Prokaryotic expression, antibody preparation and immunological identification of VASA protein in the freshwater crab (Sinopotamon henanense)

In order to study the expression pattern and function of the reproductive regulatory molecular VASA during gonadal development of the freshwater crab, Sinopotamon henanense(ShVASA), we have cloned the partial sequence of vasa gene in the freshwater crab, S. henanense (Shvasa). Based on the sequence, in the present study, we prepared the specific anti-VASA polyclonal antibody for exploring the expression pattern and function of ShVASA protein during gonadal development. A specific segment of 813 bp of Shvasa gene was selected and cloned into pET32a vector to construct the prokaryotic expression vector pET32a-Shvasa. After the recombinant vector was transferred into Escherichia coli, the recombinant VASA protein was expressed under the induction of IPTG. SDS-PAGE analysis showed that the fusion protein, which was about 47 ku, mainly existed in the supernatant. Using the fusion protein purified by Ni-NTA His-Bind affinity chromatography column as antigen, we immunized the New Zealand white rabbits, and obtained the polyclonal antibody of ShVASA protein. The titer of anti-VASA polyclonal antibody reached 1×10⁵ in ELISA assay. Furthermore, we identified the specificity of the antibody gained in this study. Immuno-adsorption and Western blot analysis indicated that the produced anti-VASA polyclonal antibody could specifically bind to the fusion protein as well as the natural ShVASA protein extracted from ovary. These results will provide a powerful tool for identification of germ cells in S. henanense and other crabs, and for the study on the function of VASA protein in decapoda.