Effects of signal peptide on secretory expression of SARS-CoV-2 S1,RBD and RBD dimer proteins in Expisf9 insect cells
Objective To compare the effects of different signal peptides on the secretion and expression of SARS-CoV-2 S1,receptor binding domain(RBD)and RBD dimer proteins in Expisf9 insect cells.Methods The gene sequences of three proteins,SARS-CoV-2 S1(M1-E661),RBD(R319-P545)and RBD dimer(R319-K537 tandem),were selected and divided into 25 groups according to the different N-terminal signal peptide sequences(Endo,honeybee melittin(HBM),GP64,GP67,chitinase(Chi)and HIV-ENV)and C-terminal label sequences.25 recombinant baculoviruses were constru-cted by Bac-to-Bac system,and 25 groups of tertiary strain banks were prepared.B2 and C4 viruses were inoculated to logarithmic prestage cells(2.8 × 106cells/mL)and logarithmic metaphase cells(1.2 × 107cells/mL),respectively.The viruses of each group were cultured to 100 mL(500 mL shaker)for protein expression,and samples were taken for SDS-PAGE electrophoresis,Western-blot and ELISA detection.Two groups with higher expression levels of S1,RBD and RBD dimer proteins were selected for repeated verification.Results When B2 and C4 were inoculated to high cell density,the secretion expression level showed no increase,while there were significant difference between 4 and 5 d after inoculation.The expression level of A7(Endo-S1-tag)was significantly lower than that of A9(HIV-ENV-S1-tag),the expression level of A4(Gp67-S1-tag)was the highest,and the secreted expression level of A1(Endo-Endo-S1-tag)was significantly lower than that of A7(Endo-S1-tag).The secretion and expression of B6(HIV-ENV-RBD-tag)was signifi-cantly higher than that of B4(Gp67-RBD-tag)and other signal peptide groups,and C4(Gp67-RBD-dimer-tag)expression was significantly higher than that of C3(Gp64-RBD-dimer-tag).Two groups with high expression of each protein were selected separately for repeated verification(A4,A9;B4,B6;C3,C4)and the results showed that A4,B6 and C4 had the highest secretion expression levels.Conclusion The signal peptide for the highest secretion expression of S1 and RBD dimer proteins is the same,which is GP67 signal peptide,while the most suitable signal peptide for RBD protein is HIV-ENV,indicating that the N-terminal sequence can affect protein secretion,signal peptide sequence is universal to a certain extent,but is also related to the target protein sequence to be expressed.
Signal peptideSecretory expressionSARS-CoV-2S1 proteinReceptor binding domain(RBD)Baculovirus-insect cell system(BICS)
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