The Screening And Identification Of Tumor Antigen Genes In Ovarian Cancer

Although there has been a long search for tumor antigens , majorprogress has only recently been made in the molecular definition of humantumor antigens. In 1995, Sahin developed an approach that can be used toidentify tumor antigens in human cancers. This approach is appointed asSEREX(Serological analysis of recombinant cDNA expression libraries),which defining antigens by the reaction with autologous patient sera. Manyantigens were defined by this approach , SEREX makes a great progress indefining human tumor antigens.Ovarian cancer is one of the most aggressive gynaecologicalmalignancies., the majority of epithelial ovarian cancers remainclinically undetected until patients have developed advanced-stage disease, which accounts for the five-year survival rate remains20 to 30% for epithelial ovarian cancer. Therefore , it is crucial to searchfor the tumor antigens for tumor diagnosis and immunotherapy.It is rarely reported about the screening of ovarian cancer antigenusing SEREX in the world. The phage cDNA expression library of ovariancancer in our study was constructed in our laboratory, and had been screenedwith the strategy of combination SSH (suppression subtractivehybridization) and SEREX , 180 positive clones of tumor antigen genes wereselected. In this study, 45 clones were evaluated for the frequency ofboth IgM and IgG antibody responses in heterologous serum from 34 ovariancancer patients and 34 normal donors. The positive rate of IgG antibodyof 6 clones in ovarian cancer patients was much higher that in normal donors,and so was the positive rate of IgM antibody of 4 clones.To provide useful information for the understanding ofthe pathogenesis of this disease, we then further investigated therelationship of autoantibodies to the antigens, taken as ameasure of the complexity of the immune response, with ovarian cancer.The results showed that there was no significantly association betweenthe autoantibody and its histopathologic type, and that differrentautoantibody existed at different stage of ovarian cancer , consistent with thefact that this disease was the result of a multistep process characterized by theaccumulation of successive molecular genetic and epigenetic abnormalities. Ourstudy showed that several IgM antibodies existed at the early stage of ovariancancer, which illustrated that autoantibodies against the antigens might be usefulas serum markers for the early diagnosis of ovarian cancer. Combination analysisof 10 antigens increased the sensitivity and specificity for detecting ovariancancer patients with the detection of IgG antibody and IgM antibody separately.If detected with IgG antibody and IgM antibody at the same time, the sensitivityand accuracy increased obviously with invariable specificity. These resultshighlighted the potential significance of this technology as a diagnostic toolthat will play a role in the discovery of new biomarkers for the detection ofovarian cancer.The sequencing results indicated 7 positive antigens represented 5 groups oftumor antigen genes after homology comparison analysis by compared with Genebankand SEREX database. They are:(1) one known ovarian carcinoma related gene ,whichis the target of oncogenic mutations in breast or ovarian cancer, (2) three geneswith special functional proteins, (3) two genes involved in the control of celldifferentiation, (4) one novel genes.To construct suitable vectors for the secretory expression of heterologousproteins in Pichia pastoris,a new vector pPEX9k derived from pPIC9K wassuccessfully constructed, and it was suitable for the secretory expression ofheterologous proteins. A restriction site XhoI in pPIC9K was eliminated in theend of secretion signal, another restriction site XhoI was added in multiplecloning site; Polyhistidine 6xHis was added before multiple cloning sites and thereading frame was reconstructed.The antigen gene Ocy-100 has the identity of 99% with interleukin enhancerbinding factor 3. The gene Ocy-100 was cloned into pPEX9k plasmid, The recombinantpPEX9k-100 was transformed into host yeast strain...