Study On Anti-human CD146 Single Domain Antibody Of Striped Bamboo Shark(Chiloscyllium Plagiosum)

Background: Glioma is the most common malignant primary brain tumor.Traditional treatments include surgery,chemotherapy and radiotherapy.The clinical prognosis is very limited.The existence of the blood-brain barrier makes the research progress of targeted drugs slow.The classification of gliomas with molecular markers is an important method to evaluate the degree of tumor malignancy and design treatment plans.CD146 is an adhesion molecule that is found to be highly expressed in a variety of tumors,including glia,and is associated with tumor invasion and infiltration.At present,drug therapy targeting CD146 has achieved good results in a variety of tumors.How to efficiently penetrate the blood-brain barrier and accurately target CD146 in gliomas is an urgent problem to be solved.The molecular weight of the variable region VNAR of Ig NAR containing only heavy chain found in cartilaginous fish is only 12-15 k Da,which can effectively penetrate the blood-brain barrier,and has extremely high antigen affinity,specificity and stability.The research and development of glioma-targeted drugs provides new ideas and methods.Methods: The CD146 epitope peptides were screened with bioinformatics software such as ABCpred and Swiss-Model,and the peptides were synthesized as antigens for immunizing the striped bamboo shark.The experimental group was immunized with 10 μg epitope polypeptide and Freund’s adjuvant mixed with emulsification.Immunization once every 30 days for a total of 4 months.During the period,blood was collected 4 times regularly.After the immunization,the striped shark was dissected to take out the spleen tissue,and the blood samples were collected for blood cell and plasma separation,and ELISA was used to detect the production of antibodies in the plasma and active.Blood cells and spleen samples were subjected to transcriptome sequencing,and total spleen RNA was extracted.After reverse transcription,specific primers were used to amplify the VNAR region sequence and high-throughput sequencing was performed to establish a VNAR library.After comparing the transcriptome and VNAR library,the anti-CD146-specific VNAR region sequence was obtained by screening.The obtained anti-CD146-specific VNAR region sequence was subjected to whole gene synthesis to obtain the recombinant plasmid p ET-32a(+)-VNAR.Using Escherichia coli BL21(DE3)as the host,prokaryotic expression was carried out,and the recombinant VNAR was purified.Elisa detects the antigen-binding specificity and activity of recombinant VNAR.Taking the A172 glioblastoma cell line as the research object,cell viability detection,cell scratching,detection of protein level and RNA level were performed to study the tumor suppressive activity and suppressive mechanism of recombinant VNAR.Results: 1)The production of anti-CD146 antibody was successfully detected in plasma,and its activity increased with the immunization cycle.2)702,755 sequences were obtained by VNAR high-throughput sequencing,and 10 pre-selected VNAR sequences were obtained by comparison and screening with the transcriptome,all of which were in line with the VNAR primary sequence and secondary structure characteristics.3)After the recombinant plasmid sequences were induced and expressed in a small test,they were all induced at 16°C for 12 h for amplification,expression and purification.4)ELISA detected that VNAR-CD146-Seq4 has good CD146 antigen binding activity.5)Tumor inhibition experiments proved that VNAR-CD146-Seq4 can effectively inhibit the activity,migration,etc.of A172 glioblastoma cell line,and inhibit the protein levels of CD146,apoptosis inhibitor Bcl-2,Mcl-1,Bax and phosphorylation of ERK in the MAPK signaling pathway.Conclusion: Striped Bamboo Shark immunized with CD146 antigenic epitope polypeptide produced a specific immune response against CD146 and produced anti-CD146 Ig NAR.The preselected VNAR sequence was obtained by comparing the transcriptome and VNAR high-throughput sequencing results.Sequence 4 has good CD146 antigen binding specificity after recombinant expression,and sequence 4 has good A172 glioblastoma inhibitory activity.