Vip3Aa Mutant Plasmid Construction

The plasmid pET28a–Vip3Aa, which carries Vip3Aa11 (NCBI accession No. AAR36859), was used as the template to generate the expression plasmids for Vip3Aa mutants. The Vip3Aa protein structure (PDB: 6TFJ) reported by Núñez–Ramírez et al. was used as a reference for selecting amino acid mutation sites [7 (link)]. Two kinds of mutant vectors (loop replacement mutations and point mutations) were constructed in this study. Take the β4–β5 loop of Domain III in Vip3Aa as an example to expound the plasmid construction of loop replacement mutations. In brief, the loop sequence substitution (mutation of residues 364–DSI–368 to 364–AAA–368) was obtained by PCR amplification using the primers loop 4–5A–F/loop 4–5A–R. T4 ligase (TransGen Biotech, Beijing, China) was then used to ligate the phosphorylated PCR products to obtain the β4–β5 loop replacement mutant plasmid pET28a–Vip3Aa–loop 4–5A. All point mutation vectors were obtained using the Fast MultiSite Mutagenesis System (TransGen Biotech, Beijing, China). All primers used for plasmid construction in this study are listed in Table S1.