| 卢康荣,白 静,张嘉宁,顾为望,黄黎珍,王万山.实验用西藏小型猪CYP3A基因的克隆及序列分析[J].,2019,19(7):1218-1221 |
| 实验用西藏小型猪CYP3A基因的克隆及序列分析 |
| Cloning and Sequence Analysis of CYP3A Gene in Tibet Minipig |
| 投稿时间：2018-08-07 修订日期：2018-08-31 |
| DOI：10.13241/j.cnki.pmb.2019.07.004 |
| 中文关键词: 细胞色素P450酶 西藏小型猪 克隆 序列分析 |
| 英文关键词: Cytochrome P450 enzyme Tibet minipig Clone Sequence analysis |
| 基金项目:广东省科技计划项目（2011B060300028; 2012B040304010） |
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| 中文摘要: |
| 摘要 目的：克隆西藏小型猪CYP3A基因并与人及其它小型猪品种进行序列比对分析。方法：根据Genebank数据库设计引物，取西藏小型猪新鲜肝脏组织，Trizol法提取肝脏总RNA，应用RT-PCR反转获得肝脏cDNA。分别扩增基因CYP3A46、CYP3A22、CYP3A29及CYP3A39，PCR片段回收后分别连接pMD-18T载体，获得重组质粒pT-CYP3A，阳性重组克隆送测序。采用NCBI Blast及vector NTI软件进行序列比对分析。结果：CYP3A46、CYP3A22、CYP3A29及CYP3A39基因编码序列全长1512 bp，编码503个氨基酸，与人CYP3A4相同。其中CYP3A22和CYP3A39与NCBI记录巴马小型猪序列相同；CYP3A46与巴马小型猪CYP3A46(NM_001134824.1)有6个位点碱基不同。CYP3A29与巴马小型猪CYP3A46(EU918131.1)亦有6个位点碱基不同。对来自2个母本后代猪的基因经过PCR扩增后测序发现CYP3A46、CYP3A29序列完全一致。结论：成功克隆西藏小型猪CYP3A的4个基因，其中CYP3A46与人CYP3A4的序列相似性最高。所得CYP3A46与CYP3A29序列可能为西藏小型猪独有。 |
| 英文摘要: |
| ABSTRACT Objective: Cloning of CYP3A from laboratory Tibet minipig and sequence comparision with human and other minipig breeds. Methods: Primers were synthesized according to Genebank data. Liver cDNA was gained by RT-PCR methods from total RNA, which was extracted from fresh Tibet minipig liver tissue, CYP3A46, CYP3A22, CYP3A29 and CYP3A39 were amplified respectively, PCR fragments were recovered and cloned into vector pMD18-T, and recombinant plasmids were obtained, the positive clones were se- quenced. NCBI Blast and vector NTI software were employed to analyze the sequences. Results: Full length of the coding sequence of CYP3A46, CYP3A22, CYP3A29 and CYP3A39 is 1512bp, coding 503 amino acids, same as human CYP3A4, of which CYP3A22 and CYP3A39 sequences were same as the NCBI-recorded Bama minipig. Different bases in 6 sites were found in CYP3A46 when compared to Bama minipigCYP3A46 (NM_001134824.1), and this was also found in CYP3A29 when compared to Bama minipig CYP3A46(EU918131.1). CYP3A46, CYP3A29 sequences of all the offspring coming from 2 female parent were in full accord. Conclusion: 4 genes of Tibet minipig CYP3A were successfully cloned, of which CYP3A46 had the highest sequence similarity to human CYP3A4. CYP3A46 and CYP3A29 sequences may be unique to Tibet minipigs. |
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