CAJ | 학술논문

目的制备携带谷氨酸转运体3( VGLUT3)基因小分子干扰RNA的重组慢病毒.方法设计合成短发卡RNA (shRNA) VGLUT3对应的两条互补的寡核苷酸链,mU6-MCS-Ubi-EGFP质粒经Hpa Ⅰ和Xho Ⅰ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(PCR)鉴定,再对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-VGL UT3-shRNA并测定病毒滴度.结果病毒滴度为1.5 × 109 TU/ml.结论成功制备携带VGLUT3-shRNA的重组慢病毒.
목적 제비휴대곡안산전운체3( VGLUT3)기인소분자간우RNA적중조만병독.방법 설계합성단발잡RNA (shRNA) VGLUT3대응적량조호보적과핵감산련,mU6-MCS-Ubi-EGFP질립경Hpa Ⅰ화Xho Ⅰ진행쌍매절여퇴화후적과핵감산련접,목적질립전화감수태세포,대극륭적균락행취합매련반응(PCR)감정,재대PCR감정양성적극륭진행측서,측서정학적중조질립전염293세포,동원중조산생Lentivirus-VGL UT3-shRNA병측정병독적도.결과 병독적도위1.5 × 109 TU/ml.결론 성공제비휴대VGLUT3-shRNA적중조만병독.
Objective To construct recombinant lentivirus with vesicular glutamate transporter 3 (VGLUT3)-short hairpin RNA (shRNA).Methods Oligonucleotide containing the small hairpin of VGLUT3 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 colony polymerase chain reaction ( PCR ) and then the positive colonies were sequenced and aligned.The 293 cells were cotransfected by the mU6-VGLUT3-shRNA-Ubi-EGFP,and the recombinant vector of Lentivirus-VGLUT3-shRNA was obtained and the titer of purified virus was determined.Results The titer of virus was 1.5 × 109 TU/ml.Conclusion The Lentivirus-VGLUT3-shRNA had been constructed successfully.