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人可溶性CD83基因的克隆、原核表达和纯化

Cloning,prokaryotic expression and purification of human soluble CD83 gene
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摘要 目的:构建人可溶性CD83基因原核表达载体,并在大肠杆菌中表达和纯化.方法:利用反转录PCR方法从正常人外周血单个核细胞中获得可溶性CD83基因,克隆到表达载体pET-32a载体,构成重组质粒,转化大肠杆菌BL21(DE3),IPTG诱导表达6×His融合蛋白,并经SDS-PAGE及Western blot检测和鉴定.表达产物包涵体经Ni-NTA亲和层析纯化.结果:经酶切鉴定及测序证实可溶性CD83蛋白基因的原核表达载体构建正确.经SDS-PAGE及Western blot显示在Mr约32000处出现融合表达条带.融合蛋白的表达量约占菌体蛋白总量的45%.重组蛋白经Ni-NTA亲和层析进行纯化后,得到了高纯度的融合蛋白.结论:成功克隆人外周血单个核细胞来源的可溶性CD83基因片段,并在大肠杆菌BL21(DE3)中高效表达,亲和层析纯化后获得高纯度融合蛋白. AIM :To construct the recombinant plasmid expressing human soluble CD83 gene and to express and purify it in E. coli. METHODS: Soluble CD83 gene was obtained from a healthy human PBMCs by RT-PCR, then cloned into expression vector pET-32a, thus forming recombinant plasmid, which was transformed into E. coli BL21 ( DE3 ) and induced by IPTG. SDSPAGE and Western blot were used to test and identify the expressed (His)6 fusion proteins. The expression product in inclusion body was purified by Ni-NTA affinity chromatography. RESULTS: Restriction analysis and sequencing confirmed that the recombinant plasmid expressing soluble CD83 gene was successfully constructed. Both SDS-PAGE and Western blot analysis showed soluble CD83 gene was expressed, and the molecular weight of the product was about 32 000 u. The fusion protein production accounted approximately for 45% of total bacterial protein. The recombinant (His)6 fused proteins were purified by Ni- NTA affinity chromatography. CONCLUSION: Soluble CD83 gene from human PBMCs can be successfully cloned and expressed in E. coli BL21 (DE3). The purified proteins are obtained by Ni-NTA affinity chromatography.
出处 《第四军医大学学报》 北大核心 2008年第21期1931-1934,共4页 Journal of the Fourth Military Medical University
基金 陕西省自然科学基金(3053)
关键词 可溶性CD83基因 树突状细胞 原核表达 纯化 soluble CD83 dendritic cell prokaryotic expression purification
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参考文献9

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二级参考文献20

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