Cellular Localization And The Binding Sites Analysis Of Rab7in The Shrimp Fenneropenaeus Chinensis Against VP28of WSSV

White spot disease is a worldwide expanding epidemic in shrimpindustry. And its pathogen, White Spot Syndrome Virus (WSSV) is one ofthe most devastating viral pathogens in shrimp culture, which causing highmortality and considerable economic loss. Nowadays, the research onWSSV has got great progress in genomics and proteomics. Someresearches focused on the characterization of envelope proteins, such asVP28, VP26, VPl9, VP466, VP37and so on. The interactions betweenviral proteins and host cell membranes are important for viruses to enterinto host cells, which trigger the replication of viral genome, then produceprogeny particles. Some host cell components, such as PmRab7, β-integrin,Hsc70, BP53, PmCBP, STAT, have been reported to interact with envelopeproteins of WSSV, so as to significantly delay or neutralize WSSVinfection. PmRab7(Penaeus monodon Rab7) appears to be one specificshrimp protein that can interact with VP28and that prior injection of bacterially expressed recombinant PmRab7could reduce shrimp mortalitycaused by WSSV. In addition, silencing of PmRab7by dsRNAdramatically inhibited replication of WSSV and yellow head virus (YHV)both at transcriptional and translational levels. These results revealed thatPmRab7should function in the endosomal trafficking pathway and playimportant roles for WSSV entry and replication in shrimp. However,whether or not PmRab7is localized on the cell surface isn’t elucidated.In this paper, we focused on the interaction between VP28andPcRab7(Fenneropenaeus chinensis Rab7), and try to analyze the motif fortheir interaction by segmental expressed PcRab7. We also identified thedistribution and localization of these two proteins by Immno-fluorescence Location Assay. These results are helpful for betterunderstanding the role of Rab7involved in WSSV infection.There are four part contents included in this research. First, theresulting full-length gene of pcrab7was obtained by rapid amplification ofcDNA ends (RACE) PCR. Second, the polyclonal antiboday againstPcRab7was prepared by using the recombinant PcRab7, which wasexpressed in bacteria of E. coli. Third, the motif of PcRab7that interactedwith VP28was analyzed by Immunoprecipitation. Forth, the distributionand localization of VP28and PcRab7was identified by Immno-fluorescence Location assay. The main results in details were as followed:(1) Thefull-length cDNA of Fenneropenaeus chinensis rab7gene(pcrab7) consisted of1369bp with a615bp Open Reading Frame (ORF),which encoded205amino acids, and the predicted molecular mass was23.2kDa. The deduced amino acid sequences contained many conservedmotifs and regions belong to Rab7, and the amino acid sequence of PcRab7showed96%and97%homology with the amino acid sequence of Rab7inL. vannamei and P. monodon separately.(2) For preparing the polyclonal antibody against PcRab7, therecombinant PcRab7was expressed in bacteria of E. coli. Firstly, therecombined vector pBAD/gIIIA-pcrab7containing the pcrab7ORF wasconstructed, and transformed into E. coli. After L-arabinose induction at37°C, the fusion protein was successfully obtained, which was confirmedby SDS-PAGE and Western blot analysis. Finally, the polyclonal antibodywas prepared by using the purified recombinant protein, which was purifiedby the antigen absorption.(3) The interaction between VP28and PcRab7was identified byimmunoprecipation. In order to get more information about the motif ofPcRab7binding to VP28, PcRab7was expressed into several segments bygene deletion technique, and followed the binding ability tests. It was foundthat there were two parts of PcRab7might have the binding sites against VP28of WSSV, one part was the amino acid from41th to the79th, anotherpart was the amino acid from125th to the160th.(4) Analysis of the distribution and localization of VP28and PcRab7by immnofluorescence in hemolymph cells was carried out. It was foundthat PcRab7localized in the cytoplasmic of the cells, but the appearance ofVP28induced PcRab7to have a distribution on the cell surface.These results above will help us better understand the role of PcRab7and VP28in the life cycle of WSSV infection.