Molecular Cloning Expressing And 3-D Structure Analyzing Of Musca Domestica Ace Gene

Pesticides contribute greatly to agriculture for controling plant diseases and insect pests , but the widespread use and disposal of pesticides causes environmental contamination, it is harmful to human and other living things.so it is necessary to detect the pesticides in environment and food.Methods traditionally used for the detection of insecticides are based on gas chromatography (GC) or high performance liquid chromatography (HPLC), which are expensive and sophisticated analytical techniques.So, we need an easier and faster way to detect the pollution of pesticides.Acetylcholinesterase (AChE , EC 3.1.1.7) plays a key role in cholinergic transmission by catalysing the rapid hydrolysis of the neurotransmitter acetylcholine into acetate and choline. The enzyme effectively terminates the chemical impulse at rates that are similar to a diffusion-controlled process allowing a rapid and repetitive response.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 the detection of insecticides.but it asks for the availability of large amounts of extremely pure, functional and stable enzyme samples at low costs.AChE can be purified from human or animal blood or even the plant, These sources are, however, limited and the purification is both time and cost intensive. To overcome this problem, we try to express AChEs by genetic engineering.We had cloned Housefly's(Musca domestica) ace gene by RT-PCR,and cloned it into the pET-22b,finally we got 22b-ace expression vector, we transformed 22b-ace into expression host BL21(DE3),A target band about 69Kd can be detected by SDS-PAGE,it indicates that we have expressed housefly's AChE in E.coli,we purified the recombined protein by QIAexpress Ni-NTA protein purification kit,and detected the protein by AChE detection kit,but the result showed that we failed to functionally express housefly's AChE in E.coli even we try to optimize the expression condition such as grew at 30℃overnight or lower concentration of IPTG.AChE is a glycoprotein and have three pairs of S-S bonds, unfortunately,E.coli expression is lacked of PTM(post translation modification) system,and it failed to make the expressed protein folded properly in nature state. so,that may be the reason why we can not functionally express AChE in E.coli. In aternative,we try to express AChE in Pichia _ pastoris,which have PTM system,we have constructed pGAP-ace and pPIC-ace expression vector hoping to functionally express AChE in P.pastoris.Some researchers...