Cells, vectors and reagents
E. coli strain DH5α, BL21-codon plus and expression vector pET28a(+) were obtained from repository of Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan. Restriction enzymes NdeI and BamHI, Taq DNA polymerase, T4 DNA ligase, RevertAid first strand cDNA synthesis kit, TA cloning kit were purchased from Fermentas Inc. Qiaquick gel extraction kit was purchased from Qiagen (USA), isopropyl-β-d-1 thiogalactopyranoside (IPTG), 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) and all other chemicals required for routine extraction and analysis of biomolecules were purchased from Sigma Aldrich (USA). Primers were synthesized by Gene link (USA).
RT-PCR
Total RNA was extracted from human leukocytes isolated from the peripheral blood of healthy person by Trizol reagent (Invitrogen, USA). RT-PCR was done using RevertAid first strand cDNA synthesis using oligo(dT)18 as reverse primer. The primers 5′ GGACATATG GCCTTGACCTTTGCTTTACT 3′ (forward primer), having NdeI site (underlined) and 5′ GGCGGATCC TCATTCCTTACTTCTTAAAC 3′ (reverse primer), having BamHI site (underlined) were designed on the basis of reported gene sequence (gi: 209413719). PCR reaction was performed in iCycler (Biorad) using 2 μl cDNA solution as template in 50 μl reaction volume containing 2.5 units of Taq DNA polymerase, 1× PCR buffer, 200 μM each dNTPs, 2 mM MgCl2, 0.5 μM of each forward and reverse primer. Thermal cycler was programmed with the following parameters: initial denaturation for 1 minute at 94°C followed by 35 cycles of denaturation for 30 seconds at 94°C, annealing for 30 seconds at 63°C and elongation for 30 seconds at 72°C with a final elongation step of 20 minutes at 72°C. The amplicon was checked on 1% agarose gel and purified by QIAquick gel extraction kit.
Characterization of cloned hIFNα-2b
The amplified hIFNα-2b gene (IAS) was ligated in pTZ57R/T vector. The recombinant vector was designated as pTA-IFN vector and transformed into chemically treated competent cells of E. coli strain DH5α. Recombinant colonies were selected by blue/white screening (Sambrook and Russell 2001). The E. coli clones having recombinant plasmid (pTA-IFN) were confirmed by colony PCR. The positive clones were further confirmed by release of insert (IAS) following digestion with NdeI/BamHI restriction enzymes. The insert IAS was processed further for DNA sequence analysis. For subcloning, the IFN vector was digested with NdeI and BamHI restriction enzymes and the released 567 bp fragment was purified. The purified fragment was ligated with the pET28a (+) expression vector. The resulting recombinant expression vector (pET-28a-IAS) was used to transform BL21-codon plus competent cells as described in Sambrook and Russell (2001). To select the transformants containing pET-28a-IAS, the cells were grown in plates containing 1% Trypton, 0.5% Yeast extract, 1% Sodium chloride and kanamycin (50 μg/ml), pH 7.4 at 37°C. The positive clones were further confirmed by colony PCR and digestion with NdeI and BamH I restriction enzymes.
Optimization of temperature and induction with IPTG for expression of hIFNα-2b
A single transformed colony was used to inoculate 5 ml LB medium containing kanamycin (50 μg/ml) and incubated in shaker water bath at 200 rpm at 37°C. When
OD600 of the bacterial culture reached 0.6, 1 ml sample from culture was removed as control. To the remaining culture, isopropyl β-d-thiogalactoside (0.2, 0.4, 0.6, 0.8 and 1.0 mM) was added independently in each culture. One ml of each induced culture was taken at 2-h intervals up to 14 h at each temperature (16, 20, 25, 30, 37 and 40°C). The induced cells were mixed with 2× SDS/PAGE sample buffer, boiled for 2 minutes and centrifuged at 5000 rpm for 5 minutes at room temperature. The cell free supernatant was loaded in 10% SDS-PAGE to check the expression of recombinant hIFNα-2b (Laemmli 1970).
Production and partial purification of antibodies
The 7–8 week old four male Balb/C mice, weighing nearly 200 gm were immunized interperitonially with denatured commercially available hIFNα-2b (Uniferon 12 μg/injection). The interferon injection was mixed with Freunds complete adjuvant in 1:1 ratio. The immunization dose was adjusted 30-40 μg of hIFNα-2b per injection at 15 days intervals with a total of four injections. The antibody titre was checked by enzyme linked immunosorbent assay by drawing 100 μl of blood from mouse orbital vein. The mice were anesthetized and whole blood was isolated. Serum was separated and stored at −20°C. The antibodies were partially purified by mixing with (NH4)2SO4 at 50% saturation. The proteins were separated by centrifuging at 5000 rpm for 10 minutes at 4°C. The separated proteins fraction pellet was dissolved in 0.05 M Tris-Cl, pH 7.4 and dialyzed against the same buffer. The dialyzed antibodies were aliquoted and stored at −20°C. Preimmune serum was used as control.
Enzyme linked immunosorbent assay
100 μl (2 μg) of commercially available hIFNα-2b (Uniferon, Interlong and Anferon) were mixed with 100 μl of 0.05 M carbonate buffer, pH 9.0 and absorbed on flat bottom microtitre plates for 2 hours at 37°C. The nonspecific binding sites in microtitre plate were blocked with blocking buffer (5% skim milk in Phosphate buffer saline-Tween 20, (PBST) by incubating at 37°C. After washing in PBST, the partially purified mouse anti-rhIFNα-2b antibody (1:100 dilution) was added and kept for one hour at 37°C with continuous shaking. Again after washing, rabbit anti-mouse IgG antibody alkaline phosphatase conjugated (1:2000 dilution) was added and incubated for 30 minutes at 37°C. After washing in PBST, 0.2 μl of para-nitrophenyl phosphate (PNPP) was added as substrate for color development. Preimmune serum was used as control. The overexpressed rhIFNα-2b produced in this study was also checked by ELISA as described above. Untransformed BL21-codon plus cells were used as control.
Computational analysis
The BLAST program was used to compare sequence of cloned hIFNα-2b with the reported IFNα-2b gene. The deduced amino acid sequence was determined by using the TRANSLATE tool available on ExPASY. The homology of the nucleotide and deduced amino acid sequence of rhIFNα-2b was evaluated and compared with reported subtypes of IFNα family members by using the CLUASTALW program. The tertiary structure of recombinant hIFNα-2b was predicted using PHYRE server on ExPASY. All the programmes used for computational analysis are freely available online. After logging in the programs, data was submitted for analysis and results were obtained.
Accession number
The full length cDNA sequence of recombinant hIFNα-2b gene was submitted to the Gene Bank database. The accession number of the gene is JN591570.