Zhang Z P, Li Y
Institute of Medicinal Biotechnology, Chinese Academy of Medical Scienes & Peking Union Medical College, Beijing.
Yi Chuan Xue Bao. 1999;26(1):8-14.
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-1RI) and type II receptor (IL-1RII). Soluble forms of IL-1R can be produced from proteolytic cleavage of the extracellular portion of the two type receptors on cell surface. In this study, mouse type I soluble IL-1R (sIL-1RI) 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 plasmid pAI (pAcGP67B-sIL-1RI) was cotransfected with wild type AcNPV DNA into insect cells Sf9, because of genetic exchange occurred by homologous recombination 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 beta biological activity demonstrated that the recombinant sIL-1RI had biological function and could be secreted into the medium.
白细胞介素1（IL-1）是一种重要的细胞因子，参与多种生理和病理过程。其作用通过存在于多种不同细胞类型上的特异性IL-1受体（IL-1R）发挥。已鉴定出两种类型的IL-1R，即I型受体（IL-1RI）和II型受体（IL-1RII）。IL-1R的可溶性形式可通过细胞表面两种类型受体细胞外部分的蛋白水解切割产生。在本研究中，小鼠I型可溶性IL-1R（sIL-1RI）已被克隆，并以杆状病毒为载体在昆虫细胞Sf9中表达。用硫氰酸胍提取NIH/3T3细胞的总RNA，然后在氯化铯溶液中进行超速离心。通过RT-PCR技术扩增sIL-1RI的cDNA。将该cDNA克隆到质粒pAcGP67B中。重组质粒pAI（pAcGP67B-sIL-1RI）与野生型AcNPV DNA共转染到昆虫细胞Sf9中，由于体内发生同源重组导致基因交换，产生了重组杆状病毒rAcNPV。用纯化的rAcNPV感染昆虫细胞Sf9，sIL-1RI基因得以表达。SDS-PAGE分析和IL-1β生物学活性阻断试验表明，重组sIL-1RI具有生物学功能，可分泌到培养基中。
