【摘要】 传染性胰腺坏死病（infectious pancreatic necrosis, IPN）是由传染性胰腺坏死病病毒（infectious pancreatic necrosis virus, IPNV）引起的一种主要危害鲑科鱼类的传染病。为克隆传染性胰腺坏死病病毒（IPNV）VP2基因和VP3基因，并构建其重组腺病毒载体，利用PCR方法分别扩增出IPNV VP2基因和VP3基因，通过多基因片段同源重组技术将IPNV VP2基因和VP3基因克隆到pAd-Track-CMV载体，经过线性化后与pAd-easy-1载体在BJ5183菌体内进行同源重组，构建出重组腺病毒质粒；通过PCR及NotⅠ和HindⅢ双酶切鉴定，再经PacⅠ线性化后用于转染293T细胞，获得重组腺病毒；通过绿色荧光蛋白（green fluorescent protein, GFP）的表达监控重组腺病毒的复制情况，用Western-blotting法分别检测IPNV VP2蛋白和VP3蛋白的表达，并测定重组病毒滴度。结果显示，分别克隆出IPNV VP2和VP3基因，基因总长度为2 211 bp,构建出目的基因重组腺病毒载体，该病毒在293T细胞中分别表达出蛋白分子质量约为54 kD和26 kD的产物，滴度为1.0×10⁹个/mL TCID₅₀。结果表明，已成功获得IPNV VP2基因和VP3基因重组腺病毒，该病毒在293T细胞中滴度较高，能够稳定表达。传染性胰腺坏死病病毒VP2蛋白和VP3蛋白重组腺病毒载体的成功构建可为大西洋鲑传染性胰腺坏死病活载体疫苗的研制提供参考。更多还原

【Abstract】 Infectious pancreatic necrosis（IPN） was an infectious disease caused by infectious pancreatic necrosis virus（IPNV）, which mainly affected salmonids.To clone the infectious pancreatic necrosis virus（IPNV） VP2 gene and VP3 gene, constructed their recombinant adenovirus vector. In this study, the IPNV VP2 gene andVP3 gene were amplified and inserted into the pAd-Track-CMV vector by PCR and multi-gene fragment homologous recombination technology. After linearization, the recombinant plasmids were recombined with the pAd-easy-1 vector in E. coli BJ5183 to construct a recombinant adenovirus plasmid, which was identified by PCR and digestion with NotⅠand HindⅢ, and then transfected into 293T cells to obtain the recombinant adenovirus after linearizing with PacⅠ. The viral replication was monitored using the expression of green fluorescent protein（GFP）. The expression of VP2 protein and VP3 protein were confirmed by the Western-blotting method, and the recombinant viral titer was detected. The results indicated that the IPNV VP2 gene and VP3 gene were cloned with a length of 2 211 bp, and the recombinant adenovirus vectors for the target genes were constructed. The virus could express a molecular mass of about 54 kD and 26 kD in 293T cells, respectively, with a titer of 1.0×10⁹ cell/mL TCID₅₀. The findings revealed that the recombinant adenovirus with the IPNV VP2 gene and VP3 gene was successfully obtained, and the virus had a high titer in 293T cells and could be expressed stably. The successful construction of recombinant adenovirus vectors with VP2 and VP3 proteins of IPNV could provide a reference for the development of the vaccine and better prevention of IPNV in Atlantic salmon.更多还原