發(fā)布時間：2018-04-09 02:15

  本文選題：Nef基因　切入點：SW細胞　出處：《湖南師范大學自然科學學報》2017年01期

【摘要】：為了構建艾滋病毒HIV-1型Nef基因重組表達載體,利用PCR從p NL4-3質粒上擴增HIV-1型Nef基因,獲得Nef基因完整的編碼區(qū),構建可表達Nef蛋白的p EB-myc-nef重組表達載體.經(jīng)鑒定正確后,利用LipofectamineTM2000將重組質粒轉染SW480細胞,通過Western Blot技術檢測Nef蛋白的瞬時表達.為了建立穩(wěn)定表達Nef基因的SW480細胞系,采用G418篩選建立穩(wěn)定表達細胞系的方法,篩選出單克隆細胞系.擴大培養(yǎng)克隆細胞后,通過RT-PCR和Western Blot分別檢測Nef的mRNA轉錄水平及蛋白表達水平,經(jīng)長達60 d的傳代,最終獲得穩(wěn)定表達Nef基因的SW480細胞系,為研究RNAi在艾滋病治療方面的應用提供了細胞模型.
[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】： 湖南師范大學生命科學學院;
【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

【相似文獻】

相關期刊論文 前2條

1 史良如,衛(wèi)丹,M.E.Schneider,H.Th.Bruesster,P.Wernet,R.Schauder,F.O.Gombert,G.Jung;HIV-1及其nef蛋白存在有與正常人組織呈交叉反應的表位[J];免疫學雜志;1991年03期

2 ;[J];;年期

，

本文編號：1724395