| 人4-1BBL胞外区/抗CD20融合蛋白的构建和表达 |
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| 引用本文： | 刘荣,姜文国,刘芳,张砚君,范冬梅,杨铭,熊冬生,杨纯正.人4-1BBL胞外区/抗CD20融合蛋白的构建和表达[J].细胞与分子免疫学杂志,2008,24(6):543-546. |
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| 作者姓名： | 刘荣 姜文国 刘芳 张砚君 范冬梅 杨铭 熊冬生 杨纯正 |
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| 作者单位： | 1. 中国医学科学院北京协和医学院,血液学研究所血液病医院,实验血液学国家重点实验室,天津,300020
2. 滨州医学院药理教研室,山东,滨州,256603 |
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| 基金项目： | 国家自然科学基金
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山东省自然科学基金 |
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| 摘 要： | 目的:构建和表达人4-1BBL胞外区/抗CD20双功能融合蛋白,并测定该融合蛋白的生物学活性.方法:采用PCR和ovelap PCR方法构建人4-1BBL胞外区/抗CD20双功能融合蛋白,并用双脱氧终止法测定DNA序列;采用亲和层析法纯化该产物,并用150 g/L SDS-PAGE和Western blot鉴定纯化产物;采用FACS法鉴定纯化产物与靶细胞的结合活性.结果:DNA序列测定结果表明:人4-1BBL胞外区/抗CD20双功能融合蛋白已构建成功,表达可溶性产物的产量达4 mg/L以上,具有与Raji细胞(CD20 )和A549(4-1BB )细胞结合的活性.结论:利用融合蛋白形式,首次成功地构建了人4-1BBL胞外区/抗CD20融合蛋白,并获得较高表达.表达产物具有与相应2个靶抗原结合的活性.
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| 关 键 词： | 4-1BBL CD20 融合蛋白 免疫治疗 胞外区 融合蛋白 构建和表达 human expression Construction 抗原结合 利用 结合活性 细胞结合 Raji 产量 可溶性 已构建 测定结果 靶细胞 FACS 纯化产物 Western blot |
| 文章编号： | 1007-8738(2008)06-0543-04 |
| 修稿时间： | 2007年12月5日 |
Construction and expression of human 4-1BBL/anti-CD20 fusion protein |
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| LIU Rong,JIANG Wen-guo,LIU Fang,ZHANG Yan-jun,FAN Dong-mei,YANG Ming,XIONG Dong-sheng,YANG Chun-zheng.Construction and expression of human 4-1BBL/anti-CD20 fusion protein[J].Journal of Cellular and Molecular Immunology,2008,24(6):543-546. |
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| Authors: | LIU Rong JIANG Wen-guo LIU Fang ZHANG Yan-jun FAN Dong-mei YANG Ming XIONG Dong-sheng YANG Chun-zheng |
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| Institution: | The State Key Laboratory of Experimental Hematology, Institute of Hematology & Hospital of Blood Disease, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin 300020, China. |
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| Abstract: | AIM: To construct and express human 4-1BBL/anti-CD20 bispecific fusion protein and identify its biological activity. METHODS: PCR and overlapping PCR were used to construct human 4-1BBL/anti-CD20 bispecific fusion protein. DNA sequencing was performed by the terminus of the fusion protein nucleotide.The product was purified by affinity chromatography and analyzed by Western blot and its antigen-binding activity was examined by FACS. RESULTS: The data of DNA sequence showed that human 4-1BBL/anti-CD20 bispecific fusion protein was correct. The fusion protein was recovered in high yield (up to 4 mg/L) after E-tag purification and predominantly(90%) as a dimer. The fusion protein could bind to Raji cells(CD20(+)) and A549 cells(4-1BB(+)), respectively. CONCLUSION: The human 4-1BBL/anti-CD20 bispecific fusion protein with high level expression was successfully obtained and could bind to Raji ceIls and A549 cells. |
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