小型猪肝脏细胞色素P450基因CYP3A29克隆与序列分析

Cloning of CYP3A29 coding region from liver of miniature pigs and sequencing analysis

设计引物通过RT-PCR扩增巴马香猪和贵州小型猪香猪药物代谢关键P450基因CYP3A29基因的全长编码区,纯化并克隆入pMD 18-T载体,通过序列测定和比对分析其序列变异情况。17头小型猪共发现CYP3A29基因的5个单核苷酸变异位点,均表现为同义替换,对编码的氨基酸序列无影响;猪CYP3A29的单核苷酸变异和品系相关,在不同品系中可表现出品系特有的单核苷酸变异;贵州小型香猪G3 a2902发现1个可变剪接,缺失89 bp并产生移码,改变编码蛋白。本研究克隆的巴马香猪和贵州小型香猪CYP3A29基因及其序列变异情况,可为其作为药物代谢模型的应用提供技术资料。

Bama and Guizhou miniature pigs are excellent breed resources, and could be good model animals for drug metabolism. But study on the cytochrome P450 gene of them is lack. In the present study, full -length coding region of CYP3A29 was amplified from liver of Bama and Guizhou miniature pigs by RT-PCR, cloned into pMD18-T vector and sequenced. Sequence variations were analyzed by alignment. Five single nucleotide substitutions were detected from 12 Bama and 5 Guizhou miniature pigs, and all of them were synonymous substitution and no changes happened for putative amino acid sequence. Single nucleotide substitutions of CYP3A29 were associated with breeds, and distinct variations could be found in different pig breeds. One alternative spliced variant was detected from Guizhou miniature pig G3a2902. A 89bp fragment were deleted and a frame shift was produced, and putative amino acid sequence was changed. Sequence variations of CYP3A29 detected from livers of Bama and Guizhou miniature pigs could provide valuable information for drug metabolism in animal models.