Molecular Cloning, Recombinant Protein Expression Of Cystatin From Stichopus Japonicus And Its Role In Autolysis

Sea cucumber is rich in nutritive and medicinal value. Whereas it is susceptible to physical, chemical and physiological factors. Changes of these factors may lead to subsequent gut vomitting and body wall softening,which would produce great difficulties to the storage,transportation and processing of sea cucumber. Previous study proved that cysteine proteinase played an important role in autolysis. But the effect of cystatin on autolysis is unknown. In this thesis, at the mRNA level, the tissue distribution, timecourse expression of cystatin, and its relative expression ratio to cathespin L was investigated to clarify the role of cystatin on autolysis of sea cucumber..Firstly, a new cystatin gene (SjCyt) was isolated using homology cloning and RACE technology. Sequence analysis revealed a 662bp cDNA containing the 22bp 5-untranslated region,292bp 3’-untranslated regions and 348bp open reading frame which encode 115 amino acid.The predicted molecular weight of the deduced amino acids was 12.9KDa with a theoretical isoelectric point of 8.65. No signal peptide was found in this hydrophilic protein. The deduced amino acid sequence of SjCyt contained a characteristic sequence VQVVSGKNYRVKFE and 3 conserved motifs of cystatin superfamily.The expression of SjCyt and SjCL mRNA was determined by fluorescence quantitative polymerase chain reaction with the primers based upon SjCyt and SjCL full-length sequence. A primer pair for cytochrome b (Cytb) gene was utilized as the internal control in the reaction. SjCyt and SjCL were constitutively expressed in respiratory tree, intestine, longitudinal muscle, coelomocytes, tube feet and dorsal epidermis, with higher expression levels in the latter three tissues. It revealed that SjCyt and SjCL had a broad distribution rang in cells and tissue and the distribution pattern showed species-specific.Moreover, the relative expression of SjCyt and SjCL in dorsal epidermis and tube feet fluctuated during autolysis and increased in varying degreescDNA of SjCyt was cloned to PET21b(+) to construct expression vector. The recombinant vector was transfomled to Ecoli BL21(DE3) and then induced with IPTG at 15 ℃ 。 Specific bands about 14kDa with the estimated molecular mass was showed bySDS-PAGE and Western blotting analysis. The optimum prokaryotic expression conditions was 15℃ with 0.4mM IPTG.