摘要
为克隆和研究猪细胞因子及相关基因，应用建库试剂盒成功构建了猪细胞因子cDNA表达文库。采取健康猪外周血及淋巴结，分离单个核细胞，经LPS＋PHA联合刺激不同时间后，提取总RNA。将各组样品混合，分离纯化mRNA。反转录合成cDNA第一链和第二链，与EcoRⅠ和HindⅢ接头连接。酶切和过柱分级分离后，与λSereen载体连接，经体外包装转染E．coli ER1647宿主菌，进行文库容量测定和扩增。以扩增文库的DNA为模板，利用已知基因引物克隆猪IL-2和IL-4的cDNA并进行测序。结果表明，成功构建了猪细胞因子cDNA文库，文库原始库容量为8×10^5，插入片段在300～2000bp，扩增得到特定的IL-2和IL-4基因，说明文库质量高、代表性强，为进一步从文库中筛选未知细胞因子及相关基因提供了有效的工具。
In order to clone and study the cytokine and related genes of porcine, a cDNA library of porcine cytokine was constructed. The mononuclear cells were isolated from the peripheral blood and lymph nodes of healthy porcine and were stimulated by PHA＋LPS at different time. The total RNA were extracted and the mRNA were isolated. Single-strand cDNA and double-strand cDNA were synthesized from mRNA, then ligated to directional EcoRⅠ and HindⅢ linkers, cDNA were ligated into λScreen vector after size fractionating by gel filtration and packaged in vitro. The cloning efficiency was evaluated and the length of the cDNA fragment was assayed by PCR. Using the amplified library as template DNA, 2 pair primers were designed according to the sequence of the porcine IL2 and IL4, then the gene were amplified by PCR. The results demonstrated that a cDNA library of porcine cytokine has been constructed and the IL2 and IL4 gene were amplified successfully. The tilter of the cDNA library was 8 × 10^5 pfu/mL and the length of inserts was about 300～2000 bp. It is helpful in the further study on screening novel cytokine and related genes.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2006年第4期352-355,共4页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
农业结构调整重大技术研究专项(04-10-03B)
国家高技术发展规划(2003AA241111)
