新型重组免疫抑制蛋白B7-2-L-PE40KDEL的分子构建与生物活性及结构特性

目的：构建重组B7－2－L－PE40KDEL融合蛋白,特异性杀伤CD28高表达的T细胞,选择性阻断T淋巴细胞活化过程中的B7：CD28／CTLA－4共刺激信号系统,由此诱导免疫耐受,特异性防治移植物抗宿主疾病（GVHD）和宿主抗移植物疾病（HCGD）。方法：采用基因融合序列重叠延伸（SOE）策略,构建新型重组pRSETA-B7-2-L-PE40KDEL外毒素融合蛋白的原核表达载体,实现融合蛋白在大肠杆菌中的高效表达,利用亲和层析等方法进行蛋白质纯化。对该融合蛋白的结构特征诸如等电点、柔性等进行模拟分析。采用MTT法对其特异性杀伤高表达CD28受体的T细胞的活性进行测定。结果：构建并高效表达了B7－2－L－PE40KDEL融合蛋白,所获融合蛋白的纯度大于95％。该融合蛋白的结构特征模拟分析的结果显示,其柔性及抗原性、亲水性表位均未显著改变。活性测定显示,该重组融合蛋白能有效特异性地杀伤高表达CD28的人T淋巴瘤细胞系,而对未来表达CD28的人白血病细胞系则无任何杀伤。结论：具有靶向性免疫抑制活性的新型重组B7－2－L-PE40KDEL毒素融合蛋白的构建、表达对特异性防治GVHD和HVGD可能产生积极的影响。

Construction,bioactivity identification and structural characteristics analysis of a novel recombinant immunosuppression protein B7-2-L-PE40KDEL

Objective To construct a novel recombinant B7 2 L PE40KDEL fusion protein used to selectively kill T cells expressing high levels of CD28 so as to induce immune tolerance and prevent graft versus host disease (GVHD) and host versus graft disease (HVGD). Methods The cDNA encoding human B7 2 was ligated with cDNA encoding PE40KDEL by using sequence overlapping extension (SOE) techniques The gene of interest was subcloned into a high output expression vector pRSETA and transformed into E coli cells Its molecular structural characteristics, such as flexibility, antigenicity, hydrophilicity, and epitope were analyzed The purification protocol of expressed protein was establishd and its cytotoxicity to selectively kill T cells expressing high levels of CD28 was measured by MTT method Results B7 2 L PE40KDEL fusion protein was expressed at high levels in E coli cells and the purified product attained over 95% of purity The structural characteristics of B7 2 L PE40KDEL were not significantly changed in comparision with B7 2 and PE40KDEL In cytotoxicity assay, B7 2 L PE40KDEL fusion protein specifically killed Jurkat cells which express high level CD28 receptor and was non cytotoxic to CD28 receptor negative cell line Raji Conclusion B7 2 L PE40KDEL novel fusion protein can selectively kill the T cells which express CD28 receptor and may become a kind of new effective drug for inducting T cell immune tolerance and preventing GVHD and HVGD