单克隆抗体亚型检测试剂盒检测其亚类,并检测其效价、特异性等。建立以双抗夹心法和间接ELISA法检测血清GST水平的方法,并探讨该方法的精密度、准确性和特异性等。结果纯化出大量的高纯度rSjGST蛋白,并且筛选出能够稳定分泌抗rSjGST单抗的杂交瘤细胞株1B11和3C12。用试剂盒检测出此单抗的亚型为IgG2b和IgG1。结论依靠大肠杆菌表达系统,本实验高效表达出了rSjGST蛋白,并以此rSjGST蛋白制备出了单克隆抗体1B11和3C12,所建立的双抗体夹心ELISA检测血清rSjGST蛋白水平的方法为今后的血吸虫病免疫诊断提供了研究基础,这项诊断技术同样可以应用于临床日本血吸虫病的血清检测。
[Abstract]:Objective to obtain the recombinant GST protein (rSjGST), from Schistosoma japonicum and prepare its monoclonal antibody, and identify its biological characteristics, and establish a method for the detection of GST protein level in schistosomiasis japonicum patients. Methods the engineering strain BL21 containing recombinant plasmid pET28a-SjGST was cultured and induced by IPTG, then the Escherichia coli cells were broken and purified by His-tag affinity chromatography and high performance liquid chromatography (HPLC). The purified protein was identified by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. B lymphocyte hybridoma technique was used to immunize Balb/c female mice with high purity rSjGST protein. The positive clones were screened by indirect ELISA method and monoclonal cell lines were screened by limited dilution subcloning. Colchicine was used to identify its karyotype and ISO-2 mouse monoclonal antibody subtype detection kit was used to detect its subclass and its titer and specificity were detected. To establish a double antibody sandwich method and indirect ELISA method to detect serum GST levels, and to explore the precision, accuracy and specificity of the method. Results A large number of high purity rSjGST proteins were purified, and hybridoma cell lines 1B11 and 3C12, which could stably secrete anti rSjGST monoclonal antibodies, were screened. The subtypes of the McAb were identified as IgG2b and IgG1 by the kit. Conclusion the rSjGST protein was highly expressed by E. coli expression system. The monoclonal antibodies 1B11 and 3C12 were prepared by using this rSjGST protein. The double antibody sandwich ELISA method for detecting the level of serum rSjGST protein provided a basis for the future diagnosis of schistosomiasis. This diagnostic technique can also be used for serum detection of clinical schistosomiasis japonicum.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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