Construction And Selection Of Phage Display ScFv Library Anti-HER-2in Human Breast Cancer

Objective:To construct human phage single-chain antibody library of anti-breast cancer HER-2, and identify anti-HER-2specific single chain Fv(scFv) antibodies from the human phage display library.Methods:Total RNA was extracted from the adjacent lymphatic tissue in the patients who has the breast cancer and reversely transcribed into cDNA. A group of the primers of VH and VL variable regions were designed. By using polymerase chain reaction(PCR), the variable genes of the full set antibody were amplified and then clone these genes into the T vector library. The pCANTAB-5E vector was constructed with pCANTAB-Linker.vector. Cut the T vector library and connect into a ScFv library. The phage display library of ScFv antibody againstHERr-2of breast cancer was constructed and used to be efficiently clone into the expression vector pCANTAB-5E. Furthermore. the products were electrically transformed in the escherichia coli TG1. Then, a primary single chain (scFv) phage display library was constructed. Recombinant phages for breast cancer cells were enrichmented in the cell SKBR-3. Then, we selected high-affinity ones by using the enzyme immunosorbent assay (ELISA).The antigen binding activity of the single chain-Fv(scFv) antibody was detected by immunohistochemical technique.Ruselts:The segment of VL and VH were about375and330bp. They were linked to form scFv of about750bp. We have successfully construct the highly affinity antibody library which resistance to the breast cancer HER-2single strand.and get the scFv whose whole length is750bp. A fusion phage display library containing total of2.4X108pfu/ml was established. ELISA also confirme that the antibody is highly affinity to the HER-2antigen. The Immunohistochemical results also show the positive clones are affinity to HER-2antigen.lt also show the positive clones are affinity to the HER-2breast cancer tissue.Conlusion:Our research has successfully construct the humanized antibody library resistance to the breast cancer and screen out the phage clones who have show high specificity to the HER-2antigen,which will provide experimental basis to achieve new targeted antibody resistance to the breast cancer for the clinical treatment.Meanwhile,It provides a carrier which use HER-2as the target of the fusion protein for the anti-tumor therapy.