摘要
目的 构建携带人 granzym e B基因的真核表达载体 ,并将其在 He L a细胞中表达 .方法 用反转录 PCR获取人 granzyme B全长 c DNA序列 ,将其活性型序列克隆进 pc D-NA3重组表达质粒 .脂质体法转染 He L a细胞 ,间接免疫荧光检测目的蛋白表达 ,HE染色观察其对转染 He L a细胞形态的影响 .结果 成功获得了野生型 granzym e B c DNA,构建了编码活性型 granzyme B的真核表达载体 pc DNA3- G.转染He L a细胞后观察到目的蛋白表达 ,转染细胞形态发生变化 ,出现多核大细胞及固缩小细胞 .结论 活性型 granzyme B哺乳动物表达系统的建立 ,为进一步研究 granzym e
AIM To construct eukaryotic expression vector carrying human granzyme B gene and express it in HeLa cells. METHODS RT PCR was used to obtain human granzyme B cDNA. Then the recombinant expression vectorpcDNA3 G was constructed by cloning active ganzyme B into eukaryotic expression vector pcDNA3. After transfection into HeLa cells through lipofectamine 2000 TM , the expression of target gene was detected by indirect immunofluorescence assay and its effect on HeLa cells was observed by HE staining. RESULTS The obtained human granzyme B cDNA was consistent with that in GenBank. The eukaryotic expression vector pcDNA3 G encoding active granzyme B was successfully constructed and expressed in HeLa cells. Some transfected cells grew bigger than the control, often with multiple nuclei , while others exhibited condensed nucleus and cytoplasm. CONCLUSION The establishment of mammalian expression system of active granzyme B is of significance in further research for granzyme B.
出处
《第四军医大学学报》
北大核心
2002年第5期394-397,共4页
Journal of the Fourth Military Medical University
基金
国家杰出青年科学基金 (3 992 5 0 3 6)
全军医药卫生科研基金重点资助项目 (0 1Z0 90 )
