Study On The Key Factors Of Omission In Biopanning Of Organophosphorus Pesticides Peptidomimetic

Immunoassay has the advantages of rapid,simple,economical,sensitive and high-throughput,which is very suitable for rapid detection of pesticide residues.The immunoassay methods of pesticide residues are usually established by using the competitive format.The competing antigen is an essential reagent for the immunoassay in this model.Phage display technology can rapidly prepare peptidomimetic of small molecules such as pesticides,which can be used as competing antigen to establish competitive immunoassays and improve the sensitivity of analysis.Biopanning,the process of separating the target phage display peptide from the peptide library,is the core step in the development of peptidomimetic.However,the current usual biopanning strategies often caused the loss of target phage in the biopanning program,which reduced the success rate of the preparation of the peptidomimetic.This study aimed at the key factors for the loss of target phage caused by the current usual biopaning methods are unknown,and chose the peptidomimetic of organophosphorus pesticides as the research object.(1)Through site-directed mutation and computer-aided simulation,the interaction mechanism between peptidomimetic and antibody was determined.(2)The phage display mutant libraries were constructed based on the interaction mechanism,and the key factors causing the loss of the target phage in the biopanning program were identified by high-throughput sequencing,which provided a theoretical basis for the development of the peptidomimetic of pesticides and other small molecular weight compounds.The main studies results are as follows:(1)M13KE phage carrier was used to carry out site-directed alanine(A)mutation at unit point on the peptidomimetic of organophosphorus pesticides(CPPWPLRPGC,with two cysteine at the ends to form disulfide bond).Phage enzyme-linked immunosorbent assay(ELISA)was used to evaluate the activity of the mutants.It was found that proline(P)at position 3,tryptophan(W)at position 4.proline(P)at position 5 and arginine(R)at position 7 could not bind to antibody after mutating into alanine.indicated the peptidomimetic was inactive.After mutating into alanine,proline(P)at position 2,leucine(L)at position 6,proline(P)at position 8 and glycine(G)at position 9 can still bind to antibody specifically and the peptidomimetic have activity.On this basis,the amino acids at positions 2,6,8 and 9 were mutated into alanine by 2,3 and 4 points,and the mutants were found to be active.In addition,positions 3,6 and 9 were selected for site-directed non-alanine mutation,and the results showed that the mutations of position 3 into phenylalanine(F),tryptophan(W),histidine(H)were inactive,while the mutations of position 6 into glutamine(Q),lysine(K),asparagine(N),aspartic acid(D),glutamate(E)and the mutations of position 9 into phenylalanine(F),histidine(H),serine(S),threonine(T),asparagine(N)were still active.By comparing with CAPWPPRPGC,another peptidomimetic of organophosphorus pesticides,it was found that the non-key amino acid of position 6 was α-imino amino acid(P),which could also bind to antibody specifically.Therefore,the core sequence of peptidomimetic of organophosphorus pesticides is PWPXR(X is any amino acid).Comparing with another peptidomimetic of organophosphorus pesticides:CSPPWPPRPC containing the core sequence PWPXR,it was found that the position of the core sequence in the peptidomimetic could be changed,but the solid phase synthesis of pentapeptides(PWPAR)containing only the core sequence had no activity.It was indicated that the amino acids of non-core sites could be changed,but could not be lost.The peptide of CAPWPLRPGC with closed loop(disulfide bond formation)and open loop(no disulfide bond formation)by solid phase synthesis,both were found to be active by ELISA.It was indicated that ring formation is not the key factor for determining the activity of peptidomimetic.In summary,the core sequence of the peptidomimetic of organophosphorus pesticides is PWPXR(X is any amino acid),and the position of the core sequence in the peptidomimetic can be changed.The positions of non-core amino acids can be changed,but not lost.Whether the cysteine at both ends of the peptide forms a ring or not does not determine the activity of the polypeptide.Gene cloning technology was used to determine the sequence of amino acids in the variable region of monoclonal antibody against organophosphorus pesticides.Then,the codes 4HXA and 1EZV were determined