本文選題：Nef基因　切入點：SW細(xì)胞　出處：《湖南師范大學(xué)自然科學(xué)學(xué)報》2017年01期

【摘要】：為了構(gòu)建艾滋病毒HIV-1型Nef基因重組表達(dá)載體,利用PCR從p NL4-3質(zhì)粒上擴(kuò)增HIV-1型Nef基因,獲得Nef基因完整的編碼區(qū),構(gòu)建可表達(dá)Nef蛋白的p EB-myc-nef重組表達(dá)載體.經(jīng)鑒定正確后,利用LipofectamineTM2000將重組質(zhì)粒轉(zhuǎn)染SW480細(xì)胞,通過Western Blot技術(shù)檢測Nef蛋白的瞬時表達(dá).為了建立穩(wěn)定表達(dá)Nef基因的SW480細(xì)胞系,采用G418篩選建立穩(wěn)定表達(dá)細(xì)胞系的方法,篩選出單克隆細(xì)胞系.擴(kuò)大培養(yǎng)克隆細(xì)胞后,通過RT-PCR和Western Blot分別檢測Nef的mRNA轉(zhuǎn)錄水平及蛋白表達(dá)水平,經(jīng)長達(dá)60 d的傳代,最終獲得穩(wěn)定表達(dá)Nef基因的SW480細(xì)胞系,為研究RNAi在艾滋病治療方面的應(yīng)用提供了細(xì)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.
【作者單位】： 湖南師范大學(xué)生命科學(xué)學(xué)院;
【基金】：湖南省科技廳重點項目(2014FJ2006) 長沙市科技局重點項目(K1205221-31)
【分類號】：R512.91;Q78

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