Interaction Research Of IHHNV Proteins And Litopenaeus Vannamei Peritrophic Proteins

The intestinal tissue cDNA library of Litopenaeus vannamei was constructed successfully by using SMART technology. A full-length sequence of peritrophic in Litopenaeus vannamei was obtained by electronic splicing and sequence analysis. The sequence encodes 275 amino acids, with a theoretical molecular weight of about 30.782 kDa. Comparative analysis of the Peritrophin gene sequence obtained in the further study showed the similarities of 77.3%, 76.1%,72.2%, and 62.3% compared to the sequences of Penaeus monodon, Penaeus semisulcatus, Chinese shrimp, and Penaeus merguiensis. According to the comparison results, a conservative segment of L.vannamei Peritrophin （LvPT） gene was chosen for subsequent protein function research. The LvPT is 687 bp in length, encodes 228 amino acids, with a theoretical molecular weight of about 25.829 kDa.The fusion expression vector primers of the open reading frame CP, NS1 and NS2 were designed according to a IHHNV Fujian strain （GenBank NC NC₀02190.2）, and yeast shuttle expression plasmid pGBKT7-CP, pGBKT7-NS1, pGBKT7-NS2 and pGADT7-LvPT were constructed. The yeast shuttle plasmids that were detected by DDO/X/A and showed no self activation phenomenon could be used in protein interaction research. The recombinant strains Y2HGold （pGBKT7-CP, pGBKT7-NS1, pGBKT7-NS2） were respectively fused to the recombinant strains Y187 （pGADT7-LvPT）, and all combinations were found to be positive by QDO/X/A filter. Further more, strong interactions were found by QDO/X/A filter between pGBKT7-CP and pGADT7-LvPT, pGBKT7-NS1 and pGADT7-LvPT, respectively. However, the interaction between pGBKT7-NS2 and pGADT7-LvPT was weak, doubting it was a false positive. Therefore, LvPT protein interacts with IHHNV, and it is likely to directly affect the process of virus invasion.In addition, we used RNAi method to study whether dsLvPT interferences LvPT gene expression and inhibites IHHNV replication in Litopenaeus vannamei, after in vitro synthesis of dsLvPT and then in vivo injection to shrimp. The results showed that after dsLvPT injection, the expression level of LvPT was relatively down and the IHHNV relative copy number significantly reduced, compared with the control group, which further illustrate the mechanism by which LvPT relates to IHHNV infection in Litopenaeus vannamei. In addition, we found that LvPT could be expressed in High-Five insect cells by Western-blot analysis, which laid the foundation for further study of the interaction mechanism between LvPT and IHHNV-encoded proteins.