果蝇乙酰胆碱酯酶基因在毕赤酵母中的表达

发布时间：2018-03-23 02:09

本文选题：果蝇　切入点：乙酰胆碱酯酶　出处：《重庆大学》2006年硕士论文　论文类型：学位论文

【摘要】：乙酰胆碱酯酶(Acetycholinesterase，AChE)是存在于中枢神经系统内的一种水解酶，经典作用是水解神经递质乙酰胆碱(ACh)，终止神经冲动的传导。常见的残留农药主要是有机磷和氨基甲酸酯类药剂，通过抑制昆虫中枢神经中的胆碱酯酶使之死亡而发挥杀虫作用。因此，AChE作为此类农药的最适反应底物，在农药残留的快速检测中得到了广泛应用，尤其是基于AChE的生物传感器已成为农残检测中的热点。目前，AChE主要从家蝇等敏感昆虫头部提取，产量低，成本高、灵敏度变幅大。因此，通过分子生物学方法克隆敏感AChE基因，构建高效表达载体表达AChE基因并且分离纯化目标蛋白，利用基因工程手段大量制备活性蛋白具有重要的研究意义和广阔的市场前景。果蝇AChE(Drosophila melanogaster AChE，DmAChE)对有机磷和氨基甲酸酯类农药敏感度极高，并且已经得到相应基因的全长核苷酸序列。毕赤酵母(Pichia pastoris)表达系统经过近十年发展，已基本成为较完善的外源基因表达系统，具有易于高密度发酵，，表达基因稳定整合在宿主基因组中，能使产物有效分泌并适当糖基化，培养方便经济等特点。我们构建DmAChE的毕赤酵母表达系统，目的就是探索AChE基因在P．pastoris表达系统中高效、稳定表达的条件，为大量制备乙酰胆碱酯酶提供有效的基因工程手段。本研究应用分子生物学PCR方法扩增DmAChE基因编码区全长1.9Kb cDNA序列；选取毕赤酵母表达载体pPIC9K，选用SnaBI／NotI内切酶把DmAChE基因克隆于表达载体的多克隆位点上，构建重组质粒pPIC9K-DmAChE。重组质粒经PCR、酶切验证并且测序后，采用电穿孔法转化入毕赤酵母表达菌株KM71，PCR方法检测重组情况，筛选阳性转化子。同时，G418抗性筛选高拷贝转化子。重组酵母进行诱导表达，并应用改良的Ellman法检测表达产物的AChE活性，于表达产物上清液中成功的检测到了乙酰胆碱酯酶活性，并对不同基因拷贝数转化子表达产物的乙酰胆碱酯酶活性作了初步比较，初步探索了果蝇乙酰胆碱酯酶基因在毕赤酵母中有效表达的条件，为下一步优化表达条件、分离及纯化目的蛋白奠定了基础。
[Abstract]:Acetylcholinesterase Acetycholinesterase (Acetycholinesterase AChE) is a hydrolase present in the central nervous system. The classical role of acetylcholinesterase is to hydrolyze the neurotransmitter acetylcholine to stop the transmission of nerve impulses. It can kill insects by inhibiting the death of cholinesterase in the central nervous system of insects. Therefore, ache, as the optimum reactive substrate of this pesticide, has been widely used in rapid detection of pesticide residues. In particular, biosensor based on AChE has become a hot spot in the field of agricultural disability detection. At present, ache is mainly extracted from the head of sensitive insects such as Musca domestica, with low yield, high cost and high sensitivity. Therefore, the sensitive AChE gene was cloned by molecular biological method, and the highly efficient expression vector was constructed to express AChE gene and to isolate and purify the target protein. The preparation of active proteins by means of genetic engineering has important significance and broad market prospect. Drosophila AChE(Drosophila melanogaster ache DmAChE is highly sensitive to organophosphorus and carbamate pesticides. The full-length nucleotide sequence of the corresponding gene has been obtained. Pichia pastoris expression system has become a perfect foreign gene expression system after nearly ten years of development, and it is easy to ferment with high density. The stable integration of the expressed gene in the host genome can effectively secrete and glycosylation of the product and facilitate the economic cultivation. We constructed the Pichia pastoris expression system of DmAChE in order to explore the high efficiency of the AChE gene in the P.pastoris expression system. The stable expression conditions provide an effective genetic engineering method for the preparation of acetylcholinesterase. In this study, the full-length 1.9Kb cDNA sequence of the coding region of DmAChE gene was amplified by molecular biological PCR method, and the expression vector pPIC9K of Pichia pastoris was selected, and the DmAChE gene was cloned into the polyclonal site of the expression vector by SnaBI/NotI endonuclease. The recombinant plasmid pPIC9K-DmAChEwas constructed. The recombinant plasmid was digested and sequenced. The recombinant plasmid was transformed into Pichia pastoris expression strain KM71PCR by electroporation. At the same time, the recombinant yeast was used to induce the expression, and the modified Ellman method was used to detect the AChE activity of the expression product. The acetylcholinesterase activity was successfully detected in the supernatant of the expression product, and the activity of acetylcholinesterase was detected successfully in the supernatant of the expression product. The acetylcholinesterase activity of different gene copy number transformants was compared, and the effective expression conditions of the acetylcholinesterase gene of Drosophila melanogaster in Pichia pastoris were preliminarily explored, which was the next step to optimize the expression conditions of acetylcholinesterase in Pichia pastoris. The separation and purification of the target protein laid the foundation.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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