Prokaryotic expression, antibody preparation and immunological characterization of VASA protein in the prawn Macrobrachium nipponense.

Vasa gene is specifically expressed in gonads and plays an important role in the germline formation and differentiation in most animals.In this study,a full open reading frame of vasa gene was amplified from the testis cDNA pool of the prawn Macrobrachium nipponense,and ligated with expression plasmid pET32a with 6×histine tag.After the recombinant plasmid was transferred into host bacteria Escherichia coli(BL21),the recombinant VASA protein was expressed under the induction of IPTG.The optimum concentration of IPTG was 0.2 mmol/L with a duration of 6 h at 30 ℃.SDS-PAGE analysis showed that the fusion protein was highly expressed as inclusion body and the yield accounted for 48.7% of the total bacterial protein.Western-blotting analysis demonstrated the target protein with the molecular weight of 85 ku.The recombinant protein was subsequently purified by Ni2+-NTA affinity chromatography.To further prepare the prawn VASA antibody,New Zealand white rabbits were immuned by the purified protein.The highest titer of the antiserums was up to 1∶160 000 as revealed by ELISA assay.The specificity of the antibodies against VASA protein was verified by Western-blotting.Immunohistochemical analyses indicated that,in oocyte VASA protein was mainly concentrated at the perinuclear region at vitellogenic oocyte;in testis,VASA protein was mainly distributed in spermatogonia.These data suggest that the VASA could play a key role in the development and differentiation of the germ cells in the prawn.