【作者】 何永刚；

【导师】 王勤； 谢幼华；

【作者基本信息】 兰州大学 ， 生物物理， 2006， 硕士

【摘要】 乙肝病毒表面蛋白PreS区在病毒成熟、装配以及感染人肝细胞等过程中发挥关键的作用,因此筛选特异性结合PreS区的短肽具有重要的理论和应用价值,将可能为寻找肝细胞表面病毒相关的结合蛋白或受体蛋白提供有用的序列和结构信息,并为研制抗乙肝病毒感染的新药提供前导分子。 基于噬菌体表面展示随机肽文库的高通量筛选是目前寻找靶蛋白结合短肽的优选策略之一。本课题中,我们构建了M13噬菌体pⅢ蛋白融合的表面展示随机肽文库。同时,将PreS1～65区融合于麦芽糖结合蛋白结合域,在大肠杆菌中进行表达,获得了可溶性和稳定性良好的融合蛋白（MBP-pres1～65）。应用亲和层析纯化的MBP-pres1～65对随机肽文库进行亲和筛选,从线形随机5肽文库中获得了一组特异性结合PreS的噬菌体。 测定噬菌体表面展示的短肽序列,对其中可能具有较高PreS结合亲和力的代表性短肽做了体外结合PreS的研究,证明这些短肽能够特异性结合PreS区。更为重要的是,这些短肽主要结合于PreS1的残基21-47区段,该区段被认为是乙肝病毒（HBV, Hepatitis B virus）附着于肝细胞质膜表面所必需的位点。上述结果提示这些短肽具有相似的PreS结合性质。对这些短肽序列的比对并结合突变分析推测出其中与PreS结合相关的保守氨基酸序列可能为-W₁T₂X₃W₄W_5-。体外结合实验表明该保守序列短肽能够结合PreS区。 该保守序列可能存在于结合PreS的细胞蛋白中。应用该序列在GeneBank中比对,发现数个细胞蛋白中含有该特征序列。对这些蛋白空间结构性质的分析提示脂蛋白脂酶（LPL）可能结合PreS。 总之,通过本项研究,1)获得了特异性结合乙肝病毒PreS区的短肽,这些短肽可能作为阻断乙肝病毒感染宿主肝细胞的抑制分子;2)发现脂蛋白脂酶能够结合乙肝病毒颗粒,这支持了本组邓强博士应用M13噬菌体pⅧ蛋白融合的表面展示随机12肽肽文库结合thio-PreS和CBD-PreS的结果,说明脂蛋白脂酶可能是一种新的乙肝病毒受体候选蛋白（1）。更多还原

【Abstract】 The PreS domain of the surface protein of hepatitis B virus (HBV) plays a key role in virus maturation, assembly and infection. Therefore, specific PreS-interacting peptide will be both a valuable tool for the study of these processes and a potential therapeutics against viral infection.Presently, high-throughput screening for target-interacting peptides with phage display is one of the most efficient methods to obtain lead molecules. In the current study, M13 pⅢ based phage display random peptide libraries were constructed. The PreS1~65 domain was produced in E.coli as a fusion protein with maltose binding protein(MBP). Then, MBP fusion protein was purified by affinity chromatography and displayed an ideal solubility and stability. The fusion protein was used as target protein to screen phage display random peptide libraries for PreS-interacting peptides.Several specific PreS-interacting phages were selected. The sequences of the peptides encoded in these phages were determined. In vitro binding assays using some of these peptides indicates that they can interact with the PreS domain specifically. Importantly, these peptides interact with the aa21-47 region of PreS1, a site thought to be necessary for HBV particles to attach to host hepatocytes. The results also indicate that these peptides interact with an identical region of PreS, which makes it possible to align their sequences to deduce a common or consensus sequence that is required for the interaction with the PreS domain. A consensus sequence -W1T2X3W4W5- is deduced from the sequence alignment. Indeed, the deduced consensus peptide can also interact with the PreS domain.It is possible that the consensus sequence also resides in PreS-interacting cellular proteins. By blasting the consensus sequence in GenBank, a few cellular proteins bearing this motif were found. A close analysis of the structures of these proteins suggest that one protein, the lipoprotein lipase may interact with the PreS domain.In summary, in the present study, 1) several specific PreS-interacting peptides have been selected. These peptides may serve as inhibitors to block viral attachment to host hepatocytes;2)we find that the lipoprotein lipase(LPL) is demonstrated to interact with HBV particles. This result may support the result of Dr Deng Qiang who is oneof our group ,which show that the lipoprotein lipase was demonstrated to interact with HBV particles from constructed M13 pVIII based phage display random peptide libraries and used thio-PreS and CBD-PreS fusion proteins as target proteins to screen phage display random peptide libraries for PreS-interacting peptides, suggesting that it is could be a new candidate for HBV cellular receptor.更多还原