β-Integrin、Hemocyanin Of Litopenaeus Vannamei Interact With White Spotsyndrome Virus And Effeciency Of VP28Vaccine Against WSSV Infection

White Spot Syndrome Virus (WSSV) is a major pathogen in shrimp culture, causing a catastrophic economical loss. Since the pathogen is worldwide expanding in1993, research on the mechanism of WSSV infection is rapidly evolved. In this study, the interaction of Litopenaeus vannamei (L. vannamei) β-integrin and hemocyanin with WSSV was investigated. Furthermore, vp28DNA vaccine combined with iel promoter was contrustrcuted. The protect effeciency of vp28vaccine against WSSV infection was evaluted.The main contents and results are as follows:(1)WSSV was used to infection L.vannamei, then the RNA from gill, blood cells and lymphoid of infected L.vannamei were extracted. Quantitative real-time PCR was used to determine the mRNA expression level of integrin in the gill tissue. The results showed that the mRNA expression level is generally lower than the control group in early stage of gills of infected L. vannamei (12-48h). But the mRNA expression level in the hemocytes was up to peak after WSSV infection for12h. The mRNA expression level in lymphoid is generally higher than the control group during the stage of WSSV infection.(2)A functional domians of L. vannamei β-integrin was cloned and ligated to the expression vector pBAD/gⅢA to constitute recombinants pBAD/gⅢA-integrin. Recombinants were transformed into E.coli Top10and induced by L-Arabinose. β-integrin was purified using Co2+affinity chromatography. Purified β-integrin was labeled with DIG and interacted with some WSSV structure protein containing motif of RGD or LDV. The result revealed that β-integrin could combine with VP26、VP31、 VP37、VP90and VP136. Elisa experiment was further confirmed the result. It is interesting that β-integrin could not combine with VP12which contains RGD motif. To define the specific binding sequences of VP26、VP31、VP37、VP90with B-integrin, peptides including RGD and LDV domain were synthesized. Competition Elisa result showed that NLDVA could block the interaction of integrin with VP26, ELDVR and KRGDT could affect integrin binding with VP31, ERGDE and LYGLR reduced VP37binding with integrin, and TRGDT affected integrin combining with VP90. The peptides with RGD and LDV motif competitivly inhibit the interaction between integrin and WSSV.(3)Hemocyanin(Hc) gene was divided into three parts according to its functional areas and ligated to the expression vector pBAD/gⅢA to constitute recombinants pBAD/gⅢA-Hc-N, pBAD/gⅢA-Hc-M, pBAD/gⅢA-Hc-C. The recombinants were induced and the expression products were purified to attain protein of Hc-N, Hc-M, Hc-C. The purified Hc-N, Hc-M, Hc-C were labeled with DIG and incubated with recombinant WSSV proteins.The result revealed that Hc-N could combine with vp12、 vp26、vp31and vp37. Hc-M could combine with vp12and vp26. Hc-C could combine with vp24、vp26、vp31and vp37. Elisa results confirmed the above conclusion. Indirect immunofluorescence assay indicated that75kD subunit of hemocyanin could combine with VP24and VP26.(4)In this study, pEGFP-Nl was reconstructed by inserting WSSV immediate early gene and vp28was cloned in new constructed vector. For WSSV challenge test, L.vannamei was divided into two groups for oral feeding and intramuscular injection, respectively.The survival rates and antiviral associated factors (Dicer, Argonaute, STAT and Lyzome) were detected in L.vannamei after injection and oral feeding immune. RT-PCR results showed that vp28can be detected in gill of shrimp by injection and oral feed immune. The relative protection rate of vp28group was reached22.4%(injection group) and36.84%(oral feeding group).The STAT and dicer mRNA expression in vp28group were significantly higher than that of control group after injection immune for7d. The argonaute mRNA expression in vp28group was significantly higher than that of control group after injection immune for3d and then decreased. The lysozyme mRNA expression in vp28injection group was consistenly significantly higher than that of control group. But the STAT and Argonaute mRNA expression in vp28group was significantly higher than that of control group on the7d after oral immune and then decreased. The dicer mRNA expression in vp28group was significantly higher than that of control group on the3d oral immune. These results demonstrated that vp28DNA vaccine has a potential utility for disease control of WSSV in shrimp.