Abstract: Objective To prepare a polyclonal antibody against SNX17, one of cell sorting nexin protein family members, and to provide a basis for future functional studies of the protein. Methods The DNA sequence encoding C-terminal 270 amino acid of Human SNX17 was obtained by PCR from cDNA library of human blood cells and was cloned into PGEM-4T-1 plasmid expressing GST and pMal-C2x plasmid expressing MBP. The GST-SNX17-C-terminal fusion protein and MBP-SNX17-C-terminal fusion protein were expressed by E.coli BL21 (DE3) after IPTG induction. The New Zealand rabbit was immunized with the purified electrophoresis GST-SNX17-C-terminal fusion protein at the 1st day, the 28th day and the 42ed day for three times. We used the ELISA assay to determine the titer of the antiserum by MBP-SNX17-C-terminal fusion protein which was purified by Affinity chromatography and Gel filtration chromatography. The rabbit blood was collected at the 56th day and IgG was purified. Effectiveness and specificity of the antiserum were identified by Western blotting. The SNX17 expression in 30 kinds of mouse organs was detected by Western blotting. Results We constructed the pGEX-4T-1/SNX17-C-terminal plasmid and pMal-C2x/SNX17-C-terminal plasmids successfully. The GST-SNX17-C-terminal fusion protein and MBP-SNX17-C-terminal fusion protein were induced and expressed successfully. The purified antibody specifically recognized GFP-SNX17 and the band was in the same position as that recognized by GFP antibody. We demonstrated that SNX17 expressed at a high level in the mouse uterus, ovary and breast, while expressed at a low level in the testis, prostate and vas deferens.Conclusion Rabbit anti-human SNX17-C-terminal polyclonal antibody was prepared successfully. This polyclonal antibody can be used for Western blotting analysis. The expression of the SNX17 in mouse organs was tested initially.

Key words: Sorting nexin 17, Fusion protein, Polyclonal antibody, Western blotting, Mouse, New Zealand rabbit