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目的:获得稳定表达卡波氏肉瘤病毒(Kaposi’s sarcoma-associated herpesvirus,KSHV)K1蛋白的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),探究K1蛋白对HUVECs增殖和迁移能力的影响。方法:从本实验室已构建好的含KSHV K1基因的重组真核表达质粒pCI-neo-K1中扩增出K1基因,克隆入慢病毒载体pHAGE-CMV-MCS-IZs-Green中构建重组质粒pHAGE-K1。利用脂质体将pHAGE-K1与包装质粒psPAX2和包膜质粒pMD2.G共转染293T细胞,收集培养上清经0.45μm滤膜过滤即获得慢病毒悬液。病毒感染HUVECs,Western blot检测K1蛋白的表达。通过绿色荧光蛋白进行流式分选、Western blot验证获得稳定表达K1蛋白的HUVECs。通过细胞增殖实验和细胞划痕实验检测K1蛋白对HUVECs增殖和迁移能力的影响。结果:核酸序列测定证实,克隆的K1基因全长906 bp,与GenBank中登记的K1基因100%同源。Western blot结果显示,含K1基因的重组慢病毒感染的HUVECs在约46 000处可观察到一特异性条带,与预期的K1蛋白大小一致。流式分选获得稳定表达K1蛋白的HUVECs,其增殖能力与对照组相比显著增强(P<0.01),细胞迁移能力亦明显增加(P<0.05)。结论:成功包装了含KSHV K1基因的重组慢病毒。KSHV K1蛋白能够促进HUVECs增殖和迁移。
OBJECTIVE: To obtain human umbilical vein endothelial cells (HUVECs) stably expressing K1 protein of Kaposi’s sarcoma-associated herpesvirus (KSHV) and investigate the effect of K1 protein on the proliferation and migration of HUVECs. Methods: The K1 gene was amplified from the recombinant eukaryotic expression plasmid pCI-neo-K1 containing KSHV K1 gene in our laboratory and cloned into lentiviral vector pHAGE-CMV-MCS-IZs-Green to construct a recombinant plasmid pHAGE-K1. The liposome was used to co-transfect 293T cells with pHAGE-K1 and packaging plasmid psPAX2 and envelope plasmid pMD2.G, and then the culture supernatant was collected and filtered through 0.45μm membrane to obtain lentivirus suspension. HUVECs were infected with virus and the expression of K1 protein was detected by Western blot. Flow cytometry was performed by green fluorescent protein, and HUVECs stably expressing K1 protein were obtained by Western blot. The effects of K1 protein on the proliferation and migration of HUVECs were detected by cell proliferation assay and cell scratch assay. Results: Nucleotide sequencing confirmed that the cloned K1 gene was 906 bp in length, which was 100% homologous with the K1 gene registered in GenBank. Western blot results showed that about 46 000 HUVECs infected with recombinant lentivirus containing K1 gene could be observed a specific band, consistent with the expected K1 protein size. HUVECs stably expressing K1 protein by flow cytometry were significantly enhanced (P <0.01) and cell migration ability (P <0.05) compared with the control group. Conclusion: The recombinant lentivirus containing KSHV K1 gene was successfully packaged. KSHV K1 protein promotes proliferation and migration of HUVECs.