Aβ阻断肽适配子TRX1-ABAD-DP-TRX2融合基因的克隆及表达

目的 将Aβ阻断肽(ABAD-DP) cDNA插入人硫氧化还原蛋白(hTRX)的活化位点,克隆融合基因TRX1-ABAD-DP-TRX2 (T-A-T) cDNA,为基因治疗阿尔茨海默病奠定基础.方法 通过PCR获得目的片段TRX1、TRX2、ABAD-DP,进一步将上述3目的片段按既定顺序插入腺相关病毒穿梭质粒pSSHG-CMV的多克隆位点,从而获得融合基因TRX1 -ABAD-DP-TRX2.经PEI介导Hela细胞包装重组腺相关病毒rAAV/T-A-T.感染NIH-3T3细胞,通过荧光免疫组织化学染色检测融合基因的表达及其与Aβ42的定位.结果 酶切后得到TRX1 -ABAD-DP-TRX2融合基因片段的大小约为435 bp,与理论值一致.荧光免疫组织化学染色检测到融合基因的表达,且共定位组T-A-T和Aβ42均位于NIH-3T3细胞胞质内.结论 成功克隆及表达了Aβ阻断肽适配子TRX1-ABAD-DP-TRX2融合基因,为进一步将阻断肽用于基因治疗阿尔茨海默病提供了基础.

Cloning and expression of fusion gene of amyloid beta binding alcohol dehydrogenase decoy peptide aptamer (TRX1-ABAD-DP-TRX2)

Objective To construct TRX-ABAD-DP-TRX (T-A-T) fusion gene of a novel ABAD-DP aptamer through the insertion of ABAD-DP into the modified human thioredoxin (hTRX) and exploit the possibility of further applications for the gene therapy of Alzheimer's disease.Methods According to the designed sequence,the target fragments of TRX1,TRX2 and ABAD-DP were created by PCR ( polymerase chain reaction) and then inserted into the multiple clone site of adeno-associated virus shuttle plasmid pSSHG-CMV with gene cloning technique.The corresponding fusion gene TRX1-ABAD-DP-TRX2 was identified by restriction enzymes digestion with EcoR Ⅰ and BamH Ⅰ.The recombinant adeno-associated virus (AAV/T-A-T) was produced in HeLa cells with linear polyethylenimine.The expression of T-A-T fusion gene and co-localization between T-A-T and Aβ peptide in NIH 3T3 cells were examined by fluorescent immunohistochemistry.Results The size of fusogenic fragment TRX1-ABAD-DP-TRX2 was approximately 435 bp.And it was consistent with our design.T-A-T fusion gene was expressed in NIH 3T3 cells.Through co-expression,T-A-T aptamer and intracellular A3 peptide were co-localized.It indicated that T-A-T aptamer could bind Aβ within NIH 3T3 cells.Conclusion The TRX1-ABAD-DP-TRX2 fusion gene is successfully cloned and expressed.And it may provide rationales for further applications in the gene therapy of Alzheimer's disease.