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目的建立稳定表达Bama小型猪细胞色素CYP3A22的HepG2细胞株。方法通过从Bama小型猪肝组织中提取tRNA、经RT-PCR得到Bama小型猪CYP3A22的基因,并将此基因克隆至亚克隆载体pMD18-T上得到重组质粒pMD-3A22;以测序确定的重组质粒pMD-3A22为模板,采用PCR扩增CYP3A22基因并在其3′添加组氨酸标签,扩增产物经BamHⅠ/XhoⅠ双酶切,将带有组氨酸标签的CYP3A22基因定向克隆至pcDNA3.1(+)中,测序表明,CYP3A22基因真核表达载体正确构建;将测序正确的CYP3A22基因通过脂质体转染至HepG2细胞,G418筛选10代,RT-PCR及Western blot分析,并用探针药物硝苯地平对重组细胞进行进行活性鉴定。结果与HepG2相比,HepG2-CYP3A22细胞株具有极显著的硝苯地平氧化活性。结论成功建立了稳定表达CYP3A22的HepG2细胞株,可用于相关的药物代谢研究。
Objective To establish a HepG2 cell line stably expressing Bama minipig cytochrome CYP3A22. Methods The gene of Bama minipig, CYP3A22, was obtained by RT-PCR from the liver tissue of Bama minipigs. The gene was cloned into the subcloning vector pMD18-T to obtain the recombinant plasmid pMD-3A22. The recombinant plasmid pMD-3A22 as a template, the CYP3A22 gene was amplified by PCR and histidine tag was added to the 3 ’end. The amplified product was double-digested with BamHⅠ / XhoⅠ, and the histidine-tagged CYP3A22 gene was cloned into pcDNA3.1 (+). Sequencing showed that the eukaryotic expression vector of CYP3A22 gene was correctly constructed. The correct sequencing of CYP3A22 gene was transfected into HepG2 cells by lipofectamine 2000. G418 was screened for 10 generations and analyzed by RT-PCR and Western blot. Nifedipine recombinant cells were identified. Results Compared with HepG2, HepG2-CYP3A22 cell line had extremely significant nifedipine oxidation activity. Conclusion The HepG2 cell line stably expressing CYP3A22 was successfully established and could be used for related drug metabolism research.