| White spot syndrome virus (WSSV) is the most important viral pathogen that causes considerable economic loss to shrimp farming industry, and it has been extensively studied since its emerging. As the receptors of WSSV target cell mediate virus attachment and entry, so receptors finding and identification are the key points to figure out the WSSV infection mechanism, which is also the theoretical foundation for WSSV disease control. Till now, several WSSV envelop proteins with RGD (Arg-Gly-AsP) motif has been found, and the antibodies against some of these envelop proteins were proved to be having neutralization ability to WSSV infection. In this paper, full length cDNA sequence ofβ-integrin was cloned from the haemocytes of F. chinensis, which was expressed in E.coli by fragments after sequence analysis.β-integrin was proved to be an important membrane receptor of WSSV by binding test in vitro and neutralization test in vivo. The monoclonal antibodies (MAbs) againstβ-integrin were also produced and used to analyze the distribution ofβ-integrin in different tissues. Moreover, for further research on other WSSV receptors, a large number of MAbs against haemocyte membrane of Fenneropenaeus chinensis were produced and a model in vitro was developed to screen the MAbs agaist the receptors of WSSV. Two strains of MAb were obtained with obvious blocking effect on the binding of WSSV to haemocyte membrane, and the receptors were identified respectively. The followings are the details:(1) A full length cDNA sequence ofβ-integrin with 2853 bp was cloned from the haemocytes of F. chinensis, containing an open read frame (ORF) of 2349 bp encoding 783 amino acids. Result of homologous alignment showed cloned sequence was highly similar toβ1-integrin. Amino acid sequence of clonedβ-integrin was identified with all the classical domains ofβ-integrin by SMART. To investigate the expression levels ofβ-integrin gene in different tissues and post WSSV infection, the semi-quantitative RT-PCR was employed. The results showed that the expression level ofβ-integrin was highest in haemocytes, then was in heart, gill, gut and gonad tissues, however, which was very low in muscle and hepatopancreas tissues. After WSSV infection, the expression level in gill was up-regulated and reached to a peak at 24 h post infection, then gradually decreased. These results showed that the tissues with high expression level ofβ-integrin were susceptible to WSSV, and the level ofβ-integrin showed up-regulation after WSSV infection, which suggested thatβ-integrin was closely related to WSSV infection.(2) The cDNA fragments encoding extracellular region or VWA domain ofβ-integrin were amplified and inserted into pET32a plasmid, then transformed into E. coli BL21. The recombinant proteins were successfully expressed in positive clones with molecular masses of 100 kDa (extracellular region ofβ-integrin) and 50 kDa (VWA domain ofβ-integrin), which both showed WSSV binding ability in vitro by ELISA and dot blotting. In vivo neutralization assays of WSSV infection by these recombinant proteins were exerted, the result of which showed the recombinant proteins of extracellular region ofβ-integrin could significantly decrease the mortality of crayfish, showing partial neutralization ability to WSSV infection. The above results proved thatβ-integrin was an important membrane receptor of WSSV, which might play an important role in the process of WSSV infection.(3) Five MAbs (2C5,2C10,1F4,1C10和1D10) againstβ-integrin were produced by immunizing mice with recombinant protein of VWA domain ofβ-integrin. Indirect immunofluensce assay showed that all of these five MAbs could react with haemocyte membrane of F. chinensis. The result of western blotting showed that all of these MAbs could react with the recombinant protein of VWA domain ofβ-integrin, and specifically recognized a protein band with a molecular mass of about 120 kDa in haemocytes proteins, which suggested that 120 kDa protein was the nativeβ-integrin of haemocyte. Tissues of healthy F. chinensis were detected by IFAT using mabs againstβ-integrin. The result showed theβ-integrin in gut, gill and heart, to which WSSV was susceptible, were much more abundant than in muscle and hepatopancreas tissues, which provided important information on the mechanism of WSSV tissue tropism. (4) A large number of MAbs against haemocyte membrane were produced by immunizing BALB/c mice with haemocyte membrane of F. chinensis prepared by differential centrifugation. Based on ELISA and dot blotting techniques, a model for screening mAbs against receptors of WSSV was established with Digoxingenin labeled WSSV (WSSV-DIG) and prepared haemocyte membrane. Two MAbs,3C4 and 2F9, were screened using this model, which showed obvious blocking effects on the binding of WSSV to the haemocyte membrane. The result of western blotting showed that mAb 3C4 could react with a protein band with a molecular mass of 42 kDa, which was identified as a beta-actin by MALDI-TOF-MS peptide mapping, and mAb 2F9 could react with two protein bands with molecular masses of 73 and 75 kDa, which were identified as the subunits of hemocyanin by MALDI-TOF-MS peptide mapping. The results of dot blotting showed that the purified beta-actin and hemocyanin subunits had WSSV binding ability, which suggested that these proteins played an important role in the process of WSSV infection.
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