中国人兽共患病学报
中國人獸共患病學報
중국인수공환병학보
CHINESE JOURNAL OF ZOONOSES
2009年
7期
640-644,649
,共6页
薛正楷%魏泓%商海涛%叶彬
薛正楷%魏泓%商海濤%葉彬
설정해%위홍%상해도%협빈
巴马小型猪%CYP3A29同工酶%HepG2%逆转录酶PCR%Western-blot%硝苯地平
巴馬小型豬%CYP3A29同工酶%HepG2%逆轉錄酶PCR%Western-blot%硝苯地平
파마소형저%CYP3A29동공매%HepG2%역전록매PCR%Western-blot%초분지평
Bama miniature pig%CYP3A29 isoenzyme%HepG2%RT-PCR%Western-blot%nifedipine
目的 建立稳定表达Bama小型猪CYP3A29的HepG2细胞株.方法 通过总RNA提取、经RT-PCR得到Bama小型猪CYP3A29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-3A29;以测序正确的重组质粒pMD-3A29为模板,采用PCR扩增CYP3A29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CYP3A29基因定向克隆至pcDNA3.1(+)中,并测序验证;将测序正确的CYP3A29基因通过脂质体转染至HepG2细胞,G418筛选10代,经RT-PCR及Western-blot分析及探针药物硝苯地平对重组细胞进行活性鉴定.结果 与HepG2相比,HepG2-CYP3A29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CYP3A29的HepG2细胞株,可用于相关药物代谢研究.
目的 建立穩定錶達Bama小型豬CYP3A29的HepG2細胞株.方法 通過總RNA提取、經RT-PCR得到Bama小型豬CYP3A29的基因,併將此基因剋隆至亞剋隆載體PMD18-T上得到重組質粒pMD-3A29;以測序正確的重組質粒pMD-3A29為模闆,採用PCR擴增CYP3A29基因併在其3′添加組氨痠標籤,擴增產物經 BamHI/XhoI雙酶切,將帶有組氨痠標籤的CYP3A29基因定嚮剋隆至pcDNA3.1(+)中,併測序驗證;將測序正確的CYP3A29基因通過脂質體轉染至HepG2細胞,G418篩選10代,經RT-PCR及Western-blot分析及探針藥物硝苯地平對重組細胞進行活性鑒定.結果 與HepG2相比,HepG2-CYP3A29細胞株具有極顯著的硝苯地平氧化活性.結論 成功建立瞭穩定錶達CYP3A29的HepG2細胞株,可用于相關藥物代謝研究.
목적 건립은정표체Bama소형저CYP3A29적HepG2세포주.방법 통과총RNA제취、경RT-PCR득도Bama소형저CYP3A29적기인,병장차기인극륭지아극륭재체PMD18-T상득도중조질립pMD-3A29;이측서정학적중조질립pMD-3A29위모판,채용PCR확증CYP3A29기인병재기3′첨가조안산표첨,확증산물경 BamHI/XhoI쌍매절,장대유조안산표첨적CYP3A29기인정향극륭지pcDNA3.1(+)중,병측서험증;장측서정학적CYP3A29기인통과지질체전염지HepG2세포,G418사선10대,경RT-PCR급Western-blot분석급탐침약물초분지평대중조세포진행활성감정.결과 여HepG2상비,HepG2-CYP3A29세포주구유겁현저적초분지평양화활성.결론 성공건립료은정표체CYP3A29적HepG2세포주,가용우상관약물대사연구.
To establish a HepG2 cell line ,which stably expressing CYP3A29 of Bama miniature pig in order to evaluate the drug metabolic characteristics of this isoenzyme,the gene for this system was obtained through total RNA extraction and RT-PCR assay.the gene was subcloned into plasmid PMD18-T,designated as pMD-CYP3A29.This gene was then amplified by PCR, and cloned into eukaryotic expression vector pcDNA3.1(+),and the recombinant plasmid was designated as pcDNA-CYP3A29. Sequencing was used to confirmed the correctness of the gene of this gene.the expressed gene was then transfected into HepG2 cells by lipid-media transfection and the transformants were screened by G418 for 10 generations ;the expression of CYP3A29 was identified by RT-PCR、West-blot and the metabolic activity of the transformant HepG2-CYP3A29 was verified by nifedipine oxidation.In comparison with HepG2, the transformant HepG2-CYP3A29 showed remarkable oxidative activity.It is apparent that the cell line stably expressing CYP3A29 isoenzyme was successfully established, and it may be used for the metabolic study of related drugs.
