| Porcine parvovirus (PPV), which belongs to parvovirus genus, carries a linear single-strandedminus DNA genome about5000nucleotides in length. PPV encodes three non-structural proteins (NS1,NS2and NS3). The viral particle consists of three proteins (VP1, VP2and VP3). VP3is formed byproteolytic cleavage of VP2. Porcine parvovirus VP2accounts for most of the virus capsid and canself-assemble virus-like particles. Transferrin receptor (TfR) locates on the cell membrane and is atransmembrane protein. TfR can transport the iron ions in biological fluids into cells for cell metabolism.TfR is presented on the surface of many cells. The cells with exuberant metabolism express more TfR.Combining with the cell surface receptors is the initiation process of virus replication which is one ofthe decisive factors that affect virus host specificity, tissue tropism and pathogenicity. Researches haveconfirmed that canine parvovirus virus and feline panleukopenia virus could infect cells throughcombining with cell surface transferrin receptors by VP2. PPV and CPV have some homology.Although the capsid protein amino acid sequences are not identical, there are four highly homologousregions which locate at the starting of the glycine-rich region of VP2protein. VP2amino acid sequencesof PPV, FPV and CPV show a homology of50~60%. Therefore, it is reasonable to speculate that VP2ofPPV could combine with porcine TfR. In order to confirm this hypothesis, this study carried outpreparatory work, including protein expression, polyclonal antibody preparation, yeast two-hybrid assayand construction of porcine transferrin receptor retrovirus packaging plasmid.The main purpose of this study is to vertify whether there are interactions between the truncatedporcine TfR and VP2. Based on online software analysis, we found the porcine transferrin receptorextracellular domain and divided it into four parts to construct truncated TfR prokaryotic expressionplasmids. After expression and purification, we acquired four purified proteins and then immunizedmouse with them to prepare the antibody to laid the foundation for the blocking experiment that canblock the cells. The VP2gene of porcine parvovirus was divided into five fragments to constructprokaryotic expression plasmids. After expression and purification, we acquired five purified proteinsand then immuned mouse with them to prepare the high titer antibody for the blocking experiment thatcan block the virus.The yeast two-hybrid technology is a simple, sensitive and efficient method to studyprotein-protein interactions. By the yeast two-hybrid technology, we wanted to examine whether therewere interactions between the truncated porcine transferrin receptor and VP2. The truncated TfR geneswere connected into the bait plasmids respectively. Meanwhile, the truncated VP2genes were connectedinto the prey plasmids. The bait plasmids and the prey plasmids were transformed into yeast strain Y2Hand Y187, respectively. Then Y2H and Y187were fused to examine whether there are interactionsbetween the truncated porcine transferrin receptor and VP2. The results showed that no interactionsoccurred between them. Retroviral vector is capable of carrying exogenous gene into the host cell andintegrates the gene into the cell genome. In the process of receptor reconstruction, through the fusionPCR and increasing the number of amplification cycles, we amplified the full-length gene of porcine TfR and inserted it into the retrovirus vector pLXRN to construct recombinant retroviral vector ofporcine TfR for building a cell line which could express stably porcine TfR.As a fundamental work, the establishment and completion of these tasks laid foundation andpreparation for the future research on the interaction between procine transferrin receptor and PPV-VP2. |
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