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| 重组人可溶性突变体BAFF蛋白的制备及生物学活性的鉴定 | |
| 引用本文： | 李鸿昌,焦玉莲,崔彬,刘晓雯,卢冰如,孙文萍,孙亚方,刘相东,赵跃然.重组人可溶性突变体BAFF蛋白的制备及生物学活性的鉴定[J].山东大学学报(医学版),2012,50(3):17-23,33. |
| 作者姓名： | 李鸿昌 焦玉莲 崔彬 刘晓雯 卢冰如 孙文萍 孙亚方 刘相东 赵跃然 |
| 作者单位： | 1. 山东省医学科学院基础医学研究所,济南,250062 2. 山东大学附属省立医院中心实验室,济南,250021 3. 山东大学附属省立医院检验科,济南,250021 4. 山东省医学科学院基础医学研究所,济南250062;山东大学附属省立医院中心实验室,济南250021 |
| 基金项目： | 山东省自然科学基金资助项目(ZR2009CM139) |
| 摘 要： | 目的制备高纯度的B细胞活化因子(BAFF)可溶性突变体(smBAFF)蛋白,鉴定其生物学活性。方法重组原核表达载体pET41a/smBAFF在大肠杆菌BL21中经IPTG诱导表达smBAFF蛋白,采用SDS-PAGE和Western blot检测表达产物,超声碎菌,提取包涵体,Ni2+-NTA亲和层析纯化,复性,鉴定其生物学活性。结果经鉴定表达出相对分子质量为1.7×104的外源蛋白smBAFF,经Ni2+-NTA亲和层析纯化出该重组蛋白,复性后的smBAFF与B细胞具有较高的亲和力,但失去共刺激B细胞增殖的能力,且能竞争性抑制天然sBAFF的作用。结论成功制备具有B细胞结合活性而失去刺激B细胞增殖活性的smBAFF,为以smBAFF为靶向载体在B细胞恶性增殖性和异常活化性疾病治疗研究奠定基础。 |
| 关 键 词： | B细胞活化因子 原核细胞 包涵体 蛋白质复性 |
Preparation and bioactivity characterization of recombinant human soluble mutant BAFF |
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| LI Hong-chang , JIAO Yu-lian , CUI Bin , LIU Xiao-wen , LU Bing-ru , SUN Wen-ping , SUN Ya-fang , LIU Xiang-dong , ZHAO Yue-ran.Preparation and bioactivity characterization of recombinant human soluble mutant BAFF[J].Journal of Shandong University:Health Sciences,2012,50(3):17-23,33. | |
| Authors: | LI Hong-chang JIAO Yu-lian CUI Bin LIU Xiao-wen LU Bing-ru SUN Wen-ping SUN Ya-fang LIU Xiang-dong ZHAO Yue-ran |
| Institution: | 1,2 (1.Institute of Basic Medicine,Shandong Academy of Medical Sciences,Jinan 250062,China; 2.Central Laboratory,Provincial Hospital Affiliated to Shandong University,Jinan 250021,China; 3.Department of Clinical Laboratory,Provincial Hospital Affiliated to Shandong University,Jinan 250021,China) |
| Abstract: | Objective To prepare highly purified human soluble mutant B cell activating factor(smBAFF) and study its biological activity.Methods Expression of the recombinant human smBAFF in E.coli BL21 was induced by IPTG,and the protein was assayed by SDS-PAGE and Western blot.The bacteria were split by sonication,inclusion bodies were extracted and dissolved,and then the smBAFF was purified by Ni2+-NTA affinity chromatography.The purified protein was refolded under specified conditions,and the bioactivity of the protein was assayed by immunofluorescence and CCK-8.Results SDS-PAGE electrophoreses displayed that 1.7×104 recombinant protein was expressed,and Western blot showed that it was the protein of interest.The highly purified protein of the recombinant human smBAFF was attained by Ni2+-NTA affinity chromatography,and the recombinant protein could bind B cells,but lost the activity of co-stimulating B cell proliferation.Also,it could competitively inhibit the function of the soluble BAFF.Conclusions The recombinant smBAFF was successfully prepared and possesses B cell binding activity but lost the activity of co-stimulating B cell proliferation.The result may lay a foundation to study the therapy for B cell malignancies and B cell abnormal activity diseases. |
| Keywords: | B cell activating factor belonging to the TNF family Prokaryotic cells Inclusion Bodies Protein renaturation |
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