| Background and ObjectiveThe morbidity and mortality of colorectal cancer (CRC), one of the most common malignant gastrointestinal tumors, were gradually increasing from the 1990s of twentith century and now are similar all over the world. The morbidity of CRC remains the fourth in that of malignant neoplasm in the world, and the same in our country. The age of CRC patients becomes younger than before. The incidence of CRC is approximately from 24 to 32 cases per one hundred thousand in the population of older than 35 years. CRC has been up to the fourth leading cause of death in cancer, and the fifth in our county. With the development of molecular biology, genetics and biotechnology, the CRC researches concerning its basis, clinics and preventions have been expanded. However, the mechanism of CRC occurrence, reoccurrence and metastasis remains unclear. Surgical resection is the main method for comprehensive CRC therapy, and chemotherapy is also important following radical resection.The mass screening in high risk population (more than 40 years old, positive family histry, or patients with ployps) could help to find CRC in the early stage. However, with a view of limited colonoscopy application, little effects caused by small CRC lesions, few clinical manifestations, and poor specificity of CEA detection, there are still lack of simple and effective methods for finding out CRC in the early stage nowadays. So it is very important of the identification of tumor markers and special or associated antigens to diagnose CRC in the early stage, treat patients by early surgery, and increase the survival rate of five years.The construction of cDNA phage expression library is essential to identification of tumor antigens. The advent of the serological identification of tumor Ags by recombinant cDNA expressing cloning (SEREX) procedure has allowed the direct molecular definition of immunogenic tumor antigens.Serveral studies showed that the method of constructing and screening cDNA expression libraries using autologous or allogeneic serum provided an effective way to find out novel tumor markers. Using fresh cancer tissues of CRC patients as materials, and A TripIExl phage as vector, human CRC cDNA expression libraries are constructed by the technique of long distance PCR (LD-PCR). After screening the libraries by SEREX and identifying the characterizations of the positive clones, we expect that a panel novel CRC tumor-associated antigens can be revealed, which will lay the foundation for the diagnosis of earlier CRC and the development of anti-cancer immunotherapies.Materials and Methods1. Synthesis of cDNA: The tumor tissues were obtained from three CRC individuals during surgical operation and frozen in liquid nitrogen. Total RNA was extracted from these tissues by the Trizol LS Reagent kit. The single-strand of cDNA was synthesized through Reverse Transcription PCR (RT-PCR), then double-strand of cDNA was amplified through Long Distance PCR (LD-PCR). After digested by proteinase K and cut by Sfi I enzyme, cDNA fragments of smaller than SOObp were removed with Chroma Spin-400 column.2. Ligation of cDNA and λ TriplEx2 phage: The remained cDNA fragments of more than SOObp were ligated to the right and left arms of dephophrylated A, TriplEx2 phage vector. The recombinant phages werepackaged in vitro with MaxPlax Packaging Extract. The packaged phages infected E.coli XL 1-blue. The human colorectal carcinoma cDNA phage expression libraries were constructed.3. Identification of cDNA library: The indexes were titer, recombinant ratio and cDNA fragment size. First, the packaged product should be diluted at 1:10 in lx lambda dilution buffer. A small portion of the diluted packaged product infected E.coli XL 1-blue, and the unamplified library was titered. The percentage of recombinant clones were determined by adding IPTG andX-gal into the phage and bacteria mixtures before incubation, by counting the white plaques(recombinant) and blue plaques(nonrecombinant) after incubation. After the reco...
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