膜联蛋白家族新成员的结构，功能和进化

发布时间：2018-11-28 13:56

【摘要】：Annexin B2(Anx B2)和Annexin B2(Anx B3)是新克隆出的两个非脊椎动物的膜联蛋白家族新成员。它的结构、功能和进化的分析研究,对与全面了解膜联蛋白家族的生物学功能以及进化具有重要意义。膜联蛋白家族是一类钙磷脂结合蛋白超家族,存在于大多数真核生物细胞内,有着十分广泛且重要的生理功能,如参与细胞骨架运动、调节细胞生长、形成膜离子通道、参与细胞信号转导等。这类蛋白结构上具有明显特征:C端保守区大多由α螺旋组成,其中含有主要的Ca~(2+)磷脂结合活性位点;N端为可变区,不同家族成员N端序列组成及长度各不相同。 Anx B2和Anx B3是我们用简并引物-PCR方法从猪囊尾蚴中克隆得到的新基因。将其核酸和氨基酸序列进行同源比较和分析,确认它们是两个新的Annexin家族成员。作为扁形动物门发现的Annexin家族成员,研究Anx B2和Anx B3不仅可以了解其对猪囊尾蚴寄生、移行、生长、发育等生命活动的重要意义,为预防和治疗该类疾病提供新的途径,而且可以为整个Annexin家族的研究提供有意义的遗传及分子进化学资料。因此我们构建了Anx B2和Anx B3的融合表达载体,在大肠杆菌中实现了高表达,利用亲和层析及离子交换层析技术纯化蛋白;采用计算机同源模建、缺失体的构建研究Anx B2和Anx B3结构与功能的关系;并对它们的抗原性和抗凝血功能等方法研究了Anx B2和Anx B3的主要生物学活性。同时与Anx B1(1997年由本教研室发现)的部分功能进行比较。本研究主要结果如下: 1.用简并引物 -PCR方法从囊尾蚴中克隆出Anx B2和Anx B3两个新基因,Anx B2和Anx B3的cDNA分别含有1065和910 bp开放阅读框,编码355、310个氨基酸,计算分子量为38682.05和35874.19 Dalton。 2.通过Blast同源性检索,表明Anx B2和Anx B3与Annexin家族成员高度同源;它具有4个典型的Annexin重复保守结构域:二级结构预测Anx B2和Anx B3均以α螺旋为主,无明显跨膜区,无信号肽,提示其为胞浆蛋白,这些与Annexin特征相符。将Anx B2和Anx B3编码序列克隆至原核表达载体pGEX-5T,亲和层析纯化后得到其融合蛋白。经N端测序证明与预测序列完全一致。根据以上的结构特点表明Anx B2和Anx B3属于Annexin家族,又由于它们N端与现有Annexin家族成
[Abstract]:Annexin B2 (Anx B2) and Annexin B2 (Anx B3) are two newly cloned members of the annexin family of non-vertebrate animals. The analysis and study of its structure, function and evolution are of great significance in understanding the biological function and evolution of integrin family. As a kind of calcium phospholipid binding protein superfamily, the membrane binding protein family exists in most eukaryotic cells and has a wide range of important physiological functions, such as participating in the cytoskeleton movement, regulating cell growth and forming membrane ion channels. Participate in cell signal transduction. The conserved region of C terminal is composed of 伪 helix, which contains the main Ca~ (2) phospholipid binding activity site, and the N terminal is variable region, and the N-terminal sequence composition and length of different family members are different. Anx B2 and Anx B 3 were cloned from cysticercus cellulosae by degenerate primer PCR method. The nucleic acid and amino acid sequences were compared and analyzed, and they were identified as two new Annexin family members. As a member of the Annexin family found in the phylum oblate, the study of Anx B2 and Anx B3 can not only understand the importance of Anx B2 and Anx B3 in the parasitism, migration, growth and development of cysticercus cellulosae, but also provide a new way for the prevention and treatment of these diseases. And it can provide significant genetic and molecular chemical data for the study of the whole Annexin family. Therefore, we constructed the fusion expression vector of Anx B2 and Anx B3, and achieved high expression in Escherichia coli. The protein was purified by affinity chromatography and ion exchange chromatography. The relationship between the structure and function of Anx B2 and Anx B 3 was studied by computer homologous modeling and deletion, and the main biological activities of Anx B2 and Anx B 3 were studied by means of their antigenicity and anticoagulant function. At the same time, it is compared with some functions of Anx B1 (discovered by our teaching and research department in 1997). The main results of this study are as follows: 1. Two new genes, Anx B2 and Anx B3, were cloned from cysticercaria by degenerate primer PCR method. The cDNA of Anx B2 and Anx B3 contained 1065 and 910 bp open reading frames, respectively, encoding 355310 amino acids. The calculated molecular weights were 38682.05 and 35874.19 Dalton., respectively. 2. Blast homology search showed that Anx B2 and Anx B3 were highly homologous to Annexin family members. It has four typical Annexin repeat conserved domains: the secondary structure predicts that both Anx B2 and Anx B 3 are 伪 helix, with no obvious transmembrane region and no signal peptide, indicating that they are cytoplasmic proteins, which are consistent with the characteristics of Annexin. The encoding sequences of Anx B2 and Anx B 3 were cloned into prokaryotic expression vector pGEX-5T, affinity chromatography to obtain the fusion protein. It was proved by N-terminal sequencing that the predicted sequence was in good agreement with the predicted sequence. According to the structural characteristics above, Anx B2 and Anx B3 belong to the Annexin family, and because of their N-terminal, they form the existing Annexin family.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：Q51

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