Cloning And Expression Of Chemokine CCL28 Gene And Development And Preliminary Application Of Its Monoclonal Antibody

CCL28 is a mucosa-associated epithelial chemokine belonging to the small molecular cytokine CC gene subfamily,and it is expressed in different level in different mucosal parts of the normal human organs and tissues such as the parotid gland,breast,trachea and colon.In addition to having a broad-spectrum antibacterial effect and participating in the immune barrier,CCL28 is also highly expressed in various epithelial tumor cell lines.Previous reports found that human-derived CCL28 protein has a homology of more than 60%identity with animal-derived CCL28 such as mice,rats,pigs and sheep.However,the interaction mechanism between CCL28 and its receptor CCR3/CCR10,whether CCL28 can be used as a mucosal vaccine adjuvant,and whether CCL28 can be used as a tumor therapy target need further to be investigated.In order to explore the relevant biological roles of CCL28,in this study,human CCL28 gene was cloned and expressed,and a specific monoclonal antibody against human CCL28 was generated and its potential for tumor therapy was tested in nude mice.1.Cloning and expression of human CCL28 gene and preparation of polyclonal antibodyIn order to obtain the immunogen and antigen for screening human CCL28 monoclonal antibody,the specific primers were designed and synthesized based on CCL28 gene sequence from Genbank,and CCL28 gene was cloned into pcDNA3.1 vector,designated as pCDNA3.1-CCL28.The positive recombinant pGEX-6P-1-CCL28 was transferred to BL21 competent cells and the expression of soluble fusion protein GST-CCL28 was induced by IPTG.The polyclonal antibody against CCL28 was prepared by immunizing mice with the purified GST-CCL28.Western Blot proved that the polyclonal antibody generated could not only react with the prokaryotic expression product GST-CCL28,but also recognize the CCL28 expressed by the eukaryotic expression plasmid.The generation of fusion protein GST-CCL28,the construction of CCL28 eukaryotic expression plasmid and the preparation of polyclonal antibody against CCL28 provided materials for the development of monoclonal antibody against CCL28.2.Preparation of monoclonal antibody against human CCL28In order to generate monoclonal antibodies against human CCL28,the purified recombinant protein GST-CCL28 was used to immunize 6-week-old BALB/c mice,and the spleen cells from the immunized mice were taken for cell fusion.293T cells transfected with pcDNA3.1-CCL28 recombinant plasmid was as the screening antigen,hybridoma cells secreting antibody against CCL28 were selected by IFA.After subcloning,a hybridoma cell line stably secreting monoclonal antibody against CCL28 was obtained and named 5A3.The subclass of 5A3 was identified as IgG2b,and the light chain was kappa chain.Both IFA and Western blot proved that monoclonal antibody 5A3 could specifically react with CCL28 expressed exogenously.and the IFA titer was more than 1:12800.Epitope identification revealed that monoclonal antibody 5A3 could recognize the important functional regions of CCL28 at positions 80aa-96aa.The development of monoclonal antibodies against CCL28 and the identification of its epitopes laid the foundation for further exploration of the biological function of CCL28 and its roles in tumor therapy.3.Preliminary study on anti-tumor effect of human CCL28 monoclonal antibodySince CCL28 is highly expressed in various epithelial tumor cell lines,and CCL28 antagonists can cooperate with the treatment of breast cancer,in order to explore whether the mouse monoclonal antibody against human CCL28 can be used as an effective antagonist in human tumors.A nude mouse colon cancer tumor model was first established by using human colon cancer cell line HCT116-P53+/+,and then the monoclonal antibody 5A3 was used for treatment through tail vein injection and intramuscular injection.The tumor growth in nude mice was monitored.Our data showed that the monoclonal antibody 5A3 had a certain inhibitory effect on tumor growth rate compared with the control monoclonal antibody.However,there was no significant difference between two groups.The above preliminary data indicate that the therapeutic effect of monoclonal antibody 5A3 against CCL28 on tumors needs to be further evaluated.