以内部核糖体进入位点序列连接两个独立表达基因构建重组腺病毒双表达载体(英文)  

Construction of recombinant adenovirus vector expressing 2 genes ligated by internal ribosome entry sites sequence

作　　者：张燕[1] 沈茜[1] 

机构地区：[1]第二军医大学长海医院实验诊断科,上海200433

出　　处：《第二军医大学学报》2004年第2期148-152,共5页Academic Journal of Second Military Medical University

基　　金：This work is supported by the NationalNatural Science Foundation of China( 3 9970 2 70 )

摘　　要：目的 :以内部核糖体进入位点 (IRES)序列连接鼠 TGF-β1 和 PL P- Ig 2基因 ,构建重组腺病毒双表达载体。方法 :采用常规分子生物学方法 ,分别从 Con A刺激的小鼠脾细胞和 WT- 1杂交瘤细胞抽提总 RNA,克隆鼠 TGF- β1 基因和 Ig重链Fc基因 ;将编码 PL P1 39- 1 51 的 DNA与信号肽设计在引物上 ,经过 2次克隆 ,形成 PL P- Ig。与 TGF-β1 以 IRES序列相连接后 ,经穿梭载体与可调控性腺病毒载体骨架连接 ,鉴定后在 HEK2 93细胞包装。获得高滴度病毒后 ,采用 EL ISA方法鉴定 TGF- β1基因的表达 ;Western印迹鉴定 PL P- Ig的表达。 结果 :该双表达重组腺病毒载体经测序、限制性内切酶酶切分析、PCR等鉴定 ,与预期结果一致 ;病毒感染细胞分别经 EL ISA和 Western印迹检测后证实 TGF-β1 和 PL P- Ig得到了独立表达。结论 :构建的可调控性鼠 TGF-β1 和 PL P- Ig重组腺病毒双表达载体的独立表达 。Objective: To construct a recombinant adenovirus vector which express mouse TGF β 1 and PLP Ig independently ligated by internal ribosome entry sites (IRES) sequence.Methods: TGF β 1 and Ig Fc fragment were cloned from C57BL/6 mice spleen cells pre treated with Con A and WT 1 hybridoma respectively.PCR was carried out to link PLP 139 151 gene and signal peptide twice.TGF β 1 and PLP Ig were ligated by IRES sequence.pTRE shuttle vector was used as mediator to ligate the backbone of the adenoviral vector.The identified adenovirus was packaged in HEK 293 cells.Supernatant of high titer adenovirus was collected to detect the TGF β 1 gene expression by ELISA kit and PLP Ig by Western blot.Results: The constructed recombinant adenovirus was identified by restriction endonucleases cutting,sequencing and PCR.In the supernatant of infected cells,TGF β 1 was expressed in high concentration of 93.77 pg/ml detected by ELISA and PLP Ig by Western blot.Conclusion: The mice PLP Ig and TGF β 1 recombinant adenovirus we constructed serve as a basis for further research of experimental allergic encephalomyelitis gene therapy and induction of immune tolerance.

关 键 词：内部核糖体进入位点 序列 基因表达 重组腺病毒 载体 转化生长因子Β 蛋白脂 

分 类 号：Q782[生物学—分子生物学]