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选取了HSP65上对自身免疫性炎症疾病有防治作用的两段功能表位P1(179-190)、P2(31-46),分别以HSP65和CTB为载体蛋白构建高效表达载体pET28a-HSP65-P1P2P1与pET28a-CTB-P1P2P1,以硫酸铵分级沉淀和包涵体洗涤、经DEAE阴离子交换柱层析分离纯化得到HSP65-P1P2P1与CTB-P1P2P1融合蛋白,并用戊二醛一步偶联法将两者偶联形成HpCp复合物。以小剂量滴鼻粘膜免疫高胆固醇膳食诱发产生动脉粥样硬化的新西兰大白兔,结果显示两组融合蛋白均表现出一定的减斑功效,与PBS对照组相比较,主动脉病斑面积分别减少了34.9%和27.9%,为进一步开发能用于临床的抗AS疫苗提供了良好的设计思路,可经进一步优化设计提高其免疫原性,研发为抗动脉粥样硬化疫苗。
Two functional epitopes, P1 (179-190) and P2 (31-46), on which HSP65 could prevent and cure autoimmune inflammatory diseases were selected and the high expression vector pET28a-HSP65-P1P2P1 And pET28a-CTB-P1P2P1, were precipitated with ammonium sulfate fractionation and inclusion body washing, purified by DEAE anion exchange column chromatography to obtain HSP65-P1P2P1 and CTB-P1P2P1 fusion protein, and glutaraldehyde coupling step by step coupling the two HpCp complex is formed. A small dose of nasal mucosa immunized with high cholesterol diet induced atherosclerosis in New Zealand white rabbits, the results showed that the two fusion proteins showed a certain effect of reducing plaque, compared with the PBS control group, aortic lesion area decreased 34.9% and 27.9%, respectively, providing a good design idea for further development of anti-AS vaccine which can be used clinically. The design can be further optimized to enhance its immunogenicity and be developed as an anti-atherosclerotic vaccine.