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  

Construction and Immunogenicity Analysis of Recombinant Replication-defective Human Adenovirus Type 5 Bearing the Porcine Circovirus Type 2 Cap Protein Gene

作　　者：宫婷[1,2] 张守峰[2] 刘晔[2] 孙程龙[2] 杨洋[2] 陈奇[2] 钱方[2] 刘博浩[1,2] 扈荣良[2] 

机构地区：[1]吉林农业大学动物科学技术学院,长春130118 [2]军事医学科学院军事兽医研究所吉林省人兽共患病预防与控制重点实验室,长春130122

出　　处：《病毒学报》2013年第1期26-31,共6页Chinese Journal of Virology

基　　金：公益性行业(农业)科研专项(201203056)

摘　　要：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。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 rAdS-Cap （wt-rAd5）, was constructed through homologous recombi- nation internally in the HEK293AD cells after co-transfection of the Pac I-linearized backbone plasmid and the shuttle plasmid pacAdSCMV-Cap containing the open reading frame （ORF2） of the porcine circovirus type 2 （PCV2） cap protein or pacAdSCMV 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 rAdS-Cap could reach up. to 108＇5 TCIDs0/mL similarly to that of wt-rAdS, indicating that there was little affect on the virus proliferation af- ter 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 107 TCIDs0 rAdS-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.

关 键 词：猪圆环病毒2型 CAP蛋白 重组腺病毒 免疫原性分析 

分 类 号：S852.65[农业科学—基础兽医学]