人巨噬细胞抑制因子-1（MIC-1）在毕赤酵母中的优化表达、蛋白纯化及多克隆抗体的制备

发布时间：2018-07-06 14:33

本文选题：巨噬细胞抑制因子-1(MIC-1) + 毕赤酵母　；参考：《中国协和医科大学》2007年硕士论文

【摘要】： 目的：构建人巨噬细胞抑制因子-1(MIC-1)优化密码子的表达载体，筛选稳定的高表达MIC-1的酵母菌株，探索MIC-1重组蛋白的纯化条件，并制备抗MIC-1多克隆抗体，为胰腺癌血清诊断试剂的研制奠定基础。方法：通过RT-PCR及基因克隆技术自人胎盘组织中获得MIC-1 cDNA的全基因片段；通过PCR技术将MIC-1 cDNA基因片段5’端的7个毕赤酵母非偏好密码子定点突变为其同义优越密码子；构建pPIC9K-mtMIC-1表达载体，转染并在毕赤酵母GS115工程菌中表达MIC-1重组蛋白，通过G418抗性筛选稳定的MIC-1高表达菌株；研究不同表达条件对MIC-1重组蛋白表达量的影响；研究和初步建立MIC-1重组蛋白的纯化方案及工艺；以纯化的重组MIC-1蛋白为抗原免疫新西兰白兔，制备抗MIC-1的多克隆抗体。结果：成功克隆MIC-1 cDNA，并构建优化密码子的MIC-1重组蛋白表达载体；获得稳定的高表达重组蛋白毕赤酵母菌株，表达量约为30～50mg／L；初步建立了MIC-1重组蛋白中试发酵工艺；建立较完整的MIC-1重组蛋白的分离纯化体系，纯化所得重组蛋白经SDS-PAGE电泳检测纯度达90％以上；获得抗MIC-1重组蛋白的多克隆抗血清，抗体效价为1：128，，000。结论：通过优化密码子的MIC-1重组蛋白表达载体，重组MIC-1毕赤酵母菌株的表达量显著高于国际上其他单位该蛋白的表达水平；优化了发酵、表达和纯化工艺，重组蛋白纯度达90％以上；以纯化蛋白为抗原获得了高效价的抗MIC-1多克隆抗体。该项研究为MIC-1蛋白的功能研究及其抗体的临床诊断试剂研制奠定了基础。
[Abstract]:Objective: to construct an optimized codon expression vector of human macrophage inhibitory factor 1 (MIC-1), screen stable yeast strain with high expression of MIC-1, explore the purification conditions of MIC-1 recombinant protein, and prepare anti-MIC-1 polyclonal antibody. To lay a foundation for the development of serum diagnostic reagent for pancreatic cancer. Methods: the whole gene fragment of MIC-1 cDNA was obtained from human placenta by RT-PCR and gene cloning, and 7 Pichia pastoris nonpreferred codon at the 5'end of MIC-1 cDNA fragment were mutated to its synonymous superior codon by PCR. PPIC9K-mtMIC-1 expression vector was constructed and transfected into Pichia pastoris GS115 to express MIC-1 recombinant protein. The stable MIC-1 high expression strain was screened by G418 resistance, and the effect of different expression conditions on the expression of MIC-1 recombinant protein was studied. The purified recombinant MIC-1 protein was used as antigen to immunize New Zealand white rabbits to prepare polyclonal antibodies against MIC-1. Results: MIC-1 cDNAwas cloned successfully and the MIC-1 recombinant protein expression vector with optimized codon was constructed, and a stable recombinant protein Pichia pastoris strain was obtained, which expressed about 30 mg / L, and a pilot fermentation process of MIC-1 recombinant protein was established. A complete purification system of MIC-1 recombinant protein was established. The purity of the purified recombinant protein was over 90% by SDS-PAGE, and the polyclonal antiserum of anti-MIC-1 recombinant protein was obtained with a titer of 1: 128000. Conclusion: the expression level of MIC-1 recombinant protein in Pichia pastoris strain was significantly higher than that of other units in the world, and the fermentation, expression and purification techniques were optimized. The purity of recombinant protein was more than 90%, and the purified protein was used as antigen to obtain a high titer polyclonal antibody against MIC-1. This study laid a foundation for the study of the function of MIC-1 protein and the development of clinical diagnostic reagent for antibodies.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392;Q78

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