Purification, CDNA Cloning And Relationship Study On Three Kind Of Immune-related Factors In Shrimp Fenneropenaeus Chinensis

Chinese shrimp (Fenneropenaeus chinensis) is one of the most economically valuable and widely cultured species in China. During the last decade its development has been seriously affected on a regular basis by the outbreak of viral and bacterial diseases. Understanding the immune defense mechanisms of shrimp may contribute to the development of management strategies for disease control and long-term sustainability of shrimp farming. The innate immune system is the only defense against pathogens in crustaceans. The innate system's recognition of non-self and danger signals is served by a limited number of pattern recognition receptors/proteins (PRPs). C-type lectin is regarded as a kind of PRPs involved in immune recognition, cellular interactions, pathogen coagulation and wound healing in crustacean. Phenoloxidase (PO) or tyrosinase (EC1.14.18.1) is a bifunctional copper-dependent enzyme. It catalyzes the oxidation of dopa rapidly to dopaquinone, which in turn polymerize non-enzymetically into insoluble melanin. PO has been found in a variety of animals including both vertebrates and invertebrates and is involved in immune responses in invertebrate. Hemocyanins(HCs),responsible for oxygen transport,are a kind of extracellular giant proteins found in the hemolymph of both mollusks and arthropods. Many studies indicate that although hemocyanins and Phenoloxidases (PO) of mollusks and arthropods serve different physiological functions,but they both have copper binding sites,and Hc can be functionally converted into a kind of Phenoloxidase-like enzyme (hemocyanin-derived Phenoloxidase,HdPO) and display PO activities under certain conditions. In this article, we purified a natural lectin FC-L from the plasma of F. chinensis by affinity chromatography. The serological and chemical characteristics, carbohydrate-binding specificity and bacterial-binding properties of this novel lectin were also studied. we cloned a prophenoloxidase (proPO)gene (FCproPO) and a hemocyanin gene (FCHC) from Fenneropenaeus chinensis. The characterization, tissue localization, expression after challenged by different pathogens and their relations were also analyzed.A natural lectin from the plasma of the shrimp F. chinensis was purified by singlestep affinity chromatography using fetuin-coupled agarose. The purified plasma lectin showed a strong affinity for human A/B/O erythrocytes (RBC), mouse RBC and chicken RBC. The hemagglutinating (HA) activity of the lectin was dependent on Ca2+ and reversibly sensitive to EDTA. This lectin was named FC-L and its inactive form had a molecular mass estimate of 168 kDa. Fifteen N-terminal amino acid sequences of this protein were determined. We performed HA-inhibition assays with several carbohydrates and glycoproteins. FC-L showed a distinct and unique specificity to N-acetylated sugars, particularly sialic acid and sialoproteins. The FC-L also has binding activity to some Gram-negative bacteria which caused disease in shrimp and fish. The activity of FC-L was inhibited at temperatures greater than 75℃and at a pH less than 7 or greater than 11. These results suggest that FC-L may play a role as pattern recognition proteins in the reorganization and clearance of invaders in shrimp F. chinensis.A prophenoloxidase gene (FCproPO) was cloned from haemocytes of Chinese shrimp by Rapid Amplification Complementary DNA Ends (RACE) method. The full length cDNA of FCproPO consisted of 2311bp with a 2061 bp Open Reading Frame(ORF),which encoded 687 amino acids,and the predicted molecular mass was 78.71KDa. It showed 84% and71% homology with those of L. vannamei and M. japonicas. The Realtime-PCR results showed that the expression level of FCproPO in haemoeytes was highest among seven studied tissue. Sequence analysis showed FCproPO contained two conserved copperbinding sites, Phylogenetic analysis revealed that FCproPO and prophenoloxidase from L. vannamei, M. japonicus and P. monodon were in the same phylogenetic branches.A hemocyanin gene (FCHC) was cloned from hepatopancreas of Chinese shrimp by Rapid Amplification Complementary DNA Ends (RACE) method. The full length cDNA of FCHC consisted of 2161bp with a 2034 bp Open Reading Frame(ORF),which encoded 678 amino acids,and a predicted signal peptide of 12 amino acids , and the predicted molecular mass was 77.59KDa. It showed 82% and85% homology with those of L. vannamei and M. japonicas. The Realtime-PCR results showed that the expression level of FCHC in hepatopancreas was highest among seven studied tissue. Sequence analysis showed that FCHC contained two conserved copperbinding sites, Phylogenetic analysis revealed that FCHC and prophenoloxidase from M. japonicus, and P. monodon were in the same phylogenetic branches.To understand theirs function,amounts of FCproPO and FCHC expressions in hepatopancreas were studied by real-time PCR after challenged by V. anguillarum and virus WSSV. After challenged by bacteria, the expressions of them were all decreased and then reached the Peak at 6 hour post injection (hpi), and then recovered to the original level. In WSSV-challenged group, the expressions of FCproPO and FCHC maintained a steady level before 6h post-injection and decreased dramatically to the bottom in the end of the experiment. It might be caused by the reproduction of WSSV. The results implied that the stable immune system balance of the animal might be destroyed. These findings provide FCproPO and FCHC might play important roles on the defense mechanism of shrimp F. chinensis innate immunity. Realtime-PCR analysis showed that the expression profiles of FCproPO and FCHC were visible similarity. Which testified there roles were correspond with etch other. FCHc was up-regulated more distinctly in the hepatopanereas of shrimp when they were challenged by microorganism, which might correlative with its multifunction in shrimp immune system.