Preparation And Functional Study Of Anti-human CTLA-4 Antibody

Cytotoxic T lymphocyte-associated antigen-4?CTLA-4?is a negative regulatory molecule that inhibits T cell activation on the surface of T cells,which plays an important role in the development of malignant tumors and some autoimmune diseases.Tumor cells can achieve immune escape through CTLA-4.The blockers of CTLA-4 such as monoclonal antibodies can reactivate the immune system and achieve anti-tumor effects.The aim of this study was to develop monoclonal antibodies targeting human CTLA-4 using mouse and human antibody library technologies.First,the extracellular domain gene fragment was amplified by human CTLA-4extracellular domain gene,insert the E.coli expression vector pET28a to construct a recombinant plasmid pET28a-CTLA4 encoding the human CTLA-4 extracellular domain gene.Transforming recombinant plasmid into E.coli Rosseta?DE3?.Under the condition of IPTG final concentration of 0.5 mmol/L and induction at 37?for 4hours,the high expression of recombinant protein in human extracellular region of CTLA-4 was successfully achieved.The expressed product was mainly inclusion body.Optimized for three factors:induction temperature,induction time and final concentration of inducer IPTG.The highest expression level of the recombinant protein was obtained under the conditions of a final concentration of IPTG of 1mmol/L and induction at 25?for 6 hours.The high-concentration urea was used to dissolve the inclusion body protein,and the recombinant protein with purity greater than 90%was obtained by Ni²⁺affinity chromatography.A recombinant antigen of the human CTLA-4 extracellular domain gene was prepared by refolding the inclusion body protein into a soluble protein.Five BALB/c mice were immunized four times using human CTLA-4extracellular domain gene recombinant antigen.The spleen was isolated from the mouse with the highest serum titer,total RNA was extracted and reverse transcribed into cDNA.The light and heavy chain variable region gene fragments of the antibody with a size of about 300 bp were amplified by using cDNA as a template.The light and heavy chain variable region genes were spliced into single-chain antibody fragment?ScFv?of about 750 bp in size by overlap extension PCR.Insert ScFv into phage vector pCANTAB-5E and transform into E.coli TG1.The insertion rate of ScFv was determined by PCR to be about 93.5%,and a mouse phage single-chain antibody library with a library capacity of about 1.4×10⁶ was finally constructed.After three rounds of enrichment screening,two mouse single-chain antibody genes targeting human CTLA-4 extracellular domain gene recombinant antigen was obtained.On the other hand,a large-volume human phage single-chain antibody library was repeatedly subjected to four rounds of panning in the order of"adsorption,elution,amplification"using the human CTLA-4 gene recombinant antigen.After the fourth round of panning was completed,24 positive phage clones were obtained by ELISA.The antibody sequences carried by the 24 positive phage clones were analyzed,and 21 of the phage clones carried the same antibody gene with a repetition rate of 87.5%,which is suggested to be a human single-chain antibody gene capable of specifically binding to antigen.The light chain variable region gene and the heavy chain variable region gene were amplified by PCR and the sizes were 324 bp and 351bp,respectively.The recombinant plasmids pABL-VL and 293H-VH were constructed and then co-transfected into 293F cells to successfully express the whole antibody with intact structure.The human monoclonal antibody with a purity greater than 90%was finally purified by Protein A affinity chromatography.The antibodies were identified by ELISA and Western blotting,and the results showed that the antibodies have good specificity.In addition,we also prepared mouse monoclonal antibodies against human synaptosome associated protein 25?SNAP25?.The SNAP25 protein was successfully expressed by E.coli expression system,and the purified protein was used to immunize BALB/c mice to prepare hybridoma cells.After 2 rounds of screening,12positive hybridoma cell lines against SNAP25 were obtained,and subtypes of antibodies were also identified.Preparation of ascites monoclonal antibody using hybridoma cell line 14 with the highest antigen binding activity.Finally,a monoclonal antibody with a purity greater than 90%was purified by Protein G affinity chromatography,which can be uesd for the detection of botulinum toxins.