Construction Of CDNA Library Of Human Epithelial Ovarian Carcinoma Tissue And Screening Of Antigenic Genes

Objective and Significance:Ovarian cancer is one of the most common malignant tumors. Onset of ovarian cancer is concealment and the prognosis of it is poor. It is crucial to search for ovarian carcinoma-associated antigens for diagnosis. We decide to construct a cDNA Library of human epithelial ovarian carcinoma tissue and screening ovarian carcinoma-associated antigens by SEREX technology.Material and Method:The total RNA was separated from human epithelial ovarian carcinoma tissue. We isolated mRNA from total RNA to synthesize the first and second cDNA. The termini of double strand cDNA were blunted with pfu DNA polymerase. The blunted cDNA was added EcoR I adaptor and then was digested by Xho I. Small cDNA molecules (<400bp) were removed through size fraction. The cDNA was ligated into the ZAP expressed vector and packaged with the host strains XL1-Blue MRF in vitro. So cDNA library of human epithelial ovarian carcinoma tissue had been constructed. Primary library can be unstable and amplified immediately. Test the efficiency and recombinant rate of the primary and amplify libraries. Then cDNA library of human epithelial ovarian carcinoma tissue had been immunoscreened by SEREX technology with autologous serum. Because autologuous serum could avoid homologuous serum reaction or cross-reaction and increase the specificity of antibody recognition to some extend.XLI-Blue was been transfected with recombinant, ZAP Express phages were plated onto LB agarplates. 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 3%BSA in Tris-buffered saline and incubated with a l:100 dilution of the patient's serum,which had been preabsorbed with XLI-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 . Positive clones were screened by first immunoscreening and secondly even more with same procedures, in order to exclude false...