摘要

摘要

肺是生物机体重要的呼吸器官，肺的主要结构由肺内导管部（支气管树）
和无数肺泡所组成。哺乳动物肺的发育通常描述为4个时期：假腺期、小管期、
原始肺泡期和肺泡期。其原始肺泡期和肺泡期与肺泡的形成关系最为密切。我
们之前通过抑制差减杂交筛选出一系列可能的肺泡发育相关调节基因，其中基
因P311同时特异性高表达于原始肺泡期和肺泡期，因此我们推测该基因表达的
蛋白P311在肺的发育中发挥着极为重要的作用。由于P311蛋白N端具有可与其他
蛋白相互作用的PEST结构域，因此我们希望通过对其结合蛋白的筛选与研究，
探知该基因在肺发育过程中的作用。我们利用酵母双杂交技术，构建小鼠肺组
织cDNA文库，并以融合Gal4 DNA结合域（DNA binding domain，BD）的P311
为诱饵蛋白筛选文库。通过将所得的432个酵母双杂交阳性克隆进行测序、排除
假阳性蛋白及回复验证，并选择其中可能对肺发育具有潜在功能的基因，利用
免疫共沉淀和双分子荧光互补等方法进行进一步验证。最终确定Galectin-1、
SP-C、Loxl-1、Tenascin-XB、Fibrillin-2、Peroxiredoxin-1和SPARC等7个P311结
合蛋白。进一步的研究发现，其中大部分基因在肺组织中具有有与P311相似的
表达时序性特征，而在细胞中的表达亦可被P311上调。因此，这些结合蛋白可
能与P311在肺发育中的作用密切相关，值得进一步深入研究。
关键词：肺泡 肺发育 P311 P311结合蛋白

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 摘要

Abstract

Lung is the critical respiratory organ of living body, which is made up of

bronchial tree and numerous alveoli. Pre- and postnatal lung development in
mammals have been described four stages: pseudoglandular, canalicular, saccular and
alveolar period. Saccular and alveolar periods are the critical stages of alveolar
formation. We used suppression subtractive hybridization to identify a series of genes
that may control alveolar generation. P311 was highly differentially expressed during
saccular and alveolar period, so we have identified P311 as a differentially expressed
gene that may play an important role in alveolar generation. Because P311 had a
PEST domain located at the N-terminus, witch can interact with other proteins, we
expected to find proteins interacting with P311, so as to discovery the role of P311 in
the development of lung. Using yeast two hybridization system, we constructed the
cDNA library of mouse lung, and used fusion of GAL4 binding domain and P311 as
the ”bait” protein to screen the library. Then we aligned the 432 positive clones’
sequence, and deleted false positive proteins. After retesting the interaction in yeast,
we chose some positive genes that might have potential function in lung development
to further verify by co-immunoprecipitation and bimolecular fluorescence
complementation (BiFC). Finally, we confirm Galectin-1, SP-C, Loxl-1, Tenascin-XB,
Fibrillin-2, Peroxiredoxin-1 and SPARC as proteins interacting with P311.
Furthermore, we found that most of those proteins had similar gene expression
feature with P311 during lung development, and could be over-expressed by P311.
Therefore, those proteins might be closely related to P311 in lung development.
Key Words: Lung alveolar development P311 P311 binding protein

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Abstract