Construction of a cytoplasmic transduction peptide-single chain antibody fusion protein
Anti-phosphoprotein scFvs have been cloned from the spleen of immunized chickens in our laboratory, as described in our previous study[14]. The CTP was added to the 3’ terminus. The forward primer was 5’-CGGAATTCGCCGTGACGTTGGAC-3’ with EcoR I; and the reverse primer was 5’-GAGTCATTCTGCGGCCGCACGGCGACGCTGGCGACGTTTCTTACGACCGTATAGGACGGTCAGGGTTGTCCC-3’ with Not I and CTP. The anti-P CTP-scFv gene was amplified by PCR. After digestion with EcoRI and NotI, the CTP-scFv gene was subcloned into pET28a(+). Thus, the recombinant expression plasmid pET28a-CTP-scFv was obtained.
Stable expression of pET28a-scFv-CTP
Trans1-Blue cells transformed with the recombinant plasmid pET28a-scFv-CTP were grown at 37°C in Luria Bertani (LB) medium supplemented with 100 mg/ml kanamycin until the bacteria reached the logarithmic growth phase (at OD600=0.7), and the cells were then induced by the addition of 1 mM isopropyl b-d-thiogalactopyranoside. Purification of the His-tagged phosphoprotein was carried out in accordance with the manufacturer’s procedures for Ni+–NTA resin-packed columns. The final protein concentration was determined by the BCA kit.
Optimal transfection of the pET28a-scFv-CTP fusion protein
BHK21 cells were passaged in 6 well plates. Cells in 100 ml cell culture bottles were washed twice using DMEM. After 1 min of digestion using tyrisin, the cells were suspended in 4 mL DMEM(10% FBS with penicillin-streptomycin), added to 6 well plates(0.6 ml of cell suspension per well), and cultured in an incubator overnight. When the cell confluence reached 80%, the cells were treated with protein at different concentrations as following: 1)1 mL of DMEM (without FBS); 2)1 mL of DMEM (without FBS) with scFv at a final concentration of 3 µM; 3) 1 mL of DMEM (without FBS) with CTP-scFv at a final concentration of 1 µM; 4) 1 mL of DMEM(without FBS)with CTP- scFv at a final concentration of 3 µM; 5) 1 mL of DMEM(without FBS) with CTP-scFv at a final concentration of 4 µM. After 2 h of culture, the cells were washed twice in DMEM (or: after 3 h of incubation) and washed twice in PBS, and the medium was replaced with new medium for another 24 h of culture. The cells were washed three times in PBS, and then, the expression was observed. After 1 min of digestion using trypsin, the cells were blown slightly and suspended in 1 mL DMEM(10% FBS with penicillin-streptomycin). The suspension were collected in EP tubes and Western blotting performed to analyse the transmembrane effect.
Cytoplasmic localization of CTP-scFv
The pET28a-scFv-CTP fusion protein was transduced into BHK21 cells as described above. After 24 h, the cells were fixed with 4 % paraformaldehyde for 30 min and incubated with 0.1 % TritonX-100 for 10 min. The cells were incubated with the primary antibody mouse anti-His tag mAb at a dilution of 1:50 for 2 h, followed by incubation with the secondary antibody FITC- conjugated goat anti-mouse IgG at 1:25 for 1 h at room temperature. The cells were analysed and photographed with confocallaser scanning microscope( Leica TCS-SP).
MTT asssy of the effect of CTP on cell viability
Cells were collected in the logarithmic phase, used to generate were made into 105 cell/ml suspensions using DMEM containing 10% FBS. Then, the cells were inoculated in 96 culture plate. After 24 h, pET28a-scFv-CTP and scFv cells were treated with 0.5 mg/ml DMEM. Every sample had 4 wells in parallel. The cells were cultured at 37℃ and 5 % CO2. After 24 h, the cells were treated with 20 µl of 5 mg/ml MTT(in PBS). After incubation for 4 h, the supernatant was removed, and the culture plate was inverted on several layers of filter paper to wipe away the supernatant. DMSO (150 µl per well) was added, and the cells were shaken in a shaker for 30 min. The OD values were detected 570nm/490nm using a microplate reader.
Virus infection
To assess the effect of the anti-P CTP-scFv antibody on NDV production, we transduced BHK21 cells with the pET28a-scFv-CTP fusion protein. Then, the cells were infected with the virus at a multiplicity of infection of 0.01. The supernatants of the cell cultures were harvested at 12, 24, 36 and 48 h. The samples were serially diluted, and assayed for virus titer by TCID50 assays[15].
Reverse transcription and real-time PCR
The transfected BHK21 cells grew exponentially in 96-well plates on glass cover-slips, and 24 h after transfection, they were infected with NDV at a multiplicity of infection of 0.1. Four hours after infection, the cullture medium was removed and the cells were collected. Total RNA was isolated from the lysed cells using TRIzol reagent according to the procedures described by the manufacturer. Reverse transcription was carried out by using reverse transcriptase in a 20 µl reaction mixture, containing 200 ng of total RNA and specific primers for P-mRNA (5’-TTTTTTTTTTTTTTTTTT-3’), P-cRNA (5’-TGGTGATCAGCCATTCAGCGCAAGGC-3’), and P-vRNA (5’- TACCCAGCAGACCAGGGCGAATATG-3’), at 42 °C for 1 h. The RT reaction mix was then used for real-time PCR using specific primers for P (sense 5’- CCTTTACAGACGCGGAGATTG-3’ and antisense 5’-GTTTTGCCTTGTGGGATTGC-3’) and β-actin (sense 5’-GCATCCACGAAACTACATTCAACTC-3’ and antisense 5’-CACTGTGTTGGCATAGAGGTCTTTG-3’), and SYBR Green I dsDNA binding dye. Real-time PCR was performed at 95 °C for 3 min for 1 cycle and then 94 °C for 45 s, 55 °C for 30 s and 72 °C for 1 min for 40 cycles. The PCR products were assessed with a Rotor-Gene 2000 Real-time Cycler and analysed with Rotor-gene software. Cycle times (Ct) were analyzed with a reader of 0.2 fluorescence units. The duplicate cycle times were averaged and normalized to the cycle time of β-actin. All reactions were performed in duplicate.