| One of the cardinal questions in tumor immunology is the identification of antigenic structures in human tumors that are recognized by host immune system. To obtain the NPC-associated antigens, a powerful new methodology was used for identifying the antigens eliciting humoral immune response, which is SEREX (serological identification of antigen by recombinant cDNA expression library).Before performing serological analysis, a high quality cDNA library which derived from human nasopharyngeal carcinoma (NPC) tissue was constructed. First, the total RNA was separated from human NPC tissue. In the next procedure, cDNA synthesis employs long-distance PCR (LD-PCR) for generating full-length cDNA, thus it was very interest for researchers who were limited by the amount of the available tissue. With the SMART (switching mechanism at 5' end of RNA transcript) technology, A modified oligo(dT) primer was used to prime the first-strand synthesis reaction ,and the SMART IV oligo served as a short ,extended template at the 5' end of the mRNA. When the RT reached the 5' end, the enzyme's terminal transferase activity added a few additional nucleotides, primarily deoxycytidine, to the 3' end, base-pairs with the deoxycytidine stretch, creating an extended template. RT then switched templates and continued replicating to the end of the oligonucleotide. The resulting full-length single-strand cDNA (ss cDNA) contained the complete 5' end of the mRNA, as well as the sequencecomplementary to the. SMART Ⅳ oligo, which then served as a universal priming site in the subsequent amplification by LD-PCR. Only those ss cDNAs having a SMART anchor sequence at the 5' end can serve as a template and can be exponentially amplified. The double-strand cDNA (ds cDNA) was digested by Sfil (IA&IB) restriction enzyme before cDNA size fractionation, ds cDNA fractionated was ligated into the λTripIEx2 vector and then was packaged in vitro.The primary library consisted of 3.64×l06 recombinants and the recombinant rate was 94%. For better preserving the cDNA library, it was amplified. As a result, the titer of the amplified cDNA library was 3.8×109 pfu/ml and the average size of the recombinant inserts is 1.2kb. The high-quality human NPC tissue cDNA library was helpful to screen NPC specific or associated antigens in followed procedures.A prerequisite for the successful application of recombinant tumor vaccines and other immunotherapeutic interventions in cancer patients is the recognition by the immune system of tumor-specific and tumor-associated antigens(i.e., of molecules that are overexpressed or specifically expressed in the tumor cells).A new methodology for identifying human tumor antigens eliciting humoral immune response-----SEREX was selected among several approaches.With this method, immunoscreening for the detection of reactive clones in the human NPC tissue cDNA library constructed immediately before was performed with autologous serum. Choosing not homologuous sera but autologuous serum could avoid homologuous serum reaction or cross-reaction, at the same time may increase the specificity of antibody recognition to some extend. XL 1-Blue transfected with recombinant, λTripIEx2 phages were plated onto Luria-Bertani agar plates. Expression of recombinant proteins was induced with IPTG.Plates were incubated at 37℃ until plaques were visible and then blotted onto nitrocellulose membranes. The membranes were blocked with 5% low-fat milk in Tris-buffered saline and incubated with a 1 : 100 dilution of the patient's serum, which had been preabsorbed with XL 1-Blue lysate. Serum antibodies binding to recombinant proteins expressed in lytic plaques were detected by incubation with an HRP-conjugated goat anti-human IgG and visualization by staining with DAB(3 , 3 ' -diaminobenzidine). Positive clones after first immunoscreening were screened secondly even more with same procedures, in order to exclude false positive clones. At last, the exact positive clones were subcloned to monoclonality and identified t...
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