Gene Cloning Of Mouse Type Ⅰ Soluble Interleukin 1 Receptor And Developing A Bioassay For Screening Its Antagonists

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-IR are identified as type I receptor (IL-IR I) and type II receptor (IL-1R II). Soluble forms of IL-IR can beproduced from proteolytic cleavage of the extracellular portion of the two type receptors on cell surface. In this study, mouse type I soluble IL-IR (sIL-1R I )has been cloned and expressed in insect cells Sf₉ with baculovirus as vector. A kind of bioassays for screening IL-IR antagonists from natural products has been developed with the recombinant sIL-1R I as targets.Total RNA from NIH/3T3 cell was extracted with guanidinium thiocyanate followed by ultra-centrifugation in cesium chloride solution. The cDNA of sIL-1R I was amplified by RT-PCR technique. The cDNA wascloned into plasmid pAcGP₆₇B and subcloned into pUC19 for DNA sequencing. Analysis result of nucleotide sequencing showed that it was the same as the IL-1R I sequence reported before. The recombinant plasmid pAI (pAcGP₆₇B-sIL-1R I ) was cotransfected with wild type AcNPV into insect cells Sf₉ with a modified method of calcium phosphate precipitation. Because of genetic exchange occured by homologous recombination in vivo, recombinant baculovirus rAcNPV was produced. The purified rAcNPV was obtained with end-point dilution-Dot blot and plaque assay. Analyses of cellular morphology, Dot blot and PCR confirmed that rAcNPV contained the sIL-IR I gene.