Expression of COVID-19 Virus N Gene in Insect Cells

doi:10.7668/hbnxb.20192941

Abstract

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 pFastBac^TMHTB vector were added to upstream and downstream primers respectively. The N gene was amplified by PCR technology,and T-Vector pMD19(simple)vector and pFastBac^TMHTB vector were connected successively and recombinant plasmids pMD19-T(simple)-COV19-N and pFastBac^TMHTB-COV19-N,and finally construct recombinant bacmid DH10Bac-pFastBac^TMHTB-COV19-N in DH10Bac 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 DH10Bac-pFastBac^TMHTB-COV19-N was constructed in DH10Bac 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.

Key words: COVID-19 virus, N protein, Insect expression system, Recombinant bacmid, Protein identification

SU Fang, HAN Diangang, YE Lingling, ZHANG Chong, YIN Shanglian, LUO Qianmin, DONG Xianlan, LI Yaoyao, LI Lingfeng, AI Jun, XIN Jige. Expression of COVID-19 Virus N Gene in Insect Cells[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(5): 174-180. doi: 10.7668/hbnxb.20192941.

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http://www.hbnxb.net/EN/Y2022/V37/I5/174

Figures/Tables 7

Fig.1 PCR verification after connecting T-Vector pMD19(simple)vector M.DL5000 DNA Marker;1—4. PCR products of pMD19-T(simple)-COV19-N.

Fig.2 Double enzyme digestion verification after connecting T-Vector pMD19(simple)vector M.DL5000 DNA Marker;1—4.pMD19-T(simple)-COV19-N double enzyme digestion results;5.PCR product control of pMD19-T(simple)-COV19-N;6.T-Vector pMD19(simple)empty vector control.

Fig.3 Electrophoresis of pFastBac^TMHTB empty carrier extracted from bacterial solution M.DL5000 DNA Marker;1—2.pFastBac^TMHTB empty vector.

Fig.4 PCR of recombinant bacmid DH10Bac-pFastBac^TMHTB-COV19-N M.DL5000 DNA Marker;1.Amplification of recombinant bacmid by M13 primer PCR.

Fig.5 Insect cell transfection experiment A.DH10Bac-pFastBac^TMHTB-GFP control group(bright field),about 12 h,40×10;B.DH10Bac-pFastBac^TMHTB-GFP control group(dark field),about 12 h,40×10;C.DH10Bac-pFastBac^TMHTB-GFP control group,120 h,40×10;D.DH10Bac-pFastBac^TMHTB-COV19-N group,120 h,40×10.

Fig.6 Electrophoresis of recombinant DH10Bac-pFastBac^TMHTB-COV19-N protein N.PageRuler pre stained protein Marker;1.DH10Bac-pFastBac^TMHTB-GFP fluorescent protein control;2.DH10Bac-pFastBac^TMHTB-COV19-N recombinant protein.The same as Fig.7.

Fig.7 DAB staining after membrane transfer

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