| The colorectal cancer (CRC) has becoming one of the most common malignant tumors, which threaten the human health. Since the development and evolution of the CRC is one kind of complicated procedure involving mult-genes, mult-factors and mult-steps, there have been no highly specific molecu-pathologic changes and tumor markers. Therefore, at CRC early stage, it is a problem to researchers that there are no special, accurate, simple and convenient biology targets and useful methods.Phage display library technique, starting emergence from 90s, has increasingly become an important tool in biotechnology, which provides a new way for antibody development. The resulting phage antibody library can be panned to select and clone rare antibodies on the basis of their binding specificities. Many domains have showed tremendous application perspective since the phage display library technique emerges. As part of the antibody fragments, Fab is one third of whole antibody mading of heavy chain VH-CH1 (Fd fragment) and integrated light chain (K or . Fab fragment is of the similar binding activity as the antibody and has some advantages, such as low molecule weight, weak heterology,strong infiltration and prokaryotic expression.At present, the antibodies that related to CRC are limited and lack specificity. So, to search for highly specific antibodies are importnt scientific and clinic issues for diagnosis and treatment of CRC. With the development of molecular biology and the applying gene chip, it is possible to find the specific antigens of CRC.In this project, by using the leading biotechnology of phage display antibody library, we constructed the naturally immunized phage display libraries expressing Fab antibodies which were derived from periphery blood lymphocytes of a patient with colorectal cancer; by using gene chip, we studied the gene expression of CRC, and found that the antigen possibly has specificity to CRC. Finaly, we panned the libraries using the antigens, which were related to CRC for selecting anti-colorectal cancer antibodies with the purpose of preparing Fab antibodies for diagnosis and therapy on CRC.METHODSConstruction of the phage display antibodies libraryAmplifying the fragments of light chain (K and and heavy (Fd) chain genes of antibodies by RT-PCR: periphery blood lymphocytes of a patient with colorectal cancer were dissociated and total RNA was extracted. After the RNA was transcripted reversely into cDNA, the fragmental groups of light chain (K and A) and heavy (Fd) chain genes were amplified with appropriate 5' and 3' primers.Cloning light chain (K and into pComb3: The Fd fragment productof PCR (isolated by agarose gel electrophoresis) was cut with an excess of the restriction enzymes Sac I /Xba I, and was ligated with Sac I /Xba I -linearized pCombS vector. The ligased DNA was transformed into XLl-Blue. After transformation, XLl-Blue samples (10) were withdrawn for plating to determine the rate of transformation. Extracting the plasmids from several random XLl-Blue monoclones to judge the recombination ratio by cutting with the restriction enzymes.Construction of antibodies Fab libraries: As mentioned above, the heavy chain Fd fragments product of PCR cloned into the pComb included light chain genes. Helper phage VCSM13 was added to XLl-Blue samples which contained Fab gene libraries. Following the superinfection, the primer Fab phage display libraries of CRC patient was constructed. Library capacity was assayed and undertaken simple identification.Screening related genes of CRC using gene chipTotal RNA was extracted from 4 well-differentiated colorectal adenocarcinomas and surrounding normal mucosa tissues.The RNA was reversely transcripted into cDNA, and labeled the probe using Cy3/Cy5.The Cy3/Cy5 labeled cDNA underwent hybridization with the gene chip. After scanning and data processing, we got the information of the CRC' s genes expression.The results of CRC gene chip verified by RT-PCR. We found that the JUNE gene was correlated with CRC.Panning of the combin...
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