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Objective To prepare camelid-derived nano antibodies with high affinity binding to programmed death receptor-1 (PD-1) antigen,and to provide experimental basis for subsequent functional studies.Methods The PD-1-Fc recombinant protein expressed in eukaryotic expression was used to immunize Xinjiang Bactrian camel 6 times.The peripheral blood was collected and the lymphocytes were isolated.Nested PCR amplification was performed to obtain the genes in variable region of camelid heavy chain antibody (VHH),and to construct a phage display library.The phage display library was screened by solid phase enzyme-linked immunosorbent assay (ELISA).The PD-1 antigen,which was sequentially reduced in mass concentration (5.00、2.50、1.00 μg/ml),was coated in an ELISA plate,and the phage display library was subjected to 3 rounds of affinity selection.Individual clones that bind to PD-1 were further screened by soluble monoclonal ELISA.According to the results of DNA sequencing,three VHH monoclonals with multiple repeats were selected and ligated into pET22b vector,and transformed into E.coli BL21 (DE3) competent cells,and then induced by isopropyl-β3-D-thiogalactoside.The recombinant VHH antibody protein was purified by nickel column affinity chromatography,and its binding activity and affinity to PD-1 antigen were detected by Western Blot and ELISA.Results After immunization of Bactrian camel 6 times with recombinant protein PD-1-Fc,high titer specific antibody was stimulated,and the immune serum titer reached 1∶32 000.A VHH phage display library with a reservoir size of 2.6×108 cfu/ml was constructed from the immunized camel lymphocytes.After 3 rounds of affinity selection,46 VHH monoclonals with absorbance (A600) values above 0.6 were obtained by soluble monoclonal ELISA.Among them,three clones of VHH-B7,VHH-H5 and VHH-H12 had higher repeats,indicating that significant enrichment was obtained.The results of Western Blot and ELISA showed that the purified B7,H5 and H12 nanobodies had good binding activity to PD-1 antigen and had high affinity.Their affinity constants were 1.19×1011 and 1.63×1011,1.59×1011 L/mol,respectively.Conclusion The anti-PD-1 camelid-derived nanobodies were obtained by affinity selection of VHH phage display library,which can bind to the PD-1 antigen with high affinity.This study can provide an experimental basis for subsequent functional studies.
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Objective To construct anti-astrocyte elevated gene-1(AEG-1)single-chain variable antibody (V23)prokaryotic ex-pression vector,and to conduct the protein purification and immunological activity detection.Methods The Primer5 software was applied to design the primers aiming at the gene sequence of the antibody anti-AEG-1 single-chain variable region for constructing the prokaryotic expression plasmid of PRsetC/V23.After the enzyme digestion by the restriction enzyme Pst1 and correct DNA se-quencing,the prokaryotic expression plasmid was led to E.coli BL21 ,the prokaryotic expression engineering strain containing the V23 gene was constructed.After the induction with IPTG,the interest protein was purified by the magnetic beads with the HIS tag,and the content of interest protein was determined by the SDS-PAGE electrophoresis.Western blot and ELISA were adopted to detect the immune activity of the nti-AEG-1 single-chain variable region antibody.Results For the constructed prokaryotic expres-sion plasmid PRsetC/V23,the single enzyme digestion and sequencing analysis displayed that the constructed V23 gene was com-pletely consistent to the designing sequences.After IPTG induction,SDS-PAGE electrophoresis showed an apparent protein band at 31×103 ,the Western blot detection showed a specific AEG-1 response band at 80 ×103 ,the ELISA test showed the positive re-sults.Conclusion The PRsetC/V23 prokaryotic expression plasmid and the V23 prokaryotic expression engineering strain are suc-cessfully constructed,this engineering strain can express anti-AEG-1 single-chain variable region antibody protein,and the protein has good immune activity.
