抗CD20单链抗体融合显性抗原肽的原核表达和其抑瘤活性研究  

Expression of a recombinant protein by fusing anti-CD20 ScFv with dominant antigen epitopes and its anti-tumor activity

作　　者：孙蕊[1,2] 朱琰[1,2] 冯海凉[1,2] 卞晓翠[1,2] 顾蓓[1,2] 王春景[1,2] 刘玉琴[1,2] 

机构地区：[1]中国医学科学院基础医学研究所 [2]北京协和医学院基础学院病理学系和细胞资源中心,北京100005

出　　处：《中国免疫学杂志》2015年第4期501-508,共8页Chinese Journal of Immunology

基　　金：国家科技部科技基础基金资助项目(NSTI-CR14)

摘　　要：目的:人工设计并表达美罗华(C2B8)单链可变区抗体与李斯特菌溶细胞素O(Listeriolysin O,LLO)显性抗原表位构成的重组融合蛋白(Sc Fv-p LLO),初步分析其抗淋巴瘤细胞活性。方法:查询数据库获得美罗华(C2B8)单抗VH和VL基因序列,构建抗CD20单链抗体(Sc Fv)基因序列,选取LLO的2个CD4+T细胞表位,构建单链抗体融合抗原表位的重组蛋白基因序列,克隆至原核表达载体,经诱导表达纯化,流式细胞术和免疫共沉淀分析其与淋巴瘤细胞结合能力,细胞增殖实验测定其对淋巴瘤细胞生长的影响,凋亡实验分析其诱导淋巴瘤细胞凋亡的能力,淋巴细胞增殖实验分析其免疫原性。结果:成功诱导表达重组蛋白Sc Fv-p LLO。Sc Fv-p LLO可不同程度地靶向结合不同淋巴瘤细胞系,并明显抑制Raji细胞生长和诱导其凋亡。Sc Fv-p LLO可刺激免疫小鼠脾细胞增殖。结论:重组蛋白Sc Fv-p LLO保留了Sc Fv的功能,可靶向结合和抑制CD20阳性淋巴瘤细胞的生长,同时可激活机体免疫应答,为进一步研究其模拟瘤细胞表面抗原和作为瘤细胞疫苗佐剂的功能奠定基础。Objective：To artificially design and express a recombinant protein named as Sc Fv-p LLO by fusing Sc Fv gene of Rituximab（ C2B8） and dominant antigen epitopes from listeriolysin O（ LLO）,and studying its anti-tumor activity. Methods： VHand VL gene sequences of C2B8 against CD20 were acquired by searching United States Patent database,and Sc Fv sequence was constructed by linking VLand VHwith a short peptide linker. Two CD4＋T cell epitopes from LLO were selected and designed to splice Sc Fv sequence. The recombinant gene of Sc Fv-p LLO was cloned into prokaryotic expression vector and purified after induction. The capacity of Sc Fv-p LLO target-binding to B-cell lymphomas was evaluated by flow cytometry（ FCM） and co-immunoprecipitation（ Co-IP）. The effects of Sc Fv-p LLO on B-cell lymphomas proliferation and apoptosis were detected respectively. The immunogenicity of Sc Fv-p LLO was assessed by lymphocyte proliferation assay. Results： Sc Fv-p LLO was successfully expressed. It could bind to different B-cell lymphomas cell lines and obviously inhibit the growth of Raji cells as well as inducing apoptosis. Moreover,Sc Fv-p LLO was able to stimulate proliferation of spleen lymphocytes of immunized mice. Conclusion： The recombinant protein Sc Fv-p LLO can target-bind to B-cell lymphomas,and perform inhibitory effect and induce apoptosis on Raji cells that indicate Sc Fv-p LLO retain the capacity of Sc Fv derived from monoclonal antibody against CD20. Besides,Sc Fv-p LLO can induce immune response. This study provides a basis for further research about the role of Sc Fv-p LLO on simulating tumor cell antigens as well as being tumor vaccine adjuvant.

关 键 词：CD20 单链抗体 B细胞淋巴瘤 抗原表位 李斯特菌溶细胞素O 

分 类 号：R392.11[医药卫生—免疫学]