Prokaryotic Expression of MGF360-13L Protein of African Swine Fever Virus and Preparation of Polyclonal Antibodies
The aim of this study was to construct the prokaryotic expression system of MgF360-13L gene of African swine fever virus(ASFV)to express 13L protein and prepare mouse polyclonal antibodies.In the present study,sequences of ASFV MGF360-13L gene were compared and analyzed for their homology using bioinformatics method and genetic evolutionary tree was constructed for optimal selection of 13L gene sequence.The optimized codon of the MGF330-13L gene was synthesized and connected to PET32a vector to construct the recombinant plasmid PET32a-13L.The recombinant plasmid PET32a-13L was then transformed into E.coli BL21(DE3)competent cells.The target protein was expressed with IPTG induction and purified using nickel columns.The purified protein was emulsified with adjuvant and injected into 8-week-old BALB/c female mice for preparation of polyclonal antibodies.The SDS-PAGE results showed that the molecular weight of the recombinant protein was 58.2 kDa,mainly in the form of inclusion body.Western blot results showed that the recombinant protein was recognized by the positive pig serum and the prepared mouse polyclonal antibodies.The mouse polyclonal antibodies were titrated in in indirect ELISA and the titer was as high as 1:25 000.The above results indicated that the recombinant ASFV protein MGF360-13L was successfully produced and polyclonal mouse antibodies were prepared with high specificity and reactivity,which laid a foundation for further elucidating the biological function of MGF360-13L protein and developing a new vaccine of African swine fever.
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维普
