摘要
目的 通过体外表达rhIL 12 ,以供研究其在体内外的生物学效应。方法 外周血单个核细胞 (PBMC)、粘附细胞和人口腔表皮样癌细胞KB分别经SAC(含A蛋白的金黄色葡萄球菌CowanⅠ菌株 )、IFN γ +LPS和PDBu(12 ,13 二丁酸佛波酯 )刺激 ,以GIT(异硫氰酸胍 )一步法提取细胞总RNA ,经RT PCR技术获取IL 12P40和P35cDNA ,将两条基因分别插入双启动子杆状病毒转染载体pAcUW5 1的多角子启动子和P10启动子下游 ,构建真核表达载体pAcUW5 1/IL 12 ,与AcNPV共转染昆虫细胞Sf9,获取表达IL 12的细胞株 ,表达产物分别经SDS PAGE ,Westernblot,ELISA和生物学活性鉴定。结果 rhIL 12产物的相对分子质量 (Mr)经SDS PAGE和Westernblot鉴定为 6 7× 10 3 ,表达水平为15 4～ 15 7μg/ 10 6细胞。MTT法检测IL 12表达产物促进NK细胞杀伤K5 6 2细胞的能力比对照可提高 6 0 % ,刺激PHA激活的PBMC增殖的能力最高可达到 5 8%。结论 成功构建了双启动子杆状病毒表达载体pAcUW5 1/IL 12 ,获得了共表达IL 12P40和P35亚单位的昆虫细胞株。
Objective Interleukin 12 (IL 12) is a heterodimeric cytokine composed of two covalently linked chains, P40 and P35. It is necessary to express recombinant human IL 12 for studying the biological effects of this cytokine in vivo and in vitro. Methods Peripheral blood mononuclear cells (PBMC), adherent cells and KB cell line were treated with Staphylococcus aureus CowanⅠ strain (SAC), IFN γ+LPS and PDBu, respectively. Total RNA was extracted from these cells by the guanidine isothiocyanate method. cDNA for both P40 and P35 subunits of IL 12 were cloned by RT PCR. Double promoter eukaryotic expression vectors, pAcUW51/IL 12 containing IL 12 P35 and P40 cDNA, were constructed. The vectors were used to co transfect the insect cells (Sf9) with linearized polyhedrosis virus genomic DNA. The cell clones expressed the rhIL 12 products were analyzed by SDS PAGE, Western blot, ELISA and biological assays. Results The molecular weight of recombinant human IL 12 products is about 67kD on SDS PAGE and Western blot in non reducing conditions. The expression level of recombinant human IL 12 was about 15.4μg 15.7μg/10 6 cells. The biological activities identified by two biological assays showed that the expressed products increased the proliferation of human PHA activated PBMC and also the human NK cell mediated cytotoxicity. Conclusion Double promoter baculovirus expression vector pAcUW51/IL 12 was successfully constructed and both P40 and P35 subunits of IL 12 were correctly coexpressed in insect cells.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2000年第6期584-587,共4页
Chinese Journal of Microbiology and Immunology
基金
山东省科委科技攻关基金资助项目!( 963 14 916)
