PMC:4576194 / 24247-25647 JSONTXT

{"project":"2_test","denotations":[{"id":"26266418-26041303-144197562","span":{"begin":314,"end":316},"obj":"26041303"}],"text":"4.3. Yeast Two-Hybrid Screen\nA Matchmaker two-hybrid system (catalog no. 630489; Clontech) was used to screen host proteins that interact with NS5A. Briefly, the bait construct pGBKT7-NS5A (BD-NS5A) was transformed into the yeast strain Y2HGold and hybridized with a cDNA library derived from porcine macrophages [39]. Transformants were selected for growth on synthetically defined (SD) medium lacking His, Leu, Trp and Ade (SD/-4) for 3 to 6 days at 30 °C. The clones were then transferred to SD/-4 medium containing 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside (X-α-Gal) and aureobasidin A (Aba) (SD/-4/X-α-Gal/Aba). Blue colonies were selected and inoculated with 5 mL of SD/-4 medium for shaking culture at 30 °C for 1 to 3 days. Yeast plasmids were extracted using a yeast plasmid kit (catalog no. D3376; Omega, Guangzhou, China) according to the manufacturer’s instructions, and the target inserts were verified by sequencing using the Gal4 AD and 3ʹAD primers. To validate the interaction between NS5A and the cellular proteins, the bait and prey plasmids were cotransformed into the Y2HGold yeast strain using a yeast transformation system 2 (catalog no. 630439; Clontech). The interaction between murine p53 and SV40 large T-antigen was used as a positive control, and the human lamin C protein, which does not interact with the SV40 large T-antigen, was included as a negative control."}