CAJ | 학술논문

目的：构建可研究Polo样激酶1（Plk1）定位的HeLa细胞系。方法：用PCR方法扩增Plk1基因，定向克隆到pRex-EGFP-IRES-Hygro载体中，构建pRex-EGFP-Plk1-IRES-Hygro表达载体；利用逆转录病毒感染的方法，向HeLa细胞系中依次转染pRex-EGFP-Plk1-IRES-Hygro、pRex-Cherry-H2B-IRES-Hygro，构建Hela/GFP-Plk1/Cher-ry-H2B稳定细胞系；激光共聚焦显微镜观察Hela/GFP-Plk1/Cherry-H2B稳定细胞系在不同有丝细胞分裂期时Plk1的定位。结果：质粒酶切及测序证明pRex-EGFP-Plk1-IRES-Hygro载体构建正确；在Hela/GFP-Plk1/Cherry-H2B稳定细胞系有丝分裂中期和末期时，观察到Plk1分别定位于着丝粒和中间体上。结论：构建了Hela/GFP-Plk1/Cherry-H2B稳定细胞系，为研究Plk1在有丝分裂不同时期的调控机制提供了细胞模型。
목적：구건가연구Polo양격매1（Plk1）정위적HeLa세포계。방법：용PCR방법확증Plk1기인，정향극륭도pRex-EGFP-IRES-Hygro재체중，구건pRex-EGFP-Plk1-IRES-Hygro표체재체；이용역전록병독감염적방법，향HeLa세포계중의차전염pRex-EGFP-Plk1-IRES-Hygro、pRex-Cherry-H2B-IRES-Hygro，구건Hela/GFP-Plk1/Cher-ry-H2B은정세포계；격광공취초현미경관찰Hela/GFP-Plk1/Cherry-H2B은정세포계재불동유사세포분렬기시Plk1적정위。결과：질립매절급측서증명pRex-EGFP-Plk1-IRES-Hygro재체구건정학；재Hela/GFP-Plk1/Cherry-H2B은정세포계유사분렬중기화말기시，관찰도Plk1분별정위우착사립화중간체상。결론：구건료Hela/GFP-Plk1/Cherry-H2B은정세포계，위연구Plk1재유사분렬불동시기적조공궤제제공료세포모형。
Objective: To construct HeLa cells available for study of localization of Polo-like kinase1(Plk1). Methods: Plk1 gene was amplified by PCR and then inserted into vector pRex-EGFP-IRES-Hygro to construct pRex-EGFP-Plk1-IRES-Hygro plasmid. Through retroviral infection, the pRex-EGFP-Plk1-IRES-Hygro and pRex-Cherry-H2B-IRES-Hygro plasmids were transfected into HeLa cells in turn to construct HeLa/GFP-Plk1/Cherry-H2B stable cells. The localization of Plk1 in different mitotic phase was observed under confocal laser scanning microscope. Results: The vector pRex-EGFP-Plk1-IRES-Hygro was verified by enzyme digestion and sequencing. In constructed HeLa/GFP-Plk1/Cherry-H2B stable cells, Plk1 was observed in kinetochores and midbody respective-ly in metaphase and telophase. Conclusion: The constructed HeLa/GFP-Plk1/Cherry-H2B stable cells will benefit the further research on the regulation mechanism of Plk1 in mitosis.