重组牛冠状病毒N蛋白在昆虫杆状病毒表达系统中的表达与鉴定OA

In this study,the insect baculovirus expression system was used to express N protein,amplify the BCoV-N gene by PCR and inserted into the insect baculovirus system transfer vector pFastBacHT B by seamless cloning to con-struct the pFastBacHT B-BCoV-N recombinant plasmid.After transforming into DH10Bac E.coli,positive recombi-nant Bacmid-BCoV-N was extracted through blue-white spot screening,and transfected into Sf9 insect cells to pro-duce baculovirus and then transited.The results showed that both validation the recombinant plasmid and the recom-binant bacmid were successfully constructed.Following transfection of Sf9 insect cells with the recombinant bacmid Bacmid-BCoV-N and five successive blind passages,recombinant baculovirus Bacmid-BCoV-N was successfully obtained,enabling the expression and purification of BCoV-N protein,and SDS-PAGE and Western blot analysis identified a molecular weight of approximately 55 kDa,demonstrating good specificity.For immunological evaluation,50 μg of the recombinant protein emulsified with Freund's complete adjuvant was administered intramuscularly to mice.Indirect ELISA results confirmed the induction of specific antibodies in immunized mice.In conclusion,the in-sect baculovirus expression system established in this study enables high-level expression of exogenous genes,ful-filling the requirements for subsequent development of immunological diagnostic assays for antibody detection.