Expression Of The Classical Swine Fever Virus E^rnsin Baculovirus Expression System And The Establishment Of An Indirect ELISA

Atypical CSF caused by low and moderate virulent CSFV is currently endangering the development of pig industry in China.It is difficult to distinguish pigs infected with CSFV and vaccined with attenuated vaccine by clinical symptoms or common serological methods.The combination of CSFV labeled vaccine and corresponding differential diagnosis methods can solve the above problems well.There is already a CSFV E2 subunit labeled vaccine on the market in China,so this study hopes to establish a corresponding differential diagnosis method.The main research contents are as follows:The E^rnsns gene of CSFV 2.1b subtype was synthesized and cloned into pFastBac1 vector to obtain recombinant plasmid pFastBac-E^rns.pFastBac-E^rnsns was transformed into DH10Bac competent cells for homologous transposition and the recombinant Bacmid rBacmid-E^rns was extracted and transfected into Sf9 insect cells to obtain recombinant baculovirus,which was amplified in Sf9 cells to obtain high titer recombinant baculovirus.Recombinant E^rnsns protein was secreted from High Five cells infected with the high titer recombinant baculovirus.Then the optimal expression MOI and time were determined,which were 5 and 96 hours,respectively.The recombinant E^rns protein purified by affinity chromatography was about 35kDa.Western blot showed that the protein had good reactivity.An indirect ELISA was established using recombinant E^rns protein by exploring various ELISA detection conditions.The sensitivity and specificity of the method were 86.14%and 88.31%,respectively.The E^rns-iELISA method was highly sensitive to E^rns antibodies of attenuated swine fever vaccine?83.47%?and domestic epidemic strain of classical swine fever?93.33%?.Using E^rns-iELISA method to detect CSFV attenuated vaccine immunized with pig blood,it was found that the E^rns antibody was detected in the serum 25 days after the first immunization.The E^rns antibody of the classical swine fever virus induced by the attenuated vaccine can maintain at least 3 months.Nonspecific response was found in detection of serum vaccined with CSFV E2 subunit vaccine by E^rns-iELISA,which indicated that improving the purity of recombinant E^rns protein can optimize the specificity of E^rns-iELISA.The results showed above indicate that the E^rns-iELISA method established by recombinant E^rns protein expressed by baculovirus expression system has a good sensitivity and specificity,which has a potential development and application value in the differential diagnosis of E2 subunit vaccine against swine and wild infected pigs.