Camelization Of Murine Anti-AFB1 Single-Domain Antibody And Its Expression

Camelid single-domain heavy chain antibody(nanobody or VHH),as the smallest complete antigen-binding domain.Compared with traditional antibodies,it has some unique properties,such as high solubility,high temperature and extreme pH resistance.However,the VHH against small molecular pollutants such as AFB1 widely has a difficulty in obtaining.Therefore,camelization of heterologous antibodies is a hot spot in this yield.However,the current researches of camelization mainly focus on the antibodies against large molecules.In this study,based on the genetic evolutionary and their sequence differences,the VH domain derived from murine anti-AFB1 scFv was camelized through site-directed mutagenesis and complementarity determining region transplantation in vitro.All the camelization of anti-AFB1 VH antibodies(cVHs)were prepared and their expression strategies were optimized.The main results are shown as follows:1.Sequence analysis of murine anti-AFB1 VHs and its camelization.Using amino acid sequence information of VH and VHH as a reference,a camelization modification plan was designed.Based on the VH domain which derived from murine anti-AFB1 scFv(VH-2E6),the cVH-FR2(Val37 Phe,Gly44 Glu,Leu45Arg and Trp47Gly)and cVH-103(Val37 Phe,Gly44 Glu,Leu45Arg,Trp47Gly And Trp103 Arg)were obtained by site-directed mutations.Meanwhile,the CDR3 of anti-AFB1 VHH Nb26(IC50 is 0.754 ng/mL)was grafted onto the cVH-103 antibody scaffold to obtain the chimeric cVH-Nb26.Preliminary expression analysis of pComb3X-VH/cVHs revealed that there were no obvious target bands at different induction times(4 h,6 h,8 h)and induction temperature(16℃,37℃).2.Optimization of prokaryotic expression strategy and characterization of antiAFB1 cVHs.In the prokaryotic system,the recombinant strains were constructed with pET-22b(+)vector.Western blot analysis found that the mutation in FR2 domain downregulated the soluble expression of antibody.And 103Trp located on the FR4 domain was a key point which affected soluble expression of antibodies.However,as the CDR3 domain of cVH-103 camelized,the extent of the effect of this site on antibody expression decreased.After optimized medium,temperature and expressing strain,the yield of VH-2E6 was 17 mg/L.The cVH-FR2 and cVH-Nb26 were obtained by refolding of inclusion bodies with yields were 4 mg/L and 5 mg/L,respectively.The expression yield of fusion expression of SUMO tag and cVH-103 was 0.3 mg/L.The binding capacity of VH and cVHs to AFB1 was determined by ELISA that murine VH2E6 was hardly binded to AFB1,while all cVHs can bind to AFB1,in which cVHNb26 was the most effective.The competitive binding reaction of cVH-Nb26 and AFB1 was constructed.When the concentration of AFB1 reached 1 μg/mL,the OD450nm of ic-ELISA decreased from 0.95±0.03 to 0.56±0.05,indicating that cVH-Nb26 could specifically bind to AFB1.3.Expression and characterization of anti-AFB1 cVHs in eukaryotic expression system.Pichia pastoris(P.pastoris)and mammalian cell were used to express cVHFR2 and cVH-Nb26,respectively.The results showed cVH-FR2 was not expressed,while cVH-Nb26 was successfully expressed with the yield in two systems were 10 mg/L and 26.7 mg/L,respectively.The most suitable coating antigen was chosen for indirect-compete ELISA.When the concentration of AFB 1 was 2 μg/mL,the OD450 nm of cVH-Nb26 expressed by P.pastoris decreased from 0.69 ± 0.00 to 0.47 ± 0.04,and cVH-Nb26 in mammalian cells decreased from 0.75 ± 0.01 to 0.64±0.03,which indicated that cVH-Nb26 expressed by eukaryotic system could effectively bind to AFB1.