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目的 研究人B7 2分子中IgV和IgC结构域何者含有与受体结合的位点 ;对由原核系统表达的B7 2蛋白体外共刺激活性作出评价。方法 用PCR方法扩增人B7 2分子胞外区的三个片段 ,即IgV、IgC和包括IgV和IgC在内的Ig(V +C)区段 ,将三片段克隆入pGEM TEasy。继而将三片段克隆入原核表达载体pGEX 4T 3,得到pGEX 4T 3 hB7 2IgV、pGEX 4T 3 hB7 2IgC和pGEX 4T 3 hB7 2Ig(V +C)三个重组体 ,将三重组体转化宿主菌DH5α。经SDS PAGE和Westernblot证实与GST融合存在的相应靶蛋白的表达。用抗CD3单抗模拟T细胞活化的第一信号 ,用 [3H] TdR掺入法观察加入目的蛋白作为第二信号可否对T细胞活化起协同效应。结果 工程菌可表达出人B7 2分子的三种融合蛋白 :GST hB7 2IgV、GST hB7 2IgC和GST hB7 2Ig(V +C) ,且以包涵体形式存在。在第一信号存在条件下 ,GST hB7 2Ig(V +C)和GST hB7 2IgV均能协同第一信号活化T淋巴细胞 ,但GST hB7 2IgC无此功能。结论 细菌可表达出具有功能活性的人B7 2融合蛋白 ,单一的人B7 2IgV结构域足以与受体结合 ,共刺激T细胞活化。
Objective To investigate which IgV and IgC domains of human B7 2 contain the binding sites to the receptor, and to evaluate the in vitro co-stimulatory activity of B7 2 protein expressed by prokaryotic system. Methods Three fragments of human B7 2 extracellular domain, namely IgV, IgC and Ig (V + C) segments including IgV and IgC were amplified by PCR. The three fragments were cloned into pGEM TEasy. Then the three fragments were cloned into the prokaryotic expression vector pGEX 4T 3 to get three recombinants, pGEX 4T 3 hB7 2IgV, pGEX 4T 3 hB7 2IgC and pGEX 4T 3 hB7 2Ig (V + C). The recombinant was transformed into host strain DH5α. The corresponding target proteins present in fusion with GST were confirmed by SDS PAGE and Western blot. The anti-CD3 monoclonal antibody was used to simulate the first signal of T cell activation. Whether [3H] TdR incorporation was used as the second signal to observe whether the addition of the target protein could have a synergistic effect on T cell activation was observed. Results The engineered bacteria can express three fusion proteins of human B7 2 molecule: GST hB7 2IgV, GST hB7 2IgC and GST hB7 2Ig (V + C), and exist as inclusion bodies. In the presence of the first signal, both GST hB7 2Ig (V + C) and GST hB7 2IgV synergized the first signal to activate T lymphocytes, but GST hB7 2IgC did not. Conclusions Bacteria can express functional human B7 2 fusion protein. A single human B7 2 IgG domain is sufficient to bind with receptor and costimulate T cell activation.