Sfii restriction enzyme

ScFv phage clones that showed high binding in phage ELISA were processed further for soluble scFv expression. The pAK100 vector carrying scFv DNA fragment and pAK400 vector were digested with SfiI restriction enzyme (New England Biolabs, USA). The scFv DNA was then ligated into pAK400 vector at a 3:1 molar ratio, using T4 DNA ligase (New England Biolabs, USA) and then transformed into E. coli HB2151. Colonies were picked and glycerol stocks were stored. These transformed cells were again cultured and induced by 1 mM IPTG, and scFv was purified from the periplasmic extract by Ni-NTA (Qiagen) affinity chromatography as described earlier42 (link). The scFv was eluted with elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 300 mM imidazole-pH-8.0). The eluted scFv protein was extensively dialysed against ice cold 1X PBS (pH 7.4), concentrated using ultrafiltration columns (Amicon) with a 10 kDa cut off, filtered through 0.2 μm sterile syringe filter and snap frozen in liquid nitrogen and stored.

By the TRIZOL method, total mRNA was extracted from the purified PBLs and the concentration was measured by optical density at 260 nm. Then, cDNA was synthesized using PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Beijing, China). The VNAR fragments were obtained after PCR amplification of the cDNA by PCR using specific primers (Table 2). The PCR products corresponding to VNAR genes were analyzed by agarose gel electrophoresis. At the same time, the PCR products were digested with the SfiI restriction enzyme (NEB, Ipswich, New England), then inserted into the phagemid pComb3XSS with T4 ligase (Thermo Scientific, Waltham, MA, USA). By electroporation, recombinant plasmids were transformed into E. coli TG1 cells and plated onto 2 × YT-Agar (1.6% tryptone, 1% yeast extract, 0.5% NaCl, 1.5% agar, 1% glucose) containing 100 μg/mL ampicillin and cultured at 37 °C overnight.

Two μg of normalized cDNA were digested by ten units of the SfiI restriction enzyme (New England Biolabs) for 2 h at 48 °C. Fragments (> 800 bp) isolated from an LMP agarose gel were purified using the MinElute Gel Extraction Kit (Qiagen). The Fast Ligation Kit (New England Biolabs) was used for ligation of 200 ng purified cDNA fragments to 100 ng SfiI using dephosphorylated pDNR-lib Vector (Clontech). The product was desalted by ethanol precipitation and re-dissolved in 10 μl water. Of this, 1.5 μl was used to transform NEB10b competent cells (New England Biolabs). To verify the success of normalization, 96 clones were randomly selected and sequenced.

Total RNA was isolated from the purified PBLs using TRIZOL, according to the manufacturer’s instructions. Then we used PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Beijing, China) to synthesize cDNA. To obtain the VNAR genes, PCR was then performed with VNAR-specific primers, which were as previously described [33 (link)]. The VNAR genes of the first PCR products were analyzed by agarose gel electrophoresis. The second round PCR products were digested with SfiI restriction enzyme (New England BioLabs, Ipswich, MA, USA) and inserted into the phagemid pComb3XSS with T4 ligase (Thermo Scientific, Waltham, MA, USA). After purification, the ligation products were transformed into E. coli TG1 cells by electroporation. The cells were plated onto 2 × YT agar medium (5 g/L NaCl, 16 g/L tryptone, 10 g/L yeast extract, and 15 g/L agar) containing 100 µg/mL ampicillin and cultured at 30 °C for 16 h. The cells were also serially diluted from 10⁶ to 10¹⁰, and the library size was determined by counting the colony number.

Recombinant adenoviruses expressing TERT and VEGFR-2 were constructed and identified in preliminary work28 (link)55 (link). Briefly, murine VEGFR-2 and TERT were amplified from the VEGFR-2 and TERT original plasmids using published primers. Using the SfiI restriction enzyme (New England Biolabs) and T4 DNA ligase (JingMei Biological Engineering Co., Ltd), the target genes were subsequently cloned into a shuttle vector (pShuttle-CMV, SinoGenoMox Research Center Co., Ltd). The recombinant vectors (pShuttle-VEGFR2 and pShuttle-TERT) were transferred into the adenoviral backbone carrier pAdxsi (SinoGenoMoxResearch Center Co., Ltd) using I-CeuI and I-SceI restriction enzymes (New England Biolabs) and T4 DNA ligase (JingMei Biological Engineering Co., Ltd). pAdxsi-VEGFR2 and pAdxsi-TERT were linearized using PacI restriction enzyme (New England Biolabs) and transfected into HEK293 cells (human embryonic kidney cell line). After 14 days, adenoviruses (AdVEGFR-2 and AdTERT) were harvested. The recombinant adenoviruses were amplified in HEK293 cells, purified with the Adenovirus Purification Miniprep Kit (Biomiga San Diego, USA), and further titred by using a plaque assay, yielding the number of plaque forming units per millilitre (PFU/ml). To investigate the process of antigen uptake, an adenovirus vector expressing green fluorescent protein (AdGFP) was also prepared.

Complex assembly was performed as described in our previous study [17 (link)]. Briefly, the reaction mixture consisting of 1 μL of 10× buffer A [10 mM HEPES (pH 7.5), 50 mM NaCl, 2 mM CaCl₂, 0.1 mM EDTA], 2 μL of DNA substrate (86 ng/μL), 1 μL 1 mM DTT, 1 μL SfiI enzyme, and 5 μL di-water was incubated for 15 min at room temperature, diluted through serial dilutions, and deposited on APS-functionalized mica [17 (link)] for AFM imaging. SfiI restriction enzyme with low BSA content (20 units/μL) was purchased from the New England Biolabs (Beverly, MA, USA).

The rabbit-human chimeric Fab library was cloned into pComb3X phagemid vector. First, the vector and the Fab DNA were digested with SfiI restriction enzyme (New England Biolabs). For digestion of DNA, 15 µg of purified PCR product or vector DNA was mixed with 50 units of SfiI, 20 µL of 10× reaction buffer, and water to a final volume of 200 µL. Digestion mixtures were incubated at 50 °C for 16 h followed by ethanol precipitation overnight at −20 °C. The precipitated DNA was centrifuged and dissolved in 100 µL nuclease-free water, and purified by agarose gel electrophoresis. Digested vector and Fab DNA fragments were ligated overnight (2 μg vector DNA and 3 μg insert DNA (~1:3 molar ratio) in 100 µL total volume) and ethanol precipitated. After centrifugation, the ligated DNA was dissolved in 10 µL of nuclease-free water and transformed to Escherichia coli (E. coli) ER2537 cells by electroporation. Transformed bacteria were plated on LB (Luria-Bertani broth)-ampicillin plates with 2% (w/v) glucose and incubated overnight. Next day, the bacterial growth was scraped and resuspended in 5 mL SB (Super Broth) medium, 15% (v/v, final concentration) glycerol was added, and 1 mL aliquots of the bacterial suspension were kept at −80 °C. The immune Fab phage library was rescued from the bacterial stock as previously reported [16 (link)].