鼠Ⅰ型可溶性白细胞介素1受体基因在昆虫细胞的克隆和表达  

Gene Cloning and Expression of Mouse Type I Soluble Interleukin 1 Receptor in Insect Cells

作　　者：张治平[1] 李元[1] 

机构地区：[1]中国医学科学院中国协和医科大学医药生物技术研究所,北京100050

出　　处：《Acta Genetica Sinica》1999年第1期8-14,共7页

基　　金：国家自然科学基金!39670016

摘　　要：白细胞介素１（ＩＬ－１）是一种重要的细胞因子，具有广泛的生物学活性。它通过与细胞表面的白细胞介素 １受体（ＩＬ－１Ｒ）结合而起作用。以杆状病毒为载体在昆虫细胞中克隆表达了小鼠Ｉ型可溶性白细胞介素１受体（ｓＩＬ－１　ＲＩ）基因。以ＮＩＨ／３Ｔ３细胞ＲＮＡ为模板，采用ＲＴ－ＰＣＲ方法扩增得到小鼠ｓＩＬ－ＩＲＩ的ｃＤＮＡ，克隆至杆状病毒转移载体ｐＡｃＧＰ６７Ｂ，将转移重组质粒与野生病毒ＡＣＮＰＶ ＤＮＡ共转染昆虫细胞Ｓｆ９，经同源重组得到重组杆状病毒ｒＡＣＮＰＶ。应用经纯化的ｒＡｃＮＰＶ感染昆虫细胞Ｓｆ９，表达获得重组的ｓＩＬ－１ＲＩ。经对亲和层析样品的ＳＤＳ－ＰＡＧＥ分析和对ＩＬ－１β生物活性阻断作用实验证实，表达产物能够与其配基结合，并且能够分泌至细胞培养上清中。Interleukin 1(IL-1), which takes part in many physiological and pathological processes, is a kind of important cytokines. Their effects are exerted via specific IL-1 receptors (IL-1R) which are present on a wide variety of different cell types. Two types of IL-1R are identified as type I receptor (IL-1R I) and type II receptor (IL-1R II). Soluble forms of IL-1R can be produced from proteolyticcs cleavage of the extracellular portion of the two type receptors on cell surface. In this study, mouse type I soluble IL-1R (sIL-1R I) has been cloned and expressed in insect cells Sf9 with baculovirus as vector. Total RNA from NIH/ 3T3 cell was extracted with guanidinium thiocyanate followed by ultra-centrifugation in cesium chloride solution. The cDNA of sIL-1RI was amplified by RT-PCR technique. The cDNA was cloned into plasmid pAcGP67B. The recombinant plashed PAl (pAcGP67B-sIL-1RI) was cotransfected with wild type AcNPV DNA into insect cells Sf9, because of genetic exchange occured by homologous recombinahon in vivo, recombinant baculovirus rAcNPV was produced. Insect cells Sf9 were infected with the purified rAcNPV and sIL-1RI gene were expressed. SDS-PAGE analyses and Blockage assay of IL-1 biological achvity demonstrated that the recombinant sIL-1RI had biological function and could be secreted into the medium.

关 键 词：受体 白细胞介素1 可溶性受体 昆虫 克隆 表达 

分 类 号：Q789[生物学—分子生物学]