重组人白细胞介素10基因的克隆及其在真核细胞中的表达  

The cloning of human interleukin-10 gene and its expression in eukaryotic cells

作　　者：卢斌[1] 卢大儒[2] 赵明[1] 

机构地区：[1]第二军医大学临床医学院(南京军区南京总医院)内分泌科,江苏南京210002 [2]复旦大学生命科学院,上海200433

出　　处：《医学研究生学报》2007年第8期796-798,805,共4页Journal of Medical Postgraduates

基　　金：南京军区南京总医院科研基金资助项目(批准号:2006026)

摘　　要：目的:克隆编码人白细胞介素10(hIL-10)基因的全长cDNA,构建真核表达载体,并在中国仓鼠卵巢(CHO)细胞中进行表达。方法:应用逆转录多聚酶链反应(RT-PCR)技术,从活化的正常人外周血单个核细胞中扩增出hIL-10 cDNA,将测序正确的hIL-10 cDNA克隆至真核表达载体pcDNA3构建重组表达载体。采用磷酸钙法转染CHO细胞,用ELISA法检测hIL-10基因的表达,用单四唑(MTT)检测hIL-10蛋白的活性。结果:RT-PCR产物插入Teasy载体,经序列测定证实hIL-10基因全序列克隆成功。在CHO细胞培养上清中有hIL-10的表达,该产物能抑制淋巴细胞转化。结论:成功构建了真核表达质粒pcDNA3-hIL-10,为进一步研究hIL-10在自身免疫、移植免疫及炎症性疾病中的作用打下了基础。Objective： To clone human interleukin 10（hIL-10） gene full-length cDNA sequence and to construct its eukaryotic expression vector in Chinese hamster ovary （ CHO ） cells. Methods ： eDNA fragment encoding hIL-10 gene was amplified from human peripheral normal white blood cells by RT-PCR and confirmed by DNA sequencing. The ds-DNA was inserted into pcDNA3 vector. This recombinant expressing vector was transfected into CHO cell. The IL-10 molecules expressed were detected by ELISA and the role of inhibition of IL-10 was determined with MTT. Results ： The eDNA encoding hIL-10 was cloned by RT-PCR and inserted into T-easy vector and sequence analysis verified that the cloned fragment was hIL-10 cDNA. The IL-10 was expressed in the CHO and it blocked the lymphocyte transformation. Conclusion： The eukaryotic expression plasmid was constructed successfully, which will contribute to further studies on the role of hIL-10 in autoimmunity, transplantation immunity and inflammatory disease.

关 键 词：人白细胞介素10 克隆 基因表达 

分 类 号：Q785[生物学—分子生物学]