摘要
通过RTPCR方法扩增小鼠41BBLcDNA,以pEGFPN1为载体,构建融合蛋白41BBLGFP重组表达质粒p41BBLGFP.采用基于流体力学原理建立的裸DNA体内转染技术,从小鼠尾静脉快速(15s)注射质粒p41BBLGFP进行体内转染.荧光显微镜观察组织切片,见小鼠肝、脾、肾及肺中均有报告基因GFP表达,尤以肝细胞中荧光最强.进一步用Western印迹和免疫组织化学染色法确定肝细胞表面表达41BBL,用Hsp70H22细胞抗原肽皮下免疫小鼠,同时尾静脉注射质粒p41BBLGFP,检测血清中IL2和IFNγ的分泌.结果显示,质粒注射联合免疫组小鼠血清IL2和IFNγ的浓度分别较生理盐水对照组增加了3倍和4倍;脾细胞对H22细胞的杀伤率则由单独免疫组的45.74%±3.27%增至86.74%±9.36%.结果表明,体内(主要在肝脏)转染质粒p41BBLGFP可以成功表达,表达产物具有41BBL的生物学活性,为进一步研究体内转染41BBL用于基因治疗奠定了基础.
The recombinant expression plasmid p4-1 BBL-GFP was constructed by inserting mouse 4-1 BBL cDNA which was amplified by RT-PCR into vector pEGFP-N1. Transfection of plasmid p4-1 BBL-GFP in vivo was performed by a single intravenous injection with a shot of plasmid p4-1 BBL-GFP via mouse tail vein. This method was a hydrodynamics-based in vivo transfection procedure. The green fluorescences in liver, spleen, kidney and lung tissue slices were observed under a fluorescence microscope, especially in liver the fluorescence intensity was very strong. The expression of 4-1 BBL on hepatic cells was detected by immunohistochemistry and Western-blot. The mice were immunized subcutaneously with Hsp70-H22 peptides complex, at the same time the mice were injected via the tail vein with a shot of plasmid p4-1 BBL-GFP, then the level of cytokine IL-2 and IFN-γ in serum was measured. The result showed that the concentration of IL-2 and IFN-γ increased by 3 and 4 times respectively compared with the normal saline control group, and the eytotoxieity of spleen lymphocytes to H22 ceils was augmented from 45.74%±3.27% to 86.74%±9.36%. All the results indicated that p4-1 BBL-GFP could be expressed in vivo （especially in liver） and could exhibit some immunological activity of 4-1 BBL. It could be useful for the further gene therapy study of 4-1 BBL in vivo.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2005年第4期482-487,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家重点基础研究发展(973)计划(No.2002CB513100)资助课题~~
