miR-TH克隆及慢病毒载体的构建

目的：克隆microRNA-酪氨酸羟化酶（TH）,构建慢病毒载体。方法：根据TH基因序列（NM_009377.1）,设计并合成4对微小RNA（miRNA）的Oligo DNA,退火形成双链后与pcDNATM 6.2-GW/EmGFP-miRNA载体相连,用Gateway技术将构建好的pcDNATM6.2-GW/EmGFP-miR-TH载体先后与中间载体pDONRTM221、慢病毒载体pLenti6/V5-DEST连接,构建慢病毒表达载体pLenti6/V5-DEST-TH,用脂质体2000（lipofectamine 2000）将慢病毒载体以及包装质粒（pLP1,pLP2和pLP/VSVG）共转染HEK-293FT细胞,收集上清,感染293T细胞株进行滴度鉴定。结果：测序结果表明,4对pcDNATM 6.2-GW/EmGFP-miR-TH表达载体序列与参考序列一致,构建出来的慢病毒载体在293FT细胞中包装成功,并通过293T细胞测得慢病毒滴度为5×106 TU/ml。结论：成功构建了TH的慢病毒干扰载体pLenti6/V5-DEST-TH,为利用RNA干扰技术进一步研究TH基因的功能和作用奠定了基础。

Cloning of miR-TH and construction of its lentiviral expression vector

Objective： To clone miR-TH and construct its lentiviral expression vector.Methods： According to the TH gene sequence（GenBank： NM_009377.1）,4 pairs of Oligo DNA sequences of miRNA were designed.The single strand of Oligo DNA was annealed to form double strand DNA,and then connected with the plasmid pcDNATM 6.2-GW/EmGFP-miRNA.By using Gateway technology,the expression vector pcDNATM6.2-GW/EmGFP-miR-TH was linked into pDONRTM221 and pLenti6/V5-DEST to construct lentiviral expression vector pLenti6/V5-DEST-TH,The lentiviral expression vector and VirapowerTM Packaging Mix（pLP1,pLP2 和pLP/VSVG） were cotransfected into HEK-293FT cells by lipofectamine 2000 transfection to produce the lentivirus,and the lentivirus titer was measured by infection of HEK-293T cells.Results： The sequence of expression vector pcDNATM 6.2-GW/ EmGFP-miR-TH was proved correct using sequencing method.The constructed lentiviral vector was successfully packaged in 293FT cells.The virus titer was 5×106 TU/ml after infection of HEK-293 cells.Conclusion： The lentiviral expression vector pLenti6/ V5-DEST-TH was constructed successfully.It could facilitate further study of the gene functions of TH by RNA interference method.