| Integrins are a superfamily of heterodimeric cell surface receptors that mediatecell–cell and cell–matrix interactions as well as bidirectional signal transductionbetween the cytosol and the extracellular matrix, which were formed by thenoncovalent association of α and β subunits belonging to type-I transmembraneglycoproteins. Integrins could recognize Arg-Gly-Asp (RGD) sequences displayed onthe viral capsid proteins or extracellular matrix (ECM) proteins, which is the mostwidespread and prominent recognition sequence identified of the known integrinrecognition motifs, so integrins become the prime examples of physiologicallyimportant receptors that have been usurped by a large number of virusesforattachment and/or cell entry, The white spot syndrome virus (WSSV) is one of themost devastating shrimp pathogens found in farmed penaeid shrimp and otherCrustacea, and it has caused serious damage to the worldwide shrimp culture industry.Previous study has shown that several major WSSV envelope proteins with theconserved integrin-binding motif RGD are involved in systemic infection of WSSV.Therefore, Integrins might act as a receptor of WSSV. In order to clarify the potentialroles of β integrin in WSSV infection, the present study intended to clone and expressthe von Willebrand factor type A domain of Chinese shrimp Fenneropenaeuschinensis β integrin (FcβInt-VWA), and produce monoclonal antibody (Mab) againstFcβInt which was employed as a probe to investigate the expression variation ofFcβInt in hemocytes following WSSV infection. Also, the dynamic state of FcβIntmRNA level and WSSV copies in hemocytes were determined by quantitativereal-time PCR. By comparative analysis of FcβInt expression kinetics and WSSVpropagation process, the resultant data would greatly facilitate the understanding ofthe potential role of FcβInt in pathogenesis of WSSV infection. Meanwhile, aplurality of WSSV envelope proteins were expressed which were further analyzed whether they had the bingding activity with FcβInt. Effect of anti-FcβInt Mabs on theblocking WSSV infection was evaluated. The results showed that FcβInt in totalhemocytes exhibited a significant response regulator to WSSV infection; Abundanceand regulation of FcβInt differed in different type cells after WSSV challenge. WSSVenvelope proteins with RGD motif, VP37, VP110, VP187could bind with FcβInt,while VP26, VP28without RGD motifdoes had no binding ativtity with FcβInt.anti-FcβInt Mabs could partially block the WSSV infection in vitro. In conclusion,this paper proved that FcβInt was a cell receptor protein but not the only one. Thedetails of research contents and results are as follows:（1） The hemocyte total RNA of F．chinensis was extracted. The frangmentFcβInt-VWA was amplified by RT-PCR with the specific primer set. The purified PCRproduct was digested with the restriction enzymes and inserted into pET32a plasmidto generate pET32a-FcβInt-VWA recombinant which was transformed intoEscherichia. coli BL21(DE3). The positive clones were screened by PCR andconfirmed by sequencing, then incubated in LB medium and induced with0.80mMisopropyl-β-D-thiogalactopyranoside (IPTG). The recombinant FcβInt-VWA(rFcβInt-VWA) with Trx/His/S-tag was purified with Ni2+-affinity column. The resultshowed that the bacteria E. coli BL21with pET32a-FcβInt-VWA successfullyexpressed with a molecular weight of approximate50kDa. After purification byNi2+-affinity chromatography, rFcβInt-VWA with high purity was obtained（2） Analyzed by western blotting, Two strains of hybridomas (2C5,2C10)secreting monoclonal antibodies (McAbs) against FcβInt all belonged to IgM class.Two Mabs were cultured and immuned to BALB/c mice. And then ascitic fluids wereobtained and purified by caprylic acid ammonium sulphate method. The purifiedMcAbs were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). It indicated that the Mab protein was pure and contained little otherprotein. The titers of Mab2C5and2C10were determined to be1:6400and1:12800by indirect ELISA. Two Mabs reacted specifically with rFcβInt-VWA analyzed bydot blotting. The result of indirect immunofluorescence assay and western blottingshowed that Mab2C5could recognize a120kDa protein in hemocytes. Finally, Mab 2C5of high titer and good bioactivity were selected as the probe for detection ofFcβInt in F．chinensis.（3） F．chinensis were intra-muscular injected with WSSV. Followingtime-course hemocytes sampling, the expression variation of FcβInt in hemocytes wasexamined by flow cytometric immunofluorescence assay (FCIFA) and enzyme linkedimmunosorbent assay (ELISA) using Mab2C5against FcβInt. Meanwhile, thedynamic state of FcβInt mRNA level and WSSV copies in hemocytes weredetermined by quantitative real-time PCR. The result of FCIFA showed that FcβIntwas mainly expressed on the semi-granular and granular cells, which wasdown-regulated at6h post infection (p.i.), and significantly increased to the peaklevel at12h p.i., then decreased to the control level by24h. However, FcβInt on thehyaline cells was lowly expressed and didn’t show active response to the viralinfection. The variation of FcβInt concentrations in total hemocytes determined byELISA was roughly in accordance with the changing tendency of FcβInt expressed onthe semi-granular and granular cells. FcβInt mRNA level in total hemocytes wassignificantly up-regulated to the peak level at12h p.i.. Moreover, the number ofWSSV copies in hemocytes began to exhibit a significant increase at24h p.i..（4） The recombinant WSSV envelope proteins VP26, VP28, VP31, VP37,VP110, VP187were induced and exprese, which were further analyzed whether theyhad the bingding activity with FcβInt using the improved VOPBA method and Dotblotting. Effect of anti-FcβInt Mabs on the blocking WSSV infection was evaluated.The results showed that WSSV envelope proteins with RGD motif, VP37, VP110,VP187could bind with FcβInt, while VP26, VP28without RGD motifdoes had nobinding ativtity with FcβInt. anti-FcβInt Mabs could partially block the WSSVinfection in vitro. |
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