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B7-H4单抗3C8的纯化及对肿瘤免疫逃逸的阻断作用研究
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引用本文:陈亚莉,李安琪,张宁玥,章良.B7-H4单抗3C8的纯化及对肿瘤免疫逃逸的阻断作用研究[J].中国药科大学学报(中文版),2017,48(4):461-468
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作者单位
陈亚莉 苏州大学药学院 
李安琪 苏州大学药学院 
张宁玥 苏州大学药学院 
章良 苏州大学药学院 
基金项目:国家自然科学基金资助项目(No.31370872)
中文摘要:采用Protein G亲和色谱及DEAE阴离子交换柱色谱分离纯化精制鼠源性B7-H4单克隆抗体3C8,结合流式检测可与B7-H4/293T转基因细胞株结合的目标抗体组分,获得了纯化的鼠源性B7-H4单克隆抗体3C8。反相柱色谱检测其纯度为93%,SDS-PAGE凝胶电泳鉴定证明经过两步柱色谱,目标抗体的纯度大大提高。流式细胞术检测发现3C8只与B7-H4/293T转基因细胞株结合,不与Mock/293T细胞结合,并且3C8也不与B7家族其他成员的转基因细胞株结合。激光共聚焦实验显示3C8可特异性染色B7-H4/293T转基因细胞,Western blot实验发现,以3C8作为一抗,B7-H4/293T转基因细胞有阳性条带而Mock/293T细胞没有条带。用3C8作为一抗进行免疫组化,结果显示,前列腺癌和肾癌组织特异性高表达B7-H4分子,而前列腺癌旁组织和肾癌癌旁组织B7-H4呈阴性表达。T细胞体外实验显示,B7-H4-Ig可特异性与活化T细胞结合,但3C8可阻断这种结合作用,并可逆转T细胞增殖抑制作用及T细胞因子分泌抑制作用。
中文关键词:单克隆抗体3C8  B7-H4  纯化  负性共刺激  免疫逃逸  阻断作用
 
Purification of monoclonal anti-B7-H4 antibody and the blockade of B7-H4-mediated tumor immune evasion by the antibody
Abstract:The purified 3C8 was obtained by two step column purification, including Protein G affinity purification and DEAE anion exchange purification. The purity of purified 3C8 was about 93% when analyzed by reverse column. SDS-PAGE showed that the purity of 3C8 was increased greatly by two step purification. By flowcytometry we found that 3C8 specifically binded with B7-H4/293T cells and did not bind with Mock/293T cells, moreover 3C8 did not bind with other B7 family members transgene cells. In confocal experiment 3C8 could specifically stained B7-H4/293T cells. In Western blot only B7-H4/293T cells showed positive band while Mock/293T cells showed negative result. The result of immunohistochemistry showed that B7-H4 was highly expressed in prostate cancer and renal cell carcinoma, while para-cancer tissues did not express B7-H4. The T cell proliferation experiment showed that B7-H4-Ig could bind to activate T cells and inhibit T cell proliferation, while 3C8 could block the binding of B7-H4-Ig and reverse the T cell proliferation inhibition effect of B7-H4-Ig by CFSE and CCK8 assay. The cytokine IFN-γ and IL-2 secreted by activating T cells was decreased by B7-H4-Ig and 3C8 could reverse the effect of B7-H4-Ig.
keywords:monoclonal antibody 3C8  B7-H4  purification  negative costimulation  immune evasion  blockade
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