Vannamei Shrimp Ig Qualitative, Functional And Immunological Molecular Evolution Studies

Shrimp aquaculture has been developed rapidly since it serves as an industrial activity. However, shrimp farms have suffered from dramatic decreases in production due to infectious diseases within the past decade. Interest has been focused on immune molecules and defense mechanisms for the establishment of strategies for disease control. Invertebrate animals lack immunoglobulin, but many molecules of the immunoglobulin super family (IgSF) and a peculiar form of adaptive immune response have been documented to be present in an invertebrate. In shrimp, one of invertebrates, there are no direct evidences of an Ig-like molecule. An attempt was made to identify the protein bound to anti-human Ig (Ig-like protein) in shrimp Penaeus vannamei using immunological, proteomic, genetic and bioinformation technologies in the current study.Using immunological methods we found that there were proteins which reacted specifically with goat anti-human Ig (IgM, IgG and IgA), namely Ig-like (IgM-like, IgG-like and IgA-like) proteins, in shrimp serum, haemocyte and tissues. The reactivity could be strongly blocked and neutralized by anti-human Ig and human Ig, respectively. Based on Western blotting analysis we estimated that there was an Ig-like protein about 70 kDa (Wp70) in shrimp serum. And the average concentrations of IgM-like, IgG-like and IgA-like protein were determined to be 0.1578 ?0.0427,1.2255 ?0.6844 and 0.3178 ?0.1243 mg/ml by direct Dot-ELISA, respectively. These findings suggest that there are Ig-like proteins with similar epitopes to human Ig, which was important to purify and identity the Ig-like proteins from shrimp.Therefore, we applied affinity chromatography to isolate IgM-like, IgG-like and IgA-like proteins from shrimp serum with the purified antibody of goat anti-human IgM, IgG and IgA respectively. Three kinds of purified Ig-like proteins were subsequently analysed using 1-DE, 2-DE and their immunoblottings. The results showed that three proteins with 75 kDa (Ap75), 73 kDa (Ap73) and 62 kDa (Ap62) were isolated, of which Ap75 was documented to be strongly reacted with goatanti-human IgQ while Ap73 was bound to both of goat anti-human IgM and IgA. The three proteins were cut out from 2-D gel and subjected to MALDI-TOF mass analysis. The database search results indicated that the Ap75, Ap73, Ap62 proteins showed homology with Litoenaeus vannimei hemocyanin of 22%, 26% and 24%, respectively. To increase our confidence peptides from the digested proteins were also analysed by tandem mass spectrometry. When the ESI tandem MS data were subjected to the database search, Ap75 and Ap73 were identified to be Penaeus vannimei hemocyanin Hcl monomer (gi|7414468) and hemocyanin precursor (gi| 1085839), respectively. Thus, the shrimp Ig-like proteins were determined to be hemocyanin and its precursor.Peptide mass fringerprints (PMF) of hemocyanin and its precursor were subjected to Mascot search, the results indicated that the two proteins shared homology with many of immune molecules of high levels of organisms or recognition molecules of low levels of organisms. Then bioinformatics technologies were used to analyse the structure and evolution of hemocyanin and its precursor. Results searched of conserved domain showed that there was an Ig-like conserved domain of 252 amino acid residues in the C terminus of hemocyanin and its precursor. Amino acid sequence alignment indicated that hemocyanin and its precursor shared seven conserved motifs with human Ig, in which both of motif 611-625 of hemocyanin precursor (HpCR 511-625 motif) and motif ei9-633of hemocyanin Hcl, one amino acid difference between them, were very similar to motif 41.55 of human Ig in their 3D structures respectively. Then we chose the HpCRei 1-625 motif to analyse its evolution status. The results showed that the HpCR6n-625 motif shared high homology with many immune molecules (eg.human Ig, TCR and MHC) or recognition molecules (eg. bacterial adhesion protein) of organisms. It lead to be indicated that HpCR6ii-625 motif is a novel conserved...