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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 To express rhIL 12 in vitro for studying its biological effects both in vitro and in vivo. Methods Peripheral blood mononuclear cells (PBMCs), adherent cells and human oral epidermoid carcinoma cells KB were transfected with SAC (Staphylococcus aureus CowanⅠ strain containing protein A), IFN γ + LPS and PDBu (12, 13, Phosphatidylcholine), total RNA was extracted by one-step GIT (guanidinium thiocyanate), IL 12P40 and P35 cDNA were obtained by RT-PCR, and the two genes were inserted into the double promoter baculovirus vector pAcUW5 1 Polyclonal promoter and P10 promoter. The eukaryotic expression vector pAcUW5 1 / IL 12 was constructed and transfected into Sf9 insect cells with AcNPV to obtain the cell line expressing IL 12. The expressed products were separated by SDS PAGE, Western blot, ELISA and biological Learning activity identification. Results The relative molecular mass (Mr) of rhIL 12 product was identified by SDS PAGE and Western blot to be 67 × 10 3 and the expression level was 15 4 ~ 15 7 μg / 106 cells. The ability of NK cells to kill K5 6 2 cells detected by MTT assay was 60% higher than that of control cells, and the highest ability of stimulating proliferation of PHA-activated PBMC was 58%. Conclusion The double-promoter baculovirus expression vector pAcUW5 1 / IL 12 was successfully constructed and the insect cell lines co-expressing IL-12 P40 and P35 subunits were obtained.