Study On The Function And Insect Inhibition Mechanism Of Recombinant Serine Protease Inhibitor ApSerpin-FA72

Protease inhibitors can inhibit proteolytic activity and interfere with the normal growth and development of insects.Insect digestive enzyme inhibitors mainly include protease inhibitors and amylase inhibitors,of which serine protease inhibitors are protease inhibitors.The serine protease inhibitor FA72（Ap Serpin-FA72）is a serine protease inhibitor cloned,expressed and purified from the Anatolica polita.In order to explore the insecticidal activity and mechanism of Ap Serpin-FA72 on Ectropis obliqua hypulina Wehrli,the protein was purified using E.coli prokaryotic expression,nickel-column affinity chromatography,and saturated ammonium sulfate precipitation method.A large amount of single soluble protein of interest band was obtained by renaturation of inclusion bodies,and its biological activity was measured.A large amount of Ap Serpin-FA72 was obtained by high-density fermentation.The optimum reaction temperature,p H and IC₅₀ of renatured serine protease inhibitors to trypsin were determined.The midgut protease activity and recombinant protein Ap Serpin-FA72 on the E.oblique midgut inhibitory enzyme activity has been detected.The insecticidal activity and feeding tendency of several insecticides and Ap Serpin-FA72 on the E.oblique were determined by the dipping method.1.Recombinant strain p ET32a-Ap Serpin-FA72/BL21 is expressed by E.coli in prokaryotic cells.The bacterial cells can be sonicated at 38%total power（342 W）in p H=8.0 Tris-HCl buffer,working for 5 s,intermittent 6 s to obtain a large amount of soluble protein.Nickel column affinity chromatography removes the imidazole concentration in the equilibration buffer,significantly improves the protein binding efficiency,and purifies the protein concentration.The ammonium sulfate saturated precipitation method,after 50%saturation ammonium sulfate treatment,obtains the target protein,which lays the foundation for the insect control experiment.2.Studies have shown that a large amount of protein can be obtained by induction with 0.4 mmol/L IPTG at 220 rpm at 37℃,and the recombinant protein exists as inclusion bodies.By screening different renaturation methods of inclusion bodies,the results showed that L-Arg had a greater effect on renaturation of inclusion bodies,and a large amount of soluble proteins were obtained.Based on the trypsin inhibitory activity curve of renatured protein Trx A-Ap Serpin-FA72 as the standard,the activity of the protease inhibitor obtained by renaturation with guanidine hydrochloride dilution was the best.3.Screen the optimal fermentation medium（silkworm pupal powder 2 g/L,soluble starch 10 g/L,Na Cl 10 g/L,tryptone 5 g/L）by single factor experiment and orthogonal experiment.High-density fermentation at 32℃and 300 rpm,to obtain a higher concentration of bacteria and protein expression,the corresponding relationship between the OD value of the bacteria and the dry weight of the bacteria is y=0.483x+0.0307,R2value is 0.9862.4.The IC₅₀ of renatured recombinant serine protease inhibitor to trypsin was 0.48μmol/L.Serine protease inhibitors are more active when the p H is 7-9 and the temperature is 60℃.This study found that the midgut of E.oblique contains different kinds of digestive enzymes.The mechanism of action of Ap Serpin-FA72 on insects is not limited to one kind of protease,but the influence on a vari-ety of proteases,which destroys the coordination between the proteases of the insects and produces an anti-nutritional effect,thereby affecting tea Inchworm gro-wth and development.The insecticidal effect of the recombinant protein obtained by different methods on E.oblique showed that the insecticidal effect of the protein purified by the nickel column at the same concentration was most significant.Under the condition of feeding recombinant protein,the three midgut enzymes of tea inchworm are inhibited,which further proves that tea inchworm causes death of insects by inhibiting the activity of various proteases.Determination of LC₅₀ of third-instar larvae by various insecticides and proteins,Emamectin>Betacypermethrin>Deltamethrin>Recombinant protein Ap Serpin FA72>Cypermethrin>Chlorpyrifos.Different kinds of LC₂₀ pesticides and LC₅₀ protein mixtures have enhanced insecticidal effects and redu-ce the environmental pollution of pesticides.The experimental results of feeding tendency of different pesticides and proteins show that feeding chlorpyrifos>protein,avermectin>protein,fenvalerate<protein,beta-cypermethrin<protein,deltamethrin<protein,insects have different feeding tendency for different kinds of insec-ticides,but the feeding index is similar,indicating that insecticides and proteins have a certain antikill effect on the insect body.