人的新基因PCIA1的全长克隆和初步功能研究
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摘要
目的：2003年底，人类基因组测序计划已经完成，2004年在Nature杂志上公布了修正的人类基因组图谱，人类基因组计划已从结构基因组时代迈入功能基因组时代，未来主要任务就是定位人类基因，研究它们的生物学功能及基因间的相互作用等。最近研究发现人类总的基因数目为20，000-25，000个，比以前所估计的要少得多，但大多数基因功能未知。因此，克隆新基因并阐明这些基因的功能及其基因间的相互作用显得尤为重要。本研究在筛选新的靶分子时获得了一条人的新基因序列PCIA1，为尽快获得新基因的功能提示，需获取新基因的全长序列，进行初步的生物信息学分析，并进行初步的功能研究。
方法：本研究在重组cDNA表达文库的血清学分析(Serological analysis of recombinant cDNA expression library，SEREX)技术基础上，筛选到一个基因部分片段，生物信息学分析发现此基因为人的新基因的片段。我们采用RACE(Rapid Amplification of cDNA Ends)技术结合EST(Expressed Sequence Tags)序列拼接技术获得了新基因PCIA1的全长cDNA序列，用RT-PCR技术检测了该新基因的人的组织表达谱，以PCIA1和GFP融合蛋白及免疫组织化学检测PCIA1基因的亚细胞定位，并结合多种生物信息学手段对其可能的性质和功能进行初步的分析和预测。进而，为进一步研究该基因的功能，我们将PCIA1基因连接至原核表达载体pQE30，
Objectives: Completed in 2003, the Human Genome Project (HGP) identified all the approximately 20,000-25,000 genes in human DNA. These data mean that human whole genes are much smaller than we expected before, and a large amount of these genes are genes of unknown function. Our further work is to identify these genes and their function. In our previous study, we identified part of a novel human cDNA using SEREX technique. In order to study the possible function of this gene, full-length cDNA must be obtained and bioinformatic analysis as well as fundermental functional analysis should be performed.
Methods: We screened part of the gene sequence of a new gene used SEREX (Serological analysis of recombinant cDNA expression library) approach. The full-length cDNA of the novel gene was obtained by RACE technology and then identified by RT-PCR. Various bioinformatic methods were used to analyze and predict the feature and possible functions of the novel gene. The tissue expression of the novel gene was identified by RT-PCR and the subcellular localization was identified by GFP fused protein as well as immunohistochemistry. The prokaryotic protein and polyclonal antibody were prepared. We further cloned it into eukaryotic expression vector and
方法：本研究在重组cDNA表达文库的血清学分析(Serological analysis of recombinant cDNA expression library，SEREX)技术基础上，筛选到一个基因部分片段，生物信息学分析发现此基因为人的新基因的片段。我们采用RACE(Rapid Amplification of cDNA Ends)技术结合EST(Expressed Sequence Tags)序列拼接技术获得了新基因PCIA1的全长cDNA序列，用RT-PCR技术检测了该新基因的人的组织表达谱，以PCIA1和GFP融合蛋白及免疫组织化学检测PCIA1基因的亚细胞定位，并结合多种生物信息学手段对其可能的性质和功能进行初步的分析和预测。进而，为进一步研究该基因的功能，我们将PCIA1基因连接至原核表达载体pQE30，
Objectives: Completed in 2003, the Human Genome Project (HGP) identified all the approximately 20,000-25,000 genes in human DNA. These data mean that human whole genes are much smaller than we expected before, and a large amount of these genes are genes of unknown function. Our further work is to identify these genes and their function. In our previous study, we identified part of a novel human cDNA using SEREX technique. In order to study the possible function of this gene, full-length cDNA must be obtained and bioinformatic analysis as well as fundermental functional analysis should be performed.
Methods: We screened part of the gene sequence of a new gene used SEREX (Serological analysis of recombinant cDNA expression library) approach. The full-length cDNA of the novel gene was obtained by RACE technology and then identified by RT-PCR. Various bioinformatic methods were used to analyze and predict the feature and possible functions of the novel gene. The tissue expression of the novel gene was identified by RT-PCR and the subcellular localization was identified by GFP fused protein as well as immunohistochemistry. The prokaryotic protein and polyclonal antibody were prepared. We further cloned it into eukaryotic expression vector and
引文
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3. Venter JC, Adams MD, Myers EW, et al. The sequence of the human genome. Science, 2001; 291:1304-1351.
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7. National Research Council. Mapping and Sequencing the Human Genome. National Academy Press, Washington DC, 1988.
8. Aparicio S, Chapman J, Stupka E, et al. Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes. Science, 2002; 297:1301-1310
9. Zhang MQ. Computational prediction of eukaryotic proteincoding genes. Nature Rev Genet, 2002; 3: 698-709.
10. Sachidanandam R,Weissman D,Schmidt SC, et al. A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature, 2001; 409: 928-933.
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19. Rehm BH. Bioinformatic tools for DNA/protein sequence analysis, functional assignment of genes and protein classification. Appl Microbiol Biotechnol, 2001; 57: 579-592.
20. Leung AK, Andersen JS, Mann M, et al. Bioinformatic analysis of the nucleolus. Biochem J, 2003; 376: 553-569.
21. Lynn DJ, Higgs R, Gaines S, et al. Bioinformatic discovery and initial characterisation of nine novel antimicrobial peptide genes in the chicken. Immunogenetics. 2004; 56: 170-177.
22. Breton G. Danyluk J, Charron JB, et al. Expression profiling and bioinformatic analyses of a novel stress-regulated multispanning transmembrane protein family from cereals and Arabidopsis. Plant Physiol, 2003; 132: 64-74.
23. Guo Y, Cheng H, Huang X, et al. Gene structure, multiple alternative splicing, and expression in gonads of zebrafish Dmrt 1. Biochern Biophys Res Commun, 2005; 330: 950-957.
24. RS Kamath, AG Fraser, Y Dong, et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature, 2003; 421: 231-237
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26. Burge, C., Karlin, S. Prediction of complete gene structures in human genomic DNA. J Mol, 1997; 268, 78-94.
27. Horton P, Nakai K. Better prediction of protein cellular localization sites with the k newest neighbor classifier. Proc. Int. Conf Intellig. Syst. Mol. Biol, 1997; 5: 147-152.
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29. Liu RS, Huang H, Yang Q, et al. Purification of alpha-sarcin and an antiftmgal protein from mold (Aspergillus giganteus) by chitin affinity chromatography. Protein Expr Purif. 2002; 25: 50-58.
30. Li L, Wang JX, Zhao XF, et al. High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris. Protein Expr Purif, 2005; 39: 144-151.
31. Feng J, Qing Li F, Li Q, et al. Expression and purification of Rhizobium leguminosarum NodD. Protein Expr Purif, 2002; 26: 321-328.
32. Yang T, Jiang Y, Jiangye Chen. The identification and subcellular localization of human MRK. Biomol Eng, 2002; 19: 1-4.
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