【作者】 何卿玮；

【导师】 彭国光；

【作者基本信息】 重庆医科大学 ， 神经病学， 2009， 硕士

【摘要】 目的:对本实验小组早期构建的表达人α-突触核蛋白(humanα-synuclein,hα-syn)的核酸疫苗进行优化。设计并构建表达由人白介素4(human IL-4,hIL-4)和人α-突触核蛋白的多个B细胞表位所组成的融合蛋白的真核表达质粒,并检测所构建核酸疫苗的正确性,研究其在哺乳动物COS-7细胞内的表达。方法:选取hα-syn蛋白的三段B细胞表位基因,其间以两个赖氨酸连接,将设计出的基因序列送往公司合成;采用RT-PCR方法从人类淋巴细胞中扩增hIL-4基因后,通过基因重组技术将其与上述合成的基因序列融合,并在两者之间引入柔性短肽(GSGGGSG);随后将融合基因产物克隆到真核表达载体pVAX1上。然后将重组质粒转化到大肠杆菌DH5α内,抽提质粒,进行限制性内切酶酶切分析和DNA测序鉴定融合质粒的大小、方向和序列。检测合格后采用脂质体转染法,将融合质粒转入COS-7细胞中,用Western blot技术检测其表达情况。结果:成功扩增了hIL-4基因,并将其与hα-syn的三段B细胞表位基因融合,并克隆到真核表达载体pVAX1上,经限制性内切酶酶切分析和DNA测序,表明重组质粒大小、方向和序列完全正确,成功构建了重组质粒pVAX1-IL-4/SYN-B,以此作为核酸疫苗。经转染哺乳动物COS-7细胞株,并在COS-7中得到有效表达,通过Western blot检测具有较好的生物学活性。结论:成功构建核酸疫苗pVAX1-IL-4 /SYN-B,并在哺乳动物COS-7细胞中有效表达。所构建的核酸疫苗具有较好的生物学活性,为研究优化后的核酸疫苗在帕金森病动物模型中的免疫治疗的防治效果奠定了基础。更多还原

【Abstract】 Objective: To construct eukaryotic expression plasmid carryed human IL-4 gene and the B cell epitopes gene ofα-synuclein protein ,and investigate the expression of the plasmid in COS-7 cells. Methods :The gene of human IL-4 was obtained by RT-PCR and then was fused with the B cell epitopes gene ofα-synuclein protein. A short flexibility peptide was inserted(GSGGGSG)between the gene of human IL-4 and B cell epitomes. The fusional product was cloned into an eukaryotic expression plasmid pVAX1.Then the fusional plasmid was confirmed by electrophoresis and DNA sequencing. The fusional plasmid was transfected into COS-7 cells by liposome transfecting technique,and then identified by Western blotting.Results : The fusional plasmid,named pVAX1-IL-4 /SYN–B,was constructed and confirmed by DNA sequencing.Meanwhile,the protein extracting in COS-7 cells transfected with the plasmid was showed reactivity with antibody againstα-synuclein. Conclusion :The pVAX1-IL-4 /SYN–B was constructed successfully and highly expressed in COS-7 cells ,which lay a foundation for further studies.更多还原