| 李珂常青△ 徐平高洪波李贞福
（青.表达增强型绿色荧光蛋白标记的hBax 和hHGF 双基因的慢病毒
载体构建[J].,2012,12(18):3473-3477 |
| 表达增强型绿色荧光蛋白标记的hBax 和hHGF 双基因的慢病毒
载体构建 |
| Construction of EGFP Labeled Lentiviral Vector CarryinghBax and hHGF Genes |
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| DOI： |
| 中文关键词: 慢病毒载体 肝细胞生长因子 冠状动脉旁路移植术 再狭窄 |
| 英文关键词: Lentiviral vector Hepatocyte growth factor Coronary artery bypass graft Restenosis |
| 基金项目:山东省自然科学基金（2005zrb104001） |
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| 中文摘要: |
| 目的：构建绿色荧光蛋白标记的hBax 和hHGF 双基因共表达的重组慢病毒并鉴定。方法：通过重叠PCR 技术构建attB1-
K-hBAX/T2A/eGFP/P2A/hHGF-attB2 基因片段，利用gateway technology 构建慢病毒载体质粒pLV.EX2d.null-EF1A>hBAX/T2A/
eGFP/P2A/hHGF 和阴性对照质粒pLV.EX2d.null-EF1A>eGFP 并测序，上述两种质粒分别与辅助质粒共转染293FT 细胞包装病
毒，荧光显微镜检测病毒滴度。结果：经鉴定慢病毒载体质粒构建正确，荧光显微镜检测hBax 和hHGF 共表达慢病毒滴度为
7.8×107 TU/mL，仅表达绿色荧光蛋白的阴性病毒滴度为9×107 TU/mL。结论：表达增强型绿色荧光蛋白标记的hBax 和hHGF
双基因的慢病毒构建成功并获得高滴度的病毒感染液。 |
| 英文摘要: |
| Objective: To construct and identify lentiviral vector expressing hBax and Hhgf fusion protein labeled with enhanced
green fluorescence protein. Methods: The attB1-K-hBAX/T2A/eGFP/P2A/hHGF-attB2 gene fragment was obtained by overlap PCR
method. Gateway technology was used to construct the pLV.EX2d.null-EF1A>hBAX/T2A/eGFP/P2A/hHGF plasmid and pLV.EX2d.
null-EF1A>eGFP plasmid. After sequencing, the lenti virus was packaged through co-transfecting above-mentioned construct into human
embryonic kidney cell line-293FT with helper plasmids. Then the virus titer was examined by fluorescence microscope. Results: The recombinant
lentiviral transfer vector plasmids were constructed correctly, the titer of lentiviral-hBAX-eGFP-hHGF was 7.8×107 TU/mL,
and the titer of lentiviral-eGFP was 9×107 TU/mL. Conclusions: The lentiviral vector was constructed and high titer of lentivirus particles
were obtained successfully. |
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