HIV-1 gp41 NC六螺旋的结构与功能研究

AIDS是严重危害人类健康的疾病,而HIV是导致这种疾病的病毒。gp41六螺旋在介导HIV-1病毒与靶细胞间的膜融合过程中起着重要作用。因此,对于gp41结合蛋白的研究有助于深入了解gp41在HIV-1感染整个过程中扮演的角色,解释gp41对靶细胞的调控机制,为寻找新的抗艾滋病药物靶点以及艾滋病抑制剂的设计提供有益的思路。作者的实验室相继发现了一批与gp41六螺旋结构相互作用的蛋白质,进而对HIV-1 gp41六螺旋介导的膜融合过程和HIV-1感染机理有了更深入的了解。     

HIV-1 CRF07 B/C亚型gp41的表达、纯化和初步结构分析

HIV-1跨膜蛋白gp41是HIV-1包膜与靶细胞膜融合过程中的关键蛋白,而且序列保守,是理想的HIV-1作用靶点。为获得HIV-1中国流行株CRF07 B/C gp41蛋白的晶体结构来指导疫苗设计及药物开发,采用CRF07 B/C gp160基因序列为模板,经PCR、酶切、连接,将gp41 helix-bundle区域克隆到pET30-his表达载体中,经表达、纯化和结晶筛选,获得了gp41 helix-bundle的晶体并解析了结构,为针对中国艾滋病病毒流行株疫苗的设计及药物开发提供了结构参考。     

HIV-1跨膜蛋白gp41与N-取代吡咯衍生物的结合模式研究

采用分子对接,分子动力学(MD)模拟和分子力学/泊松-波尔兹曼溶剂可有面积方法与分子力学/广义伯恩溶剂可及面积方法(MM-PBSA/MM-GBSA),预测两种N-取代吡咯衍生物与HIV-1 跨膜蛋白gp41疏水口袋的结合模式与作用机理．分子对接采用多种受体构象,并从结果中选取几种可能的结合模式进行MD 模拟,然后通过MM-PBSA计算结合能的方法识别最优的结合模式. MM-PBSA计算结果表明,范德华相互作用是结合的主要驱动力,而极性相互作用决定了配体在结合过程中的取向．进一步的结合能分解显示,配体的羧基与gp41残基Arg579的静电相互作用对结合有重要贡献．上述工作为进一步优化N-取代吡咯衍生物类的HIV-1融合抑制剂建立了良好的理论基础．     

HIV-1 gp41的酵母表面展示及表达优化

以His标签检测蛋白的表达, 利用酿酒酵母表面展示系统, 成功地将HIV-1 gp41片段锚定在酵母表面, 并检测到gp41的活性。以pMD18T-gp41为模板, 通过PCR技术克隆了gp41基因, 将gp41基因通过双酶切连接到载体pICAS-His上,构建了gp41酵母表面展示载体, 并将其转化至酿酒酵母(Saccharomyces cerevisiae)MT8-1中。重组菌经培养