Anti-DNase II antibody detects recombinant DNase II protein expression in the mammalian ExpiCHO cell system.

(A) Analysis of antibodies from rabbit immunized with rTsDNase II-7 by ELISA. (B-C) Agarose gel electrophoresis of double enzyme digestion identification of pcDNA 3.4-DNase II recombinants. (B. Bovine DNase II M: DL2000 DNA Marker, Lane 1: pcDNA 3.4-DNase II (BamHI and EcoRI); C. M: DL2000 DNA Marker, Lane 1: pcDNA 3.4-DNase II (BamHI and EcoRI). The specific band is indicated by the arrow. (D-E) The TsDNase II-7 protein (D) and bovine DNase II (E) were eluted with different concentrations of imidazole solutions (50 mM, 100 mM, 500 mM). (1: supernatant after centrifugation; 2: effluent after supernatant incubation with His-Trap 3–4: elution of 50 mM imidazole solution; 5–6: elution of 100 mM imidazole solution; 7–10: elution of 500 mM imidazole solution). (F-H) DNase II protein expression in CHO cells was detected by anti-rTsDNase II-7 polyclonal antibody after 12 days of culturing using Western blot. Bovine DNase II and TsDNase II-7 were analyzed using anti-HIS antibody (Lanes1, 2 denatured and nondenatured TsDNase II-7; Lanes 3, 4 denatured and nondenatured bovine DNase II) or anti-rTsDNase II-7 antibody (Lane 5: denatured TsDNase II-7). Arrowheads indicate bands recognized by only anti-His or anti-rTsDNase II-7 antibody. (I) qPCR analysis of TsDNase II-7 mRNA levels at different T. spiralis stages. TsDNase II-7 mRNA from ML, 1-day, 2-day, 3-day, 4-day, 5-day AW and NBL was isolated and amplified by qPCR. The TsDNase II-7 transcription level was calculated according to the Ct (2^-ΔΔCt) method. The fold change of TsDNase II-7 genes normalized to GAPDH served as a normalizer gene. Three repeats for each sample were performed. *** P < 0.001 compared with the ML stage.