Production And Characterization Of Lysozyme Speciifc Anti-Idiotypic Nanobody

[Background]: The part of the VH-VL antigenic determinant on the variable regionof antibody molecμLes, termed idiotypic（Id）, idiotypic epitope called idiotope（Id）, itcan induced corresponding antibody by stimμLate the body in vivo or allograft calledanti-idiotypic antibodies （Anti-Id）, Id and Anti-Id is constituted the main part ofimmune regulation network. Induced by the first antibody Ab1idiotype, the secondantibody Ab2β type anti-idiotypic antibodies as molecμLar mimics of the originalantigen, also known as the antigen within the image （internal image）. Anti-Idresearchers according to this characteristic by anti-idiotypic antibodies can beobtained with catalytic antibodies, also known as antibody enzyme.Nano-antibody found in recent years is present in the llama heavy chain variableregion of an antibody, so far the smallest functional antibody fragment.In previousstudies, we found that can use camel nano antibody priority identification enzymeactive site characteristics and we also obtained high performance with garlic enzymecatalytic activity of anti-idiotypic antibody nanoparticle. This simple structure ofantibodies, to provide convenient conditions for the study of their catalyticmechanism. At the same time, in our preliminary studies, we screened with enzymeinhibitory activity of Ab1antibody from VHH antibody library by using lysozyme.interest is one of nanometer NBL42antibody, has only a CDR3, but retained a stronginhibitory activity of lysozyme.[Objective]: In order to better validation of preliminary studies, antibody-basednano-antibody enzyme preparation strategies, as well as to the future, further clarifiedthe mechanism of antibody enzyme mimic natural enzymes, In this study, we used thelysozyme inhibitory nano antibody NBL42as an antigen, affinity panning screeningfrom the Bactrian camel immune phage display library which has already been built,expect to get the lysozyme specific anti-idiotypic nanobody, provide clues to themechanism of the antibody enzyme research and molecμLar modeling of the nano antibody.[Method]: By the determination of helper phage titer, determined the antibody libraryphage titer; Throμgh the BLAST and DNMAN analysis the lysozyme inhibitorysingle domain antibody amino acid sequence of NBL114, NBL42, abundantlyexpressed and purified the nano antibody NBL42gene, SDS-PAGE analysis andELISA detected the specific binding to lysozyme ability of NBL42,NBL114. Usingthe lysozyme inhibitory nano antibody NBL42as an antigen, affinity panningscreening from the Bactrian camel immune phage display library which has alreadybeen built. using the VHH-P1:5’-CGCGGATCCGTCCTGGCTGCTCTTCTAC-3,VHH-P2IgG3:5’ CCGCTCGAGCTTGCATACTTCATTCGTTTC-3’ tested IgG3positive cloning, and, sequencing, selecting two cloning to built the prokaryoticexpression vector, induced expression and purification, measured and simμLated theantigen binding properties of lysozyme by ELISA and Western blotting.[Results]: This experiment has measured helper phage M13K07titers of1.241×10¹³ pfu/mL,Xinjiang Bactrian camel immune library phage titer of1.4580×10¹³pfu/mL. successfμLly expression and purification the lysozyme inhibition antibodyNBL42NBL114. In immune library screening, we get35IgG3positive clones,according to the electrophoresis resμLts, selected20clones sent for sequencing. Weselected for two to construct prokaryotic expression vector, expression induced bypurified with c-Myc tag （1-6）, NBL NBL （2-3） two antibodies.ELISA and Westernblotting to detect the NBL （1-6）, the NBL （2-3） has a better antigen binding capacity,In order to detection of the NBL （1-6）, the NBL （2-3） inhibitory effect, weRespectively use the subtilis Bacillus, Staphylococcus aureus, Escherichia coli, theresμLts showed that NBL （1-6）, NBL （2-3） on Escherichia coli bacteriostasis isrelatively obvious, the OD value of respectively0.8643and0.889,（repetit3times，±0.03）， in this experiments bacteriolysis enzyme as a positive control OD valueof.855, and PBS as negative control OD value of1.065. According to the calcμLation formμLa of measuring enzyme activity, NBL （1-6） activity was7000U, NBL （2-3）was4200U.[Conclusion]:In this experiment,we successfμLly induced expression of lysozyme inhibitoryantibodies NBL42, NBL114, also obtained NBL （1-6）, NBL （2-3） antibody throμghscreening immune Library. ELISA and Western blotting technique detected the NBL（1-6）, the NBL （2-3）,resμLt shows that NBL （1-6）, NBL （2-3） has a better antigenbinding capacity, in vitro antibacterial experiments for Bacillus subtilis,Staphylococcus aureus, Escherichia coli, found the NBL （1-6）, the NBL （2-3） has acertain inhibitory effect for three of them, but more significant effects on Escherichiacoli.