| BACKGROUND / OBJECTIVESColorectal cancer (CRC) has becoming one of the most common malignant tumors which threaten the human health. Nearly 30 years,the occurrence of CRC is rising. It has been demonstrated that the prognosis of CRC is related to early diagnosis and treatment. Since the development and evolution of the CRC is one kind of complicated procedures,there have been no highly special molecular pathologic changes and tumor markers currently. Special,precise,simple biological index and practical technology for early diagnosis and treatment of CRC are still lacking.During the last few years,along with the rapid progress of molecular immunology and molecular biology,the immunodiagnosis and immunotherapy for tumor have achieved great progress. Small molecular antibodies (SMA) are turning into the focus due to their small molecular weight,weak immunogenicity and stronger penetrating capability to solid tumors and are expected to become a promising type of molecular used to administrate early diagnosis and treatment of CRC. But at present their immunogenicity is still a primary obstacle in clinical application. The approach of dealing with the problem is endowing antibody humanization and making humanized antibody. The technology of phage display library,which arised at the end of the eighties last century,possesses a great advantage of making humanized antibody and screening high affinity antibody. It can effectively finish selecting and amplifying special antibody fragments and is an advanced method of making SMA. The Fab fragment,one kind of SMA,which contains heavy chain VH-CH1 (Fd fragment) andintegrated light chain(K or X),is one third of integrated antibody,and is turning into an objective studied by many scientists. It has similar activity of specifically binding antigen in contrast to integrated antibody and higher affinity in contrast to scFv. It can express at the end of COOH termination of phage coating protein and fold automatically to turn into nature stereo figure which possesses biological activity. Human can panning positive clones through specific antigens and transformate them into bacterium to amplify,which can gain high consistency antibody Fab fragments.In this project,we want to adopt phage display library technology to express Fab fragments on phage surface from positive recombination bacterium XL1-blue-pcomb3 (containing anti-CRC antibodies' Fd fragment and K chain gene),and screen the primary library using soluble multiclonal CRC antigens derived from CRC tissues and cells. Subsequently,we want to verify the special binding activity of the Fab library screened with CRC tissues and cells and try our best to screen monoclonal phage antibodies Fab fragments which can bind with human CRC antigens. The result of this study might supply a new vector for the immunodiagnosis and immunotherapy of CRC. MATERIALS AND METHODS:1. Identifying Fab gene library's volume from XLl-blue-pcomb3 After recovering XLl-blue-pcomb3,we planted plates and cultured the recombinant E. coli overnight at 37C. Following acquiring white bacteria clones,we abstracted their plasmids and verified by PCR the simultaneous recombinant frequency of Fd +K,and calculated the volume of Fab gene library.2. Abstracting antigens of CRC tissues and cells for panning The supernates were withdrawn after CRC tissues and cells had been disintegrated and centrifugalized at a high speed and a very low temperature. These supernatants,which means antigen proteins of CRC,were preserved at -70. The antigens derived from 3 cases of immunized human colorectal cancer tissues having been used to construct Fab phagedisplay library (After mixing them according same volume,we named it AgA),13 cases of un-immunized human colorectal cancer tissues (named AgB) and 3 kinds of colorectal cancer cells in vitro:Lovo,HT-29 and LS-174T (named AgC). We also abstract a few other samples antigens,including stomach cancer,esophagus cancer,other CRC,normal intestinal mucosa and stomach cancer cell,liver cancer cell in vitro...
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