| Classical swine fever virus(CSFV),as well as bovine viral diarrhea virus(BVDV),belongs to Pestivirus Flaviviridae.The persistent infection is the main reason that prevent the elimination of Classical swine fever(CSF)and the study of the pathogenic is the foundation of prevention and treatment.Besides the proteins of virus,the help of components in host cells is indispensible in the virus duplication.Molecular chaperone contribute to the steady of cells and take part in many physiological reactions.J-domain protein interacting with viral protein(Jiv),belongs to the heat-shock proteins 40(HSP40),is very important in the infection and pathopoiesis.The study of CSFV and BVDV can draw lessons from each other.Two kinds of BVDV exist in the nature and the one which integrated the fragment of Jiv of the cells have the feature to induce cytopathic effect(CPE).The similar phenomenon was also observed in the study of CSFV.The study of BVDV indicate that Jiv promotes the duplication of virus by promote the split of NS2-3.In this study,we validate the promotion of Jiv fragment in the replication of CSFV and the interaction between Jiv and virus,preparing for the construction of recombined CSFV.In addition,we construct the full length c DNA clone of CSFV and try to rescue the virus.The main contents are as follows:1.The overexpressing recombint plasmid and three pairs of sh RNA targeting swine Jiv were cloned into p CDH-U6-MCS-EF1-Green Puro to generate the recombinant lentivectors to inhibit the expression of Jiv in PK15 cells.The detection of real time RT-PCR and Western blot indicated that the expression of Jiv were overexpressed and inhibited successfully.CSFV was then infected in the cells.The results indicate that Jiv plays an important role in the replication of CSFV.The trial of Co-IP indicate that Jiv interact with virus protain directly or indirectly.2.After analyzing the restrictions among CSFV and p BR322 plasmid,appropriate restrictions were selected to segment and construct the full-length genome of CSFV with eight segments.The amplified segments were connected into T vector.After identified by enzyme digestion the fragments were linked into p BR322.The sequence of T7 promoter and CMV promoter,Ham Rz were addited in the 5?of virus genemic respectively;T7 terminal and HDVRz,Poly A were addited in the 3?of virus genemic respectively.Finally,the full-length genome recombined plasmid p BR322-T7-CSFV and p BR322-CMV-CSFV were constructed.3.The RNA of the virus was transcribed in vitro from p BR322-T7-CSFV according to the instruction of MEGAscript T7 Kit.After detecting the concentration,the RNA was transfected into PK15 cells with optimized electroporation protocol;p BR322-CMV-CSFV was transfected into PK15 cells using turbfect.After consistent passage,nucleic acid of virus was detected by RT-PCR. |
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