Construction of transgenic silk worm that spins the cocoons containing minTBP-1 appended silk fibroin (TiBP-SF).
To introduce the RKLPDA motif (minTBP-1) into the SF heavy chain (HC), a DNA fragment coding [TS[(AGSGAG)3AS]2RKLPDA]8was synthesized (GenScript, Piscataway, NJ, USA) and inserted into theBam HI and Sal I sites of plasmid pHC-EGFP (Kojima et al., 2007). The resultant plasmid was digested with Asc I andFse I and inserted into the Asc I and Fse I sites of the plasmid pBac[3xP3-EGFPafm](Horn, Schmid, Pogoda, & Wimmer, 2002) to produce the vector plasmid pBac[HC-TiBP-3xP3EGFP] (Figure 1 b). TG silkworms were produced by injecting the vector plasmid pBac[HC-TiBP-3xP3EGFP] into the eggs of silk worms in order to produce TG silkworms, as reported previously (Tamura et al., 2000). TG silkworms were established using a non-diapausing strain (w1-pnd) and were mated with the diapausing strain (w1) to maintain TG silkworms as diapausing strains. The silkworms were reared on an artificial diet (Nosan Corporation, Yokohama, Japan) or on fresh mulberry leaves at 25 °C. Due to the poor yield and robustness of the silk of TG silkworm strains, they were backcrossed repeatedly with the commercial strains Gunma and 200, which have been shown to have high silk productivity. The resulting hybrid strains were then used for mass rearing of silkworms and collection of silk from the cocoons. The wild-type B. mori SF was used for comparison with the TG SF (TiBP-SF). The SF and TiBP-SF were dissolved by lithium bromide and desalinated using PD-10 column (GE Healthcare, Buckinghamshire, UK). Then, the samples were analyzed by 4%–12% gradient SDS-PAGE. The gel was stained with Coomassie Brilliant Blue R250 or transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was treated with an anti-His tag antibody (A190-214A, BETHYL, Montgomery, USA). The immunoreactive bands were visualized using ECL Plus (GE Healthcare, Buckinghamshire, UK) and an LAS-3000 image analyzer (Fujifilm, Tokyo, Japan).