【作者】 林育涛；

【导师】 程训佳； 邵红霞；

【作者基本信息】 复旦大学 ， 病原生物学， 2008， 硕士

【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。更多还原

【Abstract】 Objective:To predict the structure and function of Eh14-3-3proteins,clone and characterize the Eh14-3-3 gene,purify the recombinant Eh14-3-3 proteins,analyse the immunologic properties of purified products,prepare the anti-Eh14-3-3 sera,compare the Eh14-3-3 sequences from different strains of Entamoeba,and to see whether 14-3-3 proteins can be used as a phylogenetic marker.Methods:The bioinformatics prediction of the Eh14-3-3 gene was performed by employing tools at online bioinformatics websites and software packages such as Vector NTI suite.Specific primers were designed,and the Eh14-3-3 sequences were amplified based on both the cDNA cloned from the total RNA of Entamoeba histolytica at the stage of trophozoite and the genomic DNA sequence,and then cloned into the pET19b vector.The inserted fragments were confirmed by both restriction endonuclease digestion and sequencing.The Eh14-3-3 gene was then introduced into E.coli BL21(DE3) pLysS bacteria and induced to expression.Each Eh14-3-3 protein was identified by SDS-PAGE.The recombinant Eh14-3-3 proteins were purified by resin chromatography.And their immunogenic properties were identified by ELISA assay to verify its reactivity to the serum of healthy people and patients.Then,a mouse-anti-recombinant Eh14-3-3 protein serum was obtained.Finally,the 14-3-3 sequences of different Entamoeba strains were compared.Results:There are three 14-3-3 protein isoforms in Entamoeba histolytica,which were verified and identical to the sequences submitted to Genebank(Accesion No.EHU13418,EHU13419 and EHU13420).The Eh 14-3-3 protein isoforms were induced to express in E.coli.After resin chromatography,we obtained purified products with a 30kDa molecular mass.The result of ELISA assay indicated the purified protein had good reactivity to the serum of chronic patients and AC patients, but not to the serum of acute ALA patients,cyst carriers and healthy individuals. The anti-recombinant Eh14-3-3 protein serum reacts with crude antigen at a titer of 1:200.However,result of the following subcellular localization experiment turned out to be nagetive.The sequence comparison between different Entamoeba strains indicated that the 14-3-3 gene is highly conserved in Entamoeba.The bioinformatics analysis also indicated that the 14-3-3 protein can be as a phylogenetic marker.Conelusions:The 14-3-3 genes are highly conserved in Entamoeba.The 14-3-3 proteins may play an important role in the encystations of Entamoeba.The 14-3-3 protein can be used as a phygenetic marker.更多还原