Encoded gene sequence of nucleotide of acetylcholine esterase in fruit fly
A technology of acetylcholinesterase and nucleotide genes, applied in genetic engineering, plant gene improvement, hydrolytic enzymes, etc., can solve undiscovered problems
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Embodiment 1
[0089] Example 1——The activity of the recombinant AChE protein expressed by yeast is inhibited by a small amount of dichlorvos
[0090] 1. Solutions and reagents
[0091] (1) BMGY solution
[0092] Dissolve 10g of yeast extract and 20g of peptone in 700ml of water, and autoclave (20min); after the temperature of the solution drops to room temperature, add 100ml of potassium phosphate buffer (1M, pH6.0), 100ml of 10×YNB, 2ml of 500×B, 100ml 10×GY; the shelf life of the solution at 4°C is two months.
[0093] (2) BMMY solution
[0094] Change the 10×GY in the BMGY solution to 10×M, and keep the other components unchanged. The solution has a shelf life of two months at 4°C.
[0095] (3) Dichlorvos solution
[0096] 0.11 g of dichlorvos (MW=220.98) crystals were weighed and dissolved in 5 ml of acetone. At this time, the concentration of dichlorvos was 0.1M. Dilute the solution stepwise by a factor of 10 to 10 -5 、10 -6 、10 -7 M.
[0097] (4) Ellman's reagent
[0098] W...
Embodiment 2
[0107] Example 2——The activity of recombinant AChE protein expressed by yeast is inhibited by trace amounts of chlorpyrifos
[0108] 1. Solution
[0109] (1) Chlorpyrifos solution
[0110] Weigh 0.175g of chlorpyrifos (MW=350.6) crystals and dissolve in 5ml of acetone. At this time, the concentration of chlorpyrifos is 0.1M. Dilute the solution stepwise by a factor of 10 to 10 -5 、10 -6 、10 -7 M.
[0111] All the other solutions and reagent formulations are the same as in Example 1.
[0112] 2. Operation steps
[0113]Operation is basically the same as in Example 1. Dissolve chlorpyrifos crystals with acetone to prepare a concentration of 10 -5 , 10 -6 , 10 -7 M's solution. Take 3 μl and 50 μl of crude enzyme solution, mix them, and incubate (25°C, 15min). At the same time, a control group was set up, which was formed by mixing 3 μl of acetone and 50 μl of crude enzyme solution. Add the mixed solution into the microplate well containing 250 μl Ellman reagent, shak...
Embodiment 3
[0116] Example 3——The activity of the recombinant AChE protein expressed by yeast is inhibited by a small amount of pirimicarb
[0117] 1. Solution
[0118] (1) Anti-pirimicarb solution
[0119] Weigh 0.119g of pirimicarb (MW=238.33) crystals and dissolve it in 5ml of acetone. At this time, the concentration of pirimicarb is 0.1M. Dilute the solution stepwise by a factor of 10 to 10 -5 、10 -6 、10 -7 M.
[0120] All the other solutions and reagent formulations are the same as in Example 1.
[0121] 2. Operation steps
[0122] Operation is basically the same as in Example 1. Dissolve anti-pirimicarb crystals with acetone, and prepare a concentration of 10 -5 , 10 -6 , 10 -7 M's solution. Take 3 μl and 50 μl of crude enzyme solution, mix them, and incubate (25°C, 15min). At the same time, a control group was set up, which was formed by mixing 3 μl of acetone and 50 μl of crude enzyme solution. Add the mixed solution into the microwell containing 250 μl Ellman reagent...
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