基因疫苗pcDNA-AChR_(α211)免疫小鼠建立重症肌无力动物模型

Construction of a myasthenia gravis model by immunizing mice with gene vaccine pcDNA-AChR_(α211)

目的:用基因疫苗pcDNA-AChR_(α211)免疫C57BL/6小鼠建立实验性自身免疫性重症肌无力模型(EAMG)。方法:将人乙酰胆碱受体α亚基N端的主要免疫区(AChR_(α211))的基因片段插入到穿梭载体pcDNA3.0中,构建基因疫苗pcDNA-AChR_(α211)。大量提纯质粒pcDNA-AChR_(α211)后肌肉注射C57BL/6小鼠。用ELISA法检测小鼠血清中抗AChR_(α211)的IgG(ACh-RAb),并用PCR方法检测外源基因在小鼠各组织器官中的分布情况。结果:双酶切鉴定和序列测定表明构建了含正确目的基因阅读框的重组质粒pcDNA-AChR_(α211),ELISA分析表明该基因疫苗免疫的C57BL/6小鼠血清中含有AChRAb,且免疫三个月后在小鼠的肌肉、肝、脾、肾中仍可检测到目的基因AChR_(α211)的存在。结论:重组质粒pcDNA-AChR_(α211)作为基因疫苗能够诱导实验性自身免疫性重症肌无力。

Objective：To establish an experimental autoimmune myasthenia gravis by immunizing C57BL/6 mice with gene vaccine of pcDNA-AChRα211 Methods：pcDNA-AChRα211 was constructed by inserting the gene fragment of human acetylcholine receptor α-subunit N-terminal 211 residues（ AChRα211 ） into shuttle plasmid pcDNA3.0. Then the gene vaccine was directly injected intramuscularly into C57BL/6 mice. The AChRAb in serum of immunized mice were tested by ELISA. The AChRα211 DNA fragment was reamplified by PCR with total DNA of mouse tissues as the template. Results：The recombinant plasmid pcDNA-AChRα211 was testified by double enzymatic digestion to confirm the inserted site. DNA sequence analysis showed that the open reading frame of the inserted gene was the same as the original one. After immunization, the corresponding antibody, AChRAb, was detected in mice sera. The target gene could be re-amplified by PCR in muscle, liver, spleen and kidney of immunized mice. Conclusion：pcDNA-AChRα211 as gene vaccine could induce experimental autoimmune myasthenia gravis.