中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
4期
838-839
,共2页
脂肪酰胺水解酶%重组质粒%RNA干扰
脂肪酰胺水解酶%重組質粒%RNA榦擾
지방선알수해매%중조질립%RNA간우
Fatty acid amide hydrolase%Recombinant plasmid%RNA interference
目的 构建携带脂肪酰胺水解酶(FAAH)基因的小分子干扰RNA表达载体.方法 设计合成短发卡RNA(shRNA) FAAH对应的两条互补的寡核苷酸链,mU6-MCS-Ubi-EGFP质粒经HpaⅠ和XhoⅠ进行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,对克隆的菌落行聚合酶链反应鉴定,再对聚合酶链反应(PCR)鉴定阳性的克隆进行测序,测序正确的重组质粒转染293细胞,同源重组产生Lentivirus-FAAH-shRNA并测定病毒滴度.结果 经测序证实mU6-FAAH-shRNA-Ubi-EGFP表达载体构建正确,并且病毒滴度为7.0×108 TU/ml.结论 实验成果构建mU6-FAAH-shRNA-Ubi-EGFP表达载体.
目的 構建攜帶脂肪酰胺水解酶(FAAH)基因的小分子榦擾RNA錶達載體.方法 設計閤成短髮卡RNA(shRNA) FAAH對應的兩條互補的寡覈苷痠鏈,mU6-MCS-Ubi-EGFP質粒經HpaⅠ和XhoⅠ進行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,對剋隆的菌落行聚閤酶鏈反應鑒定,再對聚閤酶鏈反應(PCR)鑒定暘性的剋隆進行測序,測序正確的重組質粒轉染293細胞,同源重組產生Lentivirus-FAAH-shRNA併測定病毒滴度.結果 經測序證實mU6-FAAH-shRNA-Ubi-EGFP錶達載體構建正確,併且病毒滴度為7.0×108 TU/ml.結論 實驗成果構建mU6-FAAH-shRNA-Ubi-EGFP錶達載體.
목적 구건휴대지방선알수해매(FAAH)기인적소분자간우RNA표체재체.방법 설계합성단발잡RNA(shRNA) FAAH대응적량조호보적과핵감산련,mU6-MCS-Ubi-EGFP질립경HpaⅠ화XhoⅠ진행쌍매절여퇴화후적과핵감산련접,목적질립전화감수태세포,대극륭적균락행취합매련반응감정,재대취합매련반응(PCR)감정양성적극륭진행측서,측서정학적중조질립전염293세포,동원중조산생Lentivirus-FAAH-shRNA병측정병독적도.결과 경측서증실mU6-FAAH-shRNA-Ubi-EGFP표체재체구건정학,병차병독적도위7.0×108 TU/ml.결론 실험성과구건mU6-FAAH-shRNA-Ubi-EGFP표체재체.
Objective To construct the recombinant lentivirus vector with fatty acid amide hydrolase (FAAH)-short hairpin RNA (shRNA).Methods Oligonucleotide containing the small hairpin of FAAH was designed and synthesized,which was inserted into the mU6-MCS-Ubi-EGFP plasmid double digested by Hpa Ⅰ and Xho Ⅰ.The aim plasmids were transformed into competent cells E.coli.The grown colonies were identified by polymerase chain reaction (PCR) and then the positive colonies were sequenced and aligned.The 293 cells were cotransfected by the mU6-FAAH-shRNA-Ubi-EGFP,and the recombinant vector of Lentivirus-FAAH-shRNA was obtained and the titer of purified virus was determined.Results The gene sequencing showeded that the construction of the recombinant Lentivirus-FAAH-shRNA plasmid could be confirmed,and the titer of virus was 7.0 × 10s TU/ml.Conclusion The Lentivirus-FAAH-shRNA had been constructed successfully.
