目的构建具有免疫学活性的抗小鼠细胞毒性T淋巴细胞相关抗原4(CTLA-4)单链抗体ScFv(single chain fragment of variety region),为进一步将其应用到动物体内奠定基础。方法采用RT-PCR技术,从分泌抗小鼠CTLA-4单克隆抗体(mAb)的杂交瘤细...
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目的构建具有免疫学活性的抗小鼠细胞毒性T淋巴细胞相关抗原4(CTLA-4)单链抗体ScFv(single chain fragment of variety region),为进一步将其应用到动物体内奠定基础。方法采用RT-PCR技术,从分泌抗小鼠CTLA-4单克隆抗体(mAb)的杂交瘤细胞中扩增mAb的VH、VL基因,并进一步将其组装成VH-Linker-VL型的ScFv片段。将ScFv片段亚克隆至pGMET载体,测序正确后将其克隆至真核分泌型表达载体pSect2-GFP,将pSect2-GFP-ScFv纯化质粒转染中国仓鼠卵巢细胞(CHO)并进行表达,同时经Zeocin筛选稳定表达株。用Western blot方法检测ScFv融合蛋白的表达,采用ELISA检测ScFv的亲和活性。结果经Zeocin筛选2周后,CHO细胞株可稳定表达GFP-ScFv蛋白。Western blot法证实GFP-ScFv蛋白在细胞上清和细胞裂解液中均有表达,大小约55kD。ELISA检测表明,CHO培养上清中的GFP-ScFv蛋白经浓缩初纯后能与重组小鼠CTLA-4纯化抗原结合,并具有抑制亲本单抗与其结合的能力。结论成功构建了抗小鼠CTLA-4的ScFv真核表达载体,并能有效表达出具有免疫学活性的ScFv融合蛋白。
目的 测定慢性乙型肝炎(chb)患者外周血cd4+c25+调节性t细胞(cd4+cd25+treg细胞)的抗原诱导与抑制功能,初步探讨cd4+ cd25+ treg细胞在hbv感染慢性化过程中的作用.方法 采集22例chb患者、11例急性早期乙型肝炎患者(ahb)和20例健康对照者的外周血,分离外周血单核细胞(pbmc)后体外培养,并经重组hbsag或抗人cd3单克隆抗体诱导,流式细胞仪分析诱导前后cd4+ cd25+treg细胞比例的变化.免疫磁珠分选出cd4+ cd25+treg细胞亚群和去除了cd4+ cd25+treg细胞的效应细胞亚群(pbmc-treg),在重组hbsag刺激下进行体外单独培养或共培养,并检测效应细胞的增殖与细胞因子生成量的变化.所有患者均进行hbv血清标记物水平、血清hbv dna载量和肝功能水平的测定.结果 在重组hbsag刺激下,chb组pbmc可诱导出较高比例的cd4+cd25+ treg细胞,且与ahb组、健康对照组之间的差异有统计学意义(f值分别为3.47,4.98,p均＜0.05).体外单独培养时,患者组及健康对照组的cd4+ cd25+ treg细胞的增殖能力较弱,仅能产生微量的ifn-γ和il-10,且组间差异无统计学意义(p＞0.05).ahb组效应细胞的增殖能力和ifn-γ产量均明显高于chb组,而il-10的产量与chb组均较低,组间比较差异无统计学意义(p＞0.05).混合培养时,ahb组及chb组的cd4+ cd25+treg细胞均能显著抑制效应细胞的增殖及ifn-γ的产量(f值分别为4.66,5.24,p均＜0.01).结论 chb患者的外周血cd4+ cd25+ treg细胞可被hbsag特异性诱导,cd4+ cd25+ treg细胞在体外可显著抑制效应细胞的增殖与ifn-γ的分泌,提不cd4+ cd25+treg细胞可能通过抑制抗原特异性t细胞的应答而导致外周免疫耐受,并与hbv感染的慢性化密切相关.
abstract：
objective to investigate the induction and function of circulating cd4+ cd25+ regulatory t cells(cd4+cd25+ treg cells)in patients with chronic hepatitis b,and their role in the progress of chronic hbv *** twenty two patients with chronic hepatitis b(chb),11 patients with acute hepatitis b(ahb)and 20 healthy blood donators were enrolled in the *** stimulating with recombinant hbv antigen or anti-cd3 mab,the fraction of cd4+ cd25+treg cells in peripheral blood mononuclear cells(pbmc)from different subjects was analyzed using flow ***4+cd25+ treg cells and effector cells(pbmc-treg)were sorted out and cultured alone or together in vitro,and the cell proliferation and cytokines production were measured. serum hbv titers,hbv markers and liver function were also *** the fraction of circulating cd4+cd25+ treg cells induced in pbmc from chb patients under the stimulation of recombinant hbsag was much higher than that in ahb patients(f=3.47,p＜0.05)and normal controls(f=4.98,p＜0.05).with hbsag stimulation,cell proliferation and cytokines production (ifn-γand il-10)of cd4+ cd25+ treg cells ei
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