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[摘要]
目的：构建阳离子脂质体包裹的人促甲状腺激素受体胞外段基因真核表达质粒。
方法：PCR扩增穿梭质粒PHMCMVTSHR289目的基因并连接于真核表达质粒pcDNA3.1+上，重组质粒pcDNA3.1+/ TSHR289采用酶切及测序法鉴定。阳离子脂质体包裹重组质粒pcDNA3.1+/TSHR289。
结果：重组质粒pcDNA3.1+/TSHR289用HindIII酶切后产物经0.8%琼脂糖凝胶电泳检测显示出现512bp条带。正向测序发现AAC突变为AAT，为同义突变。反向测序发现GCG突变为GCT，亦为同义突变。阳离子脂质体与重组质粒的体积质量比例为3：1。
结论：酶切及测序鉴定重组质粒pcDNA3.1+/TSHR289构建成功。
[Key word]
[Abstract]
AIM: To construct recombination eukaryotic expression plasmid of human thyrotropin receptor extracellular domain encapsulated with cationic liposomes.
METHODS:We amplified the target gene of shuttle vector PHMCMVTSHR289, conjugated the target gene and eukaryotic expression plasmid pcDNA3.1+, and accredited whether pcDNA3.1+/TSHR289 was connected or not by enzymatic digestion and sequencing. Cationic liposomes encapsulated the recombination plasmid pcDNA3.1+/TSHR289.
RESULTS: Recombination plasmid pcDNA3.1+/TSHR289 digested with enzyme HindIII and the fragment through 0.8% gel electrophoresis showed 512bp strip. Recombination plasmid pcDNA3.1+/TSHR289 were found synonymous mutation through forward(AAC to AAT)and reverse sequencing(GCG to GCT). The volume ratio of cationic liposomes and recombinant plasmid was 3:1.
CONCLUSION: It is successful to construct the recombination plasmid pcDNA3.1+/TSHR289 by accredit it through enzymatic digestion and sequencing.
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