恶性疟原虫abstrakt同源基因的克隆及食蟹猴疟原虫eIF-4A同源蛋白的cDNA克

发布时间：2018-08-05 17:02

【摘要】：DEAD-box蛋白家族的ATP依赖的RNA解旋酶类参与细胞内几乎所有的RNA代谢过程,在几乎所有生物的细胞生长发育过程中扮演着众多不可或缺的角色。疟疾目前仍然是世界上许多地方的一个主要的健康问题;控制疟疾的一个可行的办法就是研制出通过阻断疟原虫生长有关的关键酶来抑制其生长的药物,比如通过阻断DNA/RNA解旋酶等。按照此思路,我们进行了有关疟原虫DNA/RNA解旋酶的研究。本研究主要开展了两个方面的工作:1.克隆了Plasmodium flaciparum abstrakt全长基因,分析了该蛋白的结构;并且依据不同DEAD-box蛋白的结构特征进行了初步归类。2.克隆了Plasmodium.cynomolgi eIF-4A完整cDNA,表达了该重组蛋白并对其ATPase活性进行了检测。在本实验中,通过PCR和探针杂交相结合的筛选方法,筛选恶性疟原虫(Plasmodium falciparum)的基因组文库,克隆了FH1F—abstrakt同源基因的完整序列。通过搜索已经完成测序的恶性疟原虫基因组数据库,推测FH1F序列定位在第5条连锁群上。FH1F全长2804bp,包含一个1161bp的完整阅读框,编码一个由386个氨基酸组成的蛋白。对FH1F蛋白序列用Blast P进行搜索和分析以及用DNAStar与许多典型的DEAD-box蛋白序列进行比对分析,结果均提示FH1F蛋白应该是DEAD-box家族的一个Abstrakt蛋白。另一方面,用DNAStar对已知所有完整的DEAD-box蛋白进行详细的序列分析以及用Mega对这些序列进行系统发育研究的结果都显示: DEAD-box家族的蛋白聚类成为若干不同的亚家族;与DEAD-box蛋白的一般保守序列相比,Abstrakt,eIF-4A,Vasa,P68等不同亚群的DEAD-box蛋白在保守区具有各自不同的结构特征。本文对不同的DEAD-box蛋白的结构特征进行了总结并试图给出不同亚群分类上的结构标准,对Abstrakt蛋白在本应高度保守的位点上异常于其它DEAD-box蛋白的氨基酸残基的取代也进行了相关的初步分析。采用同样的基因克隆方法筛选食蟹猴疟原虫(Plasmodium cynomolgi)的cDNA文库,克隆了一个eIF-4A同源蛋白的完整cDNA序列,命名为CH1F。CH1F全长1753bp,包含一个1197bp的完整阅读框,推测编码一个由398个氨基酸组成的蛋白。对CH1F的蛋白序列用BlastP进行搜索和分析,提示它应该是DEAD-box家族的一个eIF-4A同源蛋白;用DXAStar将其与许多典型的DEAD-box蛋白序列进行比对分析,结果显示:比起其它的DEAD-box蛋白,它与eIF-4A或eIF-4A的同源蛋白具有更高的一致性和更多序列上的相似结构域。将包含完整阅读框的片段亚克隆到表达载体pET-28a(+),在大肠杆菌DH5α中表达,产生的融合蛋白大小在45kD左右。对该融合蛋白进行纯化、重新折叠和初步鉴定。ATP酶活性检测显示,该融合蛋白只有很低的ATP酶活性,而且它的ATP酶活性似乎不依赖于核酸底物。对这一检测结果给出3种
[Abstract]:The ATP dependent RNA helicases of the DEAD-box protein family are involved in almost all RNA metabolism processes in the cells and play many indispensable roles in the cell growth and development of almost all organisms. Malaria is still a major health problem in many parts of the world; a viable way to control malaria is to develop drugs that inhibit the growth of malaria parasites by blocking the key enzymes involved in their growth. For example, by blocking the DNA/RNA helicase and so on. According to this idea, we studied the DNA/RNA helicase of Plasmodium falciparum. This study mainly carried out two aspects of work: 1. The full-length Plasmodium flaciparum abstrakt gene was cloned, the structure of the protein was analyzed, and the structural characteristics of different DEAD-box proteins were preliminarily classified. 2. The complete cDNAs of Plasmodium.cynomolgi eIF-4A were cloned, the recombinant protein was expressed and its ATPase activity was detected. In this experiment, the genomic library of Plasmodium falciparum (Plasmodium falciparum) was screened by PCR and probe hybridization, and the complete sequence of FH1F-abstrakt homologous gene was cloned. By searching the genomic database of Plasmodium falciparum which has been sequenced, we speculated that the FH1F sequence was located on the fifth linkage group. FH1 F contained a complete reading frame of 1161bp, encoding a 386-amino acid protein. The FH1F protein sequence was searched and analyzed by Blast P and compared with many typical DEAD-box protein sequences by DNAStar. The results indicated that FH1F protein should be a Abstrakt protein of DEAD-box family. On the other hand, Detailed sequence analysis of all known complete DEAD-box proteins using DNAStar and phylogenetic analysis of these sequences using Mega show that: DEAD-box The proteins of the family were clustered into several different subfamilies. Compared with the general conserved sequence of DEAD-box protein, the DEAD-box proteins of different subgroups, such as Abstraktttene eIF-4ANAP68 and so on, have different structural characteristics in the conserved region. In this paper, the structural characteristics of different DEAD-box proteins were summarized and the structural criteria of different subgroups were proposed. The substitution of amino acid residues of Abstrakt protein on the sites that were supposed to be highly conserved to other DEAD-box proteins were also analyzed. The cDNA library of Plasmodium falciparum (Plasmodium cynomolgi) was screened by the same gene cloning method, and a complete cDNA sequence of eIF-4A homologous protein was cloned, named CH1F.CH1F 1753 BP, which contains a complete reading frame of 1197bp. It is supposed to encode a protein consisting of 398 amino acids. The protein sequence of CH1F was searched and analyzed by BlastP, which suggested that it should be a eIF-4A homologous protein of DEAD-box family, and compared with many typical DEAD-box protein sequences by DXAStar, the results showed that it was better than other DEAD-box proteins. It is more consistent with eIF-4A or eIF-4A homologous proteins and has more similar domains. The fragment containing the complete reading frame was subcloned into the expression vector pET-28a (), and expressed in Escherichia coli DH5 伪. The size of the fusion protein was about 45kD. The fusion protein was purified, refolded and preliminarily identified. The results showed that the fusion protein had very low ATP activity, and its ATP activity did not seem to be dependent on the nucleic acid substrate. Three kinds of results are given for this test.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R346

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