2.2 DNA manipulation and Plasmid construction
Plasmid extraction and PCR purification used kits from CWBIO. The polymerase chain reaction (PCR) was carried out using EsTaq polymerase (CWBIO, China) or PrimerSTAR (TaKaRa, China). The restriction endonuclease was purchased from TaKaRa. Gene sequences were inserted into target vectors through ligation reaction (TaKaRa, China) or homologous recombination (Abclone, China). All experiments were performed following the standard procedure.
Table S2 lists all the primers used in this study. First, the EGFP sequence was inserted into pET28a through Nco Ⅰ andHin d Ⅲ to obtain pET28-EGFP. This plasmid was used as a skeleton to construct bicistronic T7 expression vectors containing different fore-cistronic sequences (Fig. 1A ). The construction process was as follows: The 62 bp fore-cistron sequence ended with a conserved SD2 (AAAGGAGGACAACTAATG) and TAATG translation coupling frame were obtained by primer annealing. Then, pET28-EGFP was digested withNco I, and these sixteen different sequences were inserted upstream of EGFP through homologous recombination, respectively. TheNco I cleavage site was removed from the final plasmid (Fig. 1B ).
To construct expression plasmids for PCP vaccine protein production, the Oml1, Oml7, and ApxⅡ genes were codon-optimized and synthesized by Genewiz (Suzhou, China). The sequences were listed inSupplementary material , and all three genes were appended with a 6×his tag at the 3-terminal. Then, these gene sequences and the corresponding fore-cistron sequences were inserted into the pET28a to obtain BCD expression vectors. The expression plasmid containing a monocistronic T7 promoter was used as a control.