Prokaryotic Expression And Bioactivity Analysis Of Two Recombinant Lectins From Sea Cucumber (Apostichopus Japonicus)

Sea cucumbers are economically important breeding species in East Asia. The Sea cucumbers(Apostichopus japonicus, A. japonicus) aquaculture has also rapidly developed in many Asian countries because of their nutritional and medicinal value. However, these organisms are easily infected by various bacterial, parasites and viral diseases. For instant, skin ulceration sydrome(SUS), a bacterial disease, produces ulcers in integument muscles, peristome tumescence, autolysis and death and has serverely limited further development of this important industry in China. Althought the drug treatment is usefull, it would make resistance to drugs, damage the water quality and even discruption the local environment. This is important to development a green drugs of biotherapy. As known, lectins are a group of proteins and glycoproteins with the ability to agglutinatinate cells through sugar-specific binding on the cell surface and play important roles in an innate immune of in invertebrates. Nowadays, lectins are one of the promising candidates for useful therapeutic drugs including antiviral, antifungal and antitumor therapeutics.In this study, two C-type lectins(AjL and AjMBCL) genes was cloned and characterized from the A. japonicus by rapid amplification of cDNA ends( RACE) technology. The two recombinant proteins were purified by prokaryotic expression and their bioactivities were identified. The AjL gene include a CDS(coding sequence) of 492 bp encoding a protein of 163 amino acids contained a putative signal peptide of 20 amino acids. The calculated average molecular mass of the predicted protein was 17.7 KDa with a theoretical isoelectric point of 4.48. The AjMBCL gene include a CDS(coding sequence) of 537 bp encoding a protein of 178 amino acids. The deduced amino acid sequence contained a putative signal peptide of 21 amino acids. The calculated average molecular mass of the predicted protein was 19.88 KDa with a theoretical isoelectric point of 6.98. As result, we successfully cloned the two lectin genes though the analysis of amino acid sequence, multiple sequence alignment and phylogenetic tree.The two lectin genes were expressed in Escherichia coli BL21(DE3) using the expression vector pET-32a(+). After OD600 reached 0.45 – 0.6, isopropyl-β-D-thiogalactopyranoside IPTG(final concentration of 0.5mM) was added to induce the protein. The cells were incubated at 28℃ for an additional 12 h. The expressed Aj L and AjMBCL had a molecular mass of ~34 and 30 KDa, as shown by SDS-PAGE and western-blotting. The recombinant protein was purified by affinity chromatography using a Ni-NTA column which was regarded as a simple, high efficiency and high purity method. Hemagglutination was assayed by using human erythrocytes and showed that the recombinant protein at a minimal agglutination of 0.085 and 0.03 mg/ml could induce the hemaggutination of human erythrocytes in the presence of Ca2+ ions.