猕猴抵抗素基因克隆及序列分析

Cloning and Analyzing the Sequence of Resistin Gene in Macaca mulatta

目的克隆猕猴抵抗素基因，并对其进行鉴定及序列分析。方法用猕猴腹部脂肪组织提取总RNA，采用RT—PCR进行抵抗素基因扩增，克隆至质粒载体，通过PCR和酶切鉴定后，进行测序，用DNAMAM软件进行序列分析，再用ORFfinder软件确定其编码区，并用BLAST软件比对。结果RT—PCR产物电泳检测得到了363bp目的条带，重组克隆质粒经PCR和XbaI与PstI双酶切鉴定均为阳性，测序结果经序列分析表明，其与人类抵抗素基因序列的同源性为96％，与大鼠的同源性为67％，与小鼠的同源性为65．7％，基于抵抗素基因的进化树结果表明猕猴与人类有较近的亲缘关系；ORFfinder软件的分析结果表明，编码猕猴抵抗素的氨基酸有108个，与人类抵抗素氨基酸的同源性为92％，仍然具有较高的同源性。结论成功克隆了猕猴抵抗素基因，可为下一步在猕猴中开展相关研究奠定前期基础和提供实验资料。

Objective To clone resistin gene of Macaca mulatta, its sequence has been identified and analyzed. Methods Total RNA was extracted from abdominal adipose tissue, and resistin gene was amplified by RT-PCR, cloned and inserted into vector. Sequence was identified by PCR and restriction enzyme digestion, and analyzed by DNAMAN software. The coding region was determined and compared by ORFfinder and BLAST software. Result Resistin gene （363 bp） was obtained by RT-PCR amplification and the recombinant cloning vector were positive identification by PCR and XbaI and PstI restriction enzyme digestion. Sequence analysis showed that the resistin gene homology was 96%, 67% and 65.7% with human, rat and mouse. Phylogenetic tree results showed that resistin gene of Macaca malutta has a close genetic relationship with human. The analysis results shows the resistin was coded to 108 amino acids and the homology was 92% with human. Conclusion The resistin gene of Macaca malutta was successfully cloned for laying a preliminary basis and providing experimental data in the related studies of resistin gene of Macaca malutta.