Establishment Of Non-Immunized Murine Phage Single-Chain Antibody Library And Screening Of Anti-Cimatrol Single-Chain Antibody

The purpose of this study was to solve food safety problems and improved the phenomenon that illegal additives cannot be completely banned in China.Using phage display technology to build a diversified recombinant antibody resource library with large library capacity,strong adaptability and high stability.The purpose was to screen out recombinant antibodies with specificity and affinity for food safety hazards in response to unexpected food safety events.Lay the foundation for fast and efficient detection methods.This study selected Cimaterol among?-stimulants for research.Using polymerase chain reaction and reverse transcription PCR technologies,the c DNA was obtained from the spleen of BALB/c mice without active immunization.Primers were designed to amplify the mouse antibody variable region genes of heavy chain and light chain.Using overlapping extension PCR reaction,a mouse single chain antibody gene was synthesized.Recombinant phagemids were constructed by DNA ligation,electroporation and other techniques.After rescue by the helper phage M13K07,a natural mouse phage Sc Fv library was constructed.The diversity and integrity of antibody genes were analyzed by sequencing.The artificial immune antigen CIM-BSA and the detection antigen CIM-OVA were prepared by the diazotization method,and UV,SDS-PAGE,and mass spectrometry were used to identify the complete antigen.Furthermore,the constructed complete phage Sc Fv library was subjected to affinity panning using artificial complete antigen as the target antigen,so that specific phage antibodies carrying Cimatrol surface antigen were enriched.Finally,one clone with high binding activity to cimaterol was selected.A large number of phage clones carrying the highly sensitive with anti-CIM-Sc Fv gene were prepared and infected with E.coli HB2151 to explore the best conditions for induction by isopropyl-?-D-thiogalactopyranoside?IPTG?.The collected products were identified and identified by SDS-PAGE and Western blot.Enzyme-linked immunosorbent assay?ELISA?was used to identify binding activity of anti-CIM.The assays showed that the size of the Sc Fv fragment was about 780bp by gel electrophoresis,which was consistent with the characteristics of mouse antibody genes.After reinfection with helper phage M13K07,a mouse-derived phage Sc Fv antibody library was obtained with an insertion rate of 89.47%,a diversity of 91.17%,and a storage capacity of 1.56×10¹⁰ pfu.Sequencing identified the characteristics of mouse antibody gene,the prepared Sc Fv phage display library has good abundance and the storage capacity meets specific panning requirements.The swimming speed of CIM complete antigen identified by SDS-PAGE lags behind that of carrier protein,UV scanning was performed to identify a new specific absorption peak relative to the carrier protein.And its relative molecular mass was determined by mass spectrometry.CIM-BSA and CIM-OVA were successfully coupled with that the coupling ratio were8:1 and 10:1.As the number of affinity panning for CIM antibodies increased,the phage recovery rate also increased.The fourth round recovery rate reached 4.2×10^-3%.Finally,a highly sensitive and specific anti-CIM phage clone was obtained.It was identified that no gene deletion or mutation occurred during panning.Soluble expression of Anti-CIM-Sc Fv was obtained in E.coli,and the optimal expression conditions under the induction of IPTG were 0.1 mmol L IPTG,30?,16 h.The specific Sc Fv antibody titer was 1:320.The indirect competition ELISA?ic-ELISA?detection method established with this single chain antibody has a standard curve of y=-0.2752x+1.0753 and R²=0.9848,which has a wide detection range and good fit.The antibody has a half inhibitory concentration(IC₅₀)of CIM was 101 ng/m L and the minimum detection limit?LOD?of CIM was 3.16 ng/m L.In summary,a natural mouse-derived Sc Fv phage library was successfully constructed,and an anti-CIM single-chain antibody was successfully screened from the antibody library.The antibody has good sensitivity and enriched?₂ receptor agonists such as CIM.It lays a material foundation for the further preparation of antibodies and evolution of CIM,which provides a reference for the control.