猪Ⅱ型圆环病毒ORF2与猪GM-CSF基因重组腺病毒共表达及免疫效果分析

旨在构建含融合基因pGMCSF-ORF2的重组腺病毒,并对其表达水平和免疫效果进行分析.运用PCR方法扩增PCV2 ORF2和pGM-CSF基因,拼接后克隆入pMD18-T载体,然后再亚克隆入腺病毒穿梭质粒pShuttle-CMV中,阳性穿梭质粒经PmeⅠ酶线性化后电转化含腺病毒基因组(AdEasy-1)的大肠杆菌细胞BJ5183-Ad-1,成功获得了重组腺病毒DNA.将纯化后的重组腺病毒DNA转染AD293细胞,经过病毒基因组的PCR和转录水平的RT-PCR及Western blot等方面对融合蛋白的表达进行了鉴定.以该病毒免疫Babl/c小白鼠,对免疫小鼠血清中PCV2抗体进行检测.结果显示,获得了pGMCSF-ORF2重组基因,重组腺病毒载体构建成功,获得了表达pGMCSF-ORF2融合蛋白的重组腺病毒.该病毒免疫小鼠后,在小鼠血清中检测到了PCV2的特异性抗体.获得的重组腺病毒能有效表达pGMCSF-ORF2融合蛋白,且可诱导小鼠产生针对PCV2的特异性抗体.

Co-expression of Recombinant Adenovirus Carrying pGM-CSF,ORF2 Gene of Porcine Circovirus Type2 and Immunological Analysis

The recombinant adenovirus carrying chimeric gene pGMCSF-ORF2 was constructed,and the expression level and immunogenicity was analyzedThe pGM-CSF,ORF2 gene of PCV2 was amplified by PCR.The PCR products were cloned into pMD18-T vector after chimeric.The interesting gene was subcloned into the shuttle plasmid of pAdTrackCMV.The recombinant plasmid and adenovirus backbone DNA were co-transformed into Ecoli BJ5183 after digested by PmeⅠand the recombinant adenovirus genomic DNA was obtained,the expression level of which was screened under PCR,RT-PCR and Western blot analysis.Then Babl/c mice were immunized with the recombinant adenovirus,detected sera antibody about PCV2 of the mice by ELISA method.We obtained pGMCSF-ORF2 gene,constructed the pAd-GF2 vector,and obtained the recombinant adenovirus carrying pGMCSF-ORF2 gene.The specific antibody about PCV2 has been found in serum of the immuned miceThe recombinant adenovirus which exprssed pGMCSF-ORF2 chimeric gene efficiently may induce the mice with specific antibody about PCV2.