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构建表达猪圆环病毒2型Cap蛋白重组复制缺陷型人5型腺病毒,评价其对小鼠免疫效果。本研究将编码PCV2Cap蛋白的ORF2克隆到腺病毒表达系统穿梭质粒,构建了重组穿梭质粒pacAd5CMV-Cap。将被限制性内切酶PacI线性化后的骨架质粒和重组穿梭质粒共转染HEK293AD细胞,两者在真核细胞内同源重组并包装出携带PCV2-Cap基因的复制缺陷型重组人5型腺病毒,命名为rAd5-Cap;以同样方法重组获得了不含任何靶基因的野生型重组腺病毒wt-rAd5。病毒增殖稳定后,rAd5-Cap与wt-rAd5滴度可达到108.5 TCID50/mL左右;一步增殖实验证明,相对于wt-rAd5,rAd5-Cap中Cap基因的引入几乎没有给重组腺病毒增殖造成影响。RT-PCR和间接免疫荧光实验显示,rAd5-Cap能够有效介导PCV2Cap蛋白在真核细胞HEK293AD细胞中表达。以107 TCID50的rAd5-Cap肌肉注射接种小鼠,14d后小鼠血清中产生可检测水平的针对Cap蛋白的体液免疫应答,并且至少在免疫后28d之前抗体水平持续加强。对重组腺病毒的分子生物学鉴定和在小鼠上的初步免疫试验结果表明,重组腺病毒rAd5-Cap可介导Cap蛋白在真核细胞中的表达,并且表达的靶蛋白具有较好的免疫原性,为进一步将其开发成新型PCV2疫苗奠定了基础。
Abstract：To construct a recombinant replication-defective human adenovirus type 5 expressing Cap protein of PCV2 and test the immunological efficacy in mice.In this study,the recombinant replication-defective human adenovirus type 5,named as rAd5-Cap(wt-rAd5),was constructed through homologous recombination internally in the HEK293AD cells after co-transfection of the Pac I-linearized backbone plasmid and the shuttle plasmid pacAd5CMV-Cap containing the open reading frame(ORF2) of the porcine circovirus type 2(PCV2) cap protein or pacAd5CMV without inserted fragment.Furthermore,the rAd5-Cap could induce the expression of PCV2 cap protein in the HEK293AD cells with high efficacy evaluated by the RT-PCR and indirect immunofluorescence assay(IFA).The virus titer of rAd5-Cap could reach up to 108.5TCID50/mL similarly to that of wt-rAd5,indicating that there was little affect on the virus proliferation after the insertion of PCV2 cap protein gene.The humeral immune responses could be activated and detected 14 days after the inoculation of the mice with 107TCID50 rAd5-Cap intramuscularly,and constantly in crease in another 14 days.These molecular biological and animal experiments results demonstrated that the PCV2 cap protein could be efficiently expressed by the recombinant adenovirus rAd5-Cap in eukaryotic cells and induce robust immune responses in mice,which laid a good foundation for the development of new type vaccine against porcine circovirus.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.002348
中图分类号:S852.65
引用信息:
[1]宫婷,张守峰,刘晔,等.表达猪圆环病毒2型Cap蛋白重组复制缺陷型人5型腺病毒的构建及小鼠免疫试验[J].病毒学报,2013,29(01):26-31.DOI:10.13242/j.cnki.bingduxuebao.002348.
基金信息:
公益性行业(农业)科研专项(201203056)
2013-01-31
2013-01-31
2013-01-31