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。
ORF1 of porcine circovirus （PCV） types 1 and 2 were amplified by PCR from PCV1 genomic DNA and plasmid pCI-PCV2-ORF1, 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 nag/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.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2007年第5期350-354,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(30370052)
浙江省重大攻关项目(2003C12012)
关键词
猪圆环病毒1型和2型
REP蛋白
表达
纯化
porcine circovirus types 1 and 2
Rep protein
expression
purification
