CAJ | 학술논문

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（ScFv－pLLO），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（ ScFv）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ScFv－pLLO。 ScFv－pLLO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。 ScFv－pLLO可刺激免疫小鼠脾细胞增殖。结论：重组蛋白ScFv－pLLO保留了ScFv 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。
목적：인공설계병표체미라화（C2B8）단련가변구항체여리사특균용세포소O（Listeriolysin O，LLO）현성항원표위구성적중조융합단백（ScFv－pLLO），초보분석기항림파류세포활성。방법：사순수거고획득미라화（C2B8）단항VH 화VL기인서렬，구건항CD20단련항체（ ScFv）기인서렬，선취LLO적2개CD4＋T세포표위，구건단련항체융합항원표위적중조단백기인서렬，극륭지원핵표체재체，경유도표체순화，류식세포술화면역공침정분석기여림파류세포결합능력，세포증식실험측정기대림파류세포생장적영향，조망실험분석기유도림파류세포조망적능력，림파세포증식실험분석기면역원성。결과：성공유도표체중조단백ScFv－pLLO。 ScFv－pLLO가불동정도지파향결합불동림파류세포계，병명현억제Raji세포생장화유도기조망。 ScFv－pLLO가자격면역소서비세포증식。결론：중조단백ScFv－pLLO보류료ScFv 적공능，가파향결합화억제CD20양성림파류세포적생장，동시가격활궤체면역응답，위진일보연구기모의류세포표면항원화작위류세포역묘좌제적공능전정기출。
Objective:To artificially design and express a recombinant protein named as ScFv-pLLO by fusing ScFv gene of Rituximab(C2B8)and dominant antigen epitopes from listeriolysin O(LLO),and studying its anti-tumor activity.Methods:VH and VL gene sequences of C2B8 against CD20 were acquired by searching United States Patent database,and ScFv sequence was constructed by linking VL and VH with a short peptide linker.Two CD4+T cell epitopes from LLO were selected and designed to splice ScFv sequence.The recombinant gene of ScFv-pLLO was cloned into prokaryotic expression vector and purified after induction.The capacity of ScFv-pLLO target-binding to B-cell lymphomas was evaluated by flow cytometry ( FCM ) and co-immunoprecipitation ( Co-IP ) .The effects of ScFv-pLLO on B-cell lymphomas proliferation and apoptosis were detected respectively.The immunogenicity of ScFv-pLLO was assessed by lymphocyte proliferation assay.Results: ScFv-pLLO 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,ScFv-pLLO was able to stimulate proliferation of spleen lymphocytes of immunized mice.Conclusion: The recombinant protein ScFv-pLLO can target-bind to B-cell lymphomas,and perform inhibitory effect and induce apoptosis on Raji cells that indicate ScFv-pLLO retain the capacity of ScFv derived from monoclonal antibody against CD20.Besides, ScFv-pLLO can induce immune response.This study provides a basis for further research about the role of ScFv-pLLO on simulating tumor cell antigens as well as being tumor vaccine adjuvant.