SARS病毒膜融合抑制剂鉴定、S2结构域原核表达及多克隆抗体的制备

发布时间：2018-06-27 21:40

本文选题：SARS + S蛋白　；参考：《苏州大学》2005年硕士论文

【摘要】：目的:用分子对接技术预测HIV融合抑制剂是否能对SARS起作用;原核表达SARS S蛋白S2结构域,制备抗S2的多克隆抗体。方法:搜寻HIV gp41融合抑制剂,用DOCK软件分析其与SARS S2结构域HR1三聚体晶体结构的亲和力;用PCR技术得到S2结构域编码序列,亚克隆至原核表达载体pGEX-5X-3,转化大肠杆菌BL21,IPTG诱导表达融合蛋白。所得S2融合蛋白经电泳后KCl染色割胶,免疫家兔制备多抗,分离血清并用琼脂糖双向扩散实验测定抗体效价。结果:预测了23个小分子,ADS-J1、ADS-J2、XTT Formazan等几个小分子得分较高,提示有亲和力。构建了pGEX-5X-3-S2原核表达载体,SDS-PAGE电泳显示诱导出84.2KD融合蛋白。融合蛋白免疫家兔,琼脂糖双向扩散实验证实血清有一定的抗体效价。结论:发现抗HIV的融合抑制剂确能对SARS起作用。成功表达了S2结构域,并制备了抗S2结构域的多克隆抗体。
[Abstract]:Objective: to predict whether HIV fusion inhibitor can act on SARS by molecular docking technique, and to express S 2 domain of SARS protein in prokaryotic expression and to prepare polyclonal antibody against S 2. Methods: gp41 fusion inhibitor was searched, and its affinity to HR1 trimer crystal structure of S2 domain was analyzed by dock software, and the coding sequence of S2 domain was obtained by PCR. Subcloned into prokaryotic expression vector pGEX-5X-3 and transformed into E. coli BL21 and IPTG to express fusion protein. The S 2 fusion protein was stained with KCl to prepare polyclonal antibodies. The antibody titers were determined by two dimensional agarose diffusion assay. Results: the scores of 23 small molecules ADS-J1 and ADS-J2 + XTT Formazan were higher, indicating that they had affinity. A prokaryotic expression vector pGEX-5X-3-S2 was constructed. SDS-PAGE showed that 84.2 KD fusion protein was induced by SDS-PAGE. Rabbits were immunized with the fusion protein, and the agarose diffusion assay confirmed that the serum had a certain antibody titer. Conclusion: the fusion inhibitor of anti-HIV can play an important role in SARS. The S2 domain was successfully expressed and the polyclonal antibody against S2 domain was prepared.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373;R392

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