190CT3-myc融合多肽的制备及鉴定  

Preparation and identification of 190CT3-myc fusion polypeptide

作　　者：姚云[1] 姜丹丹 孙擎[3] 刘厚奇[3] 

机构地区：[1]上海中医药大学组织胚胎学教研室,上海201203 [2]上海达安医学检测中心科研部,上海201203 [3]第二军医大学基础部组织胚胎学教研室发育生物学研究中心,上海200433

出　　处：《上海交通大学学报（医学版）》2010年第11期1348-1351,1371,共5页Journal of Shanghai Jiao tong University:Medical Science

基　　金：上海市教委优青项目(P13910);上海市教委重点学科资助项目(J50301)~~

摘　　要：目的通过基因重组的方法构建带有myc标签的190CT3功能多肽(190CT3-myc)。方法采用PCR技术对编码190CT3-myc融合多肽的cDNA序列进行扩增,克隆于T载体上,酶切及测序鉴定正确后构建带有myc标签的190CT3真核表达载体,通过脂质体转染入中国仓鼠卵巢癌(CHO)细胞中;经G418筛选,获得稳定表达目的基因的CHO细胞株,免疫荧光化学法鉴定。应用抗原抗体亲和层析柱纯化190CT3-myc融合多肽,SDS-PAGE后考马斯亮蓝染色和Westernblotting分析鉴定。结果 PCR扩增得到编码为190CT3-myc的cDNA片段,经酶切和测序鉴定正确。免疫荧光化学法鉴定显示,转染重组质粒的CHO细胞稳定表达190CT3-myc。考马斯亮蓝染色显示,融合多肽190CT3-myc表达量占细胞总蛋白的10%左右,纯度≥90%;Western blotting分析表明,纯化融合多肽的相对分子量约为17000,与目的蛋白大小相符。结论成功制备190CT3-myc重组多肽,为抗白血病分子医药研究提供了新的思路。Objective To construct a myc-tagged 190CT3 polypeptide （190CT3-myc） with genetic recombination method.Methods PCR was used to amplify cDNA sequences encoding 190CT3-myc fusion polypeptide,which were cloned to T vectors.After enzyme digestive identification of the sequences,190CT3 eukaryotic expression vector was constructed and attached with myc fusion protein tag,and was transfected into Chinese hamster ovary （CHO） cells.After screening by G418,CHO cells which stably expressed target genes were obtained,and were identified by immunofluorescence method.190CT3-myc fusion polypeptide was purified with antigen-antibody affinity chromatography column,and Coomassie brilliant blue staining and Western blotting were conducted after SDS-PAGE.Results cDNA fragment encoding 190CT3-myc was obtained by PCR amplification,and was confirmed by enzyme digestive identification of the sequences.Immunofluorescence method revealed that CHO cells transfected with recombinant plasmid stably expressed 190CT3-myc.Coomassie brilliant blue staining indicated the expression of fusion polypeptide 190CT3-myc accounted for about 10% of the total protein in cells,with the purification ≥90%.Western blotting demonstrated that the relative molecular weight of purified fusion polypeptide was 17 000,which was in accordance with target protein.Conclusion 190CT3-myc recombinant polypeptide has been successfully prepared,which may provide new ideas for the drug target research of leukemia.

关 键 词：融合多肽 真核表达载体 转染 蛋白纯化 

分 类 号：Q789[生物学—分子生物学]