
浏览全部资源
扫码关注微信
1. 重庆医科大学感染性疾病分子生物学教育部重点实验室
2. 重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆,400010
纸质出版日期:2005,
扫 描 看 全 文
[1]张林,刘杞,孙航.利用噬菌体表面展示技术构建人肝癌细胞的cDNA表达文库[J].重庆医科大学学报,2005(03):329-332+397.
ZHANG Ling, et al. Construction of a phage-displayed library with cDNA ofhuman hepatocarcinoma cell[J]. Journal of Chongqing Medical University, 2005,(3):329-332.
[1]张林,刘杞,孙航.利用噬菌体表面展示技术构建人肝癌细胞的cDNA表达文库[J].重庆医科大学学报,2005(03):329-332+397. DOI: 10.13406/j.cnki.cyxb.2005.03.006.
ZHANG Ling, et al. Construction of a phage-displayed library with cDNA ofhuman hepatocarcinoma cell[J]. Journal of Chongqing Medical University, 2005,(3):329-332. DOI: 10.13406/j.cnki.cyxb.2005.03.006.
目的:筛选对人肝再生增强因子(hALR)高反应性的肝癌细胞株
以此作为细胞来源
利用噬菌体展示技术构建出含有其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:hALR刺激不同肝癌细胞株
从中筛选出对hALR刺激有高反应性的细胞株。以PolyATtractSystem1000提取mRNA
用T7Select10-3OrientExpresscDNACloningSystem和RandomPrimer构建它的cDNA噬菌体表面展示文库;以噬菌体滴度分析和PCR评价文库质量。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。对QGY促增殖作用最强
以它作为建库细胞。对所建文库进行噬菌体滴度分析
表明原始cDNA文库含有2×107独立的重组子;随机挑取重组子进行PCR鉴定
发现插入长度在0.2~2kb
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:hALR对所检测的肝癌细胞株均有促增殖作用;QGY细胞对hALR刺激有最高的反应性;以它为基础
构建出库容量为2×107的高质量噬菌体表面展示文库。
Objective:To identify the human hepatocarcinoma cell line with the highest reactivity to hALR and to construct the phage-displayed library with its cDNA as the basis of the selection for special protein binding hALR from this library.Methods:The reactivity to hALR of HepG2
7701 and QGY was compared by 3?H-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 withT7Select10-30rient Express cDNA Cloning System and Random Primer kit.The quality of this library was evaluated by phage titer assay and PCR.Results:hALR could stimulate the proliferation of QGY
HepG2
and 7701 in dose-dependent way in vitro
but QGY showed the highest reactivity to hALR.The phage-displayed library with cDNA of QGY was successfully constructed and contained approximate 2×107 primary recombinants.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:The results show that QGY had the highest reactivity to the hALR among three strains detected.The high quality phage-displayed library is successfully constructed with cDNA of QGY.
肝再生增强因子肝癌细胞cDNA噬菌体展示文库
0
浏览量
174
下载量
3
CSCD
关联资源
相关文章
相关作者
相关机构
京公网安备11010802024621