Cloning, Expression And Function Of A Disease-resistance-related Gene Of Shrimp Penaeus Monodon

Shrimp is one of the most important species in aquaculture, but it has been puzzled by diseases especially by virus diseases in the world since 1990's, and no effective measures are available to prevent the spread of shrimp viruses. As we known, shrimp lacks adaptive immune system, and it relies on a series of factors involved in the defense mechanism. This dissertation aims at cloning and function studying of the disease-resistance-related gene from shrimp in order to help the prevention of the shrimp diseases.By applying the method of mRNA differential display (DD) to normal and disease-resistant shrimp Penaeus monodon, 9 differential bands were observed. After these cDNAs were re-amplified, labelled and then hybridized with the shrimp RNA respectively, 2 cDNA fragments were confirmed to be positive. Homology analysis of their sequences showed that only one (DDB, approximately 450bp) was a novel gene related to disease-resistance.Hepatopancreas cDNA expression libraries of normal and disease-resistant shrimp Penaeus monodon were constructed by "k ZAP Express vector. The titer, capacity, recombination ratio and the size of the inserted cDNAs of two libraries showed they were effective and could be used in the research of shrimp genes.By PCR directly from the library of disease-resistant shrimp, screening the library with the DIG-DDB probe and 5'-RACE, the full-length cDNA (approximately 800bp) of DDB was obtained. It contained an open reading frame (ORF) of 510bp. We found the ORF and its 3'-UTR were conserved, but its 5'-UTR was variable.The differential gene was inserted into prokaryotic vector pThioHisC and transformed into E.coli. A high level (more than 10% of total protein of the cell) fusion expression product was induced. The expression product was purified by affinity chromatography with Ni-NTA agarose, and efficiency of purification was more than 95%. The purified protein was successfully refolded by gradient dialysis. A control protein was also expressed from the empty vector pThioHisC and purified. By inhibiting fish virus (SGIV)-induced cytopathic effect in GP fish cells, we found the recombinant protein of the differential gene had strong antiviral activity and no cytotoxicity to cells.Mouse was immunized by the recombinant protein as an antigen, and antiserum was prepared. IgG was isolated from the antiserum by proteinA sepharose, and coupled to CNBr-activated sepharose. Two proteins were isolated from shrimp serumby antibody affinity chromatography. One was confirmed to be the target protein by Western blot. The other had hemagglutinin activity, and could strongly bind to LPS from the surface of bacteria. MS analysis and partial protein sequencing of this lectin were finished.By immuno-electron microscope technology, the differential protein was found to be located mainly in the cytoplasm of the shrimp cell, and never bind to any organelles or shrimp virus (WSBV).This differential gene is the first antiviral gene from shrimp. Further study on expression, regulation and disease-resistance mechanism of it and its protein is to becontinued.