Fig. 4

Host cell proteins interacting with the SVSP455 protein were screened using the Y2H system. A Toxicity and autoactivation analysis of pGBKT7-SVSP455 in Y2H Gold cells. B Determination of the bait plasmid transformation efficiency by PCR assay. The colonies from SDO ar plates were randomly picked and used to in a colony PCR assay in a 50-μl system with specific primers. Lanes: M, DL2,000 DNA marker; 1–18, the numbers of selected colonies grown on SDO plates transfected with the bait plasmid; NC, negative control. C Y2H screening and identification of potential host cell proteins interacting with SVSP455. The cotransformants of the bait plasmid pGBKT7-SVSP455 and the prey plasmid cDNA library of bovine B cells were grown on DDO/X/A plates, and the blue colonies from DDO/X/A plates were successively cultured on QDO/X/A plates and screened for putative prey proteins through two rounds of incubation. All blue colonies from QDO/X/A plates were selected and identified by the colony PCR assay based on the pDT7-F/R primers. Lanes: M, DL5000 DNA marker (TaKaRa; #3428A); 1, negative control; 2–18, the numbers of blue colonies from QDO/X/A plates. The positive control (pGBKT7-53 and pDT7-T plasmids) and negative control (pGBKT7-Lam and pDT7-T plasmids) were also cotransformed into Y2H Gold cells and cultured onto DDO and DDO/X/A plates. The empty pGBKT7 plasmid served as the blank control was also transformed into Y2H Gold competent cells and cultured on SDO, SDO/X and SDO/X/A plates. Abbreviations: cDNA, Complementary DNA; DDO, synthetically defined medium without tryptophan and leucine; DDO/X/A, DDO supplemented with X-α-l and aureobasidin A; QDO/X/A, DDO without adenine and histidine, supplemented with X-α-l and aureobasidin A; SDO (SD/-Trp), synthetically defined medium; SDO/X, SDO without tryptophan supplemented with X-α-l SDO/X/A, SDO without tryptophan and leucine supplemented with X-α-l and aureobasidin A; Y2H, yeast two-hybrid