Development Of Monoclonal Antibodies To The Stem Cell Secretory Factor KIAA1199 And Their Preliminary Application Tests

Objective(s): Prokaryotic expression and purification of specific polypeptide fragments of the murine hyaluronic acid binding protein KIAA1199;developing and screening of the monoclonal antibodies(m Abs)against KIAA1199;production and purification of antiKIAA1199 m Abs in large quantities;and further test the application potentials of anti-KIAA1199 m Abs.Method(s):(1)Prokaryotic expression,purification and antigenicity identification of mouse KIAA1199 protein: analyzing the immunogenic region of KIAA1199 protein,select the 31-166 aa fragment,the best predicted epitope and specificity region,including the complete G8domain;the target gene is seamlessly cloned into the p ET-28 a vector through total gene synthesis,Sequencing confirmed that the correct p ET-28 a recombinant plasmid was transformed into DH5α competent cells,induced by IPTG,purified and renatured;the purified mouse KIAA1199 recombinant protein was confirmed and identified by Western-blot analysis.(2)Development and identification of monoclonal antibody against hyaluronic acid-binding protein KIAA1199: BALB/c mice were immunized with purified KIAA1199-specific polypeptide for four weeks by subcutaneously injected for three times;and serum antibody titer was determined by indirect enzyme-linked immunosorbent assay(ELISA)after the immunization;after the titer reaches the expected value,the spleen cells of the mouse are taken for cell fusion with the myeloma cell SP2/0;the stable positive strain is screened out after hybridoma cell screening and subcloning,the stable positive clones were selected and stored;a large amount of monoclonal antibodies were produced by the ascites method;the collected ascites was subjected to two-step ammonium sulfate precipitation and protein A purification.(3)Preliminary application tests of anti-KIAA1199 m Abs: a.Detection of KIAA1199 protein by anti-KIAA1199 m Abs by Western blot;b.Effect of anti-KIAA1199 m Abs on the proliferation of lung cancer tumor cells.Result(s):(1)KIAA1199 recombinant peptide with a molecular weight of about 17 KDa was successfully prepared by prokaryotic expression and purification.The form of the target peptide was mainly as inclusion body,which was identified as the target specific antigen fragment by SDS-PAGE.(2)Preparation and identification of monoclonal antibody against hyaluronic acid-binding protein KIAA1199: BALB/C mice were successfully immunized,and hybridoma cell fusion between immunized mouse splenocytes and myeloma cells SP2/0 was completed,and the fusion rate was 51.9%(249/480).After three rounds of subcloning culture and screening,more than 50 positive monoclonal antibody clones were obtained and preserved.Three hybridoma cell lines(No.1,No.2,and No.3)with the highest titer and stable antibody secretion were selected,a large number of ascites antibodies were prepared,and antibody purification was successfully completed.(3)The purified anti-KIAA1199 m Ab can be used in Western blot to specifically recognize the expression band of KIAA1199 in tissues;the purified anti-KIAA1199 m Ab can inhibit the proliferation and growth of lung cancer cell;KIAA1199antigen polypeptide can promote obesity caused by high-fat diet.Conclusion(s): In this study,the anti-KIAA1199 m Ab was successfully developed and purified,and anti-KIAA1199 m Ab can detect KIAA1199 protein in Western blot analysis,its inhibitory effect on the growth of lung cancer cells.The development of anti-KIAA1199 m Ab provides a necessary basis for further study on the function of the antibody drug that targeting KIAA1199.