N端融合TAT穿膜肽的piggyBac转座酶的原核表达及其在HEK 293细胞中的功能验证  

Prokaryotic Expression of piggyBac Transposase with N-terminal Fused Human immunodeficiency virus(HIV) Transactivator(TAT) Peptide and Verification of Its Activity in HEK 293 Cells

作　　者：胡广东[1,2] 高元鹏[1,2] 王静[1] 郑丽明[1,2] 张涌[1,2] 

机构地区：[1]西北农林科技大学动物医学院,杨凌712100 [2]西北农林科技大学农业部动物生物技术重点实验室,杨凌712100

出　　处：《农业生物技术学报》2014年第4期406-414,共9页Journal of Agricultural Biotechnology

基　　金：国家转基因重大项目专项(No.2013ZX08007-004);国家高技术研究发展计划(863计划)(No.2011AA100303)

摘　　要：为了增加piggyBac(PB)转座系统在转基因操作中安全性和减少PB转座系统的辅助质粒带来的副作用,本研究利用N端融合人免疫缺陷病毒反式激活因子(Human immunodeficiency virus transactivator,TAT)穿膜肽的原核表达PB转座酶替代辅助质粒在人肾胚细胞(HEK293细胞)介导PB转座的发生,同时建立一种由TAT介导的外源功能蛋白进入真核细胞的方法。构建了N端和C端分别融合TAT的PB转座酶原核表达载体(pET28A-Pbase 1,pET28A-Pbase 2)。利用pET28A-Pbase1载体在大肠杆菌(Escherichia coli)BL21(DE3)中成功表达出N端含有TAT的PBase重组蛋白,纯化并对其进行蛋白定量,然后以100mg/mL的浓度加入细胞培养液中,转导进入HEK 293细胞,利用免疫荧光技术观察该酶能否进入HEK293细胞核,利用热不对称PCR(Tail-PCR)进行整合位点侧翼DNA序列检测,亚甲蓝进行阳性细胞克隆染色并利用非配对t检验统计细胞克隆数。聚丙烯酰胺凝胶电泳和Western blot鉴定结果表明PBase在大肠杆菌中成功表达,经分析发现其主要以可溶蛋白形式存在。免疫荧光结果表明,N端融合TAT穿膜肽的PBase定位于细胞核,但是整合位点检测和转座效率统计结果表明,原核表达的PBase蛋白在HEK 293细胞中无法介导转座事件的发生。该转座系统能否在其他真核细胞中正常发挥作用,还需要进一步的研究。然而,本研究为由TAT穿膜肽介导的外源蛋白在真核细胞中的跨膜转运提供了一种可靠的方法。To enhance the safety of piggyBac （PB） transposon system and weaken the genotoxicity of the helper plasmid in gene transfer, we try to use PBase N-terminal fused Human immunodeficiency virus （HIV）transactivator （TAT） peptide with prokaryotic expression to replace the helper plasmid and verify its activity in human embryonic kidney 293 cells （HEK 293 cells）. Furthermore, we hope to establish an approach that TAT peptide mediates foreign proteins into eukaryotic cells. In present study, we constructed two prokaryotic expression vectors containing the plasmid pET28A-Pbase 1 to generate PBase N-terminal fused TAT peptide and the plasmid pET28A-Pbase 2 to generate PBase C-terminal fused TAT peptide. The PBase with N-terminal fused TAT peptide was successfully expressed by the plasmid pET28A-Pbase 1 in Escherichia coli BL21 （DE3） strain. Polyacrylamide gel electrophoresis （PAGE） and Western blot （WB） were used to confirm the PBase expression. After protein quantification, the PBase with 100 mg/mL in cell culture medium was transduced with the donor plasmid into HEK 293 cells. Furthermore, HEK 293 cells transfected the dual plasmid system of PB transposon were the positive control. PBase were determined by immunofluorescent staining （IF） in HEK 293 cells. Thermal asymmetric interlaced PCR （Tail-PCR） was used to detect the flanking sequences of PB integrate sites from purified genome of HEK 293 cell clones. After methylene blue staining for HEK 293 cell clones, Student’s t-test was employed to analyze the colony count. Results showed that the PBase was successfully expressed by the form of soluble protein. Simultaneously, we found that the PBase was localized in the nucleus from the result of immunofluorescent staining. Detection of flanking sequences indicated that the integration mechanism of PB was regarded as random recombination. Compared the dual plasmid system of PB, HEK 293 cell clones generated by the PBase with prokaryotic expression were significantly reduced and similar t

关 键 词：PIGGYBAC (PB) 转座子 转座酶 原核表达 人免疫缺陷病毒反式激活因子(TAT)穿膜肽 HEK 293细胞 

分 类 号：Q813.11[生物学—生物工程]