中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2011年
11期
1981-1982
,共2页
石永勇%洪庆雄%肖建斌%招伟贤%卢传坚%项红兵
石永勇%洪慶雄%肖建斌%招偉賢%盧傳堅%項紅兵
석영용%홍경웅%초건빈%초위현%로전견%항홍병
色氨酸羟化酶%RNA干扰%表达载体
色氨痠羥化酶%RNA榦擾%錶達載體
색안산간화매%RNA간우%표체재체
Tryptophan hydroxylase%RNA interfere%Expression vector
目的 构建携带色氨酸羟化酶(TPH)-2基因的小分子干扰RNA表达载体.方法 设计并合成shRNA TPH-2对应的两条互补的寡核苷酸链,pU6-CMV-GFP质粒经Age Ⅰ和EcoRⅠ双酶切与退火后的寡核苷酸连接,目的 质粒转化感受态细胞,对长出的克隆应用菌落聚合酶链反应(PCR)鉴定,再对PCR鉴定阳性的克隆进行测序和比对分析.结果 经PCR、酶切及测序证实,pU6-CMV-GFP-TPH-2 shRNA表达载体片段大小为332 bp,其中插入的片段序列和位点与预期完全一致.结论 实验成功构建pU6-CMV-GFP-TPH2 shRNA表达载体.
目的 構建攜帶色氨痠羥化酶(TPH)-2基因的小分子榦擾RNA錶達載體.方法 設計併閤成shRNA TPH-2對應的兩條互補的寡覈苷痠鏈,pU6-CMV-GFP質粒經Age Ⅰ和EcoRⅠ雙酶切與退火後的寡覈苷痠連接,目的 質粒轉化感受態細胞,對長齣的剋隆應用菌落聚閤酶鏈反應(PCR)鑒定,再對PCR鑒定暘性的剋隆進行測序和比對分析.結果 經PCR、酶切及測序證實,pU6-CMV-GFP-TPH-2 shRNA錶達載體片段大小為332 bp,其中插入的片段序列和位點與預期完全一緻.結論 實驗成功構建pU6-CMV-GFP-TPH2 shRNA錶達載體.
목적 구건휴대색안산간화매(TPH)-2기인적소분자간우RNA표체재체.방법 설계병합성shRNA TPH-2대응적량조호보적과핵감산련,pU6-CMV-GFP질립경Age Ⅰ화EcoRⅠ쌍매절여퇴화후적과핵감산련접,목적 질립전화감수태세포,대장출적극륭응용균락취합매련반응(PCR)감정,재대PCR감정양성적극륭진행측서화비대분석.결과 경PCR、매절급측서증실,pU6-CMV-GFP-TPH-2 shRNA표체재체편단대소위332 bp,기중삽입적편단서렬화위점여예기완전일치.결론 실험성공구건pU6-CMV-GFP-TPH2 shRNA표체재체.
Objective To construct and identify recombinant TPH-2-targeted small interfering RNA (siRNA) expression vector.Methods Oligonucleotide containing the small hairpin of TPH-2 was designed and synthesized,which was inserted into the pU6-CMV-GFP plasmid double digested by Age Ⅰ and EcoR Ⅰ.The aim plasmids were transformed into competent cells E.coli DH5α.The grown colonies were identified by colony polymerase chain reaction (PCR) and then the positive colonies were sequenced and aligned.Results The result of PCR and gene sequencing confirmed that the pU6-CMV-GFP-TPH-2 shRNA expression vector with 332 bp.Conclusion The expertment had been constructed successfully.
