Construction of cytoplasmic transduction peptide-single chain antibody fusion protein
Anti-phosphoprotein scFvs have been cloned from the spleen of immunized chicken in our laboratory, referred to in our publication Biologicals. The CTP was added to 3’ terminal. The forward primer was designed as 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 digested with EcoR I and Not I, the CTP-scFv gene was subcloned into the pET28a(+). In this way, the recombinant expression plasmid pET28a-CTP-scFv was obtained.
Stable expression of pET28a-scFv-CTP
Trans1-Blue transfected with the recombinant plasmid pET28a-scFv-CTP was propagated at 37℃ in Luria Bertani (LB) medium supplemented with 100 mg/ml Kanamycin until the bacterium reached logarithmic growth phase (at OD600=0.7), and then induced by addition of 1mM 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.
The best contration of transduce of pET28a-scFv-CTP fusion protein
BHK21 cell were passaged (and immigrated) into 6 pore plate. That is : Cells in 100ml cell culture bottle were washed twice using DMEM, after 1min’s digestion using tyrisin, cells were blown and suspended in 4mLDMEM(10% FBS with penicillin-streptomycin), and were immigrated into 6 pore plate (0.6ml cell suspension per pore), then were cultured in incubator overnight. When cell’s density reached to 80%, cells were treated with protein in different concentrations respectively as following: 1) DMEM (without FBS) 1mL; 2) DMEM(without FBS) 1mL with scFv in final concentration 3uM; 3) DMEM(without FBS) 1mL with CTP- scFv in final concentration 1uM; 4) DMEM(without FBS) 1mL with CTP- scFv in final concentration 3uM; 5) DMEM(without FBS) 1mL with CTP- scFv in final concentration 4uM. 2h culture later, cells were washed twice in DMEM(or: after 3h incubating, cells were washed twice in PBS, then replaced new medium for another 24h culture. Washed three times in PBS and then observed the expression). After 1min digestion using tyrisin, cells were blown slightly and suspended in 1mLDMEM(10% FBS with penicillin-streptomycin). Suspension were collected in EP tube and taken Western blotting to analysis transmembrane effect.
Cytoplasmic localization of CTP-scFv
pET28a-scFv-CTP fusion protein was transduced into BHK21 cells as described above. After 24h, fixed with 4 % paraformaldehyde for 30 min and permeated with 0.1 % Triton-100 for 10 min. Cells were incubated with primary antibody mouse anti-His tag mAb at a dilution of 1:50 for 2 h, followed by incubation with secondary antibody FITC conjugated goat anti-mouse IgG at 1:25 for 1h at room temperature. Cells were analyzed and photographed with Confocal Laser Scanning Microscope, Leica TCS-SP.
MTT test to detect CTP influence on cell viability
Cells were collected in logarithmic phase, and were made into 105/ml suspension using DMEM containing 10% FBS, then were inoculated in 96 culture plate. After 24h, pET28a-scFv-CTP and scFv were treated to cell with final concentration 0.5mg/ml, DMEM was contrast. Every sample owned 4 holes as parallel. Cells were cultured for in 37℃ and 5%CO2. After 24h, cells were treated with 20µl 5mg/ml MTT（soluted in PBS）, after incubating for 4h, supernatant were removed, and the culture plate was inverted on several layers filter papers so to wipe put supernatant sufficiently. Added DMSO 150 µl per hole, the cells were shaked in shaker for 30min. The OD value were detected in 570nm/490nm using microplate reader.
To assess the effect of anti-P CTP-scFv antibody on NDV production, BHK21 cells were stably transduced with pET28a-scFv-CTP fusion protein, respectively. Then infected with the virus at a multiplicity infection of 0.01, supernatants of cell cultures were harvested at 12, 24, 36 and 48h. serially diluted, and assayed for virus titer by TCID50 assay.
Reverse transcription and real-time PCR
Transducted BHK21 cells grew exponentially in 96-well plates on glass cover-slips and 24 post-transducted, infected with NDV at a multiplicity of infection of 0.1, 4h post-infected, culture medium was removed and cells were collected. Total RNA was isolated from lysed cells using Trizol reagent according to the procedures described by manufacturer. Reverse transcription was carried out by using reverse transcriptase in 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 ℃ for 1 h. RT reaction mix was then used for real-time PCR using specific primers for P (sense 5’- CCTTTACAGACGCGGAGATTG-3’ and antisense 5’-GTTTTGCCTTGTGGGATTGC-3’) andβ-actin (sense 5’-GCATCCACGAAACTACATTCAACTC-3’ and antisense 5’-CACTGTGTTGGCATAGAGGTCTTTG-3’), and SYBR green I dsDNA binding dye. Real-time PCR reaction was performed at 95 ℃ for 3 min for 1 cycle and then 94 ℃ for 45 s, 55 ℃ for 30 s and 72 ℃ for 1 min for 40 cycles. PCR products were measured with Rotor-Gene 2000 Real-time Cycler and analyzed with Rotor-gene software. Cycles times (Ct) were analyzed at a reader of 0.2 fluorescence unit. The duplicate cycle times were averaged and normalized to the cycle time of β-actin. All reactions were done in duplicate.