HIV-1和SIV nef基因表达对T淋巴细胞系功能及蛋白谱改变的比较作用研究

发布时间：2018-07-04 07:35

本文选题：HIV-1 + nef　；参考：《暨南大学》2008年博士论文

【摘要】： 目的: 研究HIV-1和SIV nef基因表达对T淋巴细胞系的细胞功能及蛋白谱的作用,以探寻Nef促进HIV致病性增强的细胞内分子作用靶点。方法: 构建pAd-HIV-1 nef-EGFP和pAd-SIVmac239 nef-EGFP重组腺病毒质粒,并利用HEK293T细胞将之包装和扩增为含有目的基因的腺病毒,并检测病毒滴度,以感染T淋巴细胞系,使其表达目的蛋白。设四个实验组,正常MT-2细胞即Control组和单独表达EGFP空载体的Ad-EGFP组,表达HIV-1 nef蛋白的Ad-HIV-1 nef-EGFP组和表达SIVmac239蛋白的Ad-SIVmac239 nef-EGFP组。通过流式细胞仪和荧光显微镜检测重组腺病毒的感染率;通过Western blotting证实HIV-1和SIV nef蛋白的表达;检测HIV-1和SIV nef基因表达对T淋巴细胞系细胞增殖和细胞周期的影响,及其对细胞表面分子CD4,CXCR4,HLA-I和HLA-DR表达的影响。制备四个实验组的MT-2细胞总蛋白,利用固相PH梯度(Immobilized pHgradient,IPG)双向凝胶电泳(Two-dimensional electrophoresis,2-DE)分别对四组细胞的总蛋白进行分离,进行考马斯亮蓝染色,扫描图像并获取蛋白质双向电泳凝胶图谱。应用ImageMaster 2D Elite 5.0分析软件进行凝胶图像分析,寻找Ad-HIV-1 nef-EGFP和Ad-SIVmac239 nef-EGFP两组间差异表达的蛋白质。切取较明显表达的蛋白质点,通过胶内酶切,点样,应用基质辅助电离解析飞行时间质谱(Matrix-assitsted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF-MS)获得蛋白质的肽质量指纹图谱(Peptide mass fingerprint,PMF),再通过MALDI-TOF/TOF-MS串级质谱对蛋白质进行序列分析,得到蛋白质二级质谱图。利用Mascot软件搜索IPI Human database蛋白质数据库,将获得的肽段信息进行对比分析,明确是何种蛋白质。结果: 成功构建Ad-HIV-1 nef-EOFP和Ad-SIVmac239 nef-EGFP重组腺病毒质粒;利用HEK 293T细胞对表达目的基因的腺病毒分别包装和扩增,检测病毒滴度分别为:pAd-EGFP组:3.56×10~6活性单位/mL(IU/mL),pAd-HIV-1 nef-EGFP组:5.40×10~6活性单位/mL(IU/mL),pAd-SIVmac239nef-EGFP组:5.62×10~6活性单位/mL(IU/mL);依照重组腺病毒的滴度,对重组腺病毒进行梯度稀释感染目的细胞并进行感染效率测定,得出以下重组腺病毒的MOI值可使感染目的细胞的效率达到95%以上。本研究的实验均按照此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检测证实了HIV-1和SIV nef基因在T淋巴细胞系MT-2细胞内的表达;HIV-1和SIV nef基因表达可促进MT-2细胞和C8166细胞的增殖,并使处于DNA合成期和分裂期的细胞比率增多;HIV-1和SIVmac239 Nef蛋白表达均可下调MT-2细胞表面的CD4,CXCR4,HLA-I和HLA-DR分子,其中,SIVmac239 Nef蛋白下调CD4,CXCR4和HLA-I类分子的作用要强于HIV-1 Nef蛋白。比较分析Ad-HIV-1 nef-EGFP组和Ad-SIVmac239 nef-EGFP组两组间的双向电泳图谱,其蛋白点数分别为1002和949,匹配点为876,匹配率为89.9%。找出8个差异表达的蛋白质点,其中4个蛋白表达差异点可能与HIV致病性相关,proteasome 26S non-ATPase subunit 13 isoform 2在Ad-SIVmac239 nef-EGFP组不表达,可能与其对蛋白酶体的调节及感染SIV的T细胞活化水平较低有关,HIV-1Nef有可能在进化中丢失了这一功能而导致T活化水平升高;CMPK cytidylatekinase在Ad-HIV-1 nef-EGFP组表达可能与促进HIV复制有关;TPT1 tumorprotein,translationally-controlled 1在Ad-SIVmac239 nef-EGFP组不表达,而在Ad-HIV-1 nef-EGFP表达增加,STRAP Serine-threonine kinase receptor-associatedprotein在Ad-SIVmac239 nef-EGFP组表达下调,这两个蛋白可能与感染HIV的T细胞增殖水平升高有关。结论: 本研究成功构建Ad-HIV-1 nef-EGFP和Ad-SIVmac239 nef-EGFP重组腺病毒质粒,并证实其在T淋巴细胞系细胞中的表达具有与HIV和SIV病毒的Nef蛋白相同的功能。比较蛋白质组学的方法分析表明表达HIV-1和SIV nef基因的MT-2细胞中有四个差异表达的蛋白可能与HIV致病性相关,且均为进化过程中的高度保守蛋白。在从SIV演变和跨种传播过程中,HIV-1 Nef可能具有了选择性作用于宿主体内的某些保守蛋白的功能而导致宿主对HIV复制支持性增强。而这些保守蛋白有可能作为药物靶点来干预HIV在宿主体内的复制力和感染力,从而降低HIV在宿主体内的致病性。
[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.
【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R346;R392

【共引文献】