Development Of Receptor Mimic Polymer Nanoparticles And Used To Extract And Purify The Soil Of Bt Proteiin

Bacillus thuringiensis(Bt)is a Gram-positive soil bacterium belonging to the genus Bacillus,which is a short rod-shaped bacteria,flagella,solitary or forming short chains.During the process of forming spores,it can produce a specific Bt insecticidal activity of parasporal crystal protein.Because of its wide insecticidal activity spectrum,strong specificity,in agricululture it is often used in biological pesticide and insect-resistant transgenic crops.Bt protein can enter into the soil by transgenic crops residues,root exudates and other ways,causing the residue in the soil,enrichment,leading to potential environmental and ecological risks.Thus,in recent years the Bt protein residues in the soil has been widespread concern,how efficient,rapid and specific extraction,separation,purification and detection of Bt protein is a crucial factor in achieving effective control of Bt protein.Scientists study of Bt protein-Insect receptor recognition mechanism provides a new way of thinking for us to design synthetic Bt protein moleculular recognition material.Considering the binding sites of Bt protein and intestinal calcium receptor class cadherin are major located in domain II of the three epitopes loop 2,loop 3 and loop a-8,so we can based on the hydrophobic,hydrophilic and charge characteristics of their amino acid sequence to design synthetic polymer nanoparticles,making strong affinity and capacity for one or more epitopes,ulultimately,for the specific extraction,separation and purification of the whole Bt protein.In view of this,this paper using a radical polymerization initiator synthesized a series of different charge properties of polymeric nanoparticles,by optimizing the conditions to select the optimal polymer nanoparticles and optimal incubation conditions;Then it comparedwiththeir binging capacity of other polymeric nanoparticles which have the same,similar or different epitopes of the Bt protein,such as Cry1Ac,Cry2A,Cry 1C and Cry1F.After thatit did the inhibition force between polymer nanoparticles and insect receptor class cadherin Bt-R1 binding sites 865NITIHITDTNNK876(NK-12)when theyfunction to Bt CrylAb epitope domain Ⅱloop 2.Specific work includes the following three parts:(1)Capacities of polymeric nanoparticles with Bt CrylAb protein:static adsorption filter out the best of polymer nanoparticlesAAml8/AAc40(NP12)which has thegreatest affinity with Bt CrylAb protein,through optimizing its adsorption conditions,such as pH,buffer concentration and salt concentration,the optimum adsorption conditions were:pH 6.0 10 mM PBS buffer.Isotherm results showed that the maximum adsorption capacity of polymer nanoparticles AAm18/AAc40 with Bt CrylAb protein was 91 μg/g,the equilibrium time is about 40 min.(2)The Inhibition ability of polymer nanoparticles between insect receptor epitope NK-12 and Bt protein epitope RQ-12:Compared the remaining amount of the NK-12 in the System one RQ-12/NK-12 and the system two RQ-12 + NP12/NK-12 we found that the remaining NK-12 in the system two was significantly higher than the system one,which indicated that the addition of NP12 can obviously inhibit the binding ability between the RQ-12 and NK-12,the inhibition rate is about 67%.The results also demonstrate a certain extent,NP12 has the similarity with insects receptor class cadherin at the molecular recognition mechanism.Bt CrylAb protein binding site is indeed located in Bt CrylAb epitope domain II loop 2(RQ-12).(3)Specific adsorption Capacity of polymer nanoparticles:Compared the binding capacity between NP12 and Cry1Ab,Cry1Ac,Cry2A,Cry1C Cry1F,it found NP12 has the strongest adsorption capacity to CrylAb,besides,CrylAc also has a strong affinity,mainly because the Cry1Ab and Cry 1 Ac are homologous protein,their amino acid sequence and isoelectric point are very close,their epitopes are domain II loop 2 368RRPFNIGINNQQ379(RQ-12).But the isoelectric point,epitopes of CrylAb are different with Cry2A,Cry1C and Cry1F,so NP12 almost has no affinity with these non-homologous protein.This result indicates that,according to its design based on the Bt CrylAb protein epitope,the synthetic polymer nanoparticles have specific recognition ability to the entire Bt Cry1Ab are reasonable in theory and practical application.Likewise,this method can also filter out the polymer nanoparticles for other Bt proteins.