Transcriptome Sequencing In Sinocyclocheilus Grahami And Characterization Of A Cysteine Protease Inhibitor, Cystatin

Sinocyclocheilus grahami, has excellent edible and medicinal value, is a precious species of fish in Dianchi Lake. It usedto be a very common and important commercial fih species in Dianchi Lake around the 1950s, but because of pollution and overharvesting, it has now been categorized as a second-class, stateprotected animal on major national wildlife protection lists and endangered of China species red list, Vol.1 red list. Transcriptome sequencing in Sinocyclocheilus grahami and characterization of a cysteine protease inhibitor, Cystatin. The detailed achievements are listed as follow:First of all, the transcriptome sequenced of muscle from S. grahami using Hiseq 2500. Then bioinformatics analysis, including DENOVO assembly, functional annotation, CDS prediction, were performed. The transcriptome sequenced won 22916 proteins, contains a lot of specific protein information have not been reported in S. grahami. Among them, some bioactive peptides, such as antimicrobial peptides, antioxidant peptides, and protease inhibitors were found. Transcriptome get the information of overall gene transcription in healthy S. grahami. Our result provides a powerful resource for further study in S. grahami germplasm resources, the immune response against a pathogen, growth regulation, and many other subjects.Secondly, an inhibitor of cysteine proteases, cystatin-J, was charactered by bioinformatics analysis, including its physical and chemical properties, signal peptides, glycosylation modification, multiple sequence alignment, and three-dimensional structure prediction, and so on. All these analysis suggested that cystatin-J should be a powerful inhibitor of cysteine proteases.At last, recombinant Cystatin-J was expressed and purified from Escherichia coli using pET32a. rCystatin-J exhibits obvious inhibitory activity against cathepsin B. rCystatin-J at a concentration of 8 μM can inhibit nearly 75% of cathepsin B activity within 15 s and about 90% of cathepsin B activity within 30 min. Meanwhile, purification of rCystatin-J by the addition of different concentrations of acetic acid was performed to simplify the purification processes and reduce the cost of conventional purification by affinity chromatography.In conclusion, DENOVO transcriptome annotation of muscle from S. grahami was performed, and a lot of bioactive proteins and peptides were found. Then cystatin-J, one of the inhibitors of cysteine proteases, was expressed, purified, and charactered. All these results provide a powerful resource for further study.