Isolation And Identification Of Anti-CD47 Nanobodies By Proteomics And High Throughput Sequencing

CD47 is highly expressed on the surface of a variety of tumor cells,belongs to a member of the immunoglobulin superfamily,CD47 causes tumor cells to evade macrophage phagocytosis by binding to the SIRPαand signaling"do not eat me".At the same time,CD47 is enable to bind to TEP 1,resulting the inhibition of proliferation and activation of T lymphocytes,and the killing effect of T lymphocytes on tumor cells.Therefore,the anti-CD47 monoclonal antibody can be used as a new therapy in tumor immunotherapy.Nanobody,a single-domain antibody derived from the camel’s variable region,it is the smallest single-domain antibody with complete antigen-binding function from now.Thus it has great potential in drug development and can be used as a high-affinity reagent for research and diagnosis.In this study,high-throughput sequencing and mass spectrometry techniques were used,and then detects affinity and explore the value of proteomics and genomics in the discovery and identification.This research mainly includes the following three aspects:1 Analysis of Camel immune antibody library by High-throughput sequencing and the establishment of nanobody databaseFirst of all,prokaryotic recombinant protein CD47 in E.coil BL21 were expressed and purified,then used CD47 for immunization of Xinjiang bactrian camels and with 6 times.The fifth and sixth sera titers of were detected by indirect ELISA.Then separation of peripheral blood lymphocytes,total RNA was extracted and reverse transcription into cDNA,VHH gene was high-throughput sequenced after it had amplified by nested PCR.AbMining ToolBox was used to analyze high-throughput sequencing data,and Python was used to remove the stop codon to remove the stop codon in the database and established an anti-CD47 Nanobody amino acid database.The anti-CD47 antibody serum titers is 1:384 000,the immune response in the camel was elicited.1 054 550 VHH sequences are obtained by high-throughput sequencing and 945 061 sequences were obtained after adapters and low quality sequences were removed.The effective length of the nucleotide sequence is mainly distributed in 300-350 nt.The base quality over 20 is 97.67%,and over 30 is 95.58%,the capacity of amino acid database of anti-CD47 Nanobody is 9.19×10~5.High-throughput sequencing data is analyzed by AbMining ToolBox and 160 485specific CDRH3 sequences are obtained,the amino acid length is mainly distributed in 8-35.The isoelectric point distributions of CDRH3 is analyzed,respectively,in which pI 8-9 and pI 4-5 accounted for 22.72%and 20.1%.2 Preparation and identification of anti-CD47 specific antibody from camel immune serumAt first,IgG in camel serum was purified by Protein A and the binding activity of IgG to CD47 was measured by indirect ELISA and Western Blot,the anti-CD47specific antibody was captured by coupled CD47 protein with CNBr agarose resin and then determined CD47-CNBr Coupling efficiency was.Finally,the anti-CD47specific antibody binding activity was detected by ELISA and Western Blot.In addition,verified the binding activities of the serum,IgG and anti-CD47 specific antibodies to eukaryotic CD47 and cell for subsequently a therapeutic antibodies obtained by mass spectrometry and provide experimental basis.The experimental results show that a better binding activity of IgG to the prokaryotic expressed CD47 protein by indirect ELISA and Western Blot until IgG is diluted 1:256 000.As 15%SDS-PAGE showed,protein CD47 is successfully coupled to CNBr agarose resin and the efficiency is 67.8%.The selection elution of the purified anti-CD47 specific antibodies by indirect ELISA is eluted with Gly-HCl,pH 2.4.And then obtained anti-CD47 specific antibody successfully.by the CD47-CNBr agarose resin.The anti-CD47 specific antibody had high binding activity and specificity to prokaryotic CD47 by indirect ELISA and Western Blot.The binding activity of serum,IgG and anti-CD47-specific antibody to eukaryotic CD47 and EC9706 by ELISA demonstrated that serum,IgG enable to bind to eukaryotic CD47protein and cells,but anti-CD47 specific antibody.3 Identification and preparation of anti-CD47 Nanobody by by mass spectrometryFirst,the IgG was separated by SDS-PAGE and its heavy chain antibodies IgG2and IgG3 were cut out for detection of LC MS/MS.Then,mass spectrometry datum were matched with the camelid amino acid database by Mascot,The 7 sequences with the highest matching score were selected and constructed genes to the prokaryotic expression vector pET22b and purified them to measured the binding ability of these fantibodies to prokaryotic CD47 protein by indirect ELISA.The anti-CD47Nanobody with the highest binding activity was selected and its binding activity to eukaryotic CD47 was verified by indirect ELISA.Simultaneously,high-affinity antibody sequences screened by phage display technology were matched with mass spectrometry data and high-throughput data,respectively.The results show that seven recombinant plasmids were induced,of which the VHH-437310,VHH-737446 have expressed,ecxepted VHH-410994.And the sizes of about 20 kDa.As shown by Indirect ELISA,the anti-CD47 Nanobody VHH-737446has binding activity to prokaryotic CD47 but not to eukaryotic CD47.The antibody sequences screened by phage display technology can only be successfully matched with high-throughput databases to obtain 6 sequences and appear at low frequency in the database.