Molecular Cloning And Functional Analysis Of Invertebrate-type Lysozymes From Red Swamp Crayfish, Procambarus Clarkii

Lacking an adaptive immune system, invertebrates rely on their innate immune system to resist invasion of prokaryotic and eukaryotic pathogens. Research on the invertebrate innate immune system, especially on the innate immunity of economic invertebrates, can not only enrich the content of invertebrate innate immunology, but also be applied in disease prevention of the economic invertebrates. Red swamp crayfish (Procambarus clarkii) is a kind of important freshwater economic crustaceans, in some areas has formed mass breeding. However crayfish is vulnerable to many diseases during high temperature season, so the disease prevention and cure of crayfish had attracted people's attention. What's more, crayfish is also a model preferred in the study of innate immune system of crustacean, because it is easy to get, easy to breed and the research results can be applied in prawns breeding. We have got many immune-related genes in red swamp crayfish. In this study we focus on the invertebrate-type lysozymes from the crayfish.Lysozyme is an important immune effector and is widely distributed in many organisms. There are several types of lysozymes and recently i-type lysozyme was identified as one member of the lysozyme family. In the present study, two novel invertebrate-type lysozymes (designated as Pclysil and Pclysi2) were cloned from red swamp crayfish, Procambarus clarkii. Alignment analysis showed that these two genes were different in catalytic residues. Results of RT-PCR showed that these two genes share similar tissue distribution patterns, and both were upregulated after bacteria challenge. This result suggested the two genes participate in innate immunity of crayfish. The mature recombinant proteins were expressed in Escherichia coli system and purified, and used for activity tests. Activity analysis revealed that rPclysil had no muramidase activity and isopeptidase activity, but had antimicrobial activity. Meanwhile, rPclysi2 was muramidase-deficient and had no antimicrobial activity, but possessed isopeptidase activity. Above data suggest that Pclysi1 and 2 may offer different functions in the crayfish immunity.