生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2014年
4期
451-454
,共4页
冀全博%徐小洁%张强%康磊%李玲%黄蓉%周丽英%王荣福%王岩%叶棋浓
冀全博%徐小潔%張彊%康磊%李玲%黃蓉%週麗英%王榮福%王巖%葉棋濃
기전박%서소길%장강%강뢰%리령%황용%주려영%왕영복%왕암%협기농
人1型酪蛋白激酶%真核表达%细胞定位
人1型酪蛋白激酶%真覈錶達%細胞定位
인1형락단백격매%진핵표체%세포정위
casein kinase 1%eukaryotic expression%cellular localization
目的：构建带FLAG标签的人1型酪蛋白激酶（CK1）基因的真核表达载体，获得其表达产物，并研究该激酶在骨肉瘤U2OS、乳腺癌ZR-75-1、肝癌HepG2等多种肿瘤细胞中的表达及定位情况。方法：应用PCR技术从人乳腺文库中扩增人CK1基因的全长编码区，将其克隆到带FLAG标签的pCMV-Tag2B载体中；将重组质粒转染骨肉瘤U2OS细胞、乳腺癌ZR-75-1细胞、肝癌HepG2细胞，以SDS-PAGE和Western印迹鉴定表达情况；细胞免疫荧光观察FLAG-CK1质粒在骨肉瘤U2OS细胞、乳腺癌ZR-75-1细胞、肝癌HepG2细胞中的细胞定位。结果：双酶切和测序结果显示FLAG-CK1真核表达质粒构建成功；SDS-PAGE和Western印迹结果表明，FLAG-CK1转染骨肉瘤U2OS细胞、乳腺癌ZR-75-1细胞、肝癌HepG2细胞后成功表达；细胞免疫荧光实验显示，CK1在骨肉瘤U2OS细胞、乳腺癌ZR-75-1细胞、肝癌HepG2细胞的胞核和胞质中均有分布，且胞核信号强于胞质。结论：构建了CK1的真核表达载体，且FLAG-CK1能在不同肿瘤细胞系的细胞核和细胞质中表达，为进一步研究CK1对细胞的调控奠定了实验基础。
目的：構建帶FLAG標籤的人1型酪蛋白激酶（CK1）基因的真覈錶達載體，穫得其錶達產物，併研究該激酶在骨肉瘤U2OS、乳腺癌ZR-75-1、肝癌HepG2等多種腫瘤細胞中的錶達及定位情況。方法：應用PCR技術從人乳腺文庫中擴增人CK1基因的全長編碼區，將其剋隆到帶FLAG標籤的pCMV-Tag2B載體中；將重組質粒轉染骨肉瘤U2OS細胞、乳腺癌ZR-75-1細胞、肝癌HepG2細胞，以SDS-PAGE和Western印跡鑒定錶達情況；細胞免疫熒光觀察FLAG-CK1質粒在骨肉瘤U2OS細胞、乳腺癌ZR-75-1細胞、肝癌HepG2細胞中的細胞定位。結果：雙酶切和測序結果顯示FLAG-CK1真覈錶達質粒構建成功；SDS-PAGE和Western印跡結果錶明，FLAG-CK1轉染骨肉瘤U2OS細胞、乳腺癌ZR-75-1細胞、肝癌HepG2細胞後成功錶達；細胞免疫熒光實驗顯示，CK1在骨肉瘤U2OS細胞、乳腺癌ZR-75-1細胞、肝癌HepG2細胞的胞覈和胞質中均有分佈，且胞覈信號彊于胞質。結論：構建瞭CK1的真覈錶達載體，且FLAG-CK1能在不同腫瘤細胞繫的細胞覈和細胞質中錶達，為進一步研究CK1對細胞的調控奠定瞭實驗基礎。
목적：구건대FLAG표첨적인1형락단백격매（CK1）기인적진핵표체재체，획득기표체산물，병연구해격매재골육류U2OS、유선암ZR-75-1、간암HepG2등다충종류세포중적표체급정위정황。방법：응용PCR기술종인유선문고중확증인CK1기인적전장편마구，장기극륭도대FLAG표첨적pCMV-Tag2B재체중；장중조질립전염골육류U2OS세포、유선암ZR-75-1세포、간암HepG2세포，이SDS-PAGE화Western인적감정표체정황；세포면역형광관찰FLAG-CK1질립재골육류U2OS세포、유선암ZR-75-1세포、간암HepG2세포중적세포정위。결과：쌍매절화측서결과현시FLAG-CK1진핵표체질립구건성공；SDS-PAGE화Western인적결과표명，FLAG-CK1전염골육류U2OS세포、유선암ZR-75-1세포、간암HepG2세포후성공표체；세포면역형광실험현시，CK1재골육류U2OS세포、유선암ZR-75-1세포、간암HepG2세포적포핵화포질중균유분포，차포핵신호강우포질。결론：구건료CK1적진핵표체재체，차FLAG-CK1능재불동종류세포계적세포핵화세포질중표체，위진일보연구CK1대세포적조공전정료실험기출。
Objective: To construct the eukaryotic expression vector of human casein kinase 1(CK1) labeled with FLAG tag, obtain the expressed product, and measure its cellular localization in osteosarcoma U2OS, breast cancer ZR-75-1, hepatocellular carcinoma HepG2 cells. Methods: Human CK1 coding gene region, amplified from mammary cDNA library by PCR, was inserted into the pCMV-Tag2B vector. The resulting recombinant plas-mid pCMV-Tag2B-CK1 was transfected into U2OS, ZR-75-1 and HepG2 cells and cell lysates were examzined by SDS-PAGE and Western blotting. In addition, immunofluorescence was applied to determine the cellular local-ization. Results: The CK1 was successfully amplified by PCR and cloned into pCMV-Tag2B, as evidenced by dou-ble enzyme digestion and gene sequencing. U2OS, ZR-75-1 and HepG2 cell lines transfected by the recombinant plasmid pCMV-Tag2B-CK1 successfully expressed the protein, according to SDS-PAGE and Western blotting. Im-munofluorescence indicated that CK1 protein was expressed in all three cell lines and localized in both the nucle-us and cytoplasm, predominantly in the nucleus. Conclusion: The CK1 eukaryotic expression vector was successful-ly obtained, and the CK1 protein could be expressed in both the nucleus and cytoplasm in different tumor cell lines, which lays foundation for further research on cell regulation by CK1.
