CAJ | 학술논문

目的构建蛋白转导域(PTD)与扩增链亲和素( Strep)融合蛋白,用于转染常规操作中难于转染生物大分子的血液肿瘤细胞、神经细胞、干细胞等细胞系.方法采用聚合酶链反应( PCR)法引入PTD和G4S序列、扩增Strep序列；经酶切、连接构建重组质粒pAYZ-PTD-Strep,用低磷培养液AP5诱导表达融合蛋白,E-tag亲和层析柱纯化产物,Western blot鉴定产物,利用pAYZ-PTD-Strep将eGFP质粒转染到悬浮U937细胞内,流式细胞术测定融合蛋白转染效率.结果目的产物PTD-G4S-Strep融合蛋白以可溶形式表达,产量约为0.7 mg/L,经E-tag亲和层析柱纯化后纯度可达90％以上.融合蛋白PTD-Strep可成功将质粒运送至悬浮细胞U937内,转染效率高于PTD单体.结论构建的融合蛋白PTD-Strep具有将生物大分子转入血液肿瘤悬浮细胞内的活性,有望发展成为一种高效、可靠的真核细胞转染方法.
목적 구건단백전도역(PTD)여확증련친화소( Strep)융합단백,용우전염상규조작중난우전염생물대분자적혈액종류세포、신경세포、간세포등세포계.방법 채용취합매련반응( PCR)법인입PTD화G4S서렬、확증Strep서렬；경매절、련접구건중조질립pAYZ-PTD-Strep,용저린배양액AP5유도표체융합단백,E-tag친화층석주순화산물,Western blot감정산물,이용pAYZ-PTD-Strep장eGFP질립전염도현부U937세포내,류식세포술측정융합단백전염효솔.결과 목적산물PTD-G4S-Strep융합단백이가용형식표체,산량약위0.7 mg/L,경E-tag친화층석주순화후순도가체90％이상.융합단백PTD-Strep가성공장질립운송지현부세포U937내,전염효솔고우PTD단체.결론 구건적융합단백PTD-Strep구유장생물대분자전입혈액종류현부세포내적활성,유망발전성위일충고효、가고적진핵세포전염방법.
Objective To construct a fusion protein to transfect some cell lines that were difficult to be transfected such as neoplastic cells, nerve cells, stem cells. Methods PCR was performed to amplified protein transductions domain(PTD),G4S and streptavidin (Strep).Enzymatic digestion and ligation were used to construct pAYZ-PTD-Strep plasmid. Fusion protein was induced to express by AP5 medium and was isolated by E-tag affinity chromatography. Fusion protein was identified by Western blot. eGFP was trasfected into U937 cells by pAYZ-PTD-Strep. FACS was performed to detect transfection percentage. Results Fusion protein PTD-G4S-Strep was expressed as soluble protein.The concentration of fusion protein was about 0.7 mg/L,and purity was over 90 ％. The protein could carry plasmid into a suspended cell line, U937 cells. The transfection-efficiency of protein was higher than monometer PTD.Conclusion The protein PAYZ-PTD-Strep could carry macromolecules into blood tumor cells,and its biological activity may be expected to develop into a highly efficient and reliable transfection method.