Xhoi

The construction of expression plasmids of the Msep5-HT7L and Msep5-HT7S cDNA containing the Kozak consensus sequence (GCCACC) [62 (link)] were obtained by PCR amplification with specific primers (Table 4). The DNA fragments were double digested with KpnI and XhoI (Sangon Biotech, Shanghai, China), and after purification, they were cloned into a pcDNA3.0 vector (Invitrogen, Carlsbad, CA, USA). DNA sequencing was used to confirm the correct recombinant plasmid.

Recombinant pGEX-4T-1/USTB-05-A/BL21(DE3) and pET30a(+)/USTB-05-C/BL21(DE3) were previously studied on MCs and used in this study for NOD biodegradation [30 (link),49 (link)]. E. coli TOP10 and E. coli BL21(DE3) were purchased from Sangon Biotech (Shanghai, China). The recombinant and host strains were cultured in Luria–Bertani (LB) medium [41 (link)] on a shaker at 200 rpm at an appropriate temperature. The vectors pGEX-4T-1 and pET30a(+), restriction enzymes BamHI, XhoI, SacI and NotI, the plasmid mini-prep kit and the polymerase chain reaction (PCR) kit were obtained from Sangon Biotech (Shanghai, China). Standard NOD was purchased from Enzo Science Inc., Farmingdale, NY, USA. All other chemicals were analytical grade.