摘要
目的构建带pXJ-40-Myc标签的人肾小管间质性肾炎抗原样蛋白1(Tinagl1)真核表达载体,表达pXJ-40-Myc-Tinagl1融合蛋白,检测其对人宫颈癌细胞生长的影响。方法以人卵巢文库为模板,采用PCR技术扩增Tinagl1编码区序列,双酶切后将其插入pXJ-40-Myc载体中,构建pXJ-40-Myc重组质粒;将重组质粒与空载体分别转染人宫颈癌HeLa细胞,Western印迹鉴定目的基因表达,CCK-8法、克隆形成实验测定其对宫颈癌HeLa细胞生长的影响,划痕实验检测Tinagl1对HeLa细胞运动迁移能力的影响。结果双酶切鉴定表明,pXJ-40-Myc-Tinagl1真核表达质粒构建成功;转染HeLa细胞后融合蛋白表达,生长曲线、克隆形成及划痕实验结果表明,Tinagl1可抑制宫颈癌细胞的增殖和迁移。结论携带pXJ-40-Myc标签的人Tinagl1基因真核表达载体能在人宫颈癌HeLa细胞中表达,且能抑制该细胞的生长。该实验为进一步研究Tinagl1在肿瘤,尤其是妇科恶性肿瘤中的功能奠定了基础。
Objective To construct a eukaryotic expression vector of human tubulointerstitial nephritis antigen-like protein 1(Tinagl1)with pXJ-40-Myc tag,express the fusion protein of pXJ-40-Myc-Tinagl1,and detect its influence on the growth of human cervical cancer cells.Methods The CDS region coding sequence of Tingal1 was amplified from the human ovarian library using polymerase chain reaction(PCR)technique,and inserted into the pXJ-40-Myc vector after double enzyme digestion to construct pXJ-40-Myc recombinant plasmid.The recombinant plasmid and empty vector were transfected into HeLa cells respectively.Western blotting was used to identify the expression of the target gene.CCK-8 assay and colony formation were used to determine its effect on the growth of cervical cancer cells.Scratch test was used to detect the effect of Tinagl1 on the migration of HeLa cells.Results The result of double enzyme digestion showed that the eukaryotic expression plasmid of pXJ-40-Myc-Tinagl1 was successfully constructed.After transfection with HeLa cells,fusion protein expression,growth curves,colony formation and scratch test proved that Tinagl1 could inhibit the proliferation and migration of cervical cancer cells.Conclusion A eukaryotic expression vector carrying human Tinagl1 gene with pXJ-40-Myc tag can be expressed in human cervical cancer cells and inhibit the growth and migration of the cells.This experiment can contribute to future studies on the function of Tinagl1 in tumors,especially gynecologic malignant tumors.
作者
唐枝
高晓超
马路园
朱祥
初众
张德宇
徐小洁
闫志风
阳志军
TANG Zhi;GAO Xiao⁃chao;MA Lu⁃yuan;ZHU Xiang;CHU Zhong;ZHANG De⁃yu;XU Xiao⁃jie;YAN Zhi⁃feng;YANG Zhi⁃jun(Gynecologic Cancer Section,Cancer Hospital of Guangxi Medical University,Nanning 530021,China;Department of Allergy,the First Hospital of Hebei Medical University,Shijiazhuang 050000;Department of Medical Molecular Biology,Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Department of Gynecology and Obstetrics,the First Medical Center,General Hospital of PLA,Beijing 100853,China)
出处
《军事医学》
CAS
北大核心
2020年第7期506-509,514,共5页
Military Medical Sciences
基金
国家自然科学基金优秀青年基金项目(81822037)
国家自然科学基金面上项目(81672602,81972446)
全军后勤科研计划重点项目(BWS16J010)
作者简介
唐枝,女,硕士研究生,研究方向:妇科肿瘤;通讯作者:阳志军,E⁃mail:yzj7528@126.com;通讯作者:闫志风,E⁃mail:yanzhifeng2002@163.com;通讯作者:徐小洁,E⁃mail:miraclexxj@126.com
