Molecular Cloning And Functional Research Of The Serine Proteinase Inhibitor In Kuruma Shrimp Marsupenaeus Japonicas

The Crustacea is of the largest percentage of arthropods, which with immeasurable economic value. Among them, Marsupenaeus japonicus is an important aquaculture industry animal. In recent years, large numbers of pathogens envaded into the shrimp can cause them sick, thus lead to huge economic losses. Thus Vibrio anguillarum become the major pathogenic bacteria that await to be controlled. Study on the economic invertebrate’s innate immunity not only provide new method and basic theory to the diseases management, but also enrich the theory of the invertebrate’s innate immunity and mutually corroborate the study on the organism animals. The Serine Protease Inhibitors (Serpins) are a diverse group of inhibiors which specificly inhibit the serine proteases. Serpins involve in the innate immunity by participated in the invertebrate’s prophenoloxidase system and regulated their melanotic encapsulation. However, researches on the other function of Serpins are still in the early stages. Thus, study these genes could not only help us to better understand the new function of Serpin, but also provide theoretical basis for the prevention of shrimp disease.We identified nine serpins in the kuruma shrimp Marsupenaeus japonicus, named as MjSerpl-9. They have different tissue distribution.When challenged with V. anguillarum and WSSV, some of them could be responded to the immune challenge. We chose one from them to do further studies. The MjSerp1cDNA has a1239bp open reading frame (ORF) that encodes a412-amino acid protein with a23aa signal peptide and a classic serpin domain. MjSerp1has a calculated molecular mass of46.3kDa and a predicted isoelectric point of5.51. MjSerp1is mainly expressed in the hepatopancreas and the intestine, and is moderately expressed in hemocytes. Expression pattern analysis indicated that MjSerp1is upregulated in the hepatopancreas after Vibrio anguillarum challenge. rMjSerp1inhibits three Gram-positive bacteria and two Gram-negative bacteria, but does not inhibit phenoloxidase activity. The microorganism binding assay showed that rMjSerp1closely binds to both Gram-positive and Gram-negative bacteria. MjSerp1also exhibits inhibitory activity against microbial serine proteases, such as subtilisin A and proteinase K, indicating that MjSerp1acts as a microbial serine protease inhibitor. rMjSerp1injection into shrimp enhances V. anguillarum clearance, but MjSerp1knockdown through RNA interference impairs Vibrio clearance in vivo. These results indicate that MjSerp1functions as a direct effector in the bacterial clearance of M. japonicus. All together, our findings provide novel evidences for the serine protease inhibitors in shrimp immunity.