摘要
目的克隆鸡破骨细胞分化因子(chODF)基因,并对其进行序列测定和分析。方法根据GeneBank报道的预测的鸡ODF基因(GeneBank登录号,XM425625)序列,设计引物。从鸡胚额骨分离培养的成骨细胞中提取总RNA,逆转录合成cDNA,用SOE-PCR的方法扩增鸡ODF基因全序列。将获得的预期大小的PCR产物插入pMD18-T克隆载体,转化E.coli感受态细胞,提取质粒,进行双酶切鉴定,对含有目的片段的克隆进行序列测定。结果从成骨细胞中成功地克隆鸡ODF基因片段,其结果与GeneBank上报道的预测序列具有很高的相似性,达到99.3%,与人、小鼠、犬的一致性分别为55.4%、52%和55.7%,提示该基因在进化过程的保守。ODF基因全长1203bp,其阅读框起始于第一个氨基酸,终止于最后一个氨基酸,无信号肽,共编码400个氨基酸。结论ODF作为惟一能够直接诱导破骨细胞分化和功能的细胞因子,对它的深入研究可为探讨骨质疏松等代谢性骨病的发病机制及预防、治疗开辟出广阔的领域。
Objective To clone and sequence the gene encoding in chicken osteoclast differentiation factor （ODF）. Methods The primers were designed according to the gene sequence of chicken ODF sequence submitted by C, eneBank（ GeneBank submission number, XM425625 ）. Total RNA was isolated from osteoblast and the cDNA was synthesized by reverse transcription. The objective fragment was amplified by SOE-PCR and cloned into PMD18-T vector. E. coli was transformed with the recombinant plasmid and positive clones were selected. The inserted DNA was verified by enzyme digestion and DNA sequencing. Results A 1203 bp length ODF gene was successfully cloned and had highly similarity to the sequence reported in the C, eneBank, including the open reading frame（ORF） from 1 to 1203, encoding 400 amino acid. The sequence reported here had 99.3% identity to the documented sequence, and it also had 55.4%, 52% and 55.7% identity to human, mouse and dog respectively, which suggested that the ODF gene had high conservation in evolution. Conclusions As ODF gene is the cytokines which involved in the differentiation and activation of osteoclasts, the research of ODF gene is important to explore the mechanism and prevention of osteoporosis in layer hens.
出处
《中国骨质疏松杂志》
CAS
CSCD
2007年第10期701-703,685,共4页
Chinese Journal of Osteoporosis
基金
国家自然科学基金资助项目(30671546)
江苏省自然科学基金资助项目(BK2004099)
高校博士点基金资助项目(20040307036)
