卡波氏肉瘤病毒K9基因重组慢病毒表达载体的构建及其编码蛋白功能初探  

Construction of recombinant lentiviral expression vector carrying K9 of Kaposi's sarcoma-associated herpesvirus and preliminary exploration of K9 encoded protein

作　　者：尚元翠 卢春[1] 秦娣[1] SHANG Yuan-cui LU Chun QIN Di(Department of Microbiology, Nanjing Medical University, Nanjing Jiangsu 211166, China)

机构地区：[1]南京医科大学病原生物学系,江苏南京211166

出　　处：《江苏大学学报（医学版）》2016年第6期461-465,共5页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(81571984)

摘　　要：目的:构建卡波氏肉瘤病毒(Kaposi's sarcoma-associated herpesvirus,KSHV)K9基因的重组慢病毒载体,并探究K9基因编码的病毒干扰素调节因子1(viral IFN regulatory factor 1,v IRF1)对人脐静脉血管内皮细胞(HUVECs)增殖的影响。方法:根据K9基因的核酸序列设计聚合酶链式反应(PCR)的上下游引物。以实验室已有的真核表达载体p CI-K9-Flag为模板,PCR扩增K9基因序列。PCR产物经限制性内切酶双酶切后插入慢病毒载体p HAGE-CMVMCS-Izs-Green(简称p HAGE)中,构建重组慢病毒质粒p HAGE-K9。将p HAGE-K9质粒与包膜质粒p MD2.G、包装质粒ps PAX2共同转染人胚肾上皮细胞(293 T细胞),包装K9基因的重组慢病毒。采用病毒梯度稀释法测定病毒滴度。慢病毒感染HUVECs后,观察绿色荧光蛋白(GFP)的表达,蛋白质印迹法检测HUVECs中K9编码的v IRF1蛋白的表达。采用流式分选技术获得稳定表达v IRF1蛋白的HUVECs。运用细胞增殖实验检测v IRF1对HUVECs增殖的影响。结果:核酸序列测定结果表明,重组慢病毒质粒p HAGE-K9构建成功。蛋白质印迹结果显示,K9基因重组慢病毒感染HUVECs后其编码蛋白成功表达。细胞增殖实验结果表明,稳定表达v IRF1的HUVECs增殖能力显著强于对照组。结论:KSHV K9基因编码的v IRF1能够促进HUVECs的增殖。Objective：To construct the recombinant plasmid carrying K9 gene of Kaposi′s sarcoma-as-sociated herpesvirus（KSHV）,and to explore the effect of K9 encoded protein vIRF1 on the proliferation of human umbilical vein endothelial cells（HUVECs）.Methods：According to the sequence of K9,forward and reverse PCR primers with XhoⅠ and BamHⅠrestriction endonucleases sites were designed.Then the expression plasmid pCI-K9-Flag was used as template to amplify K9.Subsequently,PCR product digested by restriction endonucleases was inserted into pHAGE-CMV-MCS-Izs-Green vector （abbreviated as pHAGE）to construct recombinant plasmid pHAGE-K9.Then pHAGE-K9,package plasmid psPAX2 and envelope plasmid pMD2.G were co-transfected into the 293 T cells to produce lentivirus.The viral titer was determined by gradient dilution.Next,pHAGE-K9 lentivirus infected HUVECs was observed and the expression of vIRF1 protein was detected by Western blotting.Cells expressing vIRF1 were sorted by flow cytometry.Finally,the effect of vIRF1 on the proliferation of HUVECs was measured by CCK-8 assay. Results：Based on sequencing of recombinant plasmid corresponded with K9 gene,it was validated that recombinant plasmid was successful constructed.Western blotting showed lentivirus-infected HUVECs＆nbsp;expressed vIRF1 successfully.The result of CCK-8 showed that compared with control group,proliferation of HUVECs stablely expressing vIRF1 was significantly increased （P 〈0.01,n =5）.Conclusion：KSHV K9 encoded protein,vIRF1,enhanced the proliferation of HUVECs.

关 键 词：卡波氏肉瘤病毒 K9 基因 病毒干扰素调节因子 1 慢病毒 人脐静脉血管内皮细胞 细胞增殖 

分 类 号：R373[医药卫生—病原生物学]