The Method Of Selection Of The Specific Related Antigen Of Large Intestine Carcinoma By Proteomic And Phage Display Technology

Looking for the specificity related antigen and the high specificities, high affinity antibody in large intestine carcinoma has been the concern of scholars.Phage antibody library technique that developments in the late 1980's ,make the technique of antibody entered in the 3rd generation- the genetic engineering stage, which has advantage and characteristics envolving weakening antibody heterology with the technique of human source genetic engineering and Overcoming the difficulty that encounters in making human source mAb by hybridization neoplasm technique,extensioning Half-life ,imitating the natural antibody library and need not immune human and animal,imitate the mature process in affinity of antibody inside the body, acquire different affinity antibody etc.To Set up a antibody liberary large enough is the foundation of filteing the aim genes .Proteomics and its related techniques that developments since the 1990's provided the technique terrace of effectively separating and identifying proteins.phage antibody library technique and Proteomics and its related technique is currently the unique biology technique which is mutually matched in quantity, speed and its message flowing.owing to the advantages of the Proteomics technique in separating, purifying and identifying special proteins and the shortcomings of current tumor phage antibody library filtering method .We thought of the method of applying Proteomics and its related techniques to the sieving of the tumor antibody library by the Western- Blot immunity prints trials, which can not only discovers and identify tumor specific antigen but also sieve and make the homologous tumor specificity antibody 1. Materials and methods1.1 Materials1.1.1 Case selectionRandomly selects 5 patients in our department who received the radical surgery of large intestine adenocarcinoma( the period of C.D), shelling 3-5 lymph nodes which had transferred inside the shell membrane, cutting the intestine carcinoma tissue and normal large intestine mucous membrane tissues each 1g or so.Place them immediately to the liquid. nitrogekeep to back up 1.1.2 Carrier,Vaccine and Assistance phageAdopting bacteriophage carrier-pComb3, offered by The Scripps Research Institute.Host mold adopted escherichia –coli XLI—Blu,offering by The Scripps Research Institute.Assistance bacteriophage adoption VCSM13, offered by The Scripps Research Institute.Assistant phage adopted VCSM13,offered by the Scripps Research Institute. 1.1.3 Major reagentThe RT- PCR and PCR reagent box buy from the Promega, the the restriction endonucleases and ligase buy from the Promega.The pH gradient trunks , bulge liquid, PVDF membrane and mineral oil are all the products of Bi0Rad company in the United States.The Iodine ,a－nitrile－4－light cinnamon acid,the fragment of corticotropin 18-39( ACTH) are all products of the 51gma company`, three fluoride acetate ( TFA)s buy from the company of Fluka, acenitrile ( ACN) buy from the company of Fisher, the enzyme of trypsin buy from the company of Boehringer, two thiourea on behalf threose ketolase( DTT) for Promega company product,glycine,Tris,SDS,is from the living creature engineering company of Shanghai to import to load separately the product, low molecular standard protein is from a limit company in Shanghai 1.2 Method1.2.1 The distill of the total RNA in the transferred lymph node: Dissecting the fresh swollen lymph nodes which were got from the patient with large intestine cancer into two parts(half of them are proved to be transferred lymph nodes),and then grinding the surplus part into farina in liquid nitrogen to distill the total RNA of tissue through TRIZOL.1.2.2 Synthesis of the cDNA :using the RT- PCR ( the company of Promega)1.2.3 the enlargement of the heavy chain Fd and light chain gene through PCR:The design of the primer accords to the primer team provided by The Scripps Research Institute ,the method accords to the PCR reagent box manual.1.2.4 The establish of light chain g...