Construction Of Human Breast Cancer Cells CDNA Phage Expression Library And Study Of The Characteristics Of Monoclonal Antibodies

Starting in the 1990s,the development of tumor immunology and the application of tumor immunodiagnostics and immunotherapy has been driven forward by the successful cloning of many types of human tumor antigen genes. Antigens have an important targeting role in the diagnosis and treatment of tumors,so searching for a target molecules with strong specificity and definite gene function has become an important research area in the field of tumor-related molecular biology.In recent years,treatment targeted at cell receptors,key genes and key elements has become possible with technological advances in molecular biology and advances in knowledge about the role of cell receptors and the level of proliferation-regulating molecules in the pathomechanism of tumors. This is referred to as "targeted treatment." These treatments,which have developed in the last 10 years,include targeted blocking of epidermal growth factor receptors(EGFR),monoclonal antibodies directed at certain definite cell characteristics,medicines directed at certain cancer genes and cytogenetic markers for cancer,anti-tumor angionenesis,anti-tumor vaccines,genetic treatment,etcetera.The occurrence of breast cancer is intimately related to hereditary factors, and with the progress of molecular biology research,tumors have come to be recognized as a type of genetic disease.In the last several years,the frequency of occurrence of breast cancer has increased,and a lot of research has focused on finding specific antigen markers.However,there has been no reports of the discovery of new breast cancer antigens(with the exception of c-erbB-2 and EGFR,which has a relatively high specificity) with a high specificity and response rate.This study carried out immunology characterization research directed at the human breast cancer monoclonal antibodies prepared by my institute's research department.In addition,a human breast cancer cell cDNA phage expression library was created.This is in order to provide an early stage of preparation for taking a step forward in utilizing monoclonal antibodies to carry out immunology screening for the cDNA library.The experiment results are as follows:1.Determine the titer and the IgG subclass of human breast cancer monoclonal antibodies.Utilize the ELISA method to determine the titer of monoclonal antibodies, and use the double diffusion method to identify the IgG subclass.The titers of the six monoclonal antibodies were determined as follows:A₆ is 1:4000,C₈ is 1:8000,H₁₁ is 1:8000,E₆ is more than 1:32000 to 10⁶,H₆ is 1:1000,C₄ is1:2000.The IgG subclass of the six monoclonal antibodies are as follows:A₆ is IgG3,C₈ is IgG1,H₁₁ is IgG3,E₆ is IgG1,H₆ is IgG1,C₄ is IgG3.2.Immunohistochemical study of anti-human breast cancer monoclonal antibodies(E₆,H₆) in various types of tumors.Utilize immunohistochemical streptomyces antibiotic protein peroxidase (SP) method to analyze normal tissues from 3 healthy persons and tumorous tissues from 48 patients,including 12 each of breast,stomach,colon and lung cancer.Normal tissues present negative results while tumorous tissues present positive stain result in the cell membrane and cell plasma.The positive expression rate of E₆ in breast cancer is 100%(12/12),the composite expression rate of E₆ in tumorous tissues is 94%(45/48),the positive expression rate of H₆ in breast cancer is 92%(11/12),and the composite expression rate of H₆ in tumorous tissues is 83%(40/48).3.Western-blotting analysis of human breast cancer monoclonal antibody-specific antigen protein.Extract the antigen protein from the tissue of the mammary cancer,using the reaction of antigen vs.antibody to do the Western blots,and using normal mouse serum as the marker.Only two bands appeared in the serum of the mouse;one is high-chain with 54kD,and another is light-chain with 27kD. However,in the protein sample only one band appeared,and the weight is 46.414kD.4.Construction and identification of human breast cancer cell cDNA libraryA cDNA library from human breast cancer cells was constructed using the SMART(switching mechanism at the 5' end of the RNA transcript) technique.The total RNA and mRNA were separated from the human breast cancer cell,and the first-stand cDNA was synthesized through reverse transcription by a modified oligo(dT) primer while the SMART oligonuleotide was utilized as a template so that the first-stand cDNA could be extended over the 5' end of mRNA.The double-stand cDNA was amplified by LD-PCR (long-distance PCR) and then digested by sfi I(I A & I B) restriction enzyme. After cDNA size fractionation through CHROMASPIN column,the double-stand cDNA was ligated into theλTriplEx2 vector and then the recombinant DNA was packaged in vitro.The un-amplified human gland carcinoma cell cDNA library consist of 1.57×10⁷ independent clones in which the percentage of recombinant clones is about 98%.The titer of the amplified cDNA library is 4.0×10⁹pfu/mL and the average inserts of the recombinants is 2.5kb.