Construction of cDNA library of human epithelial ovarian carcinoma tissue / 第三军医大学学报

ABSTRACT

Objective To construct a cDNA library of human epithelial ovarian carcinoma tissue for screening ovarian carcinoma specific-antigen.Methods The total RNA was separated from human epithelial ovarian carcinoma tissue.The mRNA from total RNA was isolated to synthesize the first and second strand cDNA.The ds-cDNA termini were blunted with pfu DNA polymerase.The blunted cDNAs were added EcoR Ⅰ adaptor and then digested by XhoⅠ.Small cDNA molecules(less than 400 bp) were removed through size fraction.After the cDNAs were ligated into ZAP expression vector,the ligated products were packaged in vitro and the bacteriophage particles infected the host strains XL1-Blue MRF′.Results The efficiency of the primary library was 5.5?10~(6)pfu/ml and the amplified library was 3.0?10~(11)pfu/ml with 96% clones positive.The average length of the inserted fragment was over 1 kb.Conclusion The quality of the constructed human epithelial ovarian carcinoma tissue cDNA library is excellent and helpful to screen ovarian carcinoma specific-antigen.