Construction Humanize Phage Antibody Library And Screening Antibody Binding To Breast Cancer

Breast cancer is one of the most common malignant tumors of women.There are more than one point two million women were happened in breast cancer and five hundred thousand women died of this disease every year.The incidence rate in malignant tumors is seven to ten percent in fourth in the world. The incidence rate in our country is lower than that in westen country, but it display a ascensus tendency in recensy years. Now,general diagnosis methods such as hypersound and X-rays are difficile to diagnose earlier period breast cancer exactly and specificity. The same time, combined therapys that depended on operation radiotherapy and chemotherapy are confined because of side effects. So, many scholars wanted to make breakthrough in the area of antibody diagnosis and treatment .During the past ten years, cancer biotherapy has become an important therapeutic model used extensively in clinical and experimental fields. But, neither the multiclone antibody nor the monoclone antibody are fit for the diagnonsis and trementment in clinic, because of low specificity and human anti-murine antibody response(HAMA). Herceptin was Licensed to cure advanced stage breast cancer in 1998,which bring a new chanse of antibody diagnosis and treatment. However,people discovered only thirty percent breast cancer with Her2/neu masculine were efficacious to Herceptin in clinic. Micheu Presumed the too large molecular weight of monoclone antibody was one of the most important cause.Smith introduced the concept of phage display in 1985 and Huse constructed the first phage antibody library in 1990. They bring a new way of the diagnosis and treatment of tumour. Scfv was thought of one breakthrough in the diagnosis and treatment of tumour,because they have many graces such as light molecular weight and good penetrativity.at present,many phage antibody librarys reported were murine.To overcome the problem of human anti-murine antibody response and the low penetrance of intact antibody in targeted therapy, we constructed a human single-chain antibody library using phage display techniques from lymph nodes of breast cancer patients and screened this library with MCF-7 cell line.Objective: To construct human phage single-chain antibody library associated with breast cancer and to screen the specific ScFv in order to supply the new carrier for the immunoscintigraphy and immunotherapy of breast cancer.Methods and Results:Construction of phage antibody library:1. Generation of Human ScFv Antibody Gene Repertoires: Tumor adjacent lymphatic tissue of breast cancer patients were used as the B cells source, the total RNA of these B cells was extracted and prepared as the template of RT-PCR. First, the V_H and V_L fragments were first amplified from the cDNA. Second, the V_H-linker and V_L-linker were amplified from the VH and V_L fragments. Last, SOE-PCR was used to connect the V_H-linker and V_L-linker,and the Sfi I and Not I restriction site were inlet in it.PCR products were purified from gel we can get the ScFv. The results showed: The total RNA of these B cells has two bands 28s and 18s in agar gel electrophoresis. VH fragment is about 350bp, VL fragment is about 300bp and ScFv is about 750bp.2. Ligation of ScFv Gene Repertoire into pCANTAB-5E High E.coli TG1 cells with high density were prepeared, the electro competence is 6.5×10⁷cfu/μg pUC18 DNA. The gel purified ScFv gene repertoires are overdigested by Sfi I and Not I separately. The ligation mixes of ScFv and pCANTAB-5E are transformed into electro competence E.coli TG1. 4.2×10⁷cfu/μg ampicillin resistant bacteria colonies grow after overnight culture. Random digestion reaction showed that the positive insert ratio was 79% (19/24). Sequence showed that variable domain of VH and VL were correctly inserted into phagemid vector in frame.Screening of phage antibody library:1. Panning the phage antibody library using human fibroblast and breast cancer cell line MCF-7:The phage antibody library was panned with Fibroblast and breast cancer cell MCF-7 in suspension. After five rounds of panning, we gain a 4.2×10⁷ phage antibody library.2. ELISA was performed to determine the specificity of the phage antibody:we used blank human fibroblast esophageal cancer cell line Eca109 and breast cancer cell line MCF-7 to ELISA. 3/12 colones have a more than twice fold OD₄₀₅ value over control.