非洲爪蟾驱动蛋白样蛋白2靶蛋白C'末端SBP-3×Flag标记的HCT 116结直肠癌细胞模型的建立  

Establishment of A Targeting Protein for Xenopus Kinesin-like Protein 2 C' Terminal SBP-3×Flag Tagged HCT 116 Colorectal Cancer Cell Model

作　　者：黄泽彬[1] 张泽延[2] 张晓东[2] 缪时英[1] 王琳芳[1] 杜润蕾[2] 

机构地区：[1]中国医学科学院北京协和医学院基础医学研究所医学分子生物学国家重点实验室,北京100005 [2]武汉大学生命科学学院细胞与发育生物学系,武汉430072

出　　处：《中国医学科学院学报》2011年第6期624-628,共5页Acta Academiae Medicinae Sinicae

基　　金：国家重大研究计划项目(2011CB944302;2011CB944404);国家重点实验室专项基金(2060204);湖北省自然科学基金(2010CDB08704)~~

摘　　要：目的建立非洲爪蟾驱动蛋白样蛋白2靶蛋白(TPX2)C'末端SBP-3×Flag标记的HCT116结直肠癌细胞模型。方法 PCR扩增同源臂,构建TPX2的腺相关病毒打靶载体,包装病毒后打靶HCT116结直肠癌细胞,G418和PCR筛选获得含有新霉素抗性基因的阳性细胞株,最后通过Cre病毒感染去除抗性基因,并用PCR方法筛选获得TPX2C末端SBP和3×Flag内源性双标签的HCT116结直肠癌细胞株。结果筛选获得2个含有新霉素抗性基因的细胞株,随后经Cre病毒感染得到去除新霉素抗性基因的阳性细胞克隆,并经Western blot检测验证了SBP-3×Flag基因的敲入。结论成功建立了TPX2 C'末端SBP-3×Flag标记的HCT116结直肠癌细胞模型。Objective To develop a targeting protein for Xenopus kinesin-like protein 2（TPX2） C＇ terminal SBP-3×Flag-tagged HCT 116 cell model.Methods Homologous arms were amplified by polymerase chain reaction（PCR）,and then the adeno-associated virus（AAV）-targeting vector of TPX2 was constructed.HCT 116 cells were targeted after the viruses were packaged.Positive cell clones with neomycin resistance gene were obtained by G418 and PCR screening.Finally,the neomycin gene cassette was excised after the targeted clones were infected with adenovirus expressing Cre-recombinase,and the TPX2 C＇ terminal SBP and 3×Flag endogenous double-tagged HCT 116 cells were obtained by PCR screening.Results Two positive cell clones with neomycinresistance gene were obtained by PCR screening.The positive clones with neomycin resistance gene excised were obtained by Cre adenovirus infection,and the knock-in of SBP-3×Flag gene was verified by Western blot analysis.Conclusion The TPX2 C＇ terminal SBP-3×Flag tagged HCT 116 cell model was successfully established.

关 键 词：非洲爪蟾驱动蛋白样蛋白2靶蛋白 基因敲入 同源重组 HCT116 结直肠癌 

分 类 号：R735.34[医药卫生—肿瘤]