摘要
目的 构建Alzheimer病Aβ1 - 42 基因和小鼠IL 4共表达质粒 ,探讨其作为Alzheimer病特异疫苗的可能性。方法 全基因合成Aβ1 - 42 基因片段并克隆入pMD18 T载体中 ,再从Aβ1 - 42 pMD18 T重组质粒中切下Aβ1 - 42 基因并克隆入TA CE5真核双表达载体中 ,构建重组质粒Aβ1 - 42 TACE5。从pUMMV mIL4质粒中切下mIL 4并克隆到重组质粒Aβ1 - 42 TACE5中 ,构建双表达重组质粒Aβ1 - 42 mIL 4 TACE5测序鉴定克隆基因的正确性。脂质体试剂Lipofectamine 2 0 0 0体外转染Aβ1 - 42 mIL 4 TACE5重组质粒于BHK 2 1细胞中 ,用ECL Western blot方法鉴定mIL 4和Aβ1 - 42 能在该细胞中共表达。结果 正确构建了Aβ1 - 42 mIL 4 TACE5双表达重组质粒 ,并且在转化此质粒的BHK 2 1细胞中检测出了Aβ1 - 42 和mIL 4的表达。结论 双表达重组质粒Aβ1 - 42 mIL 4 TACE5基本可以满足其作为Alzheimer病特异的候选DNA疫苗的要求 。
Objective To construct the recombinant plasmid co expressing Aβ 1 42 gene of Alzheimer's disease (AD) and mouse IL 4 (mIL 4) gene to explore the possibility of the recombinant plasmid as Alzheimer's disease specific vaccine. Methods Full length Aβ 1-42 gene was synthesized and cloned into pMD18 T vector. The Aβ 1-42 gene fragment was cut out from Aβ 1-42 /pMD18 T recombinant plasimd with restriction enzymes and inserted into eukaryotic bi expressing vector TACE5 to construct recombinant plasmid Aβ 1-42 /TACE5. Mouse Il 4 gene was cut off from plasmid pUMMV mIL4 and cloned into Aβ 1-42 /TACE5 to construct recombinant Aβ 1-42 /mIL 4/TACE5 which was identified by sequencing. Recombinant Aβ 1-42 /mIL 4/TACE5 was transformed into BHK 21 cells with liposome reagent. Lipofectamine 2000 and its co expression of mouse IL 4 and Aβ 1-42 gene in the cells was identified with ECL Western blot. Results Recombinant plasmid Aβ 1-42 /mIL 4/TACE5 was constructed correctly which could express mouse IL 4 and Aβ 1-42 well in the transformed BHK 21 cells. Conclusion Bi expressing recombinant plasmid Aβ 1-42 /mIL 4/TACE5 can basically meet the requirement of Alzheimer's disease specific DNA vaccine, which can provide experimental data for the investigation of the immunological effect of this candidate genetic vaccine in transgenic mice.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2004年第15期1325-1329,共5页
Journal of Third Military Medical University
基金
国家自然科学基金资助项目 ( 30 170 338
30 10 0 0 58)
国家重点基础研究发展规划资助项目 ("973"项目 ) ( 2 0 0 1CB510 0 0 1)~~
