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Chinese Veterinary Science
2014
(5)
463-469
牛浮舰蛋白-2在真核细胞中的表达与鉴定
作 者：
孙东杰;赵峰;张杰
单 位：
中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点实验室国家口蹄疫参考实验室
关键词：
浮舰蛋白-2;中国仓鼠卵巢细胞;突变体;真核表达;融合PCR
摘 要：
采用基因重组法将牛浮舰蛋白-2(Flotillin-2)基因的整个开放阅读框(ORF)克隆到真核表达载体pcDNA3.1(-)/Myc-His(B),通过脂质体法将重组质粒转入中国仓鼠卵巢(CHO)细胞,利用Western-blot分析目的基因的表达情况。为确定35ku的小蛋白来源,采用融合PCR法构建突变体,分别利用标签抗体和针对浮舰蛋白-2的抗体鉴定突变体表达蛋白的特性。测序结果表明,成功构建了Flotillin-2基因的全长ORF及其突变体mFlotillin-2的重组质粒。Western-blot结果显示,Flotillin-2基因的全长ORF在CHO细胞中表达时除出现与预期大小相一致的48ku目的蛋白带外,还有1个35ku的条带。基因序列分析发现,在第430~438位核苷酸存在1个似Kozak序列(CGCATGGGC)。将ATG突变成GCT(丙氨酸)后,Western-blot结果显示,突变前后35ku的小蛋白始终存在。本研究以pcDNA3.1(-)/Myc-His(B)为载体,实现了牛Flotillin-2基因的全长ORF的真核表达,证明了35ku小蛋白并不是由于重新起始翻译导致的,而可能是浮舰蛋白-2在翻译过程中的裂解产物。
译 名：
Expression and identification of bovine flotillin-2 in eukaryotic cells
作 者：
SUN Dong-jie;ZHAO Feng;ZHANG Jie;State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Animal Virology,Ministry of Agriculture/National Foot-and-Mouth Disease Reference Laboratory/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences;
关键词：
Flotillin-2;;CHO cell;;mutant;;eukaryotic expression;;fusion PCR
摘 要：
In order to explore the expression characters of flotillin-2in eukaryotic cells,flotillin-2gene was amplified by PCR and cloned into eukaryotic expression vector pcDNA3.1(-)/Myc-His(B)to construct a recombinant expression vector Flotillin-2-pcDNA3.1(-)/Myc-His(B).The recombinant expression vector was transfected into CHO cells by lipofectamine method.The gene expression was analyzed by Western-blot.To determine the source of the small 35ku protein,the mutant was constructed through the fusion PCR.The characteristics of mutant proteins were identified by anti tag or flotillin-2antibodies.Sequencing showed that the recombinant vectors with flotillin-2ORF and its mutant were successfully constructed.Western-blot analysis revealed that there was an additional 35ku band except the interest protein band in 48ku.There was a Kozak-like sequence(CGCATGGGC)from 430to 438in flotillin-2gene.The 35 ku protein could be still detected when ATG was mutated to GCT.The results showed that the 35ku protein was not the result of the initial translation,but might be the product of flotillin-2dissociation during the translation.
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