Expression of COVID-19 virus N gene in insect cells

ABSTRACT

In order to obtain COVID-19 nucleocapsid protein with similar function and activity to natural protein and apply it to practical detection. Firstly, according to Bac-to-bac insect expression system and synthetic COVID-19 nucleocapsid protein(N protein)sequence, BamH I and Xba I on pFastBacTMHTB vector were added to upstream and downstream primers respectively. The N gene was amplified by PCR technology, and T-Vector pMD19(simple)vector and pFastBacTMHTB vector were connected successively and recombinant plasmids pMD19-T(simple)-COV19-N and pFastBacTMHTB-COV19-N, and finally construct recombinant bacmid DH10 Bac-pFastBacTMHTB-COV19-N in DH10 Bac cells was expressed in insect cell Sf9. The recombinant protein was obtained and analyzed by SDS-PAGE and WB. The recombinant plasmid pMD19-T(simple)-COV19-N was identified by PCR and double enzyme digestion. The recombinant bacmid DH10 Bac-pFastBacTMHTB-COV19-N was constructed in DH10 Bac cells was identified by PCR and the expected two bands were 2 430,3 690 bp, respectively, which proved that the recombinant bacmid was successfully obtained. The recombinant bacmid was transfected into Sf9 insect cells. At the same time, the recombinant GFP protein control group was established. After 120 h of transfection, the recombinant N protein and recombinant GFP protein were collected and samples were prepared;SDS-PAGE and WB analysis were carried out respectively. HRP-His labeled antibody was used to verify that the transfection was successful, and both recombinant N protein and recombinant GFP protein were successfully expressed in Sf9 cells. The experimental results were consistent with the expectation, and the size of recombinant N protein band was about 46 ku. The eukaryotic expression vector of respiratory coronavirus N gene was successfully constructed and successfully expressed in insect cells, which provides an experimental basis for the establishment of ELISA detection methods and other related research.