以β-淀粉样蛋白为靶的单价及二价真核表达载体的构建和表达  

Construction and Expression of Oligomer and Bigemina Eukaryotic Express Vector Aimed Amyloid-β as a Target

作　　者：王烈峰[1] 汪华侨[1] 张革[2] 邹俊涛[1] 谢瑶[1] 袁群芳[1] 姚志彬[1] 

机构地区：[1]中山大学基础医学院解剖学教研室,广东广州510080 [2]中山大学药学院,广东广州510080

出　　处：《中山大学学报（医学科学版）》2006年第3期250-253,317,共5页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家重点基础研究发展计划(973计划)(2006cb600700);国家自然科学基金资助项目(30400512);粤港关键领域重点突破资助项目(20054982210);广东省自然科学基金重大资助项目(20013137);广东省自然科学资金资助项目(04300218);广东省社会发展资助项目(2005B10401047);广州市科技计划资助项目(2004Z3-E0151;2005Z3-E4021);中国博士后科学基金(2004035603)

摘　　要：目的构建以β-淀粉样蛋白为靶单价及二价真核表达载体pcDNA3.1-Aβ1-42、pcDNA3.1-Aβ42×2,并在真核细胞中表达目的蛋白。方法PCR法扩增编码β-淀粉样蛋白的目的基因,利用基因克隆技术构建单价及二价真核表达载体;应用酶切及测序鉴定真核表达载体构建成功后,用Westernblot和细胞免疫组化染色检测其在真核细胞中的表达。结果相应的双酶酶切能够获得插入的目的基因片段(分别为137bp和269bp),测序未发现突变;Westernblot结果可见两条约为4ku和8ku的条带,细胞免疫组化染色可见阳性细胞。结论单价及二价真核表达载体构建成功;真核表达载体能在真核细胞中表达出目的蛋白。[Objective] To construct the oligomer and bigemina eukaryotic express vector aimed amyloid-β protein as a target and express the goal proteins in the eukaryotic cells. [Methods] Coding sequence of Aβ was amplified by PCR. The gene fragment was cloned directionally into the multi-cloning site of the plasmid and the recombine plasmid was constructed. The eukaryotic express vector was identified according to enzyme digesting and gene sequencing. The expressing proteins were determined by Western blot and the transfected cells were investigated by immunohistochemistry. [Results] The goal gene （137 bp and 269 bp） can be achieved after the restriction analysis, the expressed protein （4 ku and 8 ku） can be analyzed by Western blot and the positive cell can be seen by immunohistocbemistry. [Conclusion] The ligomer and bigemina eukaryotic express vectors were constructed successfully and the eukaryotic express vector can express the goal protein in the eukaryotic cell.

关 键 词：老年性痴呆 Β-淀粉样蛋白 Tg2576转基因鼠 重组质粒 真核表达载体 

分 类 号：R749.1[医药卫生—神经病学与精神病学]