Construction of pET22b(+)-pelB/BuLIF expression vector
Total RNA isolated from buffalo mammary epithelial cells (BuMEC)  by RNA purification kit (RNeasy Mini Kit, Qiagen, USA) and first strand of cDNA was synthesized using RevertAid Reverse Transcriptase using Oligo(dT)18 primer (Thermo Fisher Scientific, USA). The primers were designed from GenBank Acession No.: NM_001290925 nucleotides 67-606 to excluding the signal peptide coding sequence. The coding region of BuLIF was amplified using a forward primer containing NcoΙ restriction site (underlined) 5′-TAGTTCCCATGGCAAGCCCCCTTCCCATCACCCCG-3′ and reverse primer containing XhoІ restriction site (underlined) 5′-CAGTCTCGAGGAAGGCCTGGGCCAGCAC-3′. The BuLIF was amplified using Q5 High-Fidelity DNA Polymerase (New England Biolabs, Beverly, MA), in a GenePro Thermal Cycler (BIOER, China). The thermocycling conditions were pre-incubation at 98 °C for 30 s and 30 cycles at 98 °C for 7 s, 62 °C for 30 s, 72 °C for 30 s, and final extension at 72 °C for 5 min. BuLIF was subcloned into pET22b(+) expression vector using NcoΙ and XhoІ restriction endonuclease sites and the resulting vector named pET22b(+)-pelB/BuLIF. This vector encodes coding sequence of N-terminal pelB leader sequence for periplasmic expression fused with BuLIF containing His6 for affinity chromatography. The vector pET22b(+)-pelB/BuLIF was confirmed by sequencing for correct and inframe cloning of coding sequence BuLIF with pelB sequence. The recombinant plasmid was confirmed by colony PCR, double restriction enzymes digestion using NcoΙ and XhoІ enzymes followed by nucleotide sequencing.
Expression and purification of recombinant BuLIF
The vector pET22b(+) pelB/BuLIF was transformed into Lemo21 (DE3) competent E. coli cells. The positive recombinant transformants were identified by colony PCR; briefly, overnight grown colonies were picked after transformation and mixed with PCR master mix (Thermoscientific, USA). The gene specific primers were used to amplify the target BuLIF insert in the pET22b(+) construct. After confirmation of recombinant plasmids, a single colony was grown overnight at 37 °C in 5 ml LB medium containing 50 μg/ml ampicillin. The overnight grown culture was then sub-cultured into 5 ml LB broth containing 50 μg/ml ampicillin. The expression of target protein was induced with 1 mM IPTG when the optical density at 600 nm (OD 600) reached 0.6–0.8. The cells were harvested at different time intervals: 4 h, 6 h, 8 h, 12 h, and o/n. The expression of the target protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The maximum expression was observed at 12 h after IPTG induction. For purification, 5 ml of culture was inoculated into the 500 ml LB broth containing 50 μg/ml ampicillin. The culture induced with 1 mM IPTG and harvested by centrifugation at 3500×g for 30 min at 4 °C. The target protein was extracted from periplasmic space of E. coli via osmotic shock as previously described with slight modification . Briefly, the cell pellets were suspended in hypertonic solution (30 mM Tris, 20 %w/v sucrose, 1 mM EDTA, Lysozyme, pH 8, 20 ml) and incubated at 4 °C for 30 min followed by centrifugation at 4 °C for 30 min at 3500×g. The supernatant was collected and cell pellets were re-suspended in hypotonic solution (5 mM MgSO4, 20 ml) and incubated at 4 °C for 30 min. The supernatant was collected in a clean 50 ml falcon tube after centrifugation and dialyzed against binding buffer (50 mM NaH2PO4, pH 8.0, 300 mM sodium chloride) 16 h at 4 °C. The dialyzed protein filtered through 0.45 μm syringe filter (Merk, Cheongwon, Korea) and purified by immobilized metal ion affinity chromatography (IMAC). The 1 ml of Ni-NTA resin (BioRad) was loaded onto the gravity flow column and equilibrated with binding buffer (50 mM NaH2PO4, pH 8.0, 300 mM sodium chloride). The filtered lysate was loaded onto the equilibrated resin and allowed to bind for one hour. The column was washed with washing buffer (50 mM NaH2PO4, pH 8.0, 300 mM sodium chloride, 20 mM imidazole) to remove non-specific protein. The protein was eluted with elution buffer (50 mM NaH2PO4, pH 8.0, 300 mM sodium chloride, 250 mM imidazole). The eluted protein was dialysed against phosphate buffer saline for overnight at 4 °C to remove imidazole and salts. The purity of recombinant protein was analysed by SDS-PAGE followed by coomassie staining. The concentration of protein was measured using Bradford method. The purified protein was filtered through the 0.22 μm syringe filter and stored at − 80 °C.
