基于hNIS基因的辐射正反馈重组杆状病毒体系制备

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

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1×1010pfu/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。

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; Egrl was cut from plasmid PGL3-Egrl-luc; and hNIS was cut from plasmid pcDNA3, l-CMV-hNIS. Then recombinant baculovirus plasmid pFB-Egrl-hNIS was constructed. In addition, a positive control plasmid pFB-CMV-hNIS and a control plasmid pFB-0 were constructed. Baculovirus Bac-Egrl-hNIS and its controls were prepared, verified, amplified, and collected for trier determination. After glioma cell line U87 was infected with Bac-Egrl-hNIS and exposed to 131 I, the presence and enhanced expression of hNIS protein was tested by immunofluorescence using anti-hNIS antibody. To verify the function of hNIS after 13~ I stimulation, the uptake of iodide was determined after incubation of the processed cells with 125I. Results Recombinant plasmid pFB-Egrl-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 baeulovirus were packed, verified, and its titer was about 1 x 1010 Pfu/mL. After incubation with 3 MBq/mL 131I, immunofluorescence showed that the hNIS expression level of Bac-Egrl-hNIS infected U87 cells increased dramatically. In 125 I another side, 131I stimulated U87 cells accumulated more than did non stimulated cells. Conclusion The recombinant baculovirus Bac-Egrl-hNIS and its controls were constructed and verified, and high titer was obtained. This study provides a foundation of radiosensitive positive feedbaek system for further study.