重组腺病毒表达载体介导艾滋病毒Vif蛋白调控KSHV潜伏感染的初探
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国家自然科学基金(30900064),教育部高等学校博士点专项科研基金新教师基金课题(20093234120004)和江苏省高校自然科学基金项目(No. 09KJB310007)资助


Preliminary research on the regulation of the recombinant adenovirus carrying HIV-1 Vif gene upon KSHV latent infection
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    摘要:

    目的:利用AdEasy腺病毒载体系统包装含人类免疫缺陷病毒1型(艾滋病毒:HIV-1)病毒体感染性因子(Vif)的重组腺病毒,使之感染原发性渗出性淋巴瘤细胞系BCBL-1细胞,并检测Vif蛋白对细胞中卡波济肉瘤相关疱疹病毒(Kaposi′s sarcomaassociated herpesvirus,KSHV)潜伏感染与裂解性复制的影响-方法:从实验室先前构建的重组真核表达质粒pCI-neo-Vif中扩增出Vif基因,插入到腺病毒穿梭质粒pAdTrack-CMV中构建成pAdTrack-Vif,酶切线性化的重组穿梭质粒与腺病毒骨架质粒pAdEasy-1共同转化感受态BJ5183细胞构建成重组腺病毒质粒-重组腺病毒在AD293细胞中得到包装和扩增-用荧光计数法测定腺病毒滴度,以感染复数(MOI)为1的病毒量感染靶细胞BCBL-1,荧光显微镜观察靶细胞中绿色荧光蛋白(GFP)的表达,并利用逆转录PCR(RT-PCR)和免疫印记(Western blot)技术分别检测Vif基因在靶细胞中的转录和表达情况,而且采用实时荧光定量PCR(Real-time PCR)和Western blot分别检测KSHV裂解期基因ORF26 mRNA转录及裂解期蛋白vIL-6的表达-结果:经酶切鉴定-核酸序列测定-GFP表达和病毒上清液PCR证实成功包装了携带HIV-1 Vif基因的重组腺病毒,滴度为2.5×108 TU/ml-重组腺病毒感染BCBL-1细胞48 h后,80%以上的细胞中表达GFP,RT-PCR和Western blot均能够在相应位置检测到目的基因Vif的条带-Real-time PCR和Western blot结果显示,Vif降低KSHV ORF26 mRNA转录水平及vIL-6蛋白表达-结论:成功包装了含HIV-1 Vif基因的重组腺病毒且在BCBL-1细胞中表达的HIV-1 Vif蛋白能够抑制KSHV裂解性复制-增强病毒的潜伏感染-

    Abstract:

    Objective:To package the recombinant adenovirus containing HIV-1 Vif gene by using AdEasy system and detect the effect of Vif protein expression on the latent infection and lytic replication of KSHV. Methods: The fragment of Vif gene from expression vector pCI-neo-Vif was cloned into the shuttle plasmid pAdTrack-CMV. With the digested recombinant plasmid pAdTrack-Vif and the backbone plasmid pAdEasy-1, the homologous recombination took place in the E.coli BJ5183,and the recombinant adenovirus plasmid was generated. The recombination adenoviruses were packaged and amplified in the AD293 cells. Then the virus titer was checked by observing the expression of GFP. After BCBL-1 cells were infected by the recombinant adenovirus with 1 MOI, the mRNA transcripts and protein expression of Vif in BCBL-1 cells were detected by RT-PCR and Western blot. Then, the expressions of KSHV ORF26 mRNA and vIL-6 protein were detected by Real-time PCR and Western blot, respectively. Results: The recombinant adenovirus carrying HIV-1 Vif was packaged successfully with the virus titer of 2.5×108 TU/ml. After infected with adenovirus, more than 80% BCBL-1 cells could express GFP, and the exact band of Vif was detectable by RT-PCR and Western blot. Moreover, the expression of KSHV ORF26 mRNA and vIL-6 protein were down-regulated by Vif protein. Conclusion: The recombinant adenovirus carrying HIV-1 Vif was packaged successfully,and Vif protein expressed in BCBL-1 cells could inhibit KSHV lytic replication and enhance KSHV latent infection.

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贾雪梅,王 平,秦 娣,卢 春.重组腺病毒表达载体介导艾滋病毒Vif蛋白调控KSHV潜伏感染的初探[J].南京医科大学学报(自然科学版),2011,(1):13-20

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  • 收稿日期:2010-07-26
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