Cloning and expression of gene encoding anti-lipopolysaccharide factor from black tiger shrimp Penaeus monodon

Abstract

Anti-lipopolysaccharide factor (Anti-LPS factor) is the major protein of crustacean immune system. In horseshoe crabs, this protein has an antibacterial effect on the growth of Gram-negative bacteria by binding and neutralizing bacterial endotoxin, lipopolysaccharide (LPS). In the black tiger shrimp (Penaeus monodon), a cDNA encoding anti-LPS factor has been isolated from the hemocytes cDNA library. It contains an open reading frame of 372 bp coding for 123 aa residues with a predicted molecular weight of 13.7 kDa. In this study, two versions of anti-LPS factor, full-length and NH2-terminal truncated derivative, were cloned and expressed in insect cells by using baculovirus expression system to obtained large amount of recombinant proteins with similar in structure and biological activity to the naturally occurring protein. The DNA fragment of full-length and NH2-terminal truncated derivative were cloned into transfer vector (pBacPAK8) and co-transfected to baculovirus genome by homologous recombinant inside the insect host cell (Spodoptera frugiperda Sf9 cells). Analysis of the recombinant proteins from infected cell lysate by SDS-PAGE revealed over expression, both of the full-length and NH2-terminal truncated derivative. Protein bands of approximately 15 kDa were observed in the infected cells but not in uninfected cell lysate. The recombinant proteins have appropriate size corresponding to the full-length and N-terminal truncated derivative anti-LPS factor protein. The crude recombinant proteins of both of the anti-LPS factors were tested for their antibacterial effect on the growth of bacteria by measuring bacterial growth rate of Gram-negative Vibrio harveyi 1526 and Escherichia coli DH5alpa and Gram-positive Staphylococcus aureus. No significant antibacterial activity was found with crude recombinant proteins both of full-length and NH2-terminal truncated, which may be due to the recombinant proteins formed insoluble inclusion bodies when they were over expressed. Thus, purified protein is required for further antibacterial activity test. We examined the tissue expression of anti-LPS factor mRNA in normal shrimp by Northern blot and RT-PCR analysis. The result demonstrated that hemocytes were the main site of anti-LPS factor synthesis. From RT-PCR analysis, anti-LPS factor showed strong expression in hemocytes and significant amount of mRNA concentration were observed in heart, gills, intestine and lymphoid organ. No mRNA of anti-LPS factor was detected in hepatopancreas. In microbial challenge, anti-LPS factor was found increase in mRNA concentration level at 3 hours post injection and then return to initial level, indicated that anti-LPS factor is stimulated in hemocytes at early stage of infection in response to bacterial challenge.