摘要
为探讨原核表达的PCV-2ORF2-ORF1串联蛋白的抗原性及其作为基因工程亚单位疫苗的可能性,以构建好的PCV-2大庆分离株质粒pMD-18T/PCV-2为模板,PCR分别扩增去掉核定位信号的Cap蛋白基因(dNLS-ORF2)和Rep蛋白基因(ORF1),将其串联克隆到原核表达载体pET30a(+)上,经PCR、酶切和测序鉴定,成功构建了重组质粒pET30a-ORF2/ORF1。转化E.coli BL21(DE3)pLysS感受态细胞,经IPTG诱导后,进行SDS-PAGE电泳与Western blot分析。纯化的重组蛋白与PCV-2阳性血清发生特异性反应,并能与免疫小鼠的抗血清发生特异性反应。重组蛋白具有较好的免疫学活性,为基因工程亚单位疫苗的研制奠定了基础。
To analyze the antigenicity of recombinant ORF2-ORF1 protein of PCV2 and discuss the possibility as subunit vaccine. The NLS deleted ORF2 and ORF1 genes were amplified by PCR from pMD218T/ PCV-2 and cloned into the pET30a （＋ ） vector. The recombinant plasmids pET30a-ORF2/ORF1 was sueeessfully identified by PCR, enzymatic digestion and sequenced. The recombinant plasmid was transformed into E. coli BL21 （DE3） pLysS competent cells. Expression of recombinant protein was induced by IPTG. The expression of target protein was determined by SDS-PAGE and Western blot. Ther recombinant protein specifically reacted with serum against PCV-2 and the serum of mouse which immunized with dNLSORF2/ORF1 recombinant protein respectively. The recombinant protein exhibits high immunologic activity and maybe can be used as subunit vaccine.
出处
《动物医学进展》
CSCD
北大核心
2010年第2期41-44,共4页
Progress In Veterinary Medicine
基金
黑龙江省农垦总局课题项目(HNKXIV-08-07A)
黑龙江八一农垦大学博士科研启动项目(校启D-2007-1)
