| 摘要: |
| 【目的】构建含有 H5N1 型禽流感病毒 HA、NA的侵染模型病毒，并对构建的模型病毒进行侵染活性及特异性检测。【方法】采用 PCR 方法扩增出 H5N1 型 AIV-HA和,NA基因，经纯化回收后将其分别克隆至真核表达载体 pcDNA4.0 中，构建 pcDNA-HA、pcDNA-NA。然后采用磷酸钙共转染方法,将 pcDNA-HA、pcDNA-NA 和含有 LUC 报告系统的 HIV 骨架表达载体 pNL4-3.Luc.R-E，共同转染 293T 细胞，构建HIV/HA-NA模型病毒，对HIV/HA-NA模型病毒进行细胞侵染活性及特异性检测。【结果】HA、NA基因片段真核表达载体pcDNA-HA、pcDNA-NA与 HIV 骨架载体在 293T 细胞中成功表达组装，获得了HIV/HA-NA 侵染模型病毒；敏感细胞特异性检测及侵染活性分析表明，HIV/HA-NA 模型病毒与其野毒具有共同的敏感细胞。报告系统数据显示，模型病毒可以定量检测其对细胞的侵染程度。【结论】 成功构建 HIV/HA-NA模型侵染病毒，可以模拟 H5N1 病毒的侵染过程，定量报告病毒的侵染程度，并且该模型病毒不具有增殖能力，安全可靠，可以方便地用于普通实验室AIV抗体检测和特定药物的筛选。 |
| 关键词: 禽流感 模型病毒 磷酸钙共转染 报告基因 |
| DOI： |
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| 基金项目:河南省高校杰出科研人才创新工程项目(2006KYCX020) |
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| Construction and identification of infectious model virus of AIV strain H5N1 |
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| Abstract: |
| 【Objective】 HA and NA of Avian influenza virus strain H5N1 were used to construct a model virus of AIV.The activity and specificity of the model virus were tested respectively.【Method】 HA and NAgenes of AIV H5N1 were amplified and subcloned into pcDNA4.0.The recombinant plasmids were named pcDNA-HA and pcDNA-NA respectively.Using calcium phosphate cotransfection method,these plasmids together with HIV backbone pNL4 3.Luc.R-E-were transfected into 293T cell lines.The model virus was named HIV/HA-NA.The activity and specificity of the model virus HIV/HA-NA were tested subsequently.【Result】 Model viruses HIV/HA-NA were obtained after pcDNA-HA,pcDNA-NA and pNL4-3.Luc.R-E-was cotransfected into 293T cell lines.Specificity test with the sensitive cells and the activity test showed that HIV/HA-NA can mimic the infectious process of AIV.By the help of reporting system,the infectious activity of the model virus can be detected quantificationally.【Conclusion】 The model virus HIV/HA-NA can mimic the infectious process of AIV and report the infectious extent quantificationally.The model virus is safe and reliable since it can not be proliferated. It can be used as an excellent tool to screen antibody and special medicine of AIV in common laboratory. |
| Key words: Avian influenza virus model virus calcium phosphate cotransfection method gene reported |
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