摘要
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。
This study is aimed to construct eukaryotic expression vector for truncation mutants of Trim22 for further studying on the interaction of trim22 with HIV-1 gag protein.Five kinds of truncation mutants were generated by PCR with a template of eukaryotic expression vector pcDNA3.1(+)-Trim22.Subsequently,the target sequences were subcloned into the 5-Flag-pcDNA3.1(+),respectivly.The recombinant vectors were confirmed by restriction enzyme digestion,PCR and DNA sequencing,and then transfected into HEK293T cells respectively.After 24 hours,the truncation mutants of Trim22 protein were detected by Western blotting and immunoprecipitation.In conclusion,the truncation mutants of Trim22 eukaryotic expression vector are constructed successfully in our study,which will benefit the future research on the effect of Trim22 against HIV-1.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2011年第5期428-432,436,共6页
Immunological Journal
基金
滨州医学院科研启动基金项目(BY2010KYQD07)