| 师长宏江鹰毛峰峰赵勇张彩勤赵善民白冰陈涛.结核分枝杆菌Rv1759c 结构域与IL-2 融合蛋白的表达与鉴定[J].现代生物医学进展英文版,2011,11(6):1001-1004. |
| 结核分枝杆菌Rv1759c 结构域与IL-2 融合蛋白的表达与鉴定 |
| Expression and identification of Rv1759c domain of mycobacteriumtuberculosis and IL-2 fusion protein |
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| DOI： |
| 中文关键词: 结核分支杆菌 Rv1759c IL-2 潜伏感染 |
| 英文关键词: Mycobacterium tuberculosis (MTB) Rv1759c IL-2 latent infection |
| 基金项目:国家"863" 专项课题（2007AA02Z473）；国家自然科学基金（NO.30972767）；陕西省自然科学基金（2007C224，2004C125） |
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| 中文摘要: |
| 目的：构建结核分枝杆菌（MTB）Rv1759c 结构域（Rv1759cD domain, Rv1759cD）与人IL-2（hIL-2）融合基因，并在大肠杆菌
中表达获得重组的融合蛋白Rv1759cD-IL-2。方法：用PCR 方法从MTB H37Rv 基因组扩增Rv1759cD 基因片段，测序后与hIL-2
基因构建融合基因，并克隆到表达载体pProEX HTa。融合基因在大肠杆菌DH5α 中诱导表达，经SDS-PAGE 分析后，分别与
His mAb、IL-2mAb 和结核病人血清进行Western-blot 鉴定，采用Ni-NTA 亲和层析纯化蛋白。结果：获得的Rv1759cD 基因经测序
与GenBank 公布的序列完全一致，与hIL-2 基因连接后，构建的融合基因在大肠杆菌中有效表达。表达蛋白相对分子量为
30KDAa，与预测值相符；Western-blot 结果显示，在相对分子量30KDAa 处分别与His mAb 和鼠抗IL-2 mAb 形成结合带，并与结
核病人血清出现特异性结合。通过Ni-NTA 亲和层析，可得到纯化的目的蛋白。结论：成功表达、纯化和鉴定了Rv1759cD-IL-2 融
合蛋白，并有可能作为新型结核病疫苗的靶抗原。 |
| 英文摘要: |
| Objective: To construct the fused gene of Rv1759c domain (Rv1759cD) gene of Mycobacterium tuberculosis H37Rv
(MTB) and human IL-2 (hIL-2), and to express the Rv1759cD-IL-2 fusion protein in E.coil DH5α. Methods: The Rv1759cD gene was
amplified by PCR from the genome of MTB H37Rv strain. The fused gene of Rv1759cD and hIL-2 was constructed, which was cloned
into expression vector pPRO EX HTa. The Rv1759cD-hIL-2 fused gene was expressed in E.coli DH5α induced by IPTG. The recombinant
protein was identified by SDS-PAGE analysis and by Western-blot with His mAb, IL-2 mAb and sera from TB patients respectively.
The fused protein was purified by Ni-NTA purification system. Results: The PCR product was 435bp and the sequence was the same with
those of Rv1759cD gene in GenBank. The fused protein with 30KDAa relative molecular mass was consistent with that had been reported,
which reacted with the specific monoclonal antibody against His mAb and IL-2 mAb respectively at 30KDAa bandings. It also had an
apparent affinity bandings with the sera of TB patients. Conclusion: The fused protein Rv1759cD-IL-2 was expressed and purified successfully,
and it would be a target antigen of new tuberculosis vaccines. |
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