CAJ | 학술논문

设计小鼠 Txt5基因的特异性引物，将小鼠 cDNA 作为模板，用 PCR 扩增出 mTxt5编码区，并加入 HA标签序列.将这一片段插入 pMD －18T 载体，再亚克隆至 pAdtrack － cmv 穿梭载体上.线性化后，转化 BJ5183感受态细胞，在 BJ5183中发生同源重组获得 pAd － Txt5质粒.pAd － Txt5线性化后转染293A 细胞，包装得到含 Txt5基因的病毒.将病毒溶液感染大鼠原代心肌细胞，一段时间后观察绿色荧光，然后通过 RT － PCR 和免疫印迹法检测 HA 标签蛋白的表达.结果表明成功构建小鼠 Txt5腺病毒表达载体并实现在大鼠原代心肌细胞中的表达.
설계소서 Txt5기인적특이성인물，장소서 cDNA 작위모판，용 PCR 확증출 mTxt5편마구，병가입 HA표첨서렬.장저일편단삽입 pMD －18T 재체，재아극륭지 pAdtrack － cmv 천사재체상.선성화후，전화 BJ5183감수태세포，재 BJ5183중발생동원중조획득 pAd － Txt5질립.pAd － Txt5선성화후전염293A 세포，포장득도함 Txt5기인적병독.장병독용액감염대서원대심기세포，일단시간후관찰록색형광，연후통과 RT － PCR 화면역인적법검측 HA 표첨단백적표체.결과표명성공구건소서 Txt5선병독표체재체병실현재대서원대심기세포중적표체.
Specific primers for the mouse Txt5 gene were designed and employed to amplify the coding sequence using mouse cDNAs as the template .HA - tagged sequence was induced into the PCR primers .The PCR product was inserted into the pMD -18T vector and subcloned into pAd - track - cmv shuttle vector .The recombinant plasmid was linearized and transformed into bacteria BJ5183 .After homologous recombination in the bacteria ,plasmid pAd - Txt5 was obtained .Linearized pAd - Txt5 was transfected into 293 A cells .Then , the recombinant virus containing Txt5 was obtained after packaging . Cardiomyocytes were infected with the collected virus fluid .To detect the expression of Txt5 ,green fluorescence protein (GFP) was observed by green fluorescence and RT - PCR , immunoblotting was performed . The results showed that mouse Txt5 adenovirus vector was constructed successfully ,and it could induce the sequence coding Txt5 - HA into cardiomyocytes .