Studies On The Interaction Between Rabbit Immunoglobulin Light Chain Kappa And Rabbit Hemorrhagic Disease Virus

Rabbit hemorrhagic disease is an acute, highly contagious and high mortality disease caused byrabbit hemorrhagic disease virus. RHDV infects adult rabbit liver, lung, spleen and other organs, and theliver is the main organ of RHDV infection. RHDV belongs to the calicivirus. The capsid protein VP60is major structural protein and the immuno-protective antigen of the RHDV. Studies on the protein inrabbit liver interacting with VP60are of great significance to clarify the RHDV pathogenic mechanismand RHD prevention and control.Immunoglobulin is a kind of globulin with antibody activity which consists of two heavychains and two light chains. There are two types of light chain in the mammalian immune system whichare kappa and lambda. The immunoglobulin plays an important role in the process of resisting bacteriaand virus. Our laboratory have screen proteins interacting with VP60using the yeast two-hybrid andexpression cloning technology, and rabbit immunoglobulin light chain kappa is a candidate protein. Theimmunoglobulin light chain contribute to the antigen-antibody binding which also promoteB-lymphocyte receptor expression and secretion. Immunoglobulin light chain is widely involved inimmune regulation. It has been confirmed that the immunoglobulin light chain is capable of bindingmast cells. The non-specific light chain can directly inhibit the replication of virus, and also hasinteraction with amnion or virus particles. Total RNA were extracted from adult rabbit liver tissue andwere reverse transcribed into cDNA using specific primers for PCR amplification. We get a length of480bp kappa gene segment. Prokaryotic expression vector pET-32a-kappa and eukaryotic expressionvector pEGFP-N1-kappa were successfully built. The recombinant plasmid pET-32a-kappa wastransformed into E. coli BL21（DE3）plyss. We get Kappa protein after IPTG induction, purification andrefolding. The interaction between the Kappa protein and VP60was identified by GST-pulldown andELISA. Recombinant plasmid pEGFP-N1-kappa and pcDNA3.1-VP60were co-transfected intoHEK293cells and we use confocal laser assay to analyse Kappa protein and VP60co-localization. Theresults showed that the Kappa protein and VP60co-localized in the cytoplasm, indicating that there areinteraction between them.Hemagglutination inhibition tests showed that0.413μg Kappa protein can inhibit hemagglutinationactivity of8HAU RHDV. We respectively take106.1μg、212.2μg、424.4μg、848.8μg Kappa proteinand2048HAU RHDV together to inoculate SPF rabbits after incubating at room temperature. Theanimal infection experiment result show that there are an adult rabbit survival in each group120h afterinoculation, while the positive control rabbits died within96h after inoculation. The adult rabbit deadtime delayed with Kappa protein increased. We used quantitative PCR detect RHDV viral load of rabbitliver and lung tissue in each group. The rabbit liver and lung tissue viral loads of experimental groupwere lower than the positive control group. the liver tissue viral load inter groups were significantdecreased with the rabbit death time delay, while lung tissue viral load reduced not significant withrabbit death time. After comparison inner group rabbit liver and lung tissue viral load，we find that viral load of experimental group with high level Kappa protein significantly reduced as the rabbit death timedelayed. The higher Kappa protein can delay the death time of adult rabbits caused by RHDV. Theresult showed that high levels Kappa protein affect on viral replication of rabbit liver and lung infectedRHDV.In this study, we successfully expressed Kappa gene fragment in vitro and demonstrated theinteraction between Kappa protein and VP60using GST-pulldown, ELISA and laser confocal assay. Thehemagglutination inhibition result shows the interaction of kappa protein with RHDV. We seek thatKappa protein can delay the death time of adult rabbits infected with RHDV. It influences viralreplication of rabbit liver and lung infected by RHDV. It laid the foundation for further research on therole of Kappa protein in process of RHDV infection.