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转位肽/粒酶B融合蛋白基因的构建、表达及对细胞生长的抑制作用

Construction, expression and cell growth inhibition of translocating peptide/granzyme B fusion protein gene
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摘要 目的 :研究转位肽 /粒酶B融合蛋白对细胞生长的抑制作用。方法 :采用重组PCR法 ,将绿脓杆菌外毒素 (PE)部分转位肽编码序列与活性型粒酶B(GrBa)基因相融合 ,构建PEII GrBa融合蛋白基因 ,以粒酶活性中心丝氨酸突变的PEII mGrBa作为阴性对照。将所获重组基因克隆入真核表达载体pcDNA3和pIRES2 EGFP中 ,以脂质体法瞬时转染Hela等细胞系 ,通过与GFP共表达 ,用MTT比色、TUNEL及间接免疫荧光染色 ,检测PEII GrBa基因的表达对转染细胞的形态和生长的影响。结果 :表达PEII GrBa融合蛋白的细胞的细胞骨架发生异常 ,细胞生长受到抑制 ,部分细胞呈现凋亡特征。结论 :PEII Gr Ba融合蛋白的表达可抑制细胞生长。 AIM: To investigate the inhibitory effect of translocating peptide/granzyme B fusion protein on cell growth. METHODS: The translocating peptide gene of Pseudomonas exotoxin A (PE) was fused with active granzyme B gene by recombinant PCR to construct PE II-GrBa fusion protein gene. PE II-mGrBa with a mutation of serine to cystein at active center of GrB was used as negative control. The resulting PE II-GrBa and PE II-mGrBa genes were transiently transfected into mammalian cells via lipofectamine mediation. The effects of expression of PE II-GrBa gene on morphology and growth of transfected cells were detected by MTT colorimetry, TUNEL assay and indirect immunofluorescence staining. RESULTS: Transient expression of PE II-GrBa resulted in cytoskeleton abnormality, cell growth inhibition, and apoptosis in some cells. CONCLUSION: Expression of PE II-GrBa fusion protein can inhibit cell growth.
作者 赵晶 王智 许彦鸣 桂俊豪 温伟红 王成济 杨安钢
机构地区 第四军医大学生物化学与分子生物学教研室 第四军医大学免疫学教研室
出处 《细胞与分子免疫学杂志》 CAS CSCD 北大核心 2004年第4期394-397,401,共5页 Chinese Journal of Cellular and Molecular Immunology
基金 国家高技术研究发展计划 (863)项目 (No .2 0 0 1AA2 1 71 0 1 ) 国家杰出青年科学基金项目 (No.3992 50 36) 全军医药卫生科研基金重点项目资助 (No.0 1Z0 90 )
关键词 粒酶B 转位肽 生长抑制 granzyme B translocating peptide growth inhibition
分类号 Q255 [生物学—细胞生物学]
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参考文献11

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细胞与分子免疫学杂志
2004年 第4期

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