【作者】 萨仁高娃；

【导师】 张士璀；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2007， 博士

【摘要】 随着基因组测序的迅猛发展,越来越多的含有DUF985保守域的理论蛋白基因被提交到数据库。这些基因不和任何一种已知蛋白或已知功能的蛋白相关。我们实验首次表明文昌鱼中编码DUF985保守域的蛋白具有磷酸葡萄糖异构酶活性,是其家族的一个新的成员。我们成功地将BbDUF985在大肠杆菌表达系统和酵母表达系统中得到表达;酵母表达系统中表达的重组蛋白活性高达440 U/mg,远远高于大肠杆菌表达系统表达的重组蛋白。组织切片的原位杂交实验和免疫组化实验证明,BbDUF985的表达具有组织特异性,其在肝盲蘘和卵巢中的表达水平较高。在转染了表达质粒pEGFP-N1/BbDUF985的CHO细胞中,融合蛋白定位于胞质中,表明BbDUF985是一个细胞质蛋白。另一方面,Western blotting实验证明BbDUF985也可被分泌到文昌鱼的体液当中,BbDUF985的细胞质和细胞外的并存这一事实似乎表明其与已知的PGI一样,可能具有多种生物学功能。本项研究成功地描述了含有DUF985保守域的理论蛋白的表达、生物功能和细胞定位,为近一步了解这种理论蛋白的生化和生理功能奠定了基础。我们还对文昌鱼含有phd-like-VIAF保守域的理论蛋白基因进行了鉴定,许多含有phd-like-VIAF保守域的理论蛋白基因被提交到数据,这些基因和已知蛋白phosducin-like 3或未知功能的蛋白相关。我们成功地将Amphi-phdL在大肠杆菌表达系统中得到表达。组织切片的原位杂交实验和免疫组化实验证明,Amphi-phdL的表达具有组织特异性,其在肝盲蘘和卵巢中的表达水平较高。在转染了表达质粒pEGFP-N1/Amphi-phdL的CHO细胞中,融合蛋白定位于胞质中,表明Amphi-phdL是一个细胞质蛋白。Western blotting实验证明Amphi-phdL在文昌鱼的组织液和体液都存在,至于Amphi-phdL的功能正在进行鉴定中。更多还原

【Abstract】 The progress in genome sequencing has led to an increasing submission of uncharacterized hypothetical genes with the domain DUF985 in GenBank, and none of these genes is related to a known protein or a protein of known function. In this study we have shown conclusively that the DUF985-containing hypothetical gene from amphioxus Branchiostoma belcheri, BbDUF985, encodes a novel member of phosphoglucose imomerase (PGI). BbDUF985 is expressed in vitro in both Escherichia coli and Pichia pastoris, and the specific activity of the recombinant protein expressed in the eukaryotic system is approximately 440 U/mg of protein, which is significantly higher than that of BbDUF985 expressed in the prokaryotic system. Tissue-section in situ hybridization as well as immunohistochemistry demonstrate that BbDUF985 is expressed in vivo in a tissue-specific manner, with most abundant levels in the hepatic caecum and ovary. In the CHO cells transfected with the expression plasmid pEGFP-N1/BbDUF985, the fusion protein is targeted in the cytoplasm of CHO cells, suggesting that BbDUF985 is a cytosolic protein. On the other hand, western blotting indicates that BbDUF985 can also be secreted into the humoral fluids of amphioxus. The presence of both cytosolic and extracellular BbDUF985 suggests it may be like PGI playing a multiple function. These are the first report describing the successful expression, biochemical characterization and cellular localization of a hypothetical protein with the domain DUF985, providing a framework for further understanding the biochemical properties and physiological function of DUF985-domain-containing hypothetical proteins.We also report the identification of a hypothetical gene with the conserved motif Phd-like-VIAF from amphioxus Branchiostoma belcheri. Many genes with the domain Phd-like-VIAF have been deposited in GenBank, and these genes are related to a known protein, phosducin-like 3, their function remains uncertain. In this study Amphi-phdL is expressed in vitro in Escherichia coli. Tissue-section in situ hybridization as well as immunohistochemistry demonstrate that Amphi-phdL is expressed in vivo in a tissue-specific manner, with most abundant levels in the hepatic caecum and ovary. In the CHO cells transfected with the expression plasmid pEGFP-N1/Amphi-phdL, the fusion protein is targeted in the cytoplasm of CHO cells, suggesting that Amphi-phdL is a cytosolic protein. Western blotting indicates that Amphi-phdL is present in both tissue extract and humoral fluids of amphioxus. The study of function of Amphi-phdL is now under way.更多还原