猪Ⅱ型圆环病毒ORF1、ORF2基因串联重组腺病毒的构建及其免疫效果

Construction of recombinant adenovirus carrying ORF1、ORF2 gene of porcine circovirus2 and immunological study

【目的】构建含PCV2ORF1和ORF2基因的串联重组腺病毒,并对其免疫效果进行评价。【方法】扩增PCV2的ORF1和ORF2基因,串联克隆入pMD18-T载体后亚克隆入腺病毒穿梭质粒pAdTrack-CMV中,阳性穿梭质粒经PmeⅠ酶线性化后与骨架载体pAdeasy-1在细菌BJ5183内完成重组,获得重组腺病毒质粒。将重组腺病毒质粒用PacⅠ酶线性化后转染293细胞,包装成重组腺病毒,然后从致细胞病变、荧光检测及PCR检测等方面对该重组腺病毒进行了鉴定。以该重组腺病毒免疫小鼠,对免疫鼠血清中PCV2的特异性抗体进行检测。【结果】得到了PCV2的ORF1和ORF2基因,pMD18-ORF1-ORF2和pAdCMV-ORF1-ORF2载体构建成功,获得了含有PCV2ORF1和ORF2基因的重组腺病毒。该重组腺病毒免疫小鼠后,在小鼠血清中可检测到PCV2的特异性抗体。【结论】成功构建了含PCV2ORF1和ORF2基因的重组腺病毒,此腺病毒可诱导机体产生针对PCV2的特异性抗体。

【Objective】 The recombinant adenovirus carrying ORF1 and ORF2 gene of PCV2 was constructed,then the immune result was evaluated.【Method】 ORF1、ORF2 gene of PCV2 were amplified by PCR.The PCR products were cloned into pMD18-T vector one by one and recombinant plasmids were obtained.The interesting gene was subcloned into the shuttle plasmid of pAdTrack-CMV.The recombinant plasmid and adenovirus backbone DNA were cotransformated into E.coli BJ5183 and the recombinant adenovirus genomic DNA was obtained.After transfecting 293 cell lines with recombinant adenovirus the genomic DNA by the lipofectamine method,the recombinant adenovirus carrying ORF1 and ORF2 gene was screened under fluorescent microscopy and PCR analysis.The recombinant adenovirus have been immuned mice,detected sera antibody about PCV2 of the mice by using ELISA.【Result】 We obtained ORF1 and ORF2 gene of PCV2,constructed the pMD18-ORF1-ORF2 and pAdCMV-ORF1-ORF2 vector,and obtained the recombinant adenovirus carrying ORF1 and ORF2 gene.The specific antibody about PCV2 has been found in serum of the immuned mice.【Conclusion】 The recombinant adenovirus carrying ORF1 and ORF2 gene of PCV2 was obtained and it may induce the mice with specific antibody about PCV2.