【作者】 张林；

【导师】 刘杞；

【作者基本信息】 重庆医科大学 ， 内科学， 2004， 硕士

【摘要】 目的：挑选出对人肝再生增强因子(hALR)高反应性的肝癌细胞株，并以此作为细胞来源，利用噬菌体展示技术构建出人肝癌细胞cDNA表达文库，为进一步从文库中筛选hALR相互作用蛋白奠定基础。 方法：以~3H-TdR掺入法比较hALR对不同肝癌细胞株促增殖作用的大小，从数株中鉴定出一株对hALR刺激具有高反应性的细胞株，以Promega公司PolyATtract System 1000试剂盒通过磁珠分离一步法提取肝癌细胞株的mRNA，然后利用Novagen公司的T7Select10-3 OrientExpress cDNA Cloning System,Random Primer试剂盒构建出肝癌细胞cDNA的噬菌体表面展示文库，通过噬菌体滴度分析和PCR评价cDNA文库质量。 结果：hALR对不同肝癌细胞株均有不同程度的促增殖作用，且呈明显的剂量依赖关系，尤以对QGY促增殖作用最为突出；以QGY细胞作为构建文库的细胞来源，构建出它的cDNA噬菌体展示文库；对构建的肝癌细胞cDNA表达文库进行噬菌体滴度分析，结果表明构重庆医科大学硕士研究生学位论文建出的原始cDNA文J乍中包含的独立的重组子克隆数高达2x107，具有较高的库容量;随机挑取原始cDNA文库中的重组子单克隆进行PCR鉴定，结果表明插入cDNA片断长度在0.2一Zkb之间，平均为0.6kb，能够较好地代表来源细胞中mRNA所表达的几乎所有信息。 结论:QGY细胞较7701和HePGZ细胞对hALR刺激有更高的反应性;以QGY细胞为基础，构建出库容量为2x107的高质量肝癌细胞cDNA的噬菌体表面展示文库。更多还原

【Abstract】 Objective: Identify the human hepatocarcinoma cell strain with the highest reactivity to hALR and construct the phage-displayed library with its cDNA as the basis of the selection of special protein binding hALR from this library.Methods: The reactivity to hALR of HepG2, 7701 and QGY was compared by 3H-TdR methods in vitro. The mRNA of QGY with the highest reactivity to hALR was isolated with PloyATtract System 1000 kit. The phage-displayed library with cDNA fragments of QGY was constructed with T7SelectlO-3 OrientExpress cDNA Cloning System and Random Primer kit. The quality of this library was evaluated by phage titer assay and PCR.Results: hALR’s different effect on the proliferation of QGY, HepG2, and 7701 was well demonstrated. And the dose-dependent relationshiop between hALR and the proliferation of three cell strains was manifested. Particularly, it was found that QGY exerted the highest reactivity to hALR and followed the surest dose-dependent way in vitro. The phage-displayed library with cDNA of QGY was successfully constructed. According to the result of phage titer assay, approximate 2xl07 primary recombinants were measured in the cDNA library. It was indicated by PCR that the size of the cDNA inserts was from 0.2 to 2kb, the average 0.6kb. Thorough information about mRNA of QGY cells could be soundly represented by this library.Conclusion: It was showed that QGY had the highest reactivity to the hALR among three strains detected. The highly quality phage-displayed library with cDNA of QGY was successfully constructed.更多还原