人白细胞介素10基因克隆及真核表达

孙辉; 韩福生; 周令望; 高彦辉; 张新宇; 刘立志; 王宇

人白细胞介素10基因克隆及真核表达

Study on cloning and eukaryotic expression of human interleukin 10 gene

摘要

摘要:
目的建立人白细胞介素-10(hIL-10)真核表达系统并观察其在中国仓鼠卵巢(CHO)细胞中的表达。
方法用RT-PCR方法自活化的人外周血淋巴细胞扩增hIL-10cDNA, 将其克隆至pMD18-T中, 测序正确后, 再定向插入至真核表达载体pCIneo中, 经脂质体介导转染CHO细胞, 检测其在细胞内外的表达和分泌并进行活性检测。
结果 RT-PCR扩增的hIL-10cDNA序列与GenBank中hIL-10cDNA序列一致; 构建了真核重组表达载体pCIneo-hIL-10;转染CHO细胞后可检测到具生物活性的hIL-10的转录和表达。
结论成功构建了hIL-10真核表达系统, 为今后的临床研究及基因工程药物生产打下基础。

Abstract:
Objective To construct a eukary otic expression vector containingh IL-10 cDNA and express it in CHO cells.
Methods hIL-10 cDNA, which was amplified by RT-PCR from human peripheral blood mononuclear cells, was cloned to vector pMD18-T.After the sequence was confirmed, the hIL-10 cDNA was isolated and then was inserted into eukaryotic expression vector pCIneo.The recombinant plasmid pCIneo-hIL-10 was transfected into CHO cells with Lipofectamine Plus^TM Reagent.The expression of hIL-10 in CHO cells was tested by RT-PCR and immunohistochemistry assay; the secretion of hIL-10 in medium was tested by ELASA and western blot assay.
Results The sequence of hIL-10 cloned was identical with that was published on GenBank.Mammalian recombinant expression plasmid pCIneo-hIL-10 was constructed.And the transcription and expression of hIL-10 were detected.
Conclusion The eukaryotic expression system of hIL-10 was successfully established, which might provide the basis for studying affection of IL-10 in clinics and production of gene engineering medicine.

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