CAJ | 학술논문

目的构建、包装并鉴定携带红色荧光蛋白(DsRed)标记的蛋白激酶B(akt1)的慢病毒.方法通过逆转录一聚合酶链反应(RT-PCR)扩增1488 kb mouse akt1基因片段,通过TA克隆将akt1接入T载体,然后和IRES-dsred、pXZ208连接.酶切、测序鉴定正确的重组体(pXZ208-akt1-IRES-dsred).三质粒系统经磷酸钙法包装病毒,荧光显微镜和流式细胞仪(FACS)检测病毒滴度.Western blot比较病毒感染组与未感染组AKT1蛋白表达.pXZ208-akt1-IRES-dsred慢病毒和绿色荧光蛋白标记(GFP)的慢病毒共感染真核细胞,荧光显微镜观察两者的表达.结果经鉴定转移质粒构建正确,荧光显微镜观察293FT表达DsRed,FACS测得病毒滴度为(1.245±0.250)×107/ml.Western blot证实病毒感染组与未感染组比较AKT1蛋白表达增高(P＜0.05).与GFP标记的慢病毒共感染真核细胞,荧光显微镜观察到共同表达DsRed和GFP.结论 DsRed标记akt1慢病毒构建和表达成功.DsRed和GFP标记慢病毒共感染真核细胞可观察到表达两种荧光蛋白.
목적 구건、포장병감정휴대홍색형광단백(DsRed)표기적단백격매B(akt1)적만병독.방법 통과역전록일취합매련반응(RT-PCR)확증1488 kb mouse akt1기인편단,통과TA극륭장akt1접입T재체,연후화IRES-dsred、pXZ208련접.매절、측서감정정학적중조체(pXZ208-akt1-IRES-dsred).삼질립계통경린산개법포장병독,형광현미경화류식세포의(FACS)검측병독적도.Western blot비교병독감염조여미감염조AKT1단백표체.pXZ208-akt1-IRES-dsred만병독화록색형광단백표기(GFP)적만병독공감염진핵세포,형광현미경관찰량자적표체.결과 경감정전이질립구건정학,형광현미경관찰293FT표체DsRed,FACS측득병독적도위(1.245±0.250)×107/ml.Western blot증실병독감염조여미감염조비교AKT1단백표체증고(P＜0.05).여GFP표기적만병독공감염진핵세포,형광현미경관찰도공동표체DsRed화GFP.결론 DsRed표기akt1만병독구건화표체성공.DsRed화GFP표기만병독공감염진핵세포가관찰도표체량충형광단백.
Objective To constructe and identify the lentiviral vector carrying protein kinase B (akt1) gene markerd by the Discosoma red fluorescent protein (DsRed). Methods Mouse cDNAs encoding 1488 kb aktl were amplified by reverse transcription-polymerase chain reaction (RT-PCR). By using TA clone technology, aktl PCR product was cloned into T vectors, the BamH Ⅰ, Ecor Ⅰ double restriction enzymatic digesting TA clone parts were norientatial ligated with IRES-dsred, pXZ208 vector by T4 ligase.The correct recombination plasmids named pXZ208-akt1 -IRES-dsred were identified by enzymatic digestion and sequcence. The 293FT cells were co-transfected with the recombination pXZ208-akt1-IRES-dsred and the packaging mixture △NRF, VSVG by using calcium phosphate DNA precipation. The viral liter was observed by fluorescence microscope and fluorescence-activated cell sorter ( FACS) . Western blotting was used to detect AKT1 protein expression. DsRed marked lentivirus and green fluorescent protein ( GFP) marked lentivirus were co-infected into eukaryotic cells, and under the fluorescent microscopy expression efficiency of two genes was observed. Results Recombinant plasmid pXZ208-akt1-IRES-dsred was correct identified by digestion and sequcence. DsRed was observed in 293FT cells by fluorescent microscope, and the viral titer analyzed by FACS was (1. 245 ±0. 250) ×107/ml. Western blotting revealed AKT1 protein expression in infected group was higher than in control group ( P ＜ 0. 05 ). Under the fluorescent microscopy, both dsred and gfp gene expression was observed in 293 FT cells co-transfected with two lentiviruses.Conclusion The three plasmids system of lentiviral vector containing aktl gene marked by DsRed is successfully constructed. The eukaryotic cells co-infected with lentivirus marked by DsRed and GFP express DsRed and GFP.