摘要
目的构建适合融合基因pCzn1-TSP3的原核表达系统,纯化获得多房棘球蚴蛋白TSP3。方法 TSP3的基因序列在GenBank中获取,采用基于PAS(PCR-based Accurate Synthesis)的方法,构建原核表达质粒pCzn1-TSP3,并转化于大肠杆菌Arctic Express(DE3)中,经IPTG诱导,获得目的蛋白,利用Ni-IDA-Sepharose CL-6B亲和层析柱纯化获得目的蛋白。结果经生物公司测序及酶切鉴定结果证明重组质粒p Czn1-TSP3构建成功。进一步实验结果表明TSP3在原核表达系统中主要以可溶蛋白形式存在。继而经Ni-IDA-Sepharose CL-6B亲和层析柱纯化获得TSP3纯蛋白,并经过Western Blot证实。结论大肠杆菌Arctic Express(DE3)中能够成功表达蛋白TSP3,且Ni-IDA-Sepharose CL-6B亲和层析柱能够纯化目的蛋白TSP3。
Objective Constructed a prokaryotic expression system for pCznl-TSP3 and obtained Purified fu- sion protein TSP3. Method According to the sequence of Echinococcus multilocularis TSP3 antigen in GenBank, PAS ( PCR-based Accurate Synthesis) was used to construct pCznl-TSP3 plasmid of prokaryotic expression, which was transformed into escherichia coli Arctic Express (DE3). After IPTG induction of protein expression, the target protein was purified by Ni-IDA-Sepharose CL-6B column affinity chromatography.Results The results of sequen- cing and enzyme digestion identification by biological company proved that the recombinant plasmid pCznl-TSP3 was successfully constructed.Further, our results were indicated that TSP3 was expressed in soluble protein, Finally, we obtained high-purified recombinant protein TSP3 that was purified through Ni-chelating affinity chromatogra-phy, and was confirmed by Western Blot.Conclusions In this study,we successfully established the prokaryotic ex- pression system of TSP3 and obtained highly purified preparation of TSP3.
作者
刁晓艳
周虎
李润乐
彭海
李子安
汤锋
杨全余
DIAO Xiao-yan;ZHOU Hu;LI Run-le;PENG Hai;LI Zhi-an;TANG Feng;YANG Quan-yu(Qinghai Provincial People's Hospital,Qinghai,Xining 810007;Affiliated Hospital of Qinghai University,Qinghai,Xining 810001;Research Center for High Altitude Medical Sciences,Qinghai University,Qinghai,Xining 810001)
出处
《中国高原医学与生物学杂志》
CAS
2018年第3期191-196,共6页
Journal of Chinese High Altitude Medicine & Biology
基金
青海省科技厅应用基础项目(2015-ZJ-745)
关键词
多房棘球蚴
蛋白
TSP3
表达
分离
纯化
Echinococcus multilocularis
Protein
TSP3
Express
Separation
purification