The Expression And Antibody Preparation Of Natural Immune-ralated Protein SPLUNC1

Objective: To complete the eukaryotic expression of SPLUNC1-Ig fusion proteins and preparation and characterization of monoclonal antibodies against SPLUNC1 Contents: 1.The construction of SPLUNC1 eukaryotic expression vector; 2. the study to express the fusion proteins of m-SPLUNC1-Ig and h-SPLUNC1-Ig and analyze the vitro biological activity of h-SPLUNC1-Ig; 3. preparation and characterization of monoclonal antibody against SPLUNC1.Methods: SPLUNC1 gene was amplified from the lung adenocarcinoma tissues. The two steps, including megaprimer method and nested PCR, were performed to achieve the site-directed mutagenesis of 147th base in the gene SPLUNC1 in order to be successly inserted into PDH3 vector, while m-SPLUNC1 was inserted into PDM3 vector. The recombinant plasmids PDH3-SPLUNC1 and PDM3-SPLUNC1 were separately transfected into CHO/dhfr- cells by Lipofectamine. The expression of SPLUNC1-Ig fusion proteins were identified by RT-PCR,SDS-PAGE and Western-blot. By using MTX and two times cloning with limiting dilution method, individual CHO engineering cell lines, which secret SPLUNC1-Ig fusion proteins in the culture supernatant ,were obtained. The fusion proteins were purified by affinity chromatography and its vitro biological activity of binding lipopolysaccharide was analysed by ELISA and Western-blot. m-SPLUNC1-Ig fusion protein was used for immunizing Balb/c mice by the routine method. The spleen cells from immunized mice were fused with SP2/0 cells and selectively cultured with HAT medium.ELISA and Western-blot were used to screen and identify the monoclonal antidodies.These McAbs were analysed for their antibody titre,class,subclass and affinity. The antigen specificity for McAbs were evaluated and confirmed by ELISA.The binding sites and capabilities of the McAbs were observed by ELISA additive assay.Results: The SPLUNC1 gene was amplified and cloned.The mutation was achieved at the expected site by restriction enzyme digestion and sequence analysis. Each fragment of m-SPLUNC1 and h-SPLUNC1 was separately cloned into the expression vectors. RT-PCR showed that target CHO cells integrated the recombinant plasmids of PDH3-SPLUNC1 and PDM3-SPLUNC1. ELISA and Western-blot showed that the proteins purified from the cells supernatant were confirmed to be SPLUNC1-Ig fusion proteins. The fusion protein of h-SPLUNC1-Ig could bind lipopolysaccharide. Six hybridoma cell lines secreting monoclonal antibodies against SPLUNC1 were obtained. About the immunoglobulin subclasses of all McAbs were IgG1. ELISA showed that five of six McAbs reacted specifically with recombinant SPLUNC1 protein and these McAbs could bind natural SPLUNC1 protein.Conclusion: CHO cell lines which stablely express m-SPLUNC-Ig and h-SPLUNC-Ig fusion proteins were established and purified fusion proteins were obtained. We successfully obtained five specific McAbs against SPLUNC1 which could be used to detect SPLUNC1 in vitro.