發(fā)布時(shí)間：2018-01-17 19:12

  本文關(guān)鍵詞：攜帶HIV-1抗原的單純皰疹病毒載體疫苗的構(gòu)建及鑒定　出處：《生物工程學(xué)報(bào)》2015年03期 　論文類型：期刊論文

  更多相關(guān)文章： 型單純皰疹病毒 HIV-抗原 病毒載體疫苗 細(xì)菌人工染色體

【摘要】：利用細(xì)菌人工染色體技術(shù)將串聯(lián)的HIV-1 gp160、gag和protease基因以及表達(dá)元件插入1型單純皰疹病毒(Herpes simplex virus type 1,HSV-1)內(nèi)部反向重復(fù)序列區(qū),以獲得攜帶HIV-1抗原的單純皰疹病毒載體疫苗。首先將HIV-1 gp160(B型和C型)、gag和protease基因串聯(lián)克隆入pc DNA3獲得重組質(zhì)粒pc DNA/g Bgp和pc DNA/g Cgp,重組質(zhì)粒轉(zhuǎn)染293FT細(xì)胞,Western blotting檢測(cè)HIV抗原表達(dá)。繼而將pc DNA/g Bgp和pc DNA/g Cgp中包括HIV-1抗原基因和表達(dá)元件的表達(dá)框克隆入p KO5/BN獲得重組穿梭質(zhì)粒p KO5/BN/g Bgp和p KO5/BN/g Cgp,穿梭質(zhì)粒電轉(zhuǎn)含BAC-HSV的大腸桿菌,篩選重組菌,提取重組DNA并轉(zhuǎn)染Vero細(xì)胞,挑取病毒蝕斑純化重組病毒,Southern blotting鑒定重組病毒DNA,Western blotting檢測(cè)重組病毒感染細(xì)胞中HIV抗原表達(dá),并分析病毒的增殖特性。結(jié)果表明,Western blotting在pc DNA/g Bgp和pc DNA/g Cgp轉(zhuǎn)染的293FT細(xì)胞中檢測(cè)到表達(dá)的gp160和gag蛋白。p KO5/BN/g Bgp和p KO5/BN/g Cgp分別電轉(zhuǎn)獲得重組菌,并從重組DNA轉(zhuǎn)染的Vero細(xì)胞中純化獲得重組HSV,Southern blotting檢測(cè)表明重組HSV基因組發(fā)生特異性重組,重組病毒感染細(xì)胞中檢測(cè)到gp120和gp41,且重組HSV保留了在哺乳動(dòng)物細(xì)胞中的復(fù)制能力。本研究獲得攜載HIV-1 gp160、gag和protease基因的重組HSV,并保留了在哺乳動(dòng)物細(xì)胞中的復(fù)制能力,可作為HIV-1復(fù)制型病毒載體疫苗。
[Abstract]:Using bacterial artificial chromosome technique to connect the HIV-1 gp160. Gag and protease genes and expression elements were inserted into Herpes simplex virus type 1 of herpes simplex virus type 1. HSV-1) internal reverse repeat region to obtain herpes simplex virus vaccine carrying HIV-1 antigen. First, HIV-1 gp160(B and C). Gag and protease genes were cloned into PC DNA3 to obtain the recombinant plasmids PC DNA/g Bgp and PC DNA/g Cgp. The recombinant plasmid was transfected into 293FT cells. Western blotting was used to detect the expression of HIV antigen. Then PC DNA/g Bgp and PC DNA/g were detected. Recombinant shuttle plasmids p KO5/BN/g Bgp and p KO5/BN/g were obtained by cloning into p KO5/BN the expression frame containing HIV-1 antigen gene and expression element in Cgp. Cgp. The shuttle plasmid was electrotransferred to Escherichia coli containing BAC-HSV to screen the recombinant bacteria, extract the recombinant DNA and transfect it into Vero cells, and select the virus plaque to purify the recombinant virus. Southern blotting was used to identify the expression of HIV antigen in the cells infected with recombinant virus. The characteristics of virus proliferation were analyzed. Western blotting in PC DNA/g Bgp and PC DNA/g. Expression of gp160 and gag proteins. P KO5/BN/g Bgp and p KO5/BN/g were detected in 293FT cells transfected with Cgp. The recombinant bacteria were obtained by electroporation of Cgp. The recombinant Vero cells transfected with recombinant DNA were purified and purified by Southern blotting. The results of Southern blotting analysis showed that the recombinant HSV genome was specifically recombined. Gp120 and gp41 were detected in the cells infected with recombinant virus, and the recombinant HSV retained the ability of replication in mammalian cells. In this study, HIV-1 gp160 was obtained. The recombinant HSV gene of gag and protease could be used as the vector vaccine of HIV-1 replication virus, and reserved the ability of replication in mammalian cells.
【作者單位】： 新疆大學(xué)生命科學(xué)與技術(shù)學(xué)院;
【基金】：新疆維吾爾自治區(qū)高技術(shù)研究發(fā)展項(xiàng)目(No.2010016)資助~~
【分類號(hào)】：R752.11
【正文快照】： 人類免疫缺陷病毒(Human immunedeficiency virus,HIV)感染機(jī)體后主要侵犯CD4+T細(xì)胞,導(dǎo)致機(jī)體免疫缺陷并繼發(fā)各種機(jī)會(huì)性感染和腫瘤。自首例艾滋病報(bào)道以來,全球科學(xué)家一直致力于艾滋病疫苗的研制[1]。針對(duì)HIV這類高度變異、攻擊免疫系統(tǒng)且能引起持續(xù)感染和產(chǎn)生免疫病理反應(yīng)的

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