Pilot Fermentation Method for Expression of Recombinant Acetylcholinesterase in Pichia methanolica
A technology of Pichia methanolica and acetylcholinesterase, which is applied in the biological field, can solve the problems of large-scale fermentation and expression that have not been reported, and achieve the effect of low protein expression cost, convenient operation, and high expression level
Active Publication Date: 2020-02-18
ZHENGZHOU UNIV
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Problems solved by technology
However, until now, there has been no report on the large-scale fermentative expression of acetylcholinesterase in the yeast expression system
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[0019] The fermentation process of the present invention will be described in more detail below through specific examples.
[0020] The first step is to prepare genetically engineered bacteria
[0021] The strain uses red forest ants Acetylcholinesterase recombinant Pichia pastoris X33 / pPICZαA-Acetylcholinesterase strain, and its construction method is as follows:
[0022] The pMD-19T / Acetylcholinesterase and the expression vector pPICZαA were digested with EcoRI and NotI respectively, and the target product was recovered and ligated with T4DNAligase to obtain the expression vector pPICZαA-Acetylcholinesterase, which was transformed into Escherichia coli DH5α, single clone was picked, and a positive plasmid was identified.
[0023] After linearizing pPICZαA-Acetylcholinesterase with SacI, mix it with Pichia pastoris X33 competent cells for electrotransformation, spread the transformation evenly on a Zeocin (100 μg / mL) resistant YPD medium plate, and place at 29°C Cultivate at...
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The invention discloses a pilot fermentation process for expressing recombinant acetylcholinesterase (AChE) in pichia methanolica. The process is characterized by firstly culturing an activated recombinant pichia pastoris strain, thus preparing a fermentation working seed liquid, then transferring the fermentation working seed liquid to a basal salt culture medium, regulating the pH value of a fermentation system by utilizing ammonia water or phosphoric acid, then carrying out inoculation, adding proper amount of trace element solution, adding a mixed solution of glycerin and trace elements after growing to a certain stage, finally feeding a methanol solution containing trace elements to carry out protein induced fermentation, continuously maintaining the temperature at 29 DEG C and maintaining DO more than 20% in the fermentation process and carrying out induction with methanol for 120 hours, thus finishing the whole fermentation process. The pilot fermentation process has the advantages that the fermentation process is simple and is convenient to operate; the proteins are subjected to soluble expression and the expression quantities are high and the enzymatic activity is high but the protein expression cost is relatively low; large scale fermentation tank expression is carried out on AChE in a yeast expression system for the first time and lays the foundation for large scale production and final application of AChE.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a pilot-scale fermentation method for expressing recombinant acetylcholinesterase in Pichia methanolica. Background technique [0002] Pesticides are widely used in the prevention and control of agricultural and forestry crop diseases, insect pests and weeds, and play a very important role in increasing agricultural production. However, there will be some pesticide residues on crops within a certain period of time. If you eat crops with pesticide residues for a long time, it will cause harm to human health. influences. Pesticides play an insecticidal role by inhibiting the cholinesterase in the central nervous system of insects to make them die. At the same time, they also have an inhibitory effect on cholinesterase in the human body, which can block the transmission of neurotransmitters. Cause muscle paralysis, cause poisoning, carcinogenic, teratogenic, mutagenic and other hazards...
Claims
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IPC IPC(8): C12N9/18C12R1/84
CPCC12N9/18C12Y301/01007
Inventor 翁海波李倩孙召伟田柳杨
Owner ZHENGZHOU UNIV