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Objective To study the intrinsic relationships between the binding energy of the antibody light and heavy chains and the conformational characteristics , physical and chemical properties , and to establish a corresponding mathemat-ical model and evaluate the thermal stability of the antibody molecules , which contribute to the antibody design , optimiza-tion and affinity maturation .Methods Based on bioinformatics and computational biology methods , the antibody′s structur-al information with the crystal diffraction data was analyzed .The conformational character of the variable domain of the antibody was studied using distance geometry and computer graphics technology .With the aid of the intermolecular hydrogen bond formation theory and the reaction free energy theory , the dynamic structure and energy characteristics be-tween the heavy and light chain variable regions of the antibody were studied .Furthermore , using nonlinear fitting and regression analysis, a mathematical model was set up .Results According to simulation and statistic analysis , there was a linear relationship between the binding energy and the number of the intermolecular hydrogen bonding , Van der Waals interaction of the heavy and light chains of the antibody .There was polynomial correlation between the binding energy and the physicochemical properties of the antibody .Using the frequency of amino acid position and the established model , the humanized anti-ricin antibody , which could not obtain the stable engineering cell line , was evaluated and optimized .The stable engineering cell line of the humanized anti-ricin antibody was obtained in the experiment .Conclusion The self structure of the antibody variable region ( conformation and physicochemical properties ) has much effect on its stability . The antibody stability can be improved by structural optimization .
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Objective:To clone Fab genes of anti-p185 monoclonal antibody 5E12 and express it in E.coli.Methods:Fd and ? genes were cloned by RT-PCR, inserted into Fab expression vector and expressed in E.coli. The N-terminal sequences of V regions was resumed by PCR mediated mutagenesis. The antigen-binding activity of the Fab were tested by ELISA and immunohistochemistry.Results:Fd and ? genes were cloned and expressed in E.coli. But the bacterially expressed Fab fragments showed no antigen binding activity. After the N-terminal sequences of V regions was corrected to original sequences, the Fab expressed in bacterial was able to target HER2/neu-expressing cells(NIH3T3/erbB-2 cells). Correction of Fd N-terminal sequences could partially resume the antigen binding activity. But correction of ? chain N terminal sequences was shown no expected result.Conclusion:Successful in constructing and expressing anti-p185 Fab, which will benefit the construction of other engineering antibody and humanization of murine anti-p185 McAb. We also found that the V region N terminal changes introduced with PCR primers may affect antigen binding activity seriously, to which more attention should be paid during antibody engineering.
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Objective: To clone the Fab gene of a monoclonal antibody (mAb) BDI against human bladder cancer and its expression in E. coli. Methods: Fd and K genes of mAb BDI were cloned by RT-PCR and inserted into an Fab expression vector. Phage displaying Fab and soluble Fab were expressed in E. coli. The N-terminal sequence of VH region was corrected by PCR mediated mutagenesis. The antigen-binding characteristics of the Fab were tested by ELISA and immu-nohistochemistry. Results: Fd and K genes were cloned into the expressing vector p3MH and the phage displaying antibody and soluble Fab were expressed in E. coli, which showed weak binding activity to bladder cancer cells. Correction of the N-terminal sequence of the VHimproved the biding activity dramatically. The feasibility of the application of the Fab in phage antibody library screening was confirmed by a simulated panning procedure. Conclusion: The Fab gene of an anti-human bladder cancer mAb was expressed in E. coli. The importance of the N-terminal sequence on antibody binding activity was suggested.
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Objective To construct single chain antibody specific to membrane protein of Schistosoma japonicum by gonetic engineering technique. Methods The V\-H (heavy-chain variable region) and V\-L (light-chain variable region) genes were amplified by PCR from the genomic DNA of NP11-4 cell line, and sequenced by Sanger's method. The ScFv was constructed in pTHA90 vector using V\-H and V\-L genes, then expressed by IPTG. Results The V\-H and V\-L genes were obtained through PCR. The DNA sequences showed that V\-H and V\-L were new variable region genes of antibody. They were registered by GenBank. A ScFv gene with (Gly4Ser) 3 intralinker in the pTHA90 vector was successfully constructed. The ScFv was expressed as thioredoxin-fused proteins about 36^2 kDa. Conclusion A specific ScFv against the membrane protein of Schistosoma japonicum was constructed and expressed.