Prokaryotic Expression Of Swine XCL1 Gene And Preparation Of Polyclonal Antibody Against XCL1 Protein And Its Bioactivity Identification

Chemokines(chemoattractant cytokines) are cytokines of small molecular size that can induce directed chemotaxis, and play an important role in the process of cell migration and activation. Thus, chemikines are very important components in the immune system. Lymphocyte chemotactic factor (Lymphotactin, Lptn) , also known as lymphoid chemokine ligand 1 (XCL1), is a member of C chemokine family. The interaction between XCR1 and XCL1 involves in antigen presentation, T lymphocytes and NK cells activation, and the cellular immune function exertion processes, thus can regulate immune balance and induce inflammatory reaction in the process of infectious disease, and can enhance mucosal immunity and anti-tumor immunity.Liu et al(2010)had constructed eukaryotic expression vector pEGFP-PLptn, and confirmed that the fusion protein with green fluorescent marker displays chemotactic characteristic in vitro, but further study was not carried out. In this study, we first ever constructed prokaryotic expression vector pET-32a-XCL1 and found out the optimum conditions of its expression and purification. First, purify the recombinant protein using HiTrap^TM Chelating HP chromatographic column. Second, check the purified product using SDS-PAGE technique. Thirdly, detect the expression of the recombinant protein using western-blot.Immunize the experimental animals (New Zealand white rabbits) with the purified fusion protein to prepare the serum that contains the associated polyclonal antibody. The serum will then undergo double immnodiffusion test and indirect ELISA test to determine the polyclonal antibody titre. Then, test the condition of lymphocytes proliferation by MTT. This research can provide basic material for further investigation of the function of XCL1 in swine. The main contents and results of our research are as follows:1. Primers were designed according to XCL1 gene sequences on NCBI , and its ORF of about 0.27kb was amplified from the porcine lymph nodes and then integrated into the pMD-19T vector, and its positive plasmids were screened and sequenced. Sequencing results confirmed the correctness of the ORF in the recombinant vector.2. The ORF of XCL1 gene was integrated into prokaryotic expression vector pET-32a (+), and expressed in E.coli.BL21(DE3). Immunize the New Zealand white rabbits with the purified fusion protein to prepare the serum that contains the associated polyclonal antibody.3. Double immunodiffusion test showed the antigen-antibody binding ratio to be 1:8, and the titre of antibodies was 1: 12 800 detected by indirect ELISA.4. MTT method was used to study how this recombinant protein affects on lymphocytes proliferation. This result of MTT showed that the recombinant protein could stimulate lymphocytes proliferation, and that this stimulating effect could be effectively blocked by the polyclonal antibody we prepared.