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目的构建Trim22缺失突变体的真核表达载体,为进一步研究Trim22与HIV-1衣壳蛋白的相互作用奠定基础。方法以野生型Trim22真核表达质粒为模板,用PCR方法扩增出5种类型Trim22缺失突变体的基因片段,克隆到5`-Flag-pcDNA3.1(+)真核表达载体,通过酶切、菌落PCR及测序进行鉴定。将该载体转染HEK293T细胞,用蛋白印迹及免疫沉淀检测Trim22缺失突变体的蛋白表达。结果通过酶切、PCR及测序鉴定,成功构建5种人Trim22缺失突变体的表达载体,载体均可以在HEK293T细胞中表达。结论成功构建5种人Trim22缺失突变体的真核表达载体,为探寻Trim22与HIV-1衣壳蛋白相互作用的关键结构域奠定了基础。
Objective To construct eukaryotic expression vector of Trim22 deletion mutant and lay a foundation for further study on the interaction between Trim22 and HIV-1 capsid protein. Methods The wild-type Trim22 eukaryotic expression plasmid was used as a template to amplify the 5 types of Trim22 deletion gene by PCR and cloned into 5 ’-Flag-pcDNA3.1 (+) eukaryotic expression vector. , Colony PCR and sequencing were identified. The vector was transfected into HEK293T cells and the protein expression of Trim22 deletion mutant was detected by Western blotting and immunoprecipitation. Results Five recombinant human Trim22 deletion mutants were successfully constructed by restriction enzyme digestion, PCR and sequencing. The vectors were all expressed in HEK293T cells. Conclusion The eukaryotic expression vectors of five human Trim22 deletion mutants were successfully constructed, which laid the foundation for exploring the key domains of the interaction between Trim22 and HIV-1 capsid proteins.