Human/murine TLR2-cross-reactive intrabody assembly and expression. A, Primary sequence of αT2ib. Shown are the coding (lower lane) and amino acid sequence (upper lane) of αT2ib including the ER signal peptide, the myc epitope and the ER retention sequence. The complementary-determining regions (CDR1-CDR3) of the variable domains of the heavy and light chain are printed in bold. The synthetic linker (shown in bold italic letters) localized between the VH and VL domains was introduced by assembly PCR (###, stop codon). B, Transient expression of the αT2ib in HEK293 cells. Comparative immunoblot analysis of αT2ib and AdVαT2ib cosmid clones 3 and 7 (/3,/7) expression in transiently transfected HEK293 cells. As positive controls, samples of E. coli periplasmic fraction of anti-VEGFR-2 scFv A7 (E. coli αVR-ab, left lane) and anti-VEGFR-2 intrabody scFv A7, αVR-ib (lane at the right side) transfected cells were applied. As negative control, HEK293 cells transfected with the vector pCMV/myc/ER without intrabody insert (right lane,-) were applied.