| 摘要: |
| 【目的】克隆传染性造血器官坏死症病毒（IHNV）G基因，构建其重组腺病毒载体，为IHN预防及疫苗的研制提供参考。【方法】通过RT-PCR和PCR技术扩增IHNV G基因，将G基因插入到腺病毒穿梭载体中，再用带有目的基因的腺病毒载体与骨架载体在大肠杆菌BJ5183中同源重组，构建重组腺病毒质粒，通过PCR扩增及SalⅠ和XhoⅠ双酶切鉴定，经PacⅠ酶切后转染HEK-293细胞进行包装，获得带有目的基因的重组腺病毒，通过观察绿色荧光蛋白表达情况来监控重组腺病毒，用Western-blot法检测糖蛋白的表达，并测定重组腺病毒的TCID50。【结果】成功克隆出IHNV G基因，其长度为1 533 bp。成功构建了IHNV G蛋白重组腺病毒载体。GFP监测显示，重组腺病毒转染成功。重组腺病毒能在HEK-293细胞中表达出分子质量约58 ku的产物，与预期相符，其TCID50达到1.0×1010.4 mL-1。【结论】成功构建了带有IHNV G基因的重组腺病毒载体，重组腺病毒能高效表达，滴度较高。 |
| 关键词: 传染性造血器官坏死症 G基因 腺病毒载体 |
| DOI： |
| 分类号: |
| 基金项目:甘肃省兰州市科技发展计划项目（2014-RC-69） |
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| Construction of recombinant adenovirus vector of infectious hematopoietic necrosis |
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LI Shouhu,FAN Yufeng,GAO Xin,et al
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| Abstract: |
| 【Objective】This study cloned G gene of infectious hematopoietic necrosis virus (IHNV) and constructed its recombinant adenovirus vector to provide reference for IHN prevention and vaccine development.【Method】The G gene of IHNV was amplified and inserted into the adenovirus shuttle vector by RT-PCR and PCR.The adenovirus vector with the target gene was recombined with the backbone vector in Escherichia coli BJ5183 to construct the recombinant plasmid.The recombinant plasmid was transfected into HEK-293 cells after digestion with Pac Ⅰ.The recombinant adenovirus was identified by PCR amplification and digestion with Sal Ⅰ and Xho Ⅰ.The recombinant adenovirus was monitored by the expression of green fluorescence protein and its glycoprotein expression was detected by Western blot.Its TCID50 was also determined.【Result】The G gene of IHNV was cloned successfully with the length of 1 533 bp.The recombinant adenovirus vector with IHNV G protein was also successfully constructed.GFP monitoring showed that the recombinant adenovirus was transfected successfully.The recombinant adenovirus could express with molecular weight of 58 ku in HEK-293 cells,which was consistent with anticipation.Its TCID50 reached 1.0×1010.4 mL-1.【Conclusion】 The recombinant adenovirus vector with IHNV G gene was constructed successfully and it expressed efficiently with high titers. |
| Key words: infectious hematopoietic necrosis G gene adenovirus vector |