Silver staining is very sensitive method of detection of protein; it can detect 0.5–5 ng protein, so we performed silver staining to check the purity of BuLIF (23 kDa). SDS-PAGE was run and the gel was incubated in fixer (40% ethanol, 10% acetic acid), 50% water) for 1 h. Gel was washed repeatedly by water for 30 min and sensitized in 0.02% sodium thiosulfate for 1 min followed by washing with water. The gel was incubated in cold 0.1% silver nitrate solution for 20 min at 4 °C followed by washing with water. Gel was developed by 3% sodium carbonate and 0.05% formaldehyde and washed with water to avoid the excess of stain. Staining was terminated by 5% acetic acid by incubating for 5 min and gel was stored in 1% acetic acid at 4 °C.
Western blot analysis
The recombinant protein was confirmed by the western blot using antiLIF antibody. Briefly, the purified protein was separated by electrophoresis and transferred on to the PDVF membrane (Thermo Fisher Scientific, USA) in transfer buffer (25 mM Tris, 15% methanol) carried out on semi-dry blotting unit (Scie-Plas Ltd., UK.). The membrane was blocked using blocking buffer (3% bovine serum albumin in TBST) for 2 h at room temperature. The membrane was further incubated at 4 °C for overnight with primary antibody (Sigma-Aldrich, USA) in dilution 1:1000 with blocking buffer. The washing was done with TBST three times at the interval of 15 min. The membrane incubated with secondary HRP conjugated antibody (Sigma-Aldrich, USA) in dilution1:2000 for 2 h at room temperature followed by three times washing with TBST. The membrane was developed using 3, 3-Diaminobenzidine tetrahydrochloride (Merk, Sigma-Aldrich, USA).
Mass spectrometry analysis for identification of purified recombinant BuLIF
The identification of purified recombinant BuLIF was verified by matrix assisted laser desorption/ionization-time of flight Mass spectroscopy (MALDI-TOF MS) as described previously . The purified protein was separated by electrophoresis followed by coomassie staining. The band of target protein was cut into pieces and destained by adding 100 μl 40% ABC (ammonium bicarbonate, NH4HCO3) and 40% ACN (Acetonitrile, CH3CN) in the 1:1 ratio. The gel pieces were dehydrated by adding 200 μl of 100% acetonitrile and incubated for 15 min at room temperature. The rehydration solution (5 mM DTT in 40 mM ABC) was added to the sample, incubated at 60 °C for 45 min to cleave the disulfide bonds. The sample was treated with 200 μl alkylation solution (20 mM Iodoacetamide) and incubated for 10 min in dark followed by addition of 200 μl of 100% acetonitrile. The gel pieces were treated by digestion buffer (12.5 ng/μl of trypsin in 50 mM ammonium bicarbonate buffer) followed by incubation at 37 °C for overnight. The reaction was stopped by adding 5% formic acid, and the peptides were extracted from gel by adding 100 μl of extraction buffer (5% formic acid in 40% ACN). The final extraction was carried out by adding 100 μl of 100% ACN, and the extracted peptides were dried in vaccum concentrator. Peptides were desalted using Zip tip (Millipore, USA) and eluted with 20 μl of 0.1% formic acid + 60% ACN). The peptides were analyzed by Q-TOF Bruker Mass Spectrometer (Maxis HD). The generated data was interpreted by ProteinScape with Mascot.
MTT reduction assay
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was carried out to measure the activity of BuLIF. M1 cells (ATCC-TIB-192) were purchased from ATCC and routinely maintained in RPMI 1640 medium (Gibco) containing 10% FBS. Serial two-fold dilutions of different BuLIF preparation (10 to 50 ng/ml) were made in flat bottom 96-well microtitre plates (Thermoscientific, USA) using multichannel pipette. M1 cells were harvested by centrifugation and seeded per well at the density of 10,000 cells (total volume of 100 μl). The plates were then incubated at 37 °C in a humidified 5% CO2/ 95% air mixture for 24 h. Ten microliters of freshly prepared MTT solution was added to each well. The MTT solution was prepared in a concentration of 5 mg/ml in phosphate-buffered saline (PBS) and filtered through a 0.2 μm filter. The plates were incubated at 37 °C in a humidified 5% CO2/ 95% air mixture for 4 h. After formation of Formazan crystals, 75 μl DMSO was added to solubilize the MTT formazan and kept for 15 min on an orbital shaker. The absorbance was measured at 570 nm in microplate spectrophotometer. For the control, the cells were cultured in the absence of BuLIF. The data represent mean ± SD of three independent experiments.
The phagocytic assay was carried out as per manufacture’s instruction (Cayman Chemical, USA). In brief, the unstimulated M1 cells were treated with 40 ng/ml BuLIF for 4 days and seeded at 1 × 105 cells/ml in 24-well dishes. The fluorescent latex beads were added in dilution 1:300 into the cells and incubated for 24 h at 37 °C. The cells were analyzed using Inverted Fluorescence microscopy (Nikon, Japan).
Maintenance of buffalo embryonic stem cells (buESCs) in culture
Buffalo embryonic stem cells were (buESCs) were cultured as described previously . The embryos produced through IVF were used for isolation of buffalo embryonic stem cells. Briefly, the buffalo ovaries were collected from slaughter house and A-grade Cumulus–oocyte complexes (COCs) were selected and subjected to in vitro maturation (IVM) at 38.5 °C for 24 h in a humidified CO2 incubator (5% CO2 in air) in medium containing Tissue Culture Medium-199 (TCM-199) supplemented with 10% fetal bovine serum (FBS), porcine follicle-stimulating hormone (FSH; 5 μg/mL), estradiol-17b (1 μg/mL), 0.81 mM sodium pyruvate, and 50 μg/mL gentamicin sulfate. The frozen semen straws were collected from ABRC, NDRI. The semen straws were thawed for 30 s in a 38.5 °C water bath. Sperms were washed twice by centrifugation (1200 rpm) for 7 min in 10 ml of Brackett and Oliphant medium (BO medium) containing 3 mg/ml of BSA supplemented with 10 mM caffeine. The washed pellet of sperms was resuspended in 0.5–1 ml BO medium. The matured cumulus-oocyte complexes were washed twice and transferred into a 50-μl drop of BO medium (20–25 oocytes/drop) containing 6 mg/ml of BSA and 10 mg/ml of heparin, and 50 μl of sperm suspension were added to each drop. Oocytes were incubated with sperm for 16–18 h at 38.5 °C in 5% CO2 in humidified air. After fertilization oocytes were cultured in RVCL media for 7 to 8 days.
For culture of embryonic stem cells, the inner cell masses (ICMs) from hatched blastocyst were isolated mechanically by using microblades under zoom stereomicroscope (Olympus, SZ40, Japan). The ICMs were separated from trophoectoderm and seeded on to the mitomycin-C inactivated bovine fetal fibroblast cells. The ICMs were cultured in embryonic stem cells medium (KO-DMEM supplemented with 15% serum replacement FBS, 2 mM glutamine, 1000 U/ml buffalo leukemia inhibitory factor BuLIF, 4 ng/ml basic fibroblast growth factor (bFGF), 1% non-essential amino acids, 0.1 mM β-mercaptoethanol and 50 μg/ml gentamycin sulphate). Instead of mLIF or hLIF, homemade BuLIF was used in this assay. The putative ES cells with a uniform, undifferentiated morphology were selected individually using a micropipette, mechanically dissociated into two to six clumps and replated. The ES cells were passaged every 11–14 days after replating.
After 5–10 passages, in addition to morphology, buESCs were characterized by the expression of embryonic stem cell markers. The immunocytochemistry was done with specific antibodies for Oct-4 (20 μg/ml; Novus biologicals, USA), Sox-2 (20 μg/ml; Novus biologicals, USA), Nanog (20 μg/ml; Novus biologicals, USA) and a commercially available kit for detecting alkaline phosphatase (Sigma, USA).
Experiments were performed in triplicate. One-way analysis of variance (ANOVA) was performed using GraphPad Prism version 8.00 (GraphPad Software, San Diego, CA, USA Data were shown in the form of bar plot as mean with standard error of mean (± SEM) ). P values < 0.05 were considered statistically significant.