| Classical swine fever(CSF)is an acute,febrile and fatal infectious disease caused by classical swine fever virus(CSFV).Marker vaccine of CSF can differentiate between CSF vaccine antibodies and wild-virus infection antibodies for more targeted immunity.In recent years,researchers throughout the world have successively applied molecular biology and genetic engineering methods to genetically modify wild strains or attenuated strains of CSFV to construct new strains,of which the method of constructing new strains based on Erns and E2 plays an important role.The epitope of WH303 is located in the E2 protein of the CSFV.Negative mutation of WH303 have carried out on virulent basis in some studies,but the viruses recover toxicity during the passage process.Therefore,this study constructed CSF marker vaccine candidate strain based on the mutation at WH303 of the attenuated C strain of CSFV in rabbits.In this study,a 2047 bp fragment containing the minimum recognition sequence of WH303 was amplified by self-designed primers using overlapping PCR;this fragment was then ligated to the T vector to derive MT-582583;the restriction enzymes SnaBI and NgoMIV was digested and ligated into the plasmid pMD18-T-NotI-BamHI and pMD18-T-NotI-BamHI-flag constructed in the laboratory;finally it was digested with the restriction enzymes Not I and BamHI and ligated to the C and C-flag strains to complete construction in molecular biology of the mutant MC and MC-flag strains.By means of reverse genetics,the MC strain was transferred to PK15 cells by electroporation and serial passage.The virus fluids were immunohistochemically stained with monoclonal antibodies WH303 and 1C8 to determine whether the MC strains were successfully rescued.A rabbit model was established and C and MC strains were inoculated into the model to observe changes in its body temperature.Serum was used for antibody detection when the body temperature peaked.The rabbit was euthanized and its spleen and other major organs were taken for RT-PCR detection and sequencing,and tissue sections were also prepared.The proliferation of MC strains in rabbits was determined by immunohistochemistry.M-297300 positive clones were successfully screened by overlapping PCR.The positive plasmids of pMD18-T-NotI-BamHI-582583 M and pMD18-T-NotI-BamHI-flag-582583 M were successfully constructed by restriction enzyme digestion and ligation and MC and MC-flag strains were finally constructed by another enzyme digestion and ligation.The results of immunohistochemistry in passage of virus MC showed that after 48 h,6th and 12 th generation with WH303-specific antibodies was positive,while the immunofluorescence WH303 was negative,indicating that the virus was successfully rescued.Mc strain was passed in rabbits for three generations.The result of inoculation of MC into rabbits showed that the fever of rabbits was not obvious in the first two generations,and the basically typical fever reaction occurred in the third generation.RT-PCR results showed that the target band only occurred to one of the four rabbits in the first generation,and all occurred in the second and third generations,and the sequencing results were consistent with the primers IVDC582 and IVDC583,which indicated that the mutant MC maintained gene stability during the passage in rabbit body.The results of immunohistochemical staining showed that MC content in spleen and lymph nodes was significantly higher than that in other tissues.Blocking ELISA antibody detection results were negative,indicating that there is no antibody in serum to specifically recognize WH303 epitope;Indirect ELISA antibody detection results were positive,indicating the existence of antibodies in serum to recognize CSF antigen.In this study,WH303 mutation is based on the C strain,which,to a certain extent,avoided the risk of bio-safety caused by virulent reversion mutations.Complemented with the differential diagnosis method in the follow-up study,it is expected to become the latest vaccine for CSF. |
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