Cloning And Expression Of JC Virus VP2 Protein, And Identification Of NLS And Importin Protein

Background and Objective:JC virus (JCV) belongs to the polyomavirus family. JCV is widespread throughout the human population with at least 80% of adults exhibiting antibodies specific for the virus. When the immune system was suppressed, JCV will replicate in oligodendrocytes cell resulting in PML(progressive multifocal leukoencephalopathy). the purpose of the study is to clarify biological function of VP2 in the course of PML through the following aspects, such as subcellular orientation, prokaryotic expression, antibody preparation and NLS and importin protein.Methods:(1) The prokaryotic expressive vector pET-32a(+)-VP2 was constructed and transformed into the competent BL21 E. coli. The pET-32a(+)-VP2 was induced with IPTG ( Isopropylβ- D -1-thiogalactopyranoside ) , analyzed using SDS-PAGE ( sodium dodecyl sulfate polyacrylamide gel electrophoresis) and identified using Western blot. The expressed product was purified by using Ni+ affinity column chromatography.(2) The purified pET-32a(+)-VP2 fusion protein was used to immunize BALB/c mouse to produce polyclonal antibody. The specificity and potency of polyclonal antibody were evaluated by using Western blot and ELISA.(3) Construction of recombined green fluorescent protein expressive vector pEGFP-C1- VP2(complete,1-972,1-945,946-972). 7402 cells were transfected, and observed by fluorescent inverted microscope after 24hr.(4) The expressive vector pGEX-2T-importin was transformed into the competent BL21 E. coli. The importin protein was induced with IPTG and analyzed using SDS-PAGE. The expressed product was purified by using Ni+ affinity column chromatography.(5) We found the importin binding to VP2 by using the GST-pull down assay.Results:(1) The purified pET-32a(+)-VP2 fusion protein was used to immunize BALB/c mouse to produce polyclonal antibody. The ELISA manifested the titer of polyclonal antibody >1:160,000. The high specificity was testified using Western blot.(2) The eukaryotic expressive vector pEGFP-C1-VP2(complete,1-972,1-945 , 946-972) was successfully constructed. The eukaryotic expressive vector pEGFP-C1-VP2 was transfected into the 7402 cell strain, pEGFP-C1-VP2(complete) and pEGFP-C1-VP2(1-972) and (946-972)can be subcellularly located in nucleus. pEGFP- C1-VP2(1-945) can be subcellularly located in cytoplasm by its visible green fluorescent signal. These evidence indicated that its nuclear localization signal (NLS)located in 946-972bp.(3) The importin fusion protein was expressed successfully in the prokaryote expressing system through SDS-PAGE analysis.(4) We found VP2 binding to imprtinα1,α3 by GST-pull down assay, the binding complex could pass through the NPC and come into nucleus.Conclusions:1. The VP2 fusion protein was induced and expressed successfully by E. coli prokaryote expressing system. The protein provided materials for Polyclone antibody and interaction between two protein.2. Highly specific and potent polyclonal antibody of VP2 was prepared. The polyclonal antibody provided valuable material for the further study on the meaning of VP2 in the course of PML by other methods such as immune histochemistry3. Our work proved that VP2 subcellularly located in nucleus and NLS located in 946-972bp. This is the first time that we identified the VP2 NLS, and provided information for the further study.4. We found VP2 binding to imprtinα1andα3, and the binding complex could pass through NPC and come into nucleus. We found that the importin was binding to VP2.