Screening Of VEGF Nanobody

In recent years,the incidence of malignant tumors in China is on the rise.Cancer is increasingly endangering the life and health of Chinese residents and bringing a heavy economic burden to society.Cancer has become a serious challenge in the field of public health in China.Malignant tumors have the ability to infiltrate and metastasize,and often develop at a fast rate.Tumor angiogenesis plays an important role in tumor genesis,development and metastasis.It is of great significance to the treatment of tumor to inhibit the formation of tumor blood vessels and thus the blood oxygen supply of tumor.Tumor angiogenesis is a complex process promoted by a variety of cytokines,among which vascular endothelial growth factor（VEGF）is the main influencing factor.Vascular endothelial growth factor（VEGF）binds to the receptor VEGFR and thus activates intracellular tyrosine kinases that initiate downstream cell signaling and promote neovascularization.VEGF pathway is an important target in the treatment of tumor and ophthalmic macular degeneration.At present,several monoclonal antibody have been used in the blocking treatment of VEGF pathway.However,the traditional monoclonal antibodies often have large relative molecular weight,and their stability,affinity,specificity and tissue penetration ability are still lacking.Nanobody has the advantages of small relative molecular weight,penetrating blood-brain barrier,strong tissue penetration,high stability,and strong specificity,which is a potential direction of antibody development.Therefore,this dissertation study the screening of VEGF nanobody,hoping to lay a foundation for further development of nanobody for tumor treatment.In this dissertation,primers were designed based on the protein coding region of the binding site of vascular endothelial growth factor（VEGF）to the receptor VEGFR.Using laboratory human-cdna as the PCR template,V29 H vector was selected as the prokaryotic expression vector of E.coli,and plasmids for VEGF and VEGFR-2prokaryotic expression were constructed,respectively.The prokaryotic expression strains of VEGF and VEGFR-2 were cultured on a large scale and purified to obtain VEGF and VEGFR-2 proteins.At the same time,the eukaryotic expression plasmid was also constructed.PTT5 vector was selected as the expression vector of lactation system,and the eukaryotic expression plasmid of VEGF was constructed.The plasmid was transfected into cells and purified to obtain pure protein.The obtained proteins were analyzed by ultra-speed centrifugation,which proved that the VEGF protein expressed in the eukaryotic system was dimer,which was consistent with reality.In order to obtain nanobody,purified eukaryotic expression of VEGF protein was used to immunize alpaca.After the immune cycle,blood was drawn from the jugular vein of alpaca to separate peripheral blood lymphocytes.RNA was extracted from the separated peripheral blood and then reverse-transcribed into c DNA.The c DNA was used as template to amplify the VHH region of alpaca,and a phage library was constructed.Using enzyme-linked immunosorbent assay（ELISA）with low concentration of antigen coating,screening specific binding to VEGF nanobodies.Four of the nanobodies sequences were selected to construct eukaryotic expression,serialized into p TT5 vector,and then transfected into cells.No protein expression was found during purification.Prokaryotic expression was then constructed and linked to p ET22 b,in which 6F was purified to express the protein.In order to verify whether the screened nanobody binds specifically to the antigen,an ELISA experiment was designed.The results show that the screened nanobody 6F has a high affinity with the antigen VEGF.In conclusion,the experimental results indicate that VEGF can be expressed in both the prokaryotic system and the eukaryotic system,but the protein expressed in the prokaryotic system has heterozygous bands.The screened nanobody is not expressed in the eukaryotic system,but can be expressed in the prokaryotic system.The screened nanobody 6F has a high affinity with the VEGF.These results will be helpful for the development of anti-tumor nanobody.