Construction Of Cell Lines Transfected With Human CD133Gene And Preparation Of Novel Mouse Anti-human CD133Monoclonal Antibody

CD133is a membrane glycoprotein which has a5-transmembrane (5-TM) domains,and is specifically distributed in plasma membrane protrusions. It was frist identifiedand frist reported by Yin et al in1997. More and more research highlights the roles ofCD133as a surface marker of a variety of stem cells (especially cancer stem cells) inisolating and identifying those stem cells. A lot of studies have shown that CD133+tumor cells are highly proliferative, and own the potential of self-renewal anddifferentiation. So the research of CD133can make us deepen the understanding of thefeatures and functions of cancer stem cells, and also have an important prognostic valuein the diagnosis and treatment of cancer. The epitope of CD133may be a potentialtarget for cancer therapy.There has been a sea of study focus on the CD133molecule since it was identifiedin1997. Yet its exact biological function and its related signaling pathway remainsunknown due to the ligand of CD133was unclear and lack of effective research means.The function and relevant signaling of CD133need to be further explored. Nowcurrently available commercial CD133antibody (W6C3B1, AC133, AC141, MiltenyiBiotec) have their limitations because of their restricted binding to the glycosylatedepitope of the CD133molecule. Thus, to establish a novel anti-human CD133monoclonal antibodies would be helpful to broaden new prospects of its functionalcharacteristics. It will be not only of great significance in theoretical studies, but alsohas important potential value in clinical applications.This study aims to establish a stably expressed transgenic cell line L929/CD133, andthen use this cell line and cell line HCT116(high expression of CD133naturally) as immunogens to prepare mouse anti-human CD133monoclonal antibody. This researchhas provided a solid experiment foundation for further studies in the biologicalcharacteristic of CD133and its role in the development of cancer.Part Ⅰ Construction of cell lines transfected with human CD133geneObjective: To construct gene transfected cell lines that stably expressed the humanCD133. To provide the immunogen and CD133-positive screening cells for thepreparation of mouse anti-human CD133monoclonal antibodies.Methods: The human CD133gene was inserted into retroviral expressing vectorpEGZ-Term after double digestion. Then, together with two additional viral vectorspHIT456and pHIT60, the recombinant vector pEGZ-Term/CD133which has proved tobe correct by sequencing were transfected into the package cells293T withlipfectamineTM2000. These L929cells were infected with the cultural supernatant of thetransfected293T cells Collected after48h and72h, and these cells were futher selectedby Zeocin. Then their GFP and CD133expression were detected by Flow Cytometry inorder to obtain the gene transfected cells with high and stable expression of CD133.Results: Gene transfected lines L929/CD133which stably expressed the humanCD133were constructed successfully.Conclusion: The gene transfected cell lines which stably expressed the humanCD133were constructed successfully and lay foundation for preparing mouseanti-human CD133monoclonal antibody and studying the biological function of CD133molecule.Part Ⅱ Preparation of mouse anti-human CD133monoclonal antibodyObjective: To prepare mouse anti-human CD133monoclonal antibody.Methods: Choose the transgenic cell line L929/CD133and cell line HCT116(highexpression of CD133naturally) as immunogens to immunize6-8weeks old BALB/cmice. The spleen cells of immunized mouse were fused with myeloma cells Ag8, andthen were cultured with HAT selective medium. Hybridoma cells were screened withL929/CD133cells by Flow Cytometry. Meanwhile, L929/mock cells, which weretransfected with vector pEGZ-Term, were used as negative control. The positive cloneswere repeatedly subcloned, until one hybridoma cell line sustainably and stablysecreting specific anti-CD133mAb was obtained. Rapid qualitative test strip, indirect immunofluorescence assay, immunocytochemistry.etc were used to identify thespecificity of the obtained mAb. Using the mAb to detect the expressiom of CD133ondifferent tumor cell lines and tumor tissue by the indirect immunofluorescence.Results: After multiple screening and subcloning, a hybridoma cell linesustainably and stably secreting specifically anti-CD133mAb was obtained, whichnamed18E5. The hybridoma cells grew well after long-term storage in liquid nitrogenand culture in vitro. The isotype of mAb18E5was proved to be IgG2b with κ lightchain after rapid qualitative test strip. The competition test showed that the bindingactivity of AC133and AC141to L929/CD133could not be precluded by mAb18E5. Itindicated that the mAb18E5had a diferent binding site. The positive rate of ascitesformation was100%, and the average ascites productive was4ml each mouse.The mAb18E5was proved that it can identify the expression of CD133on human leukemia cellline U937, lung cancer cell line A549, NCI-H1299, colon cancer cell line SW480,Caco-2, HCT116and pancreatic cancer cell line Patu-8988. In addition, it also canrecognize the expression of CD133in lung and colon cancer tissue.Conclusion: A hybridoma cell line sustainably and stably secreting mouseanti-human CD133mAb was obtained successfully. The recognition epitope of mAb18E5was different from commercial anti-human CD133mAb AC133and AC141. Itcan be used to detect and analyze the expression of CD133by flow cytometry andimmunocytochemistry.