| Molecular imprinting technique is a kind of simulation of antigen antibody interaction theory,using artificial method to synthesize the template molecules with specific binding capacity of the polymer technology.Epitope molecular imprinting is a promising molecular imprinting technique developed in recent years.This method uses the organism when the antibody recognizes an antigen only with a particular part of the antigen,which epitope cluster effect occurs,you can recognize the entire principle of antigen,in the synthesis of the polymer to a polypeptide epitope on behalf of a template molecule,using the polypeptide epitope as a template molecule to synthesis the polymers and the template molecule will be eluted after the reaction.The synthesized molecularly imprinted polymer have specific recognition ability not only to this epitopes represented,but also biological macromolecules.If this technology can applied in the field of protein molecular imprinting,we can solve protein conformation changeable,expensive and too many binding sites and other shortcomings,and it has a good application prospect.Bt protein is a class of proteins whichhave a specific insecticidal toxicity generated in the process of sporulation parasporal crystal by Bacillus thuringiensis.Bt protein can enter the soil through transgenic plant residues,root exudates and other ways,and with mineral and humus combined firmly,long time keep insecticidal activity and effects on soil biodiversity and ecological process,resulting in enormous ecological risk.Therefore,it is very necessary to monitor the release and migration of Bt protein in the ecosystem.Bt protein are typical topology of three domains consisting of three projection.Domain ? loop structure is located at the top of ? folded to form a similar immunoglobulin epitopes section which is the determining region of Bt protein with insect midgut receptor protein specific binding and it plays a decisive role.Cry1Ac Bt protein is an important member of the Cry family,the ? loop of Domain 2 located on the top of the crystal structure,which represents the amino acid sequence,is almost completely exposed.In view of this,we use Cry1Ac protein Domain ? loop peptide 368RRPFNIGINNQQ379 on behalf of Bt protein as template,acrylamide modified carbonnanotubes as substrate,with N-isopropylacrylamide,acrylic acid(AAc),1-allyl-3-imethylimidazolium hexafluorophosphate as functional monomer.N,N'-methylene bisacrylamide as crosslinker,ammonium persulfate and N,N,N',N'-Tetramethylethylenediamine as initiator and the epitope molecularly imprinted polymers of Bt protein are initiated by free radical polymerization and the surface molecular imprinting technique.The impact of molecular imprinting effect conditions were optimizedwhile the material surface morphology,selectivity and specificity was evaluated.The main contents are as follows:(1)To reduce the irreversible adsorption of acrylamide modified carbon nanotube itself to the template molecule during the process of the molecular imprinted polymer formation,Acrylamide,we modified carbon nanotube surface by a layer of preMBA and optimized the amount preMBA.Through the performance evaluation,the added amount of preMBA was set to 20mg.Without special description,all of the acrylamide modified carbon nanotubes surface grafted MBA 20mg,and then the synthesized molecularly imprinted polymer.(2)In ultra pure water system,using RQ-12 as template molecule,with NIPAM and[AMIM][PF6]and AAC:NIPAM mixture as functional monomer,using MBA as crosslinking agent to prepare epitope molecularly imprinted polymers by surface molecular imprinting technique,and then using 10%AcOH(v/v)solution to elute template molecules.In the system,the use of single or mixed functional monomer,to optimize the content of each component.In this system,No matter a single or mixed functional monomer content of each component to be optimized,there was no obvious imprinting effect,this may be because the low elution efficiency using 10%AcOH(v/v)as eluent template peptide elution is not ideal for the strong force between the system functional monomer and template.(3)In ultra pure water system,using RQ-12 as template molecule,with hydrophilic NIPAM as functional monomer,MBA as crosslinking agent to prepare epitope molecularly imprinted polymers by surface molecular imprinting technique.and then The elution conditions were optimized.It was found that using SDS-AcOH mixed solution as eluent can elute more template molecules and forming more imprinted hole formation could improve the imprinting effect of the molecularly imprinted polymer.The content of the template molecules and the crosslinking agent were optimized and the optimal synthesis conditions were 18mg RQ-12,120mg NIPAM,and 100mg MBA.The adsorption pH value was optimized.The results showed that the adsorption capacity and imprinting effect was the best when the pH value was 5.5,the adsorption capacity of the template RQ-12 was 218.76mg/g,and the imprinting factor was 1.39.Using the best polymers to adsort different polypeptides of AE-9,FS-11,RQ-12,FR-14,RQ(o),NK-12 for preferential adsorption,the results showed that the material worked on RQ-12 has the best imprinting effect,and for the other peptides had no obvious imprinting effects.The AE-9,FS-11,RQ-12,FR-14 and RQ-12(O)peptides mixed together for competitive adsorption experiments,The results showed that the imprinting factor worked on RQ-12 can reached 2.14,while the other polypeptides showed no obvious effect of imprinting.The results of the study indicated that the synthetic epitope molecularly imprinted polymer has a good imprinting effect and selective adsorption ability.(4)Examination of the synthetic epitope molecularly imprinted polymer for Bt Cry1Ac protein adsorption capacity,imprinting effect and selectivity.The results showed that at pH 8.9 and 9.9 under alkaline conditions,the antigen surface molecularly imprinted polymer for Bt Cry1Ac had stable imprinting effect.Compared with BSA,HB,HSA,LZM and AT,the imprinting effect of BtCrylAc protein was the best,which reflected the strong selective adsorption ability of Bt protein.Finally,using molecularly imprinted and blank molecular imprinted polymer to purified Bacillus thuringiensis nutrient solution,the supernatant after adsorption and elution solution were analyzed by SDS-PAGE.The results showed that the imprinted polymers had greater adsorption capacity of the Bt Cry1Ac protein,but also greater and more pure protein in the elution liquid.It is provided that in real actual samples,the molecular imprinted polymer also showed strong selectivity for Bt Cry1Ac protein. |
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