HIV-1型Nef基因重组表达载体的构建及其稳定表达细胞系的建立

发布时间：2018-04-09 02:15

本文选题：Nef基因　切入点：SW细胞　出处：《湖南师范大学自然科学学报》2017年01期

【摘要】：为了构建艾滋病毒HIV-1型Nef基因重组表达载体,利用PCR从p NL4-3质粒上扩增HIV-1型Nef基因,获得Nef基因完整的编码区,构建可表达Nef蛋白的p EB-myc-nef重组表达载体.经鉴定正确后,利用LipofectamineTM2000将重组质粒转染SW480细胞,通过Western Blot技术检测Nef蛋白的瞬时表达.为了建立稳定表达Nef基因的SW480细胞系,采用G418筛选建立稳定表达细胞系的方法,筛选出单克隆细胞系.扩大培养克隆细胞后,通过RT-PCR和Western Blot分别检测Nef的mRNA转录水平及蛋白表达水平,经长达60 d的传代,最终获得稳定表达Nef基因的SW480细胞系,为研究RNAi在艾滋病治疗方面的应用提供了细胞模型.
[Abstract]:In order to construct the recombinant expression vector of HIV-1 type Nef gene of HIV, the HIV-1 type Nef gene was amplified from p NL4-3 plasmid by PCR, and the complete coding region of Nef gene was obtained. The recombinant expression vector of p EB-myc-nef expressing Nef protein was constructed.After being identified correctly, the recombinant plasmid was transfected into SW480 cells by LipofectamineTM2000, and the transient expression of Nef protein was detected by Western Blot technique.In order to establish a stable SW480 cell line expressing Nef gene stably, a stable expression cell line was established by G418 screening, and a monoclonal cell line was screened out.The mRNA transcription and protein expression of Nef were detected by RT-PCR and Western Blot. After 60 days of subculture, the SW480 cell lines expressing Nef gene stably were obtained.It provides a cell model for the study of the application of RNAi in the treatment of AIDS.
【作者单位】：湖南师范大学生命科学学院;
【基金】：湖南省科技厅重点项目(2014FJ2006) 长沙市科技局重点项目(K1205221-31)
【分类号】：R512.91;Q78

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