PEX慢病毒载体构建及其在293FT细胞中的分泌表达 |
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引用本文: | 赵中松,赵敬湘,张韶峰,王字玲,唐红卫,李锦毅.PEX慢病毒载体构建及其在293FT细胞中的分泌表达[J].国外医学:分子生物学分册,2010(6):488-492. |
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作者姓名: | 赵中松 赵敬湘 张韶峰 王字玲 唐红卫 李锦毅 |
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作者单位: | [1]辽宁医学院研究生学院,辽宁省锦州市121001 [2]军事医学科学院野战输血研究所,北京市100850 [3]武警总医院临床教研室,北京市100039 |
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基金项目: | 国家自然科学基金(No.30672771) |
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摘 要: | 目的 构建含人MMP-9信号肽-MMP-2-PEX片段的重组慢病毒,并在293FT细胞中分泌表达PEX蛋白.方法 利用RT-PCR、基因重组等技术,构建含MMP-9信号肽-MMP-2-PEX片段的重组慢病毒表达载体pBPLV-signal-PEX,在脂质体介导下与包装质粒(pLP1、pLP2)、包膜质粒(pLP/VSVG)共转染293FT细胞,包装产生慢病毒并进行滴度测定.慢病毒感染2931;3"细胞后,Western印迹法检测293FI"细胞培养上清中PEX的表达.结果 酶切和DNA测序表明慢病毒表达载pBPLV-signal-PEX构建正确,四质粒共转染293FT细胞成功获得慢病毒;慢病毒感染293FT细胞后,在细胞培养卜清中可检测到PEX蛋白的表达.结论 成功构建了含人PEX的重组慢病毒,在体外有效感染293fT细胞并持续分泌表达目的 蛋白,为进一步研究PEX在肿瘤侵袭与转移中的作用提供实验基础.
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关 键 词: | PEX基因 信号肽 慢病毒载体 293FT细胞 |
Construction of and Expression Lentiviral Vector Carrying PEX Gene |
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Authors: | ZHAO Zhongsong ZHAO Jingxiang ZHANG Shaofeng WANG Ziling TANG Hongwe LI Jinyi |
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Institution: | 1 School of Postgraduate, Liaoning Medical College, Jinzhou, Liaoning, 121001, China 2 Institute of Transfusion Medicine, Academy of Military Medical Sciences, Beijing, 100850, China 3 Department of Clinical Medicine, General Hospital of Chinese People' s Armed Police Force, Beijing, 100039, China) |
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Abstract: | Objective To construct a lentiviral vector containing MMP-9 signal peptide and non-catalytie carboxyl-terminal hemopexin domain of human MMP-2 gene (PEX) and observe its expression in 293FT cells. Methods Lent/viral vector containing MMP-9 signal peptide and 1VMP- 2-PEX gene was eonstructed by RT-PCR and recombinant DNA technique. Recombinant lentivirus was produced in packaging 293FF cells transfected with four plasmids of lentiviral system by lipo- fectamine 2000. Expression and secretion of PEX gene in 293FT cells were detected by Western- blot. Results Restriction endonuclease digestion and sequencing analysis verified the correct con- struetion of the recombinant lentiviral expression vector carrying PEX gene and signal peptide. West- ern blot eonfirmed the PEX expression and seeretion in 293FT cells. Conclusion The PEX-lentiviral expression veetor may be applied for further study of PEX effect on neoplastic invasion and metas- tasis. |
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Keywords: | PEX gene signal peptide lentiviral vector 293FT cells |
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