Notch1蛋白胞内段重组慢病毒载体的构建及其在原发性渗出性淋巴瘤细胞中的功能验证

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摘要目的: 构建含Notch1胞内段（intracellular Notch1，ICN）基因的重组慢病毒载体，并检测其对Notch信号通路以及卡波氏肉瘤病毒（Kaposi′s sarcomaassociated herpesvirus，KSHV）潜伏感染的原发性渗出性淋巴瘤（primary effusion lymphoma，PEL）细胞增殖能力的影响。方法: 从真核表达质粒 pIPFlagICN中扩增出ICN基因片段，并将其插入慢病毒载体pHAGECMVMCSIzsGreen（pHAGE）质粒中以构建重组质粒pHAGEICN。在人胚肾上皮细胞293T细胞中共转染重组质粒pHAGEICN、包装质粒psPAX2和包膜质粒pMD2.G，获得表达ICN的重组慢病毒，采用病毒梯度稀释法测定病毒滴度。以ICN重组慢病毒感染PEL细胞系BCP1细胞，通过蛋白质印迹法检测ICN以及Notch信号通路下游靶蛋白的表达，采用细胞计数法检测过表达ICN对BCP1细胞增殖能力的影响。结果：质粒双酶切鉴定以及核酸测序比对结果证实重组质粒pHAGEICN构建成功。通过慢病毒三质粒包装系统成功包装携带ICN基因的重组慢病毒，并经梯度稀释法测得病毒滴度约为2×107 TU/mL。以ICN重组慢病毒感染BCP1细胞，蛋白质印迹证实ICN蛋白获得有效过表达，并且能够促进Notch信号通路下游靶蛋白Hes1的表达。细胞计数结果显示，过表达ICN能显著增强BCP1细胞的增殖能力。结论：成功构建了含ICN基因的重组慢病毒载体，重组慢病毒介导的ICN过表达能够增强Notch信号通路活性，并增强PEL细胞的增殖能力。

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关键词 ： Notch1胞内段, 慢病毒, 卡波氏肉瘤病毒, 原发性渗出性淋巴瘤, 细胞增殖

Abstract：Objective: To construct the recombinant lentivirus carrying the intracellular Notch1(ICN), and to validate its role in Notch signaling pathway and the proliferative capacity of Kaposi′s sarcomaassociated herpesvirus(KSHV)infected primary effusion lymphoma (PEL) cells. Methods: The sequences of ICN were cloned from expression plasmid pIPFlagICN and inserted into lentiviral plasmid pHAGECMVMCSIzsGreen(pHAGE) to construct the recombinant plasmid pHAGEICN, after which it was cotransfected with the package plasmid psPAX2 and the envelope plasmid pMD2.G into 293 T cells. The titer of the recombinant lentivirus expressing ICN was determined by gradient dilution. The recombinant lentivirus was transduced into BCP1 cells followed by detecting the expressions of ICN and Notch downstream effector genes by Western blotting and examining the proliferation of BCP1 cells by cell counting. Results: The results of enzyme digestion and DNA sequencing indicated that the recombinant plasmid pHAGEICN was successfully constructed. The titer of the recombinant lentivirus was about 2×107 TU/mL. By Western blotting, activated Notch signaling was found in ICN overexpressed BCP1 cells.The result from cell counting showed that overexpression of ICN could promote the proliferation of BCP1 cells. Conclusion: Recombinant lentiviral vector containing ICN has been successfully constructed, which can activate Notch signaling pathway and regulate the proliferation of PEL cells.

收稿日期: 2018-01-23

通讯作者: 卢春（通讯作者），教授，博士生导师，E-mail:clu@njmu.edu.cn

作者简介: 赵润然（1994—），女，硕士研究生

引用本文:

赵润然，沈忱悠，严沁，卢春. Notch1蛋白胞内段重组慢病毒载体的构建及其在原发性渗出性淋巴瘤细胞中的功能验证[J]. 江苏大学学报:医学版, 2018, 28(02): 121-125. ZHAO Run-ran, SHEN Chen-you, YAN Qin, LU Chun. Construction of recombinant expression lentivirus vector carrying intracellular Notch1 and its function validation in primary effusion lymphoma cells . Journal of Jiangsu University(Medicine Edition), 2018, 28(02): 121-125.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2018/V28/I02/121

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