摘要
Prohibitin作为一种高度保守的蛋白广泛分布于细菌、原虫、酵母等多种生物细胞中,主要定位于细胞膜、线粒体内膜、细胞核中,参与细胞增殖、分化、衰老、凋亡等多种生物学进程。由于其结构进化上的保守性,功能的多样性,正逐渐引起广泛的关注。
本实验对日本七鳃鳗(Lampetra japonica)血液白细胞cDNA文库中2123条有效EST序列进行分析,拼接获得一条650bp核酸序列,以日本七鳃鳗外周血白细胞RNA为模板进行PCR扩增。通过cDNA3’末端快速扩增反应、CDS区的克隆最终获得全长为828bp的目的基因。
对克隆获得的目的基因进行同源搜索,搜索的结果显示我们克隆所得为Prohibitin家族中Prohibitin1基因。采用生物信息学的方法和工具对日本七鳃鳗中Prohibitin1蛋白相应的氨基酸序列的理化性质、结构特征、功能及系统演化关系等进行预测和分析,以期为该基因的进一步研究提供一定的理论依据。
将目的基因Prohibitin1与表达载体pET-32a酶切后连接。构建原核表达载体pET-32a-PHB,将其转化至宿主菌BL21中,并通过IPTG诱导使其表达。并用小鼠单克隆抗体进行免疫印迹分析对表达所得蛋白加以鉴定。
半定量的方法研究Prohibitin1在日本七鳃鳗各个组织中的特异性表达情况,并通过其在不同组织中的表达差异,推断Prohibitin蛋白是非分泌型蛋白,并有可能参与了类B淋巴细胞介导的信号通路。为进一步证实Prohibitin1与日本七鳃鳗中可能存在的免疫调节的关系,运用RT-PCR的方法检测不同组织中PHA刺激前后Prohibitin1表达量的变化。结果表明PHA刺激后白细胞、心、鳃中Prohibitin1含量有较大幅度上调。这以结果也间接的证明了在七鳃鳗这种古老的脊椎动物中存在有类B淋巴细胞调节的可能。
Prohibitin ( PHB) is a highly conserved protein and ubiquitously distributed in different cellular compartments of various organisms.Various roles have been proposed for prohibitin involving multibiological processes such as cell cycle regulation,cell proliferation,cell aging and cell apoptosis,etc. Due to its highly conserved structure in evolution and diverse functions, PHB has gradually caused widespread concern.
The purpose of this study is to obtain the full-length cDNA encoding the Prohibitin protein. According to the information from cDNA library and the primary analysis of expressed sequence tags, In final, we also cloned a Prohibitin gene, which contained a 828bp open reading frame, and encoded 275 amino acids residues, from the leukopenia of lamprey, Lampetra japonica, which is one of the most primitive jawless vertebrates still living today.
Then the cDNA was directionally cloned into the pET32a and transformed intoE. coli BL21, and expression was induced by IPTG. After the identification by SDS-PAGE, the recombinant proteins immunoblotting analysis to detecte the rcombinant protein . We take gene engineering mothod and the recombinant proteins were taken prokaryotic expression .We analysis the protein with tools of bioinformatics and check the specific expression in the different organization and Biological Activity of Prohibitin.
We detected specific expression of the different organization using the method examination of reverse transcription PCR.The result expresses that this gene is an necessary important gene in most of organizations.The Prohibitin mRNA was also detected in lamprey gut, kidney, and leukocyte, but absent in lamprey buccal gland Prohibitin Speculated may be involved in a class B-lymphocyte-mediated signaling pathway.
Design experiments,Using of RT-PCR method to detect different tissues before and after the PHA to stimulate the expression of the changes in Prohibitin1. The results show that PHA-stimulated leukocytes, heart, gill, their content in a relatively large increase This is also an indirect proof of results in a lamprey in this ancient vertebrate class B lymphocytes regulate the existence of the possible.
引文
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