人抗狂犬病毒小分子抗体ScFv-Fc在毕赤酵母中的表达与鉴定

发布时间：2018-11-27 19:09

【摘要】： 巴斯德毕赤酵母表达系统是近年来广泛用于表达外源基因的表达系统。该系统具有真核细胞和原核细胞的双重特点,既可以对外源蛋白进行适当加工、修饰,使目的蛋白的抗原性、免疫原性和活性与天然蛋白接近,也可以将外源蛋白以可溶的形式分泌到培养基中,使纯化更为简便,还可以在简单培养基中迅速生长至高密度,从而得到大量有活性的目的蛋白。本研究利用基因重组技术构建了可融合表达人抗狂犬病毒小分子抗体ScFv-Fc的表达载体pPICZα-Fc/ScFv,电转化毕赤酵母X-33后,筛选出稳定高效的分泌表达ScFv-Fc抗体的基因工程菌株,并通过Protein A层析柱纯化。经SDS-PAGE,Western blot,ELISA鉴定重组抗体的生物学活性,结果表明,在毕赤酵母菌中可经甲醇诱导产生具抗原结合活性的小分子分泌型抗体ScFv-Fc,并可方便的应用Protein A进行纯化。在抗体分子的克隆、筛选及小分子抗体的表达制备方面具有潜在价值。
[Abstract]:Pichia pastoris expression system is widely used to express foreign genes in recent years. The system has the dual characteristics of eukaryotic cells and prokaryotic cells. It can process and modify the foreign proteins properly, so that the antigenicity, immunogenicity and activity of the target proteins are close to those of natural proteins. The extraneous protein can also be secreted into the medium in soluble form, which makes purification more convenient, and can grow rapidly to a high density in the simple medium, thus a large number of active target proteins can be obtained. In this study, the expression vector pPICZ 伪-Fc/ScFv, which can express human rabies virus small molecule antibody ScFv-Fc, was constructed by gene recombination technique and transformed into Pichia pastoris X-33. A stable and efficient recombinant strain secreting ScFv-Fc antibody was screened and purified by Protein A chromatography. The biological activity of recombinant antibody was identified by SDS-PAGE,Western blot,ELISA. The results showed that small secretory antibody ScFv-Fc, with antigen-binding activity could be induced by methanol in Pichia pastoris and purified by Protein A. It has potential value in cloning, screening of antibody molecules and preparation of small molecular antibodies.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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