RESUMO
Entire 3ABC sequence of FMDV containing a 6 x his tag coding sequence at the N-terminal was obtained through PCR amplification using a pair of specific primers, subcloned into shuttle plasmid of pMelBac-B with a melittin secretion signal sequence and finally constructed recombinant plasmid of pMel-3ABC. After co-transfected the recombinant plasmid and linearized Bac-N-Blue DNA into Sf9 insect cell under intermediary agent of the Cellfectin, the result showed that we have already acquired recombinant baculovirus by screen of plaque assay and identification of PCR. Though the recombinant baculovirus infecting the Sf9 cells again, experiments indicated that 3ABC gene could express in insect cells and the expressed protein was secreted in the supernatant of Sf9 cell culture possessing favourable biological activities detected by adopting two methods of SDS-PAGE and Western blot. The result verified that the protein could respond with sera derived from FMDV infected animals, but have no responsibility with sera derived from health animals and vaccinated animals detected by indirect ELISA using antigen of expressed protein after purification with Ni-NTA his bind resin. Therefore, this study has established a solid foundation for establishing an effective diagnosis method to discriminating the FMDV infected animals from vaccinated animals.
Assuntos
Animais , Bovinos , Antígenos Virais , Genética , Alergia e Imunologia , Metabolismo , Western Blotting , Linhagem Celular , Clonagem Molecular , Meios de Cultivo Condicionados , Metabolismo , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Febre Aftosa , Alergia e Imunologia , Virologia , Vírus da Febre Aftosa , Genética , Alergia e Imunologia , Metabolismo , Regulação Viral da Expressão Gênica , Soros Imunes , Alergia e Imunologia , Plasmídeos , Genética , Proteínas Recombinantes , Alergia e Imunologia , Secreções Corporais , Ovinos , Spodoptera , Suínos , Transfecção , Métodos , Proteínas não Estruturais Virais , Genética , Alergia e Imunologia , Secreções CorporaisRESUMO
A study was performed to validate 3 FMDV 3ABC-I-ELISA kits developed in China for the differentiation of FMDV infected and vaccinated animals.Sets of sera from naive and vaccinated cattle as well as from cattle that had been infected were tested for antibodies against nonstructural proteins (NSPs) of FMDV by commercial diagnosis kits,Ceditest(R)FMDV-NS (Ceditest(R) kit),UBI(R) FMDV NONSTRUCTURAL PROTEIN ELISA DIRECTION INSERT (UBI(R) kit) and a FMDV 3ABC-I-ELISA kitdeveloped at the Lanzhou Veterinary Research Institute.The test parameters (sensitivity and specificity) of the three kits were determined,and the result obtained from FMD 3ABC-I-ELISA kit was compared with that obtained from two foreign kits.The results indicated that the coincidence rate between the FMDV 3ABC-I-ELISA and Ceditest(R) kits was 98.05%,and the coincidence rate between the FMDV 3ABC-I-ELISA and UBI(R) kits was 94.4%; the sensitivity of both Ceditest(R) and FMDV 3ABC-I-ELISA kit was 100%.However,the sensitivity of the UBI(R) kit was only 81.8%.With sera from naive or vaccinated non-infected animals,the specificity of all tests exceeded 90%.