Recombinant Expression Of Acetylcholinesterase Of Housefly(Musca Domestica)

With the development of agriculture, the problem of pesticide residues which is caused by the excessive and unreasonable use of pesticides, has been a serious threat to human health. Therefore, it is meaningful to detect the pesticide in environment and agricultural products. Acetylcholinesterase (AChE, EC3.1.1.7) plays a key role in cholinergic transmission. Inhibitors of AChE, such as organophosphates and carbamates, interfere with this process and may lead to a severe impairment of nerve functions or even death. Therefore, as an alternative, AChE inhibition tests have been shown to be suitable for detection of insecticides. At present, AChEs used for detection of pesticide residues are mainly extracted from animal tissue or blood, but this method is complex, low yield and poor stability, which greatly affected. the detection of pesticide residues. With the development of gene engineering technology, we can resolve this problem by cloning AChE genes and efficiently expressing them in heterologous expression systems.Total RNA was extracted from Housefly (Musca domestica) using total RNA purification kit and cDNA was synthesized. A1737bp fragment of AchE gene was successfully amplified using RT-PCR. A target protein of92kDa was successfully abtained from the host strain BL(DE3) containing prokaryotic expression vector pDEX-4T-1-A8. The optimal conditions were determined by the optimization on the time and concentration of IPTG induction. It was proved that the recombinant proteins formed inclusion bodies. The pure recombinant protein was obtained after further denaturation, renaturation, dialysis, purification. The concentration protein was determined as10μg/mL with Brandford method. Activity of recombinant protein examined by improved Ellman method showed that the protein has a certain activity,but it is low. The recombinant plasmid of pPIC9K-A8were successfully constructed and transformed into Pichia pastoris GS115by electrotransformation. Positive transformants were screened by PCR with the yeast genome as a template. The verified yeast transformants were picked and induced with methanol in BMMY medium. Results of SDS-PAGE indicated that the recombinant protein was secreted into the culture. The protein was primaryly purified by ammonium sulfate precipitation method, and its activity was detected by improved Ellman method, which showed that the protein has no acetylcholinesterase activity.