溶组织内阿米巴14-3-3蛋白功能初探

发布时间：2018-12-08 21:33

【摘要】： 目的:预测溶组织内阿米巴14-3-3(Eh14-3-3)蛋白的结构与功能,克隆与鉴定溶组织内阿米巴14-3-3基因,并纯化重组Eh14-3-3蛋白,分析纯化产物的免疫学特性,制备抗重组Eh14-3-3蛋白血清,比较不同内阿米巴属虫株14-3-3蛋白序列,探讨14-3-3蛋白作为研究遗传进化标记分子的可能性。方法:利用生物信息学网站的各种在线工具和Vector NTI Suite软件包对溶组织内阿米巴14-3-3(Eh14-3-3)基因进行分析和功能预测,分别以溶组织内阿米巴HM1∶IMSS株滋养体总RNA逆转录合成的cDNA链和基因组DNA为模板,设计合成引物,PCR扩增Eh14-3-3蛋白编码基因序列,克隆入pET19b载体,并用双酶切法和DNA测序进行鉴定。在大肠杆菌BL21(DE3)中进行表达,用树脂层析纯化目的蛋白。SDS-PAGE鉴定,并用ELISA鉴定纯化产物的免疫学特性,检测病人和正常人血清对其的反应性,继而以之免疫小鼠制备抗血清。随后分别扩增各不同内阿米巴虫株滋养体的14-3-3蛋白编码基因序列,并对它们进行了比较分析。结果:溶组织内阿米巴具有三种Eh14-3-3蛋白编码基因的异构体,与GenBank收录(编号分别为EHU13418,EHU13419,EHU13420)的Eh14-3-3蛋白编码基因一致。重组Eh14-3-3蛋白,通过纯化层析,30kD附近得到单一蛋白条带。ELISA等实验证实纯化得到的Eh14-3-3蛋白与慢性反复发作者血清和阿米巴结肠炎病人血清有反应,与包囊携带者血清、急性阿米巴肝脓疡患者血清和正常人血清无明显反应。抗重组Eh14-3-3蛋白血清的与溶组织内阿米巴粗抗原ELISA反应的效价为1∶200,但随后的细胞定位实验为阴性。不同内阿米巴属虫株的14-3-3蛋白序列比较结果提示内阿米巴属14-3-3基因高度保守,生物信息学分析还提示该蛋白是研究物种进化的理想分子。结论:溶组织内阿米巴含有三个14-3-3蛋白的异构体,它们可能在溶组织内阿米巴的成囊过程中发挥重要作用。14-3-3蛋白是研究物种进化有用的靶分子。
[Abstract]:Objective: to predict the structure and function of endomoeba 14-3-3 (Eh14-3-3) protein, clone and identify the 14-3-3 gene, and purify the recombinant Eh14-3-3 protein. The immunological characteristics of the purified product were analyzed and the anti-recombinant Eh14-3-3 protein serum was prepared. The 14-3-3 protein sequences of different Entamoeba strains were compared and the possibility of 14-3-3 protein being used as a genetic and evolutionary marker was discussed. Methods: using various online tools of bioinformatics website and Vector NTI Suite software package to analyze and predict the function of Amiba 14-3-3 (Eh14-3-3) gene in tissues. Using the reverse transcription synthesis of cDNA strand and genomic DNA of total RNA of HM1:IMSS strain as templates, primers were designed to amplify the encoding gene sequence of Eh14-3-3 protein by PCR and cloned into pET19b vector. It was identified by double enzyme digestion and DNA sequencing. E. coli BL21 (DE3) was expressed. The target protein. SDS-PAGE was purified by resin chromatography, and the immunological properties of the purified product were identified by ELISA. Then the mice were immunized with it to prepare antiserum. The 14-3-3 protein coding gene sequences of trophozoites of different endoamoeba strains were amplified and compared with each other. Results: amoeba had three isoforms of Eh14-3-3 protein encoding genes, which were consistent with the Eh14-3-3 protein encoding genes included in GenBank (numbered EHU13418,EHU13419,EHU13420). The recombinant Eh14-3-3 protein was purified and a single protein band was obtained near 30kD by purification chromatography. ELISA and other experiments confirmed that the purified Eh14-3-3 protein reacted with the sera of patients with chronic recurrent episodes and the sera of patients with amoeba colitis. There was no significant response to the serum of patients with acute amoebic liver abscess and normal persons. The titer of anti-recombinant Eh14-3-3 protein serum was 1: 200 and the titer of ELISA was 1: 200, but the later cell localization test was negative. The comparison of 14-3-3 protein sequences among different strains of Entamoeba indicates that the 14-3-3 gene of Entamoeba is highly conserved, and the bioinformatics analysis also indicates that the 14-3-3 protein is an ideal molecule for studying the evolution of species. Conclusion: Entamoeba in tissue contains three isomers of 14-3-3 protein, which may play an important role in the formation of endomoeba cysts. 14-3-3 protein is a useful target molecule to study the evolution of species.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R383

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