摘要
目的表达、纯化Tgo DNA聚合酶,并检测其扩增性能。方法采用PCR技术从嗜热古细菌Thermococcus gorgonarius中扩增Tgo DNA聚合酶基因,并克隆至含His-Tag靶序列的pET101载体中,转化E.coliBL21Sta(rDE3),IPTG诱导表达。目的蛋白纯化后经SDS-PAGE分析,并利用标准pfu酶,采用对比法初略测定酶活性;以质粒pUC19为模板,用Tgo酶和pfu酶进行PCR扩增,比较二者的扩增效率,采用改进的蓝白试验法检测Tgo酶的扩增忠实性。结果 PCR扩增的Tgo DNA聚合酶基因大小约2300bp,根据测序获得的基因序列推导出的氨基酸序列与GenBank中公布的序列一致。表达的目的蛋白为可溶性蛋白,相对分子质量约为89000,表达量为350μg/gE.coli,50%甘油保存的酶活性约为5U/μl。Tgo酶和pfu酶的扩增产出率和忠实性均相当。结论成功表达并纯化了Tgo DNA聚合酶,其扩增性能与pfu酶相当。
Objective To express and purify Tgo DNA polymerase and study its amplificatioin performance.Methods The gene encoding Tgo DNA polymerase was amplified from Thermococcus gorgonarius by PCR and cloned into vector pET101 containing His-Tag target sequence.The constructed recombinant plasmid pET101-Tgo was transformed to E.coli BL21 Star（DE3）for expression under induction of IPTG.The expressed protein was purified,analyzed by SDS-PAGE,and determined for enzyme activity using standard pfu as control.Tgo and pfu were amplified by PCR using plasmid pUC19 as template,and their proliferation efficacies were compared.The fidelity of amplification of Tgo was verified by modified blue-white selection.Results The Tgo DNA polymerase gene at a length of about 2 300 bp was amplified by PCR,of which the amino acid sequence deduced based on the nucleotide sequence was consistent with that reported in GenBank.The expressed protein was in a soluble form with a relative molecular mass of about 89 000,of which the expression level was 350 μg/g E.coli,and the enzyme activity after storage in 50% glycerin was about 5 U/μl.Both the yield and fidelity of amplification of Tgo were equivalent to those of pfu.Conclusions Tgo DNA polymerase was successfully expressed and purified,of which the amplification performance was equivalent to that of pfu.
出处
《中国生物制品学杂志》
CAS
CSCD
2010年第11期1182-1184,1189,共4页
Chinese Journal of Biologicals
基金
国际合作项目(20062200135)
吉林省自然科学基金项目(20060723)
关键词
TGO
DNA聚合酶
基因表达
纯化
扩增性能
Tgo DNA polymerase
Gene expression
Purification
Amplification performance
