SMART技术构建人肝癌抗原基因cDNA噬菌体表达文库
肝细胞癌;噬菌体;抗原基因;cDNA文库,丁世华,杨冬华,汤绍辉,叶刚,通讯作者:,Switchmechanismat5'endofmRNAtemplatetechnologyusedtoconstructacDNAphageexpress
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丁世华, 杨冬华, 汤绍辉, 叶刚, 暨南大学附属第一医院消化科 广东省广州市 510630
广东省科技攻关项目, No. 2006B19901014
广东省名医工程研究项目, No. 2004-199
通讯作者: 杨冬华, 510630, 广东省广州市暨南大学附属第一医院消化科. thdyang@163.com
收稿日期: 2007-04-04 修回日期: 2007-08-08
Switch mechanism at 5' end of mRNA template technology used to construct a cDNA phage expression library of hepatocellular carcinoma antigens
Shi-Hua Ding, Dong-Hua Yang, Shao-Hui Tang, Gang Ye
Shi-Hua Ding, Dong-Hua Yang, Shao-Hui Tang, Gang Ye,Department of Gastroenterology, the First Affiliated Hospital of Ji’nan University, Guangzhou 510630, Guangdong Province, China
Supported by:the Key Program of Science and Technology in Guangdong Province, No. 2006B19901014 and Famous Doctor Project Research Program of Guangdong Province, No. 2004-199
Correspondence to:Dong-Hua Yang, Department of Gastroenterology, the First Affiliated Hospital of Ji’nan University, Guangzhou 510630, Guangdong Province, China. thdyang@163.com
Received:2007-04-04 Revised:2007-08-08
Abstract
AIM: To construct a cDNA phage expression library of hepatocellular carcinoma antigens.
METHODS: Total RNA was extracted from hepatocellular carcinoma cells and mRNA was purified. Single- and double-stranded cDNA were synthesized through reverse transcription polymerase chain reaction (RT-PCR) and long distance polymerase chain reaction (LD-PCR). cDNA fragments, after removing those smaller than 500 bp, were recombined to a λTripLEx2 phage vector. The recombined cDNA was packaged in vitro with Gigapack Ш Gold packing extract. A small portion of package phage was then used to infect Escherichia coli XL1-Blue for titration and determination of the percentage of recombinant clones. PCR was used to identify the size of the inserted cDNA.
RESULTS: The constructed cDNA phage expression library of hepatocellular carcinoma antigens consisted of 3.4×106 pfu/mL independent clones with a recombination rate of 98 ......
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