as templates through PDB database comparison,and homologous modeling were conducted for light chain and heavy chain of antibodies.The evaluation results by Verify Score and Procheck showed that the structure of homologous module was reasonable and reliable.The modeling structures of four peptidomimetics of organophosphorus pesticides were constructed by multiple simulated annealing dynamics(SA-MD).and the evaluation results by Procheck and Zscore showed that the structures of homologous module were reasonable and reliable.By using Autodock and Gold to perform molecular docking,it was found that cyclic 8-amino-acid peptidomimetic of organophosphorus pesticides bind with antibody in an active pocket consisting of Tyr35,Tyr53,Asn60,Arg100,Tyr162,that form weak interaction.At the same time,the proline(third),tryptopha(fourth),proline(fifth)and arginine(seventh)of the peptidomimetic form hydrogen bonding interaction with antibody.These weak forces greatly reduce the free energy between peptidomimetic and antibody,to make the conformation is more stable.However,the software Autodock and Gold were used to determine that methyl-parathion molecules bind with antibody in a hydrophobic cavity formed by Tyr34,Trp48,Tyr51,Tyr108,Phe109.There is T-π bonding with Phe109,π-π bonding with Tyr108,but also hydrogen bonding with Asn36.Although the weak forces of the peptidomimetic and analyte binding to antibody are different,the binding pocket of the peptidomimetic is substantially coincident with the hydrophobic cavity of methyl-parathion.These results confirmed the interaction mechanism between the core sequence PWPXR of cyclic 8-amino-acid peptidomimetic of organophosphorus pesticides and antibody,and explained the mechanism of peptidomimetic as competing antigens.(2)Based on the interaction mechanism between peptidomimetic and antibody,the phage display system was used to construct two mutant phage libraries with core sequence characteristic(CXPWPXRXXC,X for any amino acid)and non-core sequence characteristic(CAXXXAXAAC,X for any amino acid).The number of positive clones of the mutant library containing the core sequence characteristic sequence was 1.28×107 pfu,which was about 12 times of the theoretical library capacity.The insertion rate of this library reached 100%,and the titer after amplification was 8.54×1013 pfu/mL.The insertion rate of the mutant library containing the non-core sequence characteristic sequence was 90%.The number of transformed clons was 2.07 ×107 pfu,and the number of positive clons was 1.86 ×107 pfu,which was about 18 times of the theoretical library capacity.After amplification the titer was 7.0×1013 pfu/mL.The results of the mutation libraries by high-throughput sequencing showed that the mutation libraries basically cover all the theoretical sequences.Monoclonal antibodies against organophosphorus pesticides were used to perform affinity screening of the two mutant libraries and the mutant library mixed 1:1 with each other.The results showed that a large number of positive clones could be obtained from both the mutant library containing characteristic sequence and the mixed library,and all of them contained the core sequence,while no positive clones were obtained from the mutant library with non-core sequence characteristic.The results are consistent with the conclusion of interaction mechanism between peptidomimetic and antibody.The phages of each operating step in biopanning program from mutation library with the core sequence characteristic and hybrid library were collected for high-throughput sequencing(impurity removal,incubation,washing,elution,amplification).Through the comparison of sequences types and abundance,it was found that amplification would significantly reduce the types of sequences and significantly change the abundance of sequences,indicating that the genetic preference of phages would lead to the loss of positive sequences or the decrease of positive sequences abundance.In addition,washing also would result in the loss of peptidomimetics with weak affinity or low abundance.Gradient oriented competing elution based on analytes can exclude some peptidomimetics with high affinity(resulting in low sensitivity of competitive immunoassay).Therefore,the main factors affecting the loss of the target peptidomimetic are the genetic preference of the phage and the affinity of the phage with the antibody.According to the research results,the biopanning strategy should be adopted to avoid the genetic preference of phages as much as possible,such as using the phagemid display system.