Expression And Detected Of Nonstructural Protein Gene 3ABC Of Foot-and-Mouth Disease Virus In Sf9 Cells

Non-structural protein 3ABC of Foot-and-Mouth Disease Virus (FMDV) was expressed by baculovirus expression system. The pure target protein 3ABC was purified with Ni-NTA his bind resin and detected with Western blot and indirect ELISA, the results showed that the protein has good biological activity which responded with positive sera derived from FMDV infected animals, but have no responsibility with sera derived from healthy animals and vaccinated animals. Then, FMDV NSP 3ABC approximating the natural protein was obtained and an indirect ELISA diagnosis method to discriminate the FMDV infected animals from vaccinated animals was established.3ABC gene of FMDV containing a 6×his tag coding sequence at the 3'-terminal was obtained by PCR amplification using a pair of specific primers, and then PCR products were subcloned into shuttle plasmid of pMelBac-B with a melittin secretion signal sequence to construct in a recombinant plasmid pMel-3ABC. After co-transfecting the recombinant plasmid and linearized Bac-N-BlueTM DNA into Sf9 insect cell under intermediary agent of the Cellfectin(?), the results showed that the recombinant baculovirus was acquired by screen of plaque assay and identification with PCR. The Sf9 cells was infected with the recombinant baculovirus agsin under the optimal multiplicity of infection (MOI) 10 and cells cultural supematant was harvested at 71 h. The insert gene was expressed in insect cells and the target protein was secreted in the supematant of Sf9 cells culture. The protein was detected by SDS-PAGE and Western blotting, results showed that it has favourable biological activities. Results suggested that the protein could react with sera derived from FMDV infected animals, while not with sera derived from health animals and vaccinated animals. The rate of the expressed dissoluble 3ABC protein in the gross cell's secreted protein was about 80%, the absolute density was 2.076mg/mL.The Ni-NTA His binding resin protein purification system in the natural condition provides a simple, rapid and reliable method for the purification of recombinant secreted proteins in high efficiency and low background. After optional reaction conditions was determined: coating antigen at 37℃for 1 h and 4℃24 h, serum sample (1:20) and HRP labeled anti-porcine IgG (1:30000) being incubated at 37℃for 45 minutes, reacting with chromogenic substrate at 37℃for 15 minutes. Using the purified proteins as antigen, an indirect ELISA to detecting of anti-FMDV NSP antibodies was established. The ELISA assay was confirmed to have a good reiteratively, specificity and sensitivity. This ELISA method was also compared with the routine ELISA test kit which was developed by using the recombinant protein expressed with E. coli to detect the serum samples from swine and sheep on farms. It was showed that the coincidence was 90%.