上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

基于hNIS基因的辐射正反馈重组杆状病毒体系制备

郭 睿，李 彪   

  1. 上海交通大学 医学院附属瑞金医院核医学科， 上海 200025
  • 出版日期:2013-12-28 发布日期:2014-01-02
  • 通讯作者: 李 彪, 电子信箱: lb10363@rjh.com.cn。
  • 作者简介:郭 睿（1980—）， 男， 住院医师， 博士; 电子信箱: gr11734@rjh.com.cn。
  • 基金资助:

    国家自然科学基金（81101071）；上海市优秀学科带头人计划项目（11XD1403700）；上海高校青年教师培养资助计划（2011）

Preparation of recombinant baculovirus based on the human sodium iodide symporter which participates radiosensitive positive feedback system

GUO Rui, LI Biao   

  1. Department of Nuclear Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
  • Online:2013-12-28 Published:2014-01-02
  • Supported by:

    National Natural Science Foundation of China, 81101071; Shanghai Outstanding Academic Leaders Program, 11XD1403700; Program of Training Youth Scholars of Shanghai Institutions of Higher Learning, 2011

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摘要：

目的 构建含早期生长应答因子1 （Egr1）辐射敏感启动子调控的人钠碘同向转运体（hNIS）基因重组杆状病毒，为进一步延长核素在细胞中的滞留时间，提高hNIS介导的恶性肿瘤放射性核素基因靶向治疗疗效提供理论基础。方法 空载体pFB取自质粒pFB-CMV-EGFP，Egr1启动子取自质粒PGL3-Egr1-luc，hNIS取自质粒pcDNA3.1-CMV-hNIS，构建重组杆状病毒载体质粒pFB-Egr1-Hnis；同时构建含CMV启动子的质粒pFB-CMV-hNIS作为阳性对照，并将杆状病毒空载体质粒pFB-0作为阴性对照。随后制备各组杆状病毒，扩增收集重组杆状病毒并进行滴度测定。为进一步鉴定病毒功能，一方面通过体外感染U87脑胶质瘤细胞并采用131I刺激，通过免疫荧光检测131I刺激后的hNIS蛋白表达；另一方面通过摄碘实验进一步验证所表达的hNIS蛋白的摄碘功能和特性。结果 成功构建重组杆状病毒载体质粒pFB-Egr1-hNIS和pFB-CMV-hNIS；转座成功并提取了重组Bacmid；重组Bacmid转染Sf 9细胞后扩增收集的重组杆状病毒滴度可以达到1×1010 pfu/mL。体外感染U87细胞后，免疫荧光检测表明131I刺激后可增加hNIS的表达，感染细胞表达的hNIS蛋白位于细胞膜上，且具有摄碘的功能。结论 成功构建了重组杆状病毒载体质粒pFB-Egr1-hNIS及pFB-CMV-hNIS，获得高滴度的重组杆状病毒Bac-Egr1-hNIS、Bac-CMV-hNIS和Bac-0并得到验证；建立了基于hNIS基因的辐射正反馈重组杆状病毒体系，为进一步提高核素靶向治疗疗效奠定了重要的理论基础。

关键词: 人钠碘同向转运体, 早期生长应答因子1, 重组杆状病毒, 正反馈

Abstract:

Objective To prepare and verify a recombinant baculovirus harboring the human sodium iodide symporter (hNIS) under control of the early growth response-1 (Egr-1) promoter, and to provide the foundation for the research on radionuclides gene targeted treatment of malignant tumors. Methods The vector pFB was cut from plasmid pFB-CMV-EGFP; Egr1 was cut from plasmid PGL3-Egr1-luc; and hNIS was cut from plasmid pcDNA3.1-CMV-hNIS. Then recombinant baculovirus plasmid pFB-Egr1-hNIS was constructed. In addition, a positive control plasmid pFB-CMV-hNIS and a control plasmid pFB-0 were constructed. Baculovirus Bac-Egr1-hNIS and its controls were prepared, verified, amplified, and collected for titer determination. After glioma cell line U87 was infected with Bac-Egr1-hNIS and exposed to 131I, the presence and enhanced expression of hNIS protein was tested by immunofluorescence using anti-hNIS antibody. To verify the function of hNIS after 131I stimulation, the uptake of iodide was determined after incubation of the processed cells with 125I. Results Recombinant plasmid pFB-Egr1-hNIS, pFB-CMV-hNIS, and pFB-0 were correctly constructed and confirmed by DNA sequencing. Transposition was correct and recombinant Bacmid was extracted. The recombinant baculovirus were packed, verified, and its titer was about 1×1010 Pfu/mL. After incubation with 3 MBq/mL 131I, immunofluorescence showed that the hNIS expression level of Bac-Egr1-hNIS infected U87 cells increased dramatically. In another side, 131I stimulated U87 cells accumulated more 125I than did non stimulated cells. Conclusion The recombinant baculovirus Bac-Egr1-hNIS and its controls were constructed and verified, and high titer was obtained. This study provides a foundation of radiosensitive positive feedback system for further study.

Key words: human sodium iodide symporter, early growth response-1, recombinant baculovirus, positive feedback