中华普通外科杂志
中華普通外科雜誌
중화보통외과잡지
CHINESE JOURNAL OF GENERAL SURGERY
2010年
3期
243-245
,共3页
白燕南%严茂林%王耀东%李德华
白燕南%嚴茂林%王耀東%李德華
백연남%엄무림%왕요동%리덕화
DNA,重组%吲哚胺吡咯2,3双加氧酶%白蛋白类%启动区(遗传学)%腺病毒类
DNA,重組%吲哚胺吡咯2,3雙加氧酶%白蛋白類%啟動區(遺傳學)%腺病毒類
DNA,중조%신타알필각2,3쌍가양매%백단백류%계동구(유전학)%선병독류
DNA,recombinant%Indoleamine pyrrole 2,3-dioxygenase%Albumins%Promoter%Adenoviridac
目的 构建携带小鼠白蛋白启动子和IDO基因的重组腺病毒载体,研究肝脏Hepa l石细胞的IDO基因mRNA及蛋白表达情况.方法 酶切含有小鼠全长IDO cDNA的IDO质粒,亚克隆至穿梭载体pAdTrack.ALB上,在BJ5183细菌中和AdEasy-1进行同源重组,生成并筛选阳性克隆,测序、鉴定正确后,转染AD-293细胞进行包装、扩增,检测病毒滴度,RT-PCR和荧光显微镜鉴定重组腺病毒转染AD-293细胞后IDO的表达.重组腺病毒进一步感染Hepa 1-6细胞,RT-PCR和WesternBlot法分别检测IDO基因在细胞内表达情况.结果 经酶切及测序证实携带白蛋白启动子和IDO基因重组腺病毒载体构建成功,RT-PCR检测到转染后AD-293细胞内IDO的表达,病毒感染滴度为2.9×10~6pfu/ml.感染Hepa 1-6细胞后,RT-PCR和Western Blot可以检测到IDO mRNA水平和蛋白水平表达.结论 构建了携带白蛋白启动子和IDO基因的重组腺病毒载体.
目的 構建攜帶小鼠白蛋白啟動子和IDO基因的重組腺病毒載體,研究肝髒Hepa l石細胞的IDO基因mRNA及蛋白錶達情況.方法 酶切含有小鼠全長IDO cDNA的IDO質粒,亞剋隆至穿梭載體pAdTrack.ALB上,在BJ5183細菌中和AdEasy-1進行同源重組,生成併篩選暘性剋隆,測序、鑒定正確後,轉染AD-293細胞進行包裝、擴增,檢測病毒滴度,RT-PCR和熒光顯微鏡鑒定重組腺病毒轉染AD-293細胞後IDO的錶達.重組腺病毒進一步感染Hepa 1-6細胞,RT-PCR和WesternBlot法分彆檢測IDO基因在細胞內錶達情況.結果 經酶切及測序證實攜帶白蛋白啟動子和IDO基因重組腺病毒載體構建成功,RT-PCR檢測到轉染後AD-293細胞內IDO的錶達,病毒感染滴度為2.9×10~6pfu/ml.感染Hepa 1-6細胞後,RT-PCR和Western Blot可以檢測到IDO mRNA水平和蛋白水平錶達.結論 構建瞭攜帶白蛋白啟動子和IDO基因的重組腺病毒載體.
목적 구건휴대소서백단백계동자화IDO기인적중조선병독재체,연구간장Hepa l석세포적IDO기인mRNA급단백표체정황.방법 매절함유소서전장IDO cDNA적IDO질립,아극륭지천사재체pAdTrack.ALB상,재BJ5183세균중화AdEasy-1진행동원중조,생성병사선양성극륭,측서、감정정학후,전염AD-293세포진행포장、확증,검측병독적도,RT-PCR화형광현미경감정중조선병독전염AD-293세포후IDO적표체.중조선병독진일보감염Hepa 1-6세포,RT-PCR화WesternBlot법분별검측IDO기인재세포내표체정황.결과 경매절급측서증실휴대백단백계동자화IDO기인중조선병독재체구건성공,RT-PCR검측도전염후AD-293세포내IDO적표체,병독감염적도위2.9×10~6pfu/ml.감염Hepa 1-6세포후,RT-PCR화Western Blot가이검측도IDO mRNA수평화단백수평표체.결론 구건료휴대백단백계동자화IDO기인적중조선병독재체.
Objective To construct a recombinant adenovirus vector encoding for indoleamine 2,3-dioxygenase(IDO)and chimetric albumin promoter,evaluate the mRNA and protein expression levels in Hepa 1-6cell.Methods Full-length mouse derived IDO cDNA was subeloned into pAdTraek-ALB shuttle Plasmid.The product was linearized to homologous recombination with AdEasy-l vector in BJ5183 bacteria.The positive clone was identified by restriction endonuclease digestion and further confirmed by sequencing.The recombined adenoviruses DNA were transfected into AD-293 cells for packaging and amplification of Ad-ALB/IDO.The expression of IDO was monitored by RT-PCR and EGFP fluorescence in infected cells.The recombinant viruses with Hepa 1-6 cells were cultured and the mRNA and protein expression levels monitored bv RT-PCR and Western blot, respectively. Results Construction of recombinant andenoviruses containing IDO and albumin promoter was confirmed by restriction endonuclease digestion and sequencing.The expression of IDO was identified by RT-PCR in transfected AD-293 cell.The virus titer was 2.9×10~6 pfu/ml.The IDO mRNA and protein expression levels were detectable after transfected Hepa 1-6 cells by RT-PCR and Western blot. Conclusion A recombinant adenovirus Ad-ALB/IDO was susceessfullyconstructed.
