[Preparation and application of rabbit polyclonal antibody against mouse Tubby-like protein 2 (TULP2)]

Objective To generate rabbit polyclonal antibody against mouse Tubby(Tub)-like protein 2 (TULP2) and detect the expression of TULP2 in mouse testis. Methods pET30a (+)-TULP2 and pET30(+)-TULP2-C recombinant plasmids were constructed by inserting TULP2 full-length gene fragment and TULP2-C gene fragment containing Tub domain into pET30a (+). pET30a (+)-TULP2 and pET30(+)-TULP2-C were transformed into E. coli BL21, and the prokaryotic protein expressions were induced with the supplementation of IPTG. The prokaryotic recombinant proteins were purified with His-Binding-resin, and denaturation was performed by adding urea with gradient concentration. Adult male New Zealand white rabbits were inoculated with recombinant TULP2 and TULP2-C proteins as immunogens to generate two kinds of TULP2 polyclonal antibodies. Titers of antibodies were detected by ELISA. The efficiency and specificity of antibodies were determined by Western blot and immunofluorescence (IF) staining. Results pET30a (+)-TULP2 and pET30a (+)-TULP2-C recombinant plasmids were constructed successfully, and the protein expressions of TULP2 and TULP2-C could be induced by adding IPTG. The titers of polyclonal antibodies were 1:1 000 000. Western blot and IF staining showed poor specificity of TULP2-C antibody. TULP2 antibody could specifically recognize the endogenous TULP2 protein in the testes of adult wild-type mice, and IF staining showed that TULP2 was expressed specifically in the round spermatids and elongating spermatids of mice. Conclusion A rabbit anti-mouse TULP2 polyclonal antibody is generated successfully using TULP2 full-length protein, which can be used for detecting TULP2 expression by Western blot and IF staining.