The single chain fragment variable (scFv) is a small molecule composed of the variable region of the heavy chain and the variable region of the light chain of the antibody under the connection of a peptide chain. It is the smallest functional structural unit with antibody activity. It is a recombinant antibody that has been studied more and widely at present. It does not contain the antibody constant region, but still shows high affinity with antigen. The Y2H system is used to screen scFv in eukaryotic cell yeast, The antibodies produced by it may maintain their natural folding state, which is closer to the true level in the body.
Firstly, construct the bait plasmid pGBKT7-bait in the yeast two hybrid system, transform it into yeast strain AH109, and detect its self activation in a culture medium containing nutrient deficient forms.
2. Extract total RNA from mouse spleen tissue immunized with bait protein, and construct a VH linker VL type scFv gene library using reverse transcription PCR and overlapping PCR methods. Connect it to carrier pGADT7
3. pGADT7-scFV was transformed into yeast AH109 containing bait vector, and positive clones were screened in nutrient deficient plates.
4. Successfully screened scFv fragments against bait and expressed recombinant antibodies secreted by yeast
5. Use indirect ELISA and immunocytochemistry methods to analyze the affinity characteristics of antibodies
Nanjing Ruiyuan Biology has a complete yeast expression platform and a research team that utilizes a yeast two hybrid system to obtain scFVs with good affinity against bait, providing new ideas for screening scFVs.
ScFv has the advantages of low molecular weight, strong penetration, and high specificity, and has important applications in targeted therapy, imaging diagnosis, intracellular immunity, and biological detection