KSHV K15P及其突变体的慢病毒包装与稳定细胞株的筛选及其对内皮细胞增殖迁移的影响  

Lentivirus packaging of KSHV K15P and its mutants and screening of stable cell lines and their effects on proliferation and migration of endothelial cells

作　　者：周畅[1] 许常青[1] 许方玮 方圆[1] 陈伟[1] 文志 林康 王林定[1] Zhou Chang;Xu Changqing;Xu Fangwei(Dept of Microbiology,Anhui Medical University,Hefei 230032;Dept of Foreign Languages,University of International Relations,Beijing 100089)

机构地区：[1]安徽医科大学微生物学教研室,合肥230032 [2]国际关系学院英语系,北京100089

出　　处：《安徽医科大学学报》2019年第11期1765-1770,共6页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81271837);安徽高校自然科学研究项目(编号:KJ2012A161);安徽省自然科学基金(编号:1708085MH193)

摘　　要：目的包装包含卡波肉瘤相关疱疹病毒(KSHV)K15P及其突变体K15P(YF)基因的慢病毒颗粒,并对此基因在EA.hy926细胞内稳定表达进行研究。方法通过PCR方法扩增获得KSHV K15P、K15P(YF)基因片段,使用限制性内切酶Not I与BamH I酶切和TA克隆法构建T载体与慢病毒表达载体pHAGE-CMV-MCS-K15P-Puro、pHAGE-CMV-MCS-K15P(YF)-Puro,通过lip2000将构建好的慢病毒表达载体与两种辅助包装质粒PSPAX2和pMD2.g共转染HEK 293T细胞,48 h后收集慢病毒。慢病毒感染EA.hy926细胞并通过嘌呤霉素筛选稳定表达株,Western blot检测K15P与K15P(YF)蛋白的表达。Transwell与CCK8实验检测K15P对内皮细胞的增殖迁移影响。结果成功构建慢病毒表达载体,筛选出稳定表达K15P、K15P(YF)蛋白的EA.hy926细胞。在EA.hy926细胞Transwell迁移实验中,与空载体组和K15P突变体组(0.98±0.23)比较,K15P组(2.25±0.15)的EA.hy926细胞的迁移能力明显增强(P<0.01),三组细胞迁移量差异有统计学意义(F=113.5,P<0.01);CCK8增殖活性实验中,与空载体组和K15P(YF)组(109.23±12.58)比较,K15P组(138.69±7.47)的EA.hy926细胞的增殖能力明显增强(P<0.01),三组细胞增殖活性差异有统计学意义(F=40.78,P<0.01)。结论K15P蛋白增强了内皮细胞的增殖与迁移,而K15P(YF)突变体的促进细胞增殖迁移能力减弱,提示K15P蛋白可能在KS的发生发展中参与重要作用。Objective To package lentivirus particles containing Kaposi's sarcoma-associated herpesvirus(KSHV)K15P and its mutant K15P(YF)gene and to study its stable expression in EA.hy926 cells.Methods KSHV K15P and K15P(YF)gene fragments were amplified by PCR method.The T vector and lentivirus expression vector pHAGE-CMV-MCS-K15P-Puro were constructed by restriction endonuclease Not I and BamH I digestion and TA cloning.PHAGE-CMV-MCS K15P(YF)Puro,co-transfected the constructed lentivirus expression vector with two auxiliary packaging plasmids PSPAX2 and pMD2.g into HEK 293T cells through lipo2000,and collected lentivirus after 48 hours.The expression of K15P and K15P(YF)protein was detected by purine mycin screening and stable expression strain,Western blot in EA.hy926 cells infected with lentivirus.Transwell and CCK8 assay were used to detect the effect of K15P on proliferation and migration of endothelial cells.Results Lentivirus expression vector was successfully constructed by restriction enzyme digestion and sequencing.EA.hy926 cells infected with lentivirus could survive in culture medium containing Puromycin,indicating that K15P,K15P(YF)gene was stably expressed in cells.In EA.hy 926 cells,the proliferation activity and migration ability of K15P group were significantly higher than those of control group and K15P mutant group.Conclusion The pHAGE-CMV-MCS-K15P-Puro,pHAGE-CMV-MCS-K15P(YF)Puro lentivirus expression vector is successfully constructed,and the lentivirus granules expressing K15P,K15P(YF)gene are packaged.EA.hy926 cells expressing K15P,K15P(YF)stably are obtained.K15P protein enhanced the proliferation and migration of endothelial cells.

关 键 词：卡波肉瘤相关疱疹病毒 K15P K15P(YF) 慢病毒 EA.hy926 

分 类 号：R394.3[医药卫生—医学遗传学] R373.9[医药卫生—基础医学]