抗GST标签蛋白单克隆抗体的制备及其活性鉴定

发布时间：2018-02-12 15:24

本文关键词： 单克隆抗体谷胱甘肽S-转移酶亲和层析原核表达　出处：《华中农业大学》2008年硕士论文　论文类型：学位论文

【摘要】： 在基因工程中,蛋白质的表达经常用到各种融合表达系统。GST基因融合表达体系具有蛋白表达产率高、表达产物纯化方便以及利于抗体制备等优点,得到越来越广泛的应用。用GST融合表达系统表达外源基因时,对融合表达产物的检测和纯化非常重要。目前GST基因融合表达系统表达的融合蛋白均使用市售的特定的亲和层析系统纯化如Glutathione Sepharose-4B亲和层析柱,Glutathione亲和柱存在价格昂贵、使用寿命有限、不适合大规模应用等不足。为此,本试验拟制备抗GST标签蛋白的特异性单克隆抗体,为利用单抗建立亲和层析、免疫磁珠等表达产物大规模提纯系统的研制做好前期工作,研究结果如下。 1.抗GST单克隆抗体的研制用含pGEX质粒的表达菌BL21表达,并经Glutathione Sepharose-4B亲和层析柱纯化得到高纯度的GST标签蛋白,用纯化的GST蛋白免疫Balb/c小鼠,制备免疫脾细胞,通过常规的细胞融合技术与SP2/0骨髓瘤细胞融合;用相同的纯化的GST蛋白进行筛选,所得的阳性杂交瘤细胞株,采用有限稀释法进行了克隆,建立了2株能持续分泌抗GST蛋白的McAb的杂交瘤细胞株3E和10G。 2.GST单克隆抗体的特性鉴定 3E和10G株单抗腹水与GST蛋白反应的间接ELISA效价均在1×10~6以上,鉴定其亚类类型均为IgG1,染色体数目为80～94条。10G株细胞腹水与含GST标签的融合蛋白(AIV-NS1、IBDV-VP2、IBV-NP)的反应性都很低,而3E株细胞腹水与三种融合蛋白反应的间接ELISA效价均达到2×10~5以上。 3.抗GST单克隆抗体的初步应用用辛酸-硫酸铵法纯化出3E株细胞的腹水单抗,测得纯化后单抗对GST蛋白和IBDV-VP2蛋白的效价分别为1.28×10~5和6.4×10~4。而且3E株细胞单抗对GST蛋白和IBDV-VP2蛋白的相对亲和力差别不大。3E株细胞单抗可用于GST融合蛋白的检测,以及后续的利用单抗作为配基来纯化GST融合蛋白。
[Abstract]:In gene engineering, various fusion expression systems. GST gene fusion expression system has the advantages of high protein expression yield, easy purification of expression products and easy preparation of antibodies. When using GST fusion expression system to express foreign gene, It is very important to detect and purify the fusion expression products. At present, the fusion proteins expressed by the fusion expression system of GST gene are all purified by specific affinity chromatography system sold on the market, such as Glutathione Sepharose-4B affinity chromatography column Glutathione affinity column. Because of its limited service life, it is not suitable for large-scale application and so on. Therefore, we intend to prepare specific monoclonal antibodies against GST tagged proteins to establish affinity chromatography by using McAbs. The large scale purification system of immunomagnetic beads and other expression products has been developed and the results are as follows. 1. Development of monoclonal antibody against GST. High purity GST tag protein was obtained by BL21 expression with pGEX plasmid and purified by Glutathione Sepharose-4B affinity chromatography. Balb/c mice were immunized with purified GST protein to prepare spleen cells. SP2/0 myeloma cells were fused with conventional cell fusion techniques, and the positive hybridoma cell lines were cloned by using the same purified GST protein. Two hybridoma cell lines 3e and 10G were established which could continuously secrete McAb resistant to GST protein. 2. Characterization of GST monoclonal antibody. The indirect ELISA titers of 3e and 10G McAb ascites reacted with GST protein were above 1 脳 10 ~ (-6). The subtypes were all identified as IgG1. The number of chromosomes was 800.94. The reactivity of AIV-NS1 / IBDV-VP2IBV-NPs was very low in AIV-NS1 / IBDV-VP2IBV-NPs. The indirect ELISA titers of the three fusion proteins in the ascites of 3e cell line were above 2 脳 10 ~ (5). 3. Application of monoclonal antibody against GST. The ascites monoclonal antibody of 3e cell line was purified by octanoic acid-ammonium sulfate method. The titers of purified McAbs to GST protein and IBDV-VP2 protein were 1.28 脳 10 ~ (5) and 6.4 脳 10 ~ (4) respectively. Moreover, the relative affinity of 3e cell McAb to GST protein and IBDV-VP2 protein was not different. 3e cell McAb could be used for the detection of GST fusion protein. GST fusion protein was purified by using monoclonal antibody as ligand.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【引证文献】