| 重组sCR1在毕赤酵母细胞中的表达、纯化及鉴定 |
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| 引用本文： | 王广兰,宫璀璀,王文丽,刘亚利,刘琰,邰苏豫.重组sCR1在毕赤酵母细胞中的表达、纯化及鉴定[J].河南医学研究,2008,17(1):1-5. |
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| 作者姓名： | 王广兰 宫璀璀 王文丽 刘亚利 刘琰 邰苏豫 |
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| 作者单位： | 解放军第153中心医院济南军区检验中心,河南郑州,450042 |
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| 摘 要： | 目的:酵母细胞SMD 1168表达人sCR1,并对重组蛋白进行纯化。方法:从人外周血中提取总RNA,应用RT-PCR获得人sCR1全长cDNA,将其克隆入真核表达载体pPIC9K,构建含人sCR1的重组质粒(pPIC9K-sCR1);经测序鉴定正确后,将重组质粒转化入毕赤酵母菌细胞SMD 1168中,经甲醇诱导,表达产物经SDS-PAGE分析及W estern b lot鉴定,并通过N i2+-NTA agarose亲和层析纯化。结果:经甲醇诱导的含pPIC9K-sCR1的酵母细胞表达出重组人sCR1的融合蛋白,48~72 h sCR1融合蛋白表达量最高。此蛋白在凝胶上表现为M r大于31 KDa的蛋白区带,在W estern b lot分析中可被sCR1的CD35单克隆抗体(mAb)识别。经N i2+-NTA agarose亲和层析纯化后得到较纯的sCR1融合蛋白。结论:人sCR1融合蛋白在酵母细胞表达系统中的高水平表达,并且有与人体天然蛋白相同的抗原活性。
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| 关 键 词： | 毕赤酵母细胞 酵母载体 可溶性补体受体1 蛋白表达 |
| 文章编号： | 1004-437X(2008)01-0001-05 |
| 修稿时间： | 2008年3月2日 |
Expression, purification and identification of recombinant sCR1 in Pichia pastoris |
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| WANG Guang-lan,GONG Cui-cui,WANG Wen-li,LIU Ya-li,LIU Yan,TAI Su-yu.Expression, purification and identification of recombinant sCR1 in Pichia pastoris[J].Henan Medical Research,2008,17(1):1-5. |
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| Authors: | WANG Guang-lan GONG Cui-cui WANG Wen-li LIU Ya-li LIU Yan TAI Su-yu |
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| Institution: | WANG Guang-lan; GONG Cui-cui; WANG Wen-li; LIU Ya-li; LIU Yan; TAI Su-yu(Center Laboratory Medicine of Jinan Command; NO.153 Hospital of PLA; Zhengzhou 450042; China); |
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| Abstract: | Objective: To express human sCR1 in Pichia pastoris SMD1168 and purify the recombinant protein product.To provide convenience for clinical diagnosis and therapy with the expression similar to human natural protein.Methods: Human total RNA was extracted from peripheral blood.The full length cDNA of human sCR1 gene was obtained using RT-PCR,cloned into eukaryotic expression vector pPIC9K to construct the recombinant plasmid pPIC9K-sCR1 containing human sCR1.Identified by DNA sequencing,then the recombinant pl... |
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| Keywords: | Pichia pastoris Soluble complement receptor type 1 Protein expression Identification |
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