摘要
从水囊引产的4～5个月孕龄胎儿肝脏中提取mRNA,合成单、双链CDNA,用离心式柱层析去除短的cDNA片段,加上EcoRI接头,与去磷酸化的λgt 11 DNA-EcoRI长、短臂连接,体外包装后感染Y1090,建成含4.05×10~5重组子的表达型人胎肝cDNA文库。白斑噬菌体DNA经酶切鉴定表明均含1kb以上大小不等的CDNA片段。
In order to clone novel cytokines of human fetal liver origin we constructed an expressing cDNA library. In brief, mRNA was extracted from liver tissue derived from a fetus of 4-5 month gestation, ss-and ds-cDNA were then synthesized successfully. After removing the short cDNA fragments less than 200bp by spin-column chromatography, EcoRI adaptors were added on the remaining cDNA and they were recombined with phosphorylated λgtll-EcoRI arms. The recombined DNAs were packaged in vitro, and used to infect Y1090 host for amplification. There were 4.1×10~5 recombinant bacteriophages as recognized by their ability to form white plaques over 3.5×10~6 dark plaques when plated on lac host in the presence of both IPTG and X-Gal. It was confirmed that all randomly selected white plaques contained inserts of cDNA. The library constructed can be used to screen interested cDNA both by oligonucleotide probe and by monoclonal antibody.
出处
《军事医学科学院院刊》
CSCD
北大核心
1992年第2期86-90,共5页
Bulletin of the Academy of Military Medical Sciences
基金
国家青年自然科学基金 青年课题基金
军事医学科学院青年基金
关键词
CDNA文库
人胎肝
cDNA library
human fetal liver
λgtll
