| White spot syndrome virus is a major pathogen of shrimp farming, and hamper the shrimp aquaculture industry severely. The whole genome sequence of the virus provides the basis of the research on the mechanism of the virus infection, and the center of the research has been transferred to the function genomic research, that is the interpretation of the each gene function in the infection. The virus attachment protein as the first protein interacts with the host cell, intrigues the cycle of the virus entry and is one of the hits for the control of the virus. WSSV VAP gene has not been well known yet. VP28, a major envelop protein of WSSV, has been reported playing important role in virus entry, and was suggested as a potential VAP of the WSSV; Another reported envelop protein, VP281 is found containing a cell attachment sequence by protein sequence analysis and may play a role in the virus attachment. This report developed the phages displaying the two envelop proteins to verify their binding ability to host cell. Furthermore, by screening the WSSV genome, we selected several putative ORFs that may code envelop protein and phage display to analyze their binding ability to host cell.In order to locate the virus proteins, rabbit polyclonal antibody against WSSV was prepared by immunizing of rabbit with purified WSSV particles. Cell membrane extracted from shrimp gill was coated on 96 well plate and bound with DIG labeled WSSV, then detected with Anti-DIG-Fab-HRP system, meanwhile taking the cell membrane from hepatopancreasx muscle, and E. coli for control group, the result verified the specific binding between WSSV and gill cell membrane. The binding assay can be employed as a convenient and rapid way to evaluate the binding nature between WSSV and the shrimp cell membrane.Designed the PCR primers which encompass the whole gene of VP28, VP281 in WSSV genome .meanwhile included the endonuclease site Not I , Sfi I ; After digestion with endonuclease, the PCR products were ligated into phage display vector pCANTAB 5E, and then transformed E. coli TGI, and therecombinant phage which display the VP28 and VP281 was rescued by infection of helper phage M13K07; Anti-E-tag antibody was used to detect the expression of the insert gene, and the WSSV polyclonal antibody was used to detect the antigen nature of displayed VP28 and VP281; VP28 and VP281 was successfully displayed, and the binding assay of the displayed VP28 and VP281 and shrimp gill cell membrane indicated that VP28 and VP281 could not bind to shrimp gill cell membrane, which mean that VP28 and VP281 may not be the virus attachment protein of WSSV.By screening the WSSV genome, selected 8 open reading frames and design the PCR primers respectively, then the PCR products was phage displayed; the displayed ORFs were detected by Anti-E-tag antibody to verify the expression of the insert genes; the verified displayed clones were carried out the binding assay with shrimp gill cell membrane; the binding assay positive ORFs were then used to competitively bind to shrimp gill cell membrane against WSSV. Except ORF5#, ORFs were successfully phage displayed , 4 of which could bind to coated shrimp gill cell membrane; and when the 4 ORFs undertake competitive binding assay against WSSV , only ORFlft could bind to gill cell membrane against WSSV, which indicated that the ORF1# perhaps code a virus attachment protein of WSSV.The recombinant phage infected E. coli HB2151, and colons from SOBAGN plates were selected for plasmid extraction to detect the insert gene; the positive clones were amplified and induced by IPTG; the periplasmic protein was extracted and employed for westernblot analysis in which the anti-E-tag antibody was used to detect the expression of the E-tag sequence and he WSSV polyclonal antibody was used to identify the ORFs. ORFltt was successfully expressed by E~tag expression detection, and the expressed protein was 35.5KD and accompanied with a protein 29.8KD, but it was negative result by WSSV polyclonal antibody. The rest of the ORFs w... |
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