Design Of Insect Receptor Aminopeptidase-N Biomimetic Polymeric Nanoparticles For The Selective Recognition Of Bt Proteins

Bacillus thuringiensis is a gram-positive bacterium that acts on the midgut of susceptible insects.During the process of sporulation,bacillus thuringiensis releases a parasporal crystal with high virulence and high specific insecticidal activity against insect larvae which is usually called Bt protein.Bt protein has the advantages of wide spectrum of insecticidal activity and strong effect,therefore it has been widely used in the cultivation of insect-resistant transgenic crops and the production of biological insecticides.Although the use of Bt protein has achieved an increase in the production and income of crops,its residue and enrichment in the environment can also lead to potential ecological and environmental risks,which has aroused widespread concern in society.Therefore,it is necessary to develop a molecular recognition material with specific recognition,extraction,separation and purification effects on Bt protein in order to study the migration,transformation and residue of Bt protein in the environment.Polymer nanoparticles have been widely used for the separation,purification and detection of biological macromolecules as well as the capture and removal of biotoxins due to the advantages of simple synthesis,controllable conditions,small particle size,large specific surface area and good biocompatibility.Studies on the insecticidal mechanism of Bt protein have indicated that the midgut receptor aminopeptidase-N?APN?of Spodoptera litura is bound to Bt Cry1C protein through its receptor epitope ¹²⁸HLHFHLP¹³⁴?HP-7?.Therefore,in this study polymer nanoparticles were engineered by simulating the molecular recognition mechanism between Bt Cry1C protein and APN.The peptide HP-7representing the receptor binding site was acrylamided on the amino terminal and used as the functional monomer,and acrylamide?AAm?,isopropyl acrylamide?NIPAm?and tert-butyl acrylamide?TBAm?were used as coexisting monomers.The hydrophilicity,hydrophobicity and charge of polymer nanoparticles are controlled by adjusting the types and proportion of peptide monomers and other coexisting monomers.Polymer nanoparticles obtained by this strategy would have complementary multiple interactions with the epitopes of Bt proteins,so as to display the bionic characteristics of the insect receptor APN and thus realize specific recognition of the Bt proteins.The work completed mainly includes:synthesis and screening of polymer nanoparticles with the best affinity for Bt proteins;optimization of factors affecting the adsorption of Bt protein;evaluation of the adsorption thermodynamics,kinetics and selectivity of the polymer nanoparticles to Bt protein;investigation and comparison of the interaction mechanism between polymer nanoparticles and Bt protein with the two polymer nanoparticles APhe-NP2 and NK-12-NP4 screened earlier in our lab.The main results are as follows:1. The optimal polymer nanoparticle for Bt Cry1C protein was NP3?20%HP-7,38%AAm,40%TBAm,2%BIS?,and the optimal adsorption condition was p H 8.0 10 m M PBS buffer solution.2. Selective adsorption experiments showed that the NP3 had a very low affinity for several conventional proteins.Although it had a strong affinity for Bt Cry1C protein,the adsorption capacity was far less than that of Bt Cry1F and Bt Cry1Ac proteins.Further experiments confirmed that the polymer nanoparticle with the strongest affinity for Bt Cry1F proteins was still NP3,and the optimal adsorption condition was still p H 8.0 10 m M PBS buffer solution.These results indicated that the adsorption mechanism of polymer nanoparticles to Bt Cry1F was similar to Bt Cry1C.The maximum theoretical adsorption capacity for Bt Cry1F protein can reach 646.4 mg/g,and the adsorption equilibrium can be reached within 20 minutes.3. Adsorption experiment for the pepsin hydrolysate of Bt Cry1F showed that the chromatographic peak signals of some peptides in the pepsin hydrolysate were significantly weakened after adsorption by NP3.Therefore,these peptide fragments may be the potential sites for binding of Bt Cry1F protein to NP3,and Bt Cry1F protein to Spodoptera litura receptor APN,but the amino acid sequence has yet to be identified.4. Adsorption experiment for the epitopes and their mutants of Bt Cry1Ac showed thatAPhe-NP2 and NK-12-NP4 had a very strong affinity for the three naturally occurring peptides RQ-12,FR-14 and TS-14 in Bt Cry1Ac protein.Among them,RQ-12 may play a more significant role in the binding of Bt Cry1Ac protein to polymer nanoparticles since it is at the top of Domain II in the crystal structure of Bt Cry1Ac proteins and has smaller steric hindrance for binding.By comparing the adsorption results of RQ-12,TS-14 and their mutated peptides on polymer nanoparticles,we found that electrostatic interaction and hydrogen bonding play a crucial role in the binding of Bt Cry1Ac protein to APhe-NP2 and NK-12-NP4.Whether the polypeptide was linear or circular had little effect on its affinity to polymer nanoparticles.The binding mechanism between polymer nanoparticles NP3 and Bt Cry1Ac protein was very different from that of APhe-NP2 and NK-12-NP4.The affinity of RQ-12,FR-14 and TS-14 naturally present in Bt Cry1Ac to the polymer nanoparticle NP3 was significantly lower than that of APhe-NP2 and NK-12-NP4,which mainly because the HLHFHLP introduced to the side chain of NP3 contained three positively charged histidines,while RQ-12,FR-14 and TS-14 also contained multiple positively charged amino acid residues.The electrostatic repulsion between these positively charged groups may be the main reason for the weak binding force.The specific binding sites need to be further analyzed by other research methods.