【摘要】 分别以猪圆环病毒1型基因组和重组质粒pCI-PCV2-ORF1为模板,利用PCR扩增了猪圆环病毒1型和2型的ORF1基因,随后克隆到pET-28a(+)和pET-32a(+)两种原核表达载体上。测序正确的重组质粒分别转化E.coli BL21(DE3),进行目的蛋白的诱导表达。SDS-PAGE和Western blot分析表明,猪圆环病毒1型和2型的ORF1均能在pET-28a和pET-32a中分别以重组蛋白His-Rep(40 Ku)和Trx-His-Rep(54 Ku)的形式表达。进一步纯化后测定重组蛋白的浓度,推算出猪圆环病毒1型和2型的His-Rep蛋白产量分别为34 mg/L菌液和14 mg/L菌液,Trx-His-Rep蛋白产量则为12 mg/L菌液和10 mg/L菌液。这些纯化的蛋白在Western blot中能够与猪抗猪圆环病毒2型阳性血清发生特异性反应,显示其具有良好的免疫学活性。本研究建立的猪圆环病毒重组Rep蛋白的原核表达系统及其纯化方法,为下一步开展Rep蛋白功能等相关研究奠定了基础。更多还原

【Abstract】 ORF1 of porcine circovirus(PCV)types 1 and 2 were amplified by PCR from PCV1 genomic DNA and plasmid pCI-PCV2-ORFI,respectively,and cloned into expression vectors pET-28a(+)and pET-32a(+).The recombinant plasmids were transformed into E.coli strain BL21(DE3)and protein expressed by IPTG induction.SDS-PAGE and Western blot analysis showed that ORF1 proteins of PCV 1 and 2 were expressed as His-Rep(40 Ku)in pET28a and Trx-His-Rep(54 Ku)in pET32a.The His-Rep proteins were expressed at a yield of 34 mg/L for PCV 1 and 14 mg/L for PCV 2 in primary cultures,while Trx-His-Rep of PCV 1 and 2 were expressed at 12 mg/L and 10 mg/L,respectively.The purified proteins reacted specifically with swine anti-PCV2 serum by Western blot,which confirmed their immunogenicity.The Rep proteins of PCV 1 and 2 expressed in E.coli provided useful candidates for immune diagnosis and for function study of PCV ORF1 protein.更多还原