| Lung cancer is a kind of serious disease worldwide which endangers human life and health. It has the features of insidious onset, low survival rate and high morbidity and mortality. According to pathology, lung cancer can be divided into small cell lung cancer and non-small cell lung cancer, in which the proportion of non-small cell lung cancer is up to 80% - 85%, and the morbidity of the adenocarcinoma of lung makes up 60% - 80% of non-small cell lung cancer. Up to now, the 5-year survival rate of lung cancer is still to remain 10% or a little more. Although medical workers have been committing to finding ways to improve the prognosis of lung cancer, the morbidity and mortality in China are still rising gradually. Finding effective ways for early diagnosis has been the concern for medical workers.It is worthwhile to find out the early diagnosis method of malignant cancer including lung cancer from elucidating the origin of tumor. There are many hypotheses on tumor pathogenesis, and the cancer stem cell theory is the widely accepted one. According to the theory, tumor may be a stem cell disease. Cancer stem cells are cell populations existing in tumor with stem cell properties, such as self-renewal and unlimited proliferation, generating daughter cells with identical properties to previous generation, having a variety of differentiation potential, producing different phenotype of tumor cells, resulting in tumor cell population drift, thus formed heterogeneity, which causing expanding constantly or forming new tumors nidus. Malignant phenotype features of tumor such as recurrence, invasion, metastasis, drug resistance are relevant to cancer stem cells. Scholars speculated: eradication of cancer stem cells maybe mean elimination of the tumor since cancer stem cells are the source of tumors. Thus, the membrane markers of lung cancer stem cells can be used for early diagnosis, so as to promote early diagnosis of cancer, thereby improving the prognosis.It's now known that surface markers of lung cancer such as CEA, CA-199, NSE, etc. have some contribute to the diagnosis of lung cancer, but lacking of specificity. Epithelial cell adhesion molecule (EpCAM), also known as ESA, or CD326, is a common high-level expression of the epithelial tumor-associated antigen whose expression in most human epithelial tumor is far stronger than in normal tissue, which can be used as the specific marker of diagnosis and prognosis of epithelial tumor, and it has already been used in a variety of tumors based on antibodies or vaccine-related clinical trials. Recently study indicate that EpCAM is the molecular marker of variety epithelial origin tumor stem cell. It has been identified as an additional marker of stem cells– the origin of the tumor, which make it become a more meaningful target of epithelial origin tumor for diagnosis and treatment. For example, it has been confirmed that the cells separated from lung carcinoma stem cell by means of molecular phenotype(EpCAM+CD133+)which is conformed of EpCAM highly expressed in lung carcinoma stem cell of human associated with CD133, have the features of stem cell, such as self-renewal and unlimited proliferation, by Eramo with a varity of methods. Thus, if specific antibody against EpCAM can be prepared, it will play an important role in targeted identification of lung cancer stem cells and then early diagnosis of lung cancer.Though hybridoma technology has generated a lot of valuable monoclonal antibodies, it must go through a cumbersome and time-consuming process which include animal immunity, cell fusion and clone screening. Phage-displaly technology successfully simulates the generation process of antibody in vitro and can be prepared to produce antibodies without immune, especially single-chain antibodies which have small molecular weight, Fc-free antibody molecules fragment, weak immunogenicity, better tissue penetration ability, and short half-life in vivo, etc. Therefore, in the condition of no effective ways to prepare specific antibodies targeted lung cancer stem cells, this study is to use phage-display technology to explore the new avenue to preparing humanized lung cancer antibodies and make it an effective method for the diagnosis of tumors.ObjectiveThe aim of this study is to construct mouse anti-human EpCAM single-chain antibody (ScFv) by phage display technology, and study preliminarily its immunity activity. It is expected to provide preliminary experimental work for the following construction of phage antibody library of lung adenocarcinoma and screening specific antibodies of lung adenocarcinoma stem cells.Methods1.To construct EpCAM ScFv by phage-display technology. 2 . To analyze the EpCAM ScFv sequence by randomly selected recombinant pCANTAB-5E/ EpCAM ScFv.3.The immunity activity identifying of EpCAM ScFv.Results1.The EpCAM ScFv sequence was desgined and synthesized successfully.2.The EpCAM ScFv was displayed successfully on phage surface.3.Positive reactions to EpCAM antigen were detected in 1 of 5 randomly-selected clones, with a positive rate of 20% using ELISA. The selected ScFv specifically bound to human lung adenocarcinoma A549 cells.Conclusion1.The monoclonal phage antibody EpCAM (ScFv) to aim directly at epithelial antigen was prepared successfully by the means of phage-display technology. Compared with traditional immunized animals method, this method has advantages such as being simple and time-saving.2.The EpCAM ScFv has good immunity activity, and could be an important useful tool to targeted diagnosis tumor cells of epithelial origin and even cancer stem cells. It provide good pre-experimental basis for the next construction of phage antibody library associated with lung adenocarcinoma and screening specific antibodies of lung adenocarcinoma stem cells. |
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