卵巢上皮性癌相关抗原cDNA表达文库的构建

CONSTRUCTING cDNA LIBRARY OF OVARIAN CANCER

目的:构建人卵巢上皮性癌cDNA表达文库。方法:提取卵巢上皮性癌患者腹腔积液肿瘤细胞mRNA,反转录合成双链cDNA,末端补平后连接EcoRI接头,末端磷酸化,XhoI消化,将双末端黏性化的双链cDNA经过凝胶层析柱,收集大于500bp的cDNA片段,加工后与Uni-ZAP XR Vector连接,经过体外包装,感染宿主菌XL1-blueMRF′,构建cDNA文库,检测文库容量和质量。结果:cDNA表达文库原始库容为1.82×106,扩增后文库的库容为7.5×109,重组效率约为90%,插入片段大小主要集中在1.0～2.0kb。结论:成功构建了一个卵巢上皮性癌cDNA表达文库,为下一步筛选、鉴定卵巢上皮性癌相关抗原奠定了基础。

Objective： This study is to construct a cDNA expression library of epithelial ovarian cancer. Methods： Messenger RNA was extracted from mixture of epithelial ovarian cancer patients＇ ascites tumor cells by PolyATtract System 1000 kit according to the manufacturer＇s instruction. Double-stranded（ds） cDNA was synthesized by ZAP-cDNA Synthesis kit. Uneven ends of the ds cDNA were filled in with Pfu DNA polymerase. EcoRI adapters were ligated to blunt ends and the ends were phosphorylated. Products were digested with restriction enzym XhoI. The fragments smaller than 500bp were re moved by a drip column containing Sepharose CL-2B gel and the fragments longer than 500bp were precipitated and ligated to the Uni ZAP XR vector. The lambda library is packaged in vitro and is plated on the E. coli cell line XL1 Blue MRF＇. The con tent and quality of cDNA library is tested. Result： The primary cDNA library consisted of 1.82×10^6 recombinants and the recombinantion rate was 90%. The content of the amplified cDNA library was 7.5 × 10^9 pfu/ml. The majority length of the inserted cDNA fragments were from 1.0kb to 2.0kb. Conclusion： The cDNA expression library of epithelial ovarian cancer is successfully constructed for screening and identifying associ ated antigens of epithelial ovarian cancer.