Resumen de Immunolocalization of Jian Carp (Cyprinus CarpioVar. Jian) Cathepsin B: Cloning, Expression, Characterization, and Antibody Preparation
Shu‐Hong Li, Ran Li, Hai‐Xia Zhong, Xiu‐Hua Chen, Ai‐Ping Liu, Juan Yang, Qiang Hu, Qin‐Qin Ke, Mei‐Liang Li
Cathepsin B (CatB) cDNA of 759 bp from Jian carp (Cyprinus carpiovar. Jian) with amino acid similarity of 99.6% to common carp was cloned. The mature CatB was expressed in Escherichia coliBL21 transferred with vector CatB‐pET‐30a. It was purified and identified as a single band (29 kDa) on SDS‐PAGE. Optimum CatB activity was observed at 40 °C and pH 5.5. Mouse anti‐CatB polyclonal antibody with a high titer of 1:256000 was prepared successfully and shown to specifically recognize the antigen both in prokaryotic cells and in the tissues of Jian carp according to western blotting and immunohistochemistry results. Immunolocation analysis showed that CatB distribution at protein level varied among the tested tissues. The results presented in this study may provide a significant reference for future research on the inherent relationship between CatB and the quality of fish or fish products at both the gene and protein levels. The investigation on the cDNA cloning and biochemical characteristics of Jian carp CatB and its immunolocalization is essential for utilizing piscine Cathepsins, or researching the inherent relationship between CatB and the quality of fish or fish products.
