IL-22基因克隆及其真核表达质粒pcDNA3/hIL-22的构建

CLONING OF HUMAN INTERLEUKIN-22 GENE AND CONSTRUCTING OF ITS EUKARYOTIC EXPRESSION PLASMID pcDNA3/hIL-22

目的克隆人白细胞介素 2 2 (humaninterleukin 2 2 ,hIL 2 2 )基因及构建其真核表达质粒 pcDNA3 /hIL 2 2。方法从人外周血分离淋巴细胞 ,加刀豆蛋白A培养 ;提取总RNA ,进行逆转录 聚合酶链反应 (reversetranscriptase polymerasechainreaction ,RT PCR)扩增 ;获得目的基因片段后 ,与 pGEM T载体连接成 pGEM T/hIL 2 2 ,转化大肠杆菌E .coli DH5a大量繁殖后 ,提取质粒DNA ,酶切鉴定、测序。用BamH1和Xho1将目的片段从pGEM T/IL 2 2切下 ,与 pcDNA3连接成pcDNA3 /hIL 2 2 ,转化大肠杆菌E .coli DH5a大量繁殖后 ,提取质粒DNA ,酶切鉴定。结果 pGEM/hIL 2 2内插入片段序列与hIL 2 2基因序列完全一致 ;pcDNA3 /hIL 2 2经酶切鉴定与预期结果一致。 结论本实验成功完成了hIL 2 2基因克隆和pcDNA3 /hIL 2

ObjectiveTo clone the gene of human interleukine 22(hIL 22) and to construct its eukaryotic expressing plasmid pcDNA3/hIL 22. MethodsThe human lymphocytes were isolated from peripheral blood and cultured with ConA. Total RNA were extracted from the cultured lymphocytes, and reverse transcriptase polymerase chain reaction(RT PCR) was performed using a pair of primer specific to hIL 22.The fragment obtained were inserted into pGEM T plasmid to form pGEM T/hIL 22 and then E.coli DH5a was transformed. The plasmid DNA obtained was sequenced and cut with BamH1 and Xho1 to get the objective fragment. The fragment was religated with pcDNA3 that was cut with the same enzymes to form pcDNA3/hIL 22 and then E.coli DH5a was transformed. The DNA was extracted from the transformed E.coli DH5a and identified by different enzyme cutting. ResultsThe sequence of the inserted fragment in pGEM T/hIL 22 was identical to hIL 22 and the pcDNA3/hIL 22 contained the same gene fragment. ConclusionThe gene of hIL 22 was cloned and the eukaryotic expressing plasmid of pcDNA3/hIL 22 was constructed succesffuly.