人牙髓细胞DPDP-2基因的克隆、测序及序列分析

Sequence analysis of DPDP-2 gene cloned from human dental pulp cells

目的:在人牙髓细胞中克隆DPDP-2基因,研究其可能的功能.方法:体外培养HDPC和人牙龈成纤维细胞(HGF),应用基于PCR的改良消减杂交技术构建HDPC和HGFcDNA消减文库,批量克隆HDPC和HGF的差异表达基因并测序,使用GenBank的BLAST对测序结果进行同源序列比较,并运用计算机软件PC/GENE6.60版对筛选到的DP-DP-2基因进行结构和功能分析.结果:DPDP-2基因序列全长810bp,没有一个完整的编码区.可与KIAA0265gene拼接成一个长6172bp的完整序列,编码一个含有83个氨基酸的疏水性蛋白,其二级结构由螺旋结构、卷曲结构和伸展部分所组成.它们可以通过α,β或α/β折叠而形成三级结构.抗原表位分析发现它可能有3种抗原决定簇结构,即Glu-Asn-Lys-Arg-Thr-Gly,Leu-Ile-Glu-Arg-Leu-Lys和Glu-Leu-Ala-Trp-Glu-Lys.该蛋白存在一种跨膜螺旋结构和线粒体运输肽结构.进一步的分泌信号分析结果也证实它是一种非分泌蛋白.经BLAST同源蛋白质搜索,在Non-redundantSwissProtse-quences和PDBproteindatabase中均未查询到同源蛋白质.结论:DPDP-2所编码的蛋白是一种跨膜蛋白,具有信号转导分子的结构特征,又具有CKⅡ的磷酸化位点,可能与细胞分裂、转录调节过程有关.

AIM： To study the function of DPDP-2, a new gene cloned from human dental pulp cell （HDPC）. METHODS： A modified PCR-based subtractive hybridization technique was used to establish a subtractive cDNA library. Genes differentially expressed in HPDC and human gingival fibroblasts （HGF） were cloned and sequenced. A new gene named DPDP-2 was cloned and structural and functional analyses were performed by PC/ GENE 6. 60 software. RESULTS： The fragment of DPDP-2 cloned from the present subtractive eDNA library was 810 bp, containing an incomplete coding region. However, after splicing with an EST sequence named KIAA0265, a 6172 bp complete eDNA sequence was achieved which encodes a 83aa hydrophobic protein with a secondary structure characterized by a helical conformation, an extended conformation and a coil conformation. The secondary structures could assemble a three-dimensional structure through folding of α, β or α/β sheets. Antigen epitope analysis implied three possible antigenic determinants： Glu-Asn-Lys-Arg- Thr-Gly, Leu-Ile-Glu-Arg-Leu-Lys and Glu-Leu-Ala-Trp-Glu- Lys. A transmembrane helical structure and a mitochondria trans- portation structure were also found. Signal peptide analysis further indicated that DPDP-2 was a non-secretory protein. No homogeneous protein was found in Non-redundant SwissProt sequences and PDB protein database. CONCLUSION： DPDP-2 encodes a transmembrane protein that has both signal transduction structural features and CKⅡ phosphorylation site, indicating its possible functional relationship with cell mitosis and transcription.