SECRETED EXPRESSION OF LITOPENAEUS VANNAMEI LYSOZYME GENE IN PICHIA PASTORIS AND ITS BIOACTIVITY DETECTION

Litopenaeus vannamei, commonly known as White shrimp, belong to Arthropoda, is a widely eurythermal and euryhaline tropical shrimp. Since the late 80s of last century, L.vannamei has become the major species of cultured shrimp in China. However, during the last decade, the outbreak of infectious diseases has caused significant losses of farmed prawns. The latest research shows that, immunological system of L.vannamei are powerful and useful to increase its resistance and evaluate the health state. The immunologies of shrimp mainly consist of cellular immunity and humoral immunity and the existing studies have shown that, shrimp lysozyme, as an important factor for non-specific immune, plays an important role in the process of body’s humoral immunity. Up to present, a variety of shrimp lysozyme gene has been cloned and the recombinant lysozymes have been expressed. However, all the obtained recombinant lysozymes were using the prokaryotic expression system to achieve the expression of lysozyme genes. The resulting problem is that: when expressed in procaryotic expression system, the L. vannamei recombinant lysozyme protein was mainly insoluble inclusion bodies with low bioactivity. Therefore, it is necessary to test the other expression system,such as Pichia pastoris eukaryotic expression system, which is one of the most perfect expression system in eukaryotic gene expression. The object of this study was thus to construct a L. vannameilysozyme gene (lvlyz gene) expression vectors of Pichia pastoris and express a new recombinant lysozyme protein with high bioactivity. A DNA fragment coding lvlyz gene was obtained from the plasmid pMD18-T/Lvlyz and cloned into the Not Ⅰ site of the P. pastoris expression vector pPIC9K. The constructed plasmid, pPIC9K/Lvlyz, was transformed into the P. pastoris strain GS115 by electroporation. The positive transformants, screened out from histidine-deficient medium plates, were confirmed by polymerase chain reaction amplification. The recombinant strains were induced continuously by methanol and the induced expression product was centrifugated to collect supernatant. The supernatant collected in different time periods were detected by SDS-PAGE and western blot. Both SDS-PAGE and western blot indicated that the induced product of about 19.3 kD was the recombinant lysozyme protein. The recombinant protein was suggested to be high antimicrobial activity by Micrococcus lysodeikticus bacteriostatic method. In this research, the L.vannamei soluble lysozyme protein was first expressed in the P.pastoris eukaryotic expression system and the detection results demonstrated that the recombinant lysozyme protein has a high bioactivity.