传染性造血器官坏死症重组腺病毒载体的构建

Construction of recombinant adenovirus vector of infectious hematopoietic necrosis

【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TCID_(50)。【结果】成功克隆出IHNVG基因,其长度为1 533bp。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58ku的产物,与预期相符,其TCID_(50)达到1.0×10^(10.4)mL^(-1)。【结论】成功构建了带有IHNVG基因的重组腺病毒载体,重组腺病毒能高效表达,滴度较高。

【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 Ggene 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 Ⅰand 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 Ggene 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 monitoringshowed 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 1.0×10^（10.4）mL^（-1）.【Conclusion】The recombinant adenovirus vector with IHNVGgene was constructed successfully and it expressed efficiently with high titers.