本文選題：HIV-1 + nef　； 參考：《暨南大學(xué)》2008年博士論文

【摘要】： 目的: 研究HIV-1和SIV nef基因表達(dá)對T淋巴細(xì)胞系的細(xì)胞功能及蛋白譜的作用,以探尋Nef促進(jìn)HIV致病性增強(qiáng)的細(xì)胞內(nèi)分子作用靶點(diǎn)。 方法: 構(gòu)建pAd-HIV-1 nef-EGFP和pAd-SIVmac239 nef-EGFP重組腺病毒質(zhì)粒,并利用HEK293T細(xì)胞將之包裝和擴(kuò)增為含有目的基因的腺病毒,并檢測病毒滴度,以感染T淋巴細(xì)胞系,使其表達(dá)目的蛋白。設(shè)四個實(shí)驗(yàn)組,正常MT-2細(xì)胞即Control組和單獨(dú)表達(dá)EGFP空載體的Ad-EGFP組,表達(dá)HIV-1 nef蛋白的Ad-HIV-1 nef-EGFP組和表達(dá)SIVmac239蛋白的Ad-SIVmac239 nef-EGFP組。通過流式細(xì)胞儀和熒光顯微鏡檢測重組腺病毒的感染率;通過Western blotting證實(shí)HIV-1和SIV nef蛋白的表達(dá);檢測HIV-1和SIV nef基因表達(dá)對T淋巴細(xì)胞系細(xì)胞增殖和細(xì)胞周期的影響,及其對細(xì)胞表面分子CD4,CXCR4,HLA-I和HLA-DR表達(dá)的影響。 制備四個實(shí)驗(yàn)組的MT-2細(xì)胞總蛋白,利用固相PH梯度(Immobilized pHgradient,IPG)雙向凝膠電泳(Two-dimensional electrophoresis,2-DE)分別對四組細(xì)胞的總蛋白進(jìn)行分離,進(jìn)行考馬斯亮藍(lán)染色,掃描圖像并獲取蛋白質(zhì)雙向電泳凝膠圖譜。應(yīng)用ImageMaster 2D Elite 5.0分析軟件進(jìn)行凝膠圖像分析,尋找Ad-HIV-1 nef-EGFP和Ad-SIVmac239 nef-EGFP兩組間差異表達(dá)的蛋白質(zhì)。切取較明顯表達(dá)的蛋白質(zhì)點(diǎn),通過膠內(nèi)酶切,點(diǎn)樣,應(yīng)用基質(zhì)輔助電離解析飛行時間質(zhì)譜(Matrix-assitsted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF-MS)獲得蛋白質(zhì)的肽質(zhì)量指紋圖譜(Peptide mass fingerprint,PMF),再通過MALDI-TOF/TOF-MS串級質(zhì)譜對蛋白質(zhì)進(jìn)行序列分析,得到蛋白質(zhì)二級質(zhì)譜圖。利用Mascot軟件搜索IPI Human database蛋白質(zhì)數(shù)據(jù)庫,將獲得的肽段信息進(jìn)行對比分析,明確是何種蛋白質(zhì)。 結(jié)果: 成功構(gòu)建Ad-HIV-1 nef-EOFP和Ad-SIVmac239 nef-EGFP重組腺病毒質(zhì)粒;利用HEK 293T細(xì)胞對表達(dá)目的基因的腺病毒分別包裝和擴(kuò)增,檢測病毒滴度分別為:pAd-EGFP組:3.56×10~6活性單位/mL(IU/mL),pAd-HIV-1 nef-EGFP組:5.40×10~6活性單位/mL(IU/mL),pAd-SIVmac239nef-EGFP組:5.62×10~6活性單位/mL(IU/mL);依照重組腺病毒的滴度,對重組腺病毒進(jìn)行梯度稀釋感染目的細(xì)胞并進(jìn)行感染效率測定,得出以下重組腺病毒的MOI值可使感染目的細(xì)胞的效率達(dá)到95%以上。本研究的實(shí)驗(yàn)均按照此MOI值。pAd-EGFP:17.8 IU/cell,pAd-HIV-1 nef-EGFP:27 IU/cell,pAd-SIVmac239 nef-EGFP:28.1 IU/cell。 Western blotting檢測證實(shí)了HIV-1和SIV nef基因在T淋巴細(xì)胞系MT-2細(xì)胞內(nèi)的表達(dá);HIV-1和SIV nef基因表達(dá)可促進(jìn)MT-2細(xì)胞和C8166細(xì)胞的增殖,并使處于DNA合成期和分裂期的細(xì)胞比率增多;HIV-1和SIVmac239 Nef蛋白表達(dá)均可下調(diào)MT-2細(xì)胞表面的CD4,CXCR4,HLA-I和HLA-DR分子,其中,SIVmac239 Nef蛋白下調(diào)CD4,CXCR4和HLA-I類分子的作用要強(qiáng)于HIV-1 Nef蛋白。 比較分析Ad-HIV-1 nef-EGFP組和Ad-SIVmac239 nef-EGFP組兩組間的雙向電泳圖譜,其蛋白點(diǎn)數(shù)分別為1002和949,匹配點(diǎn)為876,匹配率為89.9%。找出8個差異表達(dá)的蛋白質(zhì)點(diǎn),其中4個蛋白表達(dá)差異點(diǎn)可能與HIV致病性相關(guān),proteasome 26S non-ATPase subunit 13 isoform 2在Ad-SIVmac239 nef-EGFP組不表達(dá),可能與其對蛋白酶體的調(diào)節(jié)及感染SIV的T細(xì)胞活化水平較低有關(guān),HIV-1Nef有可能在進(jìn)化中丟失了這一功能而導(dǎo)致T活化水平升高;CMPK cytidylatekinase在Ad-HIV-1 nef-EGFP組表達(dá)可能與促進(jìn)HIV復(fù)制有關(guān);TPT1 tumorprotein,translationally-controlled 1在Ad-SIVmac239 nef-EGFP組不表達(dá),而在Ad-HIV-1 nef-EGFP表達(dá)增加,STRAP Serine-threonine kinase receptor-associatedprotein在Ad-SIVmac239 nef-EGFP組表達(dá)下調(diào),這兩個蛋白可能與感染HIV的T細(xì)胞增殖水平升高有關(guān)。 結(jié)論: 本研究成功構(gòu)建Ad-HIV-1 nef-EGFP和Ad-SIVmac239 nef-EGFP重組腺病毒質(zhì)粒,并證實(shí)其在T淋巴細(xì)胞系細(xì)胞中的表達(dá)具有與HIV和SIV病毒的Nef蛋白相同的功能。比較蛋白質(zhì)組學(xué)的方法分析表明表達(dá)HIV-1和SIV nef基因的MT-2細(xì)胞中有四個差異表達(dá)的蛋白可能與HIV致病性相關(guān),且均為進(jìn)化過程中的高度保守蛋白。在從SIV演變和跨種傳播過程中,HIV-1 Nef可能具有了選擇性作用于宿主體內(nèi)的某些保守蛋白的功能而導(dǎo)致宿主對HIV復(fù)制支持性增強(qiáng)。而這些保守蛋白有可能作為藥物靶點(diǎn)來干預(yù)HIV在宿主體內(nèi)的復(fù)制力和感染力,從而降低HIV在宿主體內(nèi)的致病性。
[Abstract]:Objective:
To investigate the effect of HIV-1 and SIV nef gene expression on the cell function and protein spectrum of T lymphocyte line, in order to explore the target of Nef to promote the intracellular molecular action of HIV pathogenicity.
Method:
The recombinant adenovirus plasmid of pAd-HIV-1 nef-EGFP and pAd-SIVmac239 nef-EGFP was constructed, and HEK293T cells were used to package and amplify the adenovirus containing the target gene, and the virus titer was detected to infect the T lymphocyte line to express the target protein. Four experimental groups were set up, the normal MT-2 cells, Control group and single expression EGFP empty body, were set up. The Ad-EGFP group, the Ad-HIV-1 nef-EGFP group expressing the HIV-1 Nef protein and the Ad-SIVmac239 nef-EGFP group expressing the SIVmac239 protein. The infection rate of the recombinant adenovirus was detected by flow cytometry and fluorescence microscopy, and the expression of HIV-1 and SIV proteins was confirmed by Western blotting. Effects of cell proliferation and cell cycle on cell surface molecules CD4, CXCR4, HLA-I and HLA-DR expression.
The total protein of the MT-2 cells of the four experimental groups was prepared. The total protein of the four groups was separated by the solid-phase PH gradient (Immobilized pHgradient, IPG) two-dimensional gel electrophoresis (Two-dimensional electrophoresis, 2-DE). The gomas blue staining was performed, the images were scanned and the two-dimensional gel electrophoresis of the protein was obtained. ImageMaster 2D E was used. Lite 5 analysis software carries out gel image analysis to find proteins expressed differentially between groups of Ad-HIV-1 nef-EGFP and Ad-SIVmac239 nef-EGFP. The protein points that are clearly expressed are cut out, by gel enzyme digestion, point samples, and matrix assisted ionization analytical flight time mass spectrometry (Matrix-assitsted laser desorption/ionization time-of-flight m). Ass spectrometry, MALDI-TOF-MS) obtained the peptide mass fingerprint of protein (Peptide mass fingerprint, PMF), and then sequence analysis of protein by MALDI-TOF/TOF-MS cascade mass spectrometry, the protein two level mass spectrogram was obtained. The IPI Human database protein database was searched by Mascot software, and the peptide segment information was obtained. Analysis, what protein is clear.
Result:
The recombinant adenovirus plasmid of Ad-HIV-1 nef-EOFP and Ad-SIVmac239 nef-EGFP was successfully constructed, and HEK 293T cells were used to package and amplify the adenoviruses expressing the target genes respectively. The virus titers were respectively: pAd-EGFP group: 3.56 x 10~6 active unit /mL (IU/mL), pAd-HIV-1 nef-EGFP group: 5.40 x 10~6 active unit. Group: 5.62 x 10~6 active unit /mL (IU/mL); in accordance with the titer of recombinant adenovirus, the recombinant adenovirus was infected with gradient dilution of the infected cells and the infection efficiency was measured. The MOI value of the recombinant adenovirus could make the efficiency of the infected target cells more than 95%. This study was conducted in accordance with this MOI value,.PAd-EGFP:17.8 IU/cell, pAd-H IV-1 nef-EGFP:27 IU/cell, pAd-SIVmac239 nef-EGFP:28.1 IU/cell.
Western blotting detection confirmed the expression of HIV-1 and SIV nef gene in T lymphocyte line MT-2 cells, HIV-1 and SIV nef gene expression promoted the proliferation of MT-2 cells and C8166 cells, and increased the ratio of cells in the period of synthesis and division. -I and HLA-DR molecules, in which SIVmac239 Nef protein down CD4, CXCR4 and HLA-I molecules are stronger than HIV-1 Nef protein.
The two-dimensional electrophoresis maps between group Ad-HIV-1 nef-EGFP and Ad-SIVmac239 nef-EGFP group were compared and analyzed. The number of protein points was 1002 and 949, the matching point was 876, and the matching rate was 89.9%. to find out 8 differentially expressed protein points, and 4 protein expression differences may be related to the pathogenicity of HIV, proteasome 26S non-ATPase subunit 13 isofor. The non expression of M 2 in the Ad-SIVmac239 nef-EGFP group may be related to the regulation of proteasome and the lower activation level of T cells in SIV. HIV-1Nef may lose this function in the evolution and lead to the increase of T activation level; CMPK cytidylatekinase in Ad-HIV-1 nef-EGFP group may be related to the promotion of HIV replication. N, translationally-controlled 1 was not expressed in the Ad-SIVmac239 nef-EGFP group, but the expression of Ad-HIV-1 nef-EGFP increased, and the STRAP Serine-threonine kinase receptor-associatedprotein was down regulated in the Ad-SIVmac239 nef-EGFP group. These two proteins may be related to the increase in the proliferative level of the infected cells.
Conclusion:
This study successfully constructed recombinant adenovirus plasmid of Ad-HIV-1 nef-EGFP and Ad-SIVmac239 nef-EGFP, and confirmed that its expression in T lymphocyte line cells had the same function as Nef protein of HIV and SIV virus. Analysis of proteomics methods showed that there were four differentially expressed eggs expressed in MT-2 cells expressing HIV-1 and SIV Nef basis. White may be associated with the pathogenicity of HIV and are highly conserved proteins in the process of evolution. In the course of SIV evolution and trans species transmission, HIV-1 Nef may have the function of selectively acting on certain conserved proteins in the host, leading to the host's support for HIV replication. These conservative proteins may be used as drug targets. The replication and infectivity of pre HIV in the host can reduce the pathogenicity of HIV in the host.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R346;R392

【共引文獻(xiàn)】

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