Gene Cloning,Protein Expression And Expression Analysis Of Cathepsin B And L In Roughskin Sculpin(Trachidermus Fasciatus)

Roughskin Sculpin(Trachidermus fasciatus) is one of the four most famous fishes in China. They have high economic and nutritional value. Body length of roughskin Soulpin is usually12-17cm, and weight is about50-100g. Roughskin Soulpin was used to find in the Bohai Sea, Yellow Sea, the East China Sea coasts and estuaries of some rivers. The environment was destroyed with rapid economic growth of our country in the past decade, especially many factories discharged sewage into the rivers. Nevertheless, roughskin Soulpin relys on the habitat at estuaries and pollution of environment compresses their habitat in no doubt. Now it distributes only in the Yalu River, Qinglong River and Fuchun River. Roughskin soulpin is protected in order II in China. The first reserve of roughskin Soulpin were built at estuary of Qinglong River, Wendeng city, Shandong Province in January,2007. The roughskin Soulpin samples were collected from this area.Cathepsins belong to hydrolase, and they can hydrolyze proteins or peptides at some specific bindings. Cathepsin was found in1920s, and made a rapid progress was made in the follwing century. Several kinds of cathepsins were discovered by now. Cathepsins are essential in metabolism in bacteria, plants and animals. They have many functions, for example, playing a role in immune response, degrading proteins to maintain celluar homeostasis, digesting foods in alimentary canal and degrading yolk proteins for growth of embryos and so on. In our experiments we obtained the cDNA sequence of cathepsin B and L, constructed recombinant vector, expressed and purified their proteins. In addition, we analysed the expression of two cathepsins in normal condition and challenged with Vibrio anguillarum in liver, gill, blood and spleen through qRT-PCR. The results indicated that cathepsin B and L played a key role in iummne reponse of Roughskin soulpin.We obtained nucleotide sequence of cathepsin B and L. The full-lengh of cathepsin B is1226bp. It has a118bp5’-UTR,115bp3’-UTR and single typical polyadenylation. The993bp open reading frame(ORF) encodes330amino acids. The N-terminal of amino acids sequence contains a18aa singal peptides, and the mature peptide contains a proper functional area,21aa occluding loop. Cathepsin L has a1404bp full-lengh cDNA, a53bp5’-UTR, a340bp3’-UTR and single typical polyadenylation. The1011bp ORF encodes336amino acids. The N-terminal of amino acids sequence also contains a singal peptides, but it is16aa. In addition, it has a Inhibitor_I29area, an ERF/WNIN motif and EF hand Ca2+binding area. The results indicate that the cathepsin L belongs to calmodulin.We analysed the expression of two cathepsins using (3-actin through qRT-PCR. mRNA of cathepsin B and L ubiquitously expressed in all analyzed tissues, namely blood, heart, liver, gill, intestine, skin, kidney, spleen, brain and ovary. The results suggested that the two cathepsins might play an important role in metabolism. On the other side, this experiment determined the relative expression of liver, gill, blood and spleen with post-injection at different times, lh,2h,6h,12h,24h,48h and72h. The up-regulation at1h and2h indicated that cathepsin B and L had an extremely possible key function in immune response of Roughskin soulpin.Cathepsin B and L are immune-related enzymes like other studied animals. The achievement of cDNA sequence, analysis of protein structure, forecasting of function and discussion of expression could provided the first-hand information for disease control and domestication of Roughskin soulpin.