Recombinant Expression Of Shrimp Lysozyme In Escherichia Coli And Product Analysis

The aquaculture of Marsupenaeus japonicus and Fenneropenaeus chinensis has rapidly grown to a major economic activity in China. Shrimp can provide a high quality of food product and is very popular to customers. However, the rapidly expanding fleshy prawn industry has caused many problems since1990s. Diseases have emerged as a major constraint to the sustainable growth of shrimp aquaculture, especially the infection of virus, causing severe lost. Many studies have been done, but the immunology mechanism of shrimp is not well understood. In order to prevent and cure diseases, it is thus important and necessary to develop some effective methods for the pathogen control, Lysozyme is an important effective factor in shrimp innate immune system which is involved in a variety of immune response. And it forms a hydrolytic system in the process of bacteriolysis which destroys and eliminates the invasive pathogens in vivo. In recent years, researches of lysozyme from many sources have been done widely, but the report of shrimp lysozyme was very little.The total RNA from blood of Marsupenaeus japonicus was extracted. The complete lysozyme gene from M. japonicus was cloned by RT-PCR and named Mj-lysozyme (MjLys in short). The open reading frame of the gene consisted of477bp and encoded158amino acids (aa), including a signal peptide of18aa and a mature peptide of140aa. The molecular weight of the mature protein was of16.4kD, and the isoelectric point was8.80. It was found that structure of the MjLys gene contained a domain (residue1-130aa) of conserved c-type lysozyme, including the two catalytic residues GIU33and Aspso and the eight cysteine residue motif. In addition, the complete lysozyme gene from Fenneropenaeus chinensis was synthetized by genes custom services and named Fc-lysozyme (FcLys in short). After then these two genes of mature peptides of MjLys and FcLys was subcloned into the expression vector pET-32a(+) and transformed into E. coli BL21(DE3)pLysS. As a result, the recombinant protein of MjLys and FcLys was strongly expressed, but it is in insoluble form. Using two methods of purification and renaturation of the inclusion body protein, more than90%of refolded protein was obtained, and the MjLys yield was114mg of every litres of fermented liquid, and the FcLys yield was96mg of every litres of fermented liquid The present study suggested the MjLys and FcLys possesses significant activities against Gram-positive bacteria Micrococcus lysodeikticus and Staphyloccocus aureus.