生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2014年
4期
455-459
,共5页
李超%祖勉%连雯雯%刘艾林%杜冠华
李超%祖勉%連雯雯%劉艾林%杜冠華
리초%조면%련문문%류애림%두관화
Cdc2-like kinase 1%真核表达%重组质粒
Cdc2-like kinase 1%真覈錶達%重組質粒
Cdc2-like kinase 1%진핵표체%중조질립
Cdc2-like kinase 1%eukaryotic expression%recombinant plasmid
目的：合成真核细胞CLK1（Cdc2-like kinase 1）编码基因，构建CLK1/pEGFP-N2真核表达载体并在真核细胞HEK293A中过表达，为CLK1的生物学功能研究奠定基础。方法：从人脐静脉血管内皮细胞中提取总RNA，采用RT-PCR技术用已知引物合成cDNA，将CLK1基因扩增后插入真核细胞表达载体pEGFP-N2，将重组质粒热转化至大肠杆菌感受态Trans 10细胞中获得重组菌株，提取质粒进行酶切鉴定及插入基因测序；将构建的重组质粒转染HEK293A细胞，用Western印迹及免疫荧光检测CLK1的表达水平，同时对其下游的磷酸化SF2/ASF蛋白进行检测。结果：构建了CLK1/pEGFP-N2真核表达载体，将其转染HEK293A细胞后24 h，CLK1蛋白表达水平最高；同时，CLK1过表达后使得下游的SF2/ASF蛋白磷酸化水平升高。结论：构建了人CLK1基因的真核细胞表达载体CLK1/pEGFP-N2，并在HEK293A细胞中过表达，其生物活性也得到了验证。本研究为外源性CLK1基因在真核细胞中过表达提供了一种途径，为CLK1的生物学功能研究奠定了基础，也可为真核细胞其他蛋白表达体系的构建提供借鉴。
目的：閤成真覈細胞CLK1（Cdc2-like kinase 1）編碼基因，構建CLK1/pEGFP-N2真覈錶達載體併在真覈細胞HEK293A中過錶達，為CLK1的生物學功能研究奠定基礎。方法：從人臍靜脈血管內皮細胞中提取總RNA，採用RT-PCR技術用已知引物閤成cDNA，將CLK1基因擴增後插入真覈細胞錶達載體pEGFP-N2，將重組質粒熱轉化至大腸桿菌感受態Trans 10細胞中穫得重組菌株，提取質粒進行酶切鑒定及插入基因測序；將構建的重組質粒轉染HEK293A細胞，用Western印跡及免疫熒光檢測CLK1的錶達水平，同時對其下遊的燐痠化SF2/ASF蛋白進行檢測。結果：構建瞭CLK1/pEGFP-N2真覈錶達載體，將其轉染HEK293A細胞後24 h，CLK1蛋白錶達水平最高；同時，CLK1過錶達後使得下遊的SF2/ASF蛋白燐痠化水平升高。結論：構建瞭人CLK1基因的真覈細胞錶達載體CLK1/pEGFP-N2，併在HEK293A細胞中過錶達，其生物活性也得到瞭驗證。本研究為外源性CLK1基因在真覈細胞中過錶達提供瞭一種途徑，為CLK1的生物學功能研究奠定瞭基礎，也可為真覈細胞其他蛋白錶達體繫的構建提供藉鑒。
목적：합성진핵세포CLK1（Cdc2-like kinase 1）편마기인，구건CLK1/pEGFP-N2진핵표체재체병재진핵세포HEK293A중과표체，위CLK1적생물학공능연구전정기출。방법：종인제정맥혈관내피세포중제취총RNA，채용RT-PCR기술용이지인물합성cDNA，장CLK1기인확증후삽입진핵세포표체재체pEGFP-N2，장중조질립열전화지대장간균감수태Trans 10세포중획득중조균주，제취질립진행매절감정급삽입기인측서；장구건적중조질립전염HEK293A세포，용Western인적급면역형광검측CLK1적표체수평，동시대기하유적린산화SF2/ASF단백진행검측。결과：구건료CLK1/pEGFP-N2진핵표체재체，장기전염HEK293A세포후24 h，CLK1단백표체수평최고；동시，CLK1과표체후사득하유적SF2/ASF단백린산화수평승고。결론：구건료인CLK1기인적진핵세포표체재체CLK1/pEGFP-N2，병재HEK293A세포중과표체，기생물활성야득도료험증。본연구위외원성CLK1기인재진핵세포중과표체제공료일충도경，위CLK1적생물학공능연구전정료기출，야가위진핵세포기타단백표체체계적구건제공차감。
Objective: To investigate synthesis of the gene sequence encoding eukaryotic Cdc2-like kinase 1 (CLK1), construction of the eukaryotic vectors CLK1/pEGFP-N2 and its over-expression in HEK293A cells. This work provides guideline for research in CLK1 biological function. Methods: Total RNA was extracted from human umbilical vein endothelial cells. The CLK1 gene was amplified by RT-PCR, and then was inserted reversely into the eukaryotic expression vector pEGFP-N2. The recombined plasmid was sequenced and transformed into E.coli Trans 10. Subsequently, the plasmid was transfected into HEK293A cells. The expression level of CLK1 was mea-sured by Western blot and immunofluorescence. Then the phosphorylation level of SF2/ASF protein was detected. Results: The CLK1/pEGFP-N2 eukaryotic over-expression vector was successfully constructed. The optimum time of highest expression level of CLK1 in HEK293A cells was 24 h. And the phosphorylation level of SF2/ASF pro-tein was increased. Conclusion: A recombinant eukaryotic expression plasmid containing CLK1 gene was success-fully constructed and expressed in HEK293A. This research provides a means for the exogenous CLK1 gene ex-pression in eukaryotic cells, and laid the foundation for the further study of CLK1 biological function. At the same time, it makes a reference for the construction of other protein expression system in eukaryotic cells.
