Molecular Cloning,Recombinant Protein Expression Of Serpin From Stichopus Japonicus And Its Expression Profile During Autolysis

Sea cucumber, one of highest item of marine food, has great nutritional and therapeutic value. However, sea cucumbers were subjected to autolysis after stimulated by external adverse environmental factors. One of our research demonstrated that endogenous protease played a key role during autolysis of sea cucumber. Serine protease(SEP) is one of the main proteases involved in autolysis. SEP is regulated strictly by its endogenous serine protease inhibitors(serpin, SPI) in vivo. The expression change and interaction of SPI and SEP at mRNA level during autolysis have not been reported until now. In this paper, SPI gene was cloned from sea cucumber, and the expression pattern of SPI and SEP gene during autolysis was studied during autolysis in order to clarify the mechanism of autolysis from the respective of relationship between SPI and SEP.The total RNA of intestine from sea cucumber Stichopus japonicus was extracted, and full-length cDNA sequence of SPI gene was obtained by RT-PCR and 3’-RACE. The SPI gene, with a total length of 846 bp, contained a 402 bp open reading frame(ORF), which encoded a theoretical 133 amino acid protein with a predicted isoelectric point of 7.36 and molecular mass of 14.48 kDa. The amino acid sequence had two connected Kazal domains,which is a typical structure of Kazal-type serine protease inhibitor family.Tissue distribution and expression profile of SPI and SEP gene during autolysis was evaluated by quantitative real-time PCR. Results showed both SPI and SEP gene expressed in all the six tissues(longitudinal muscle, respiratory tree, intestine, abdominal epidermis, dorsal epidermis, coelomocyte), but the highest expression level for both of them were fond in intestine. During autolysis, the expression level of SPI and SEP decreased significantly from 0h to 3 h in dorsal epidermis, but a maximal value occurred at 5 h, afterwards decreased sharply. In abdominal epidermis, the expression of SPI decreased before 3 h, then increased after 5 h and reached a maximal value at 7-9 h, afterwards decreased again at 11 h. The expression of SEP decreased sharply and kept obvious low level before 7 h, then reached a maximal value at 9 h, but the expression level still lower than that of 0 h. The ratio of SEP/SPI was higher than that of 0 h, indicating the relative expression level of SEP was higher than that of SPI in dorsal epidermis. The relative expression level of SEP was higher than that of SPI at 3 h in abdominal epidermis, in contrast the relative expression level was higher for SPI in other stages. In addition, a recombinant plamid pET-21b-SPI wasconstruction and transformed to E.coli Arctic Express cells. Recombinant SPI was induced by IPTG(final concentration 0.5 mM).The above results showed that SPI and SEP gene distributed in all tissues of sea cucumber, with a characteristic of tissue specificity. The expression profile of SPI and SEP demonstrated different fluctuation mode, which might relevant with their different response mechanism to external stimulus. The changes of SEP/SPI ratio indicated that the balance between SEP and SPI was broken during autolysis, which might lead to protein degradation, a critical reason for autolysis. The obtained recombinant SPI protein provided a prerequisite foundation for the future research on the interactions between SPI and SEP at protein level during autolysis of sea cucumber.