Expression of recombinant classical swine fever virus E2 glycoprotein by endogenous Txnip promoter in stable transgenic CHO cells

FIGURE 1. Txnip promoter TSS prediction, PCR amplification and expression vectors construction. (A) Schematic illustration of different Txnip promoter fragments and expression vectors construction. According to the promoter prediction score, four different promoter fragments were amplified by PCR and used to construct different expression vectors. (B) Four PCR products of different Txnip promoter sequences. M: DNA marker, Txnip‐1–4: PCR products amplified with different pairs of primers as shown in materials and methods section

FIGURE 2. Development and selection of target cell clone. (A) Initial selection of recombinant CHO cell clones with transgenic expression of recombinant CSFV‐E2 protein driven by different promoters after second G418 screening. * P < 0.05 was considered statistically significant compared with parental CHO cells. (B) Effects of methotrexate on recombinant E2 productivity in five specific cell clones from each transfected cell pool with the increasing of MTX concentration from 200 to 1200 nM (B). (C and D) Measurement of viable cell density of top five cell clones from CHO pCMV‐rE2 cell pool and CHO pTxnip‐4‐rE2 cell pool respectively each day. (E and F) Measurement of rE2 expression level of top five cell clones from CHO pCMV‐rE2 cell pool and CHO pTxnip‐4‐rE2 cell pool respectively each day. Results were presented as average values ± SD from three independent samples of each cell clone

FIGURE 3. Expression patterns of recombinant CSFV‐E2 protein caused by different promoter within 6 days batch‐wise culture process. (A) Expression profile of rE2 protein in CHO pTxnip‐4‐rE2 (F12) cells CHO pCMV‐rE2 (A11) cells cultured in 1 L shaking flasks. Statistically significant differences between two cells were analyzed by t‐test (* P < 0.05). (B) The gradual upward trend of mRNA level of rE2 driven by Txnip‐4 promoters in CHO pTxnip‐4‐rE2 (F12) cells. (C) Dynamic trend of endogenous Txnip transcription in parental CHO cells, CHO pTxnip‐4‐rE2 (F12) cells CHO pCMV‐rE2 (A11) cells. Results were presented as average values ± SD from three independent cultures of each cell clone

FIGURE 4. Comparison of time‐course profile of viable cell density (A), glucose (B), lactate (C), and glutamine (D) concentration in parental CHO cells, CHO‐pCMV‐rE2 (A11) cells, and CHO‐pTxnip‐4‐rE2 (F12) cells within 6 days batch‐wise culture process. Results were presented as average values ± SD from three independent cultures of each cell clone

FIGURE 5. Inducible expression strategy of recombinant CSFV‐E2 protein by addition of adenosine‐containing molecules in single cell suspension culture of CHO pTxnip‐4‐rE2 (F12) cells. (A) Time course profile of mRNA changing folds induced by 0.1 mM NADH and 0.1 mM ATP on endogenous Txnip and recombinant CSFV‐E2 in CHO pTxnip‐4‐rE2 (F12) cells. (B–D) Comparison of viable cell density and rE2 productivity by mixed addition of 0.1 mM NADH and 0.1 mM ATP at days 2–4 of batch culture in CHO‐pTxnip‐4‐rE2 (F12) cells, respectively. (E) Viable cell density and rE2 productivity of CHO‐pTxnip‐4‐rE2 (F12) cells without induction. Results are presented as average values ± SD from three independent experiments

FIGURE 6. Sequencing results of Txnip‐4 fragment from Chinese hamster and potential cis‐regulatory elements in this region. Numbers indicate the upstream distance to the starting codon, ATG