摘要
根据GenBank中I群禽腺病毒(AAV)基因组序列,设计了1对特异性引物。以CELO毒株基因组DNA为模板,通过PCR扩增出结构蛋白五邻体基因(penton),将其克隆到原核表达载体pGEX-4T-1的EcoR I和Xho I位点,构建了原核表达载体pGEX-penton。将其转化到感受态细胞DH5α中,经IPTG诱导,SDS-PAGE分析,获得了45.9 ku的融合蛋白。用不同的IPTG浓度和时间诱导表达,得出诱导的最佳IPTG浓度为1.5 mmol/L,最佳时间为4 h。用尿素提取包涵体表达产物,得到penton纯化蛋白,浓度为2.98 mg/mL。经Western-bolt分析,该蛋白具有良好的反应原性。
According to the aviadenoviruses group I （AAV） genome sequence in the GenBank, a pair of specific primers were designed to amplify the penton gene of structure protein and CELO strain genomic DNA as templates by PCR, and then cloned into the EcoR I and Xho I sites of pGEX-4T-1 vector, constructing the prokaryotic expression vector named pGEX-penton.The recombinant plasmid was transformed into competent cells DH5a for expression and induced with IPTG. The results showed that 45.9 ku of fusion protein was obtained followed by SDS-PAGE analysis. Expression inducing with different IPTG concentration and time found that the best IPTG induction concentration was 1.5 mmol/L, the optimal time was 4 h. Purified penton protein was acquired by urea extracting the inclusion body of expressed products and its concentration was 2.98 mg/mL, the expressed protein had a good reactionogenicity by followed western-bolt analysis.
出处
《广东农业科学》
CAS
CSCD
北大核心
2011年第7期154-157,共4页
Guangdong Agricultural Sciences
基金
新世纪百千万人才工程专项(945200603)
广西科技攻关计划(0815009-3-6)
广西自然科学基金(2010GXNSFA013090)
