人granzyme B基因在HeLa细胞中的表达

目的 构建携带人granzyme B基因的真核表达载体，并将其在HeLa细胞中的表达。方法 用反转录PCR获取人granzyme B全长cDNA序列，将其活性型序列克隆进pcD-NA3重组表达质粒。脂质体法转染HeLa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HeLa细胞形态的影响。结果工成功获得了野生型granzyme B cDNA，构建了编码活性型granzyme B的真核表达载体pcDNA3-G。转染HeLa细胞后观察到目的的蛋白表达，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granzyme B的生物学效应奠定了基础。

Expression of human granzyme B gene in HeLa cells

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.