从人末梢血T细胞cDNA文库中筛选表达人白细胞介素4基因的克隆

ISOLATION AND IDENTIFICATION OF A HUMAN INTERLEUKIN 4 cDNA CLONE FROM A PHORBOL MYRISTATE ACETATE AND PHYTOHEMAGGLUTININ ACTIVATED HUMAN PERIPHERAL BLOOD LYMPHOCYTE cDNA LIBRARY

白细胞介素4具有多种生物功能,是调节免疫应答的重要淋巴因子之一。本文从以表达型质粒pcD为载体的人末梢血T细胞cDNA文库中,筛选出人IL-4cDNA克隆pcD-X-IL-4。经限制性内切酶酶切及Southern杂交鉴定,结果表明,pcD-X-IL-4包含有人IL-4基因的片段;转染真核COS7细胞后,在细胞培养上清测出T细胞生长因子样活性,说明所含IL-4基因可表达人IL-4完整分子。

From the library which is constructed on the eukaryotic expression vector pcD and includes thecDNAs derived from the perip heral blood lymphocytes activated by PHA and PMA, we have isolateda clone, named pcD-X-IL-4 and identified it as having the homologues with IL-4 gene, and successfullyexpressed it in COS 7 cells. The expressed human rIL-4 exhibited BCGF-like and TCGF-like activities. The probe used for cloning in situ hybridization was the EcoRI-EcoRV fragment harboring humanIL-4 cDNA. A positive clone was initially screened out from 2.4×10~5 clones. This clone was furtherpurified via two consecutive cloning and hybridization. The purified clone was identified by enzymerestriction map, Southern Blotting and hybridization analysis. The results obtained were consistent withthose expected, and thus the purified pcD-X-?IL-4 harbors human IL-4 gene, which was furtherevidenced by the expressions of human rIL-4 with biological activity in the supernatant of COS 7 cellstransfected with the isolated plasmid.