Prokaryotic Expression Of Fenneropenaeus Chinensis Lysozym Point Mutation Gene

The aquaculture of the shrimp 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. 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. Lysozyme plays a special role in hydrolysis of microbial cell wall. It is a natural safety microbicides and preservatives, so it is used in many fields, such as food quality and drug development. In recent years, researches of lysozyme from many sources have been widely done, but the report of shrimp lysozyme was very little. In one of the few studies, it is report pointed out that shrimp lysozyme of recombinant expression is severely constrained by the protein amount of expression and soluble, thus seriously has affected the reorganization of the industrialized production of aquatic animals lysozyme.The study was done to construct the recombinant expression plasmid of Fenneropenaeus chinensis lysozyme (FcLys) point mutation gene in order to induce its soluble expression in E. coli Rosetta(DE3). The gene of FcLysM was amplified by PCR from the recombinant cloning vector pMD18-T-FcLys stored by our lab and mutated the nucleotides of some hydrophobic amino acids (aa) to hydrophilic aa without changing active centres and disulfide bonds of the lysozyme. The FcLysM gene was inserted into plasmid pET-39b(+) to construct the recombinant plasmid pET-39b(+)-FcLysM and transformed into E. coli Rosetta(DE3). Sequencing assays showed that the recombinant plasmid pET-39b(+)-FcLysM was constructed successfully. By the induction of IPTG, the recombinant protein FcLysM was obtained in E. coli Rosetta(DE3). The analysis of SDS-PAGE indicated that the fusion protein FcLysM was about43ku. Further research analysis proved that the amount of soluble lysozyme protein expression via gene point mutations was higher than the amount of non-mutated lysozyme expression. These results will simplified the subsequent experiments to purify the product of the shrimp lysozyme and provide a way to obtain the efficient amount of soluble expressed protein through gene point mutation.