Abstract: To screen receptor of vesicular stomatitis virus (VSV), the calves oral mucosal epithelial cells T7 phage display library was constructed. The total RNA of calves oral mucosal epithelial cells was extracted using Trizol reagent. Then, mRNA was separated and purified by mRNA isolation kit and the ds cDNA were synthesized by reverse transcription. ds cDNA ends were ligated EcoR Ⅰ/Hind Ⅲ linkers, then ds cDNA were digested by EcoRⅠ/Hind Ⅲ, so that we got ds cDNA ends containing EcoR Ⅰ/Hind Ⅲ sticky ends. All digested ds cDNA were separated by Mini Column, only ds cDNA fragments more than 400 bp were collected. Then, the collected ds cDNA were ligated into the T7 Select 10-3b vector. After packaging in vitro, the recombinant T7 Select 10-3b vectors were transformed into BLT5403, so we could construct the T7 phage display library. The results indicated that the titer of un-amplified library was 1.3×10⁷ PFU/mL, and the recombination rate was 95.8%. After amplification, titer of library was 2.6×10¹⁰ PFU/mL. Randomly picked 100 plaques were identified by PCR, 95% of the inserted cDNA fragments were longer than 400 bp in length. These results indicated that the calves oral mucosal epithelial cells T7 phage display library was successfully constructed.

Key words:

calves oral mucosal epithelial cells; T7 phage display library; vesicular stomatitis virus