ABSTRACT
AIM: To obtain purificated his-XCL1 recombinant protein of porcine in vitro and prepare the associated polyclonal antibody; To study how the recombinant protein affects on lymphocytes proliferation. METHODS: Purify the recombinant protein using HiTrap(TM); Chelating HP chromatographic column; Check the purified product using SDS-PAGE; Detect the expression of the recombinant protein using Western blot; Immunize the experimental animals with the purified fusion protein to prepare the serum containing the associated polyclonal antibody. The serum will then undergo double immnodiffusion test and indirect ELISA test to determine the polyclonal antibody titer. Then, test the condition of lymphocytes proliferation by MTT. RESULTS: Single target strip could be seen under conducting the SDS-PAGE electrophoresis when the concentration of the binding buffer is 40 mmol/L and the concentration of the elution buffer is 500 mmol/L; Western blot test showed that the recombinant protein could be successfully expressed; 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. The result of MTT showed that both the native XCL1 and the recombinant protein could stimulate lymphocytes proliferation, and this stimulating effect could be effectively blocked by the polyclonal antibody we prepared. CONCLUSION: To conclude, this recombinant protein has biological activity and this research can provide basic material for further investigation of the function of XCL1 in swine.
Subject(s)
Lymphokines/pharmacology , Sialoglycoproteins/pharmacology , Animals , Antibodies/immunology , Electrophoresis, Polyacrylamide Gel , Lymphocyte Activation/drug effects , Lymphokines/immunology , Lymphokines/isolation & purification , Recombinant Proteins/pharmacology , Sialoglycoproteins/immunology , Sialoglycoproteins/isolation & purification , SwineABSTRACT
AIM: To clone a novel swine gene P58(IPK)[58-kDa(inhibitor of protein kinase) protein] and prepare its polyclonal antibody for further research of influenza and host interaction. METHODS: The swine P58(IPK); gene was first identified in silico through homology searching in the swine EST database. Then this gene was amplified by reverse transcription polymerase chain reaction (RT-PCR). The cDNA of the gene contained the complete open reading frame(ORF) of 1 518 bp, and encoded 505 amino acid residues (Accession No.HQ287801). The gene was first analyzed using bioinformatics methods. Then P58(IPK) was cloned into a prokaryotic expression vector pET-32a to construct a recombinant plasmid named as pET-P58(IPK). The fusion protein his-P58(IPK) was expressed in E.coli BL21 and purified using a his-tag protein purification column. Subsequently rabbits were immunized with the purified protein. RESULTS: Specific polyclonal antibody against the fusion protein his-P58(IPK) was obtained. The activity of the antibody was determined through double-immunodiffusion test. The titer of the antibody was 1:20 000 as shown by ELISA. specifically recognized the protein P58(IPK) by Western blot and immunofluorescence assay. CONCLUSION: The novel swine gene P58(IPK) has been successfully cloned and its polyclonal antibody has been prepared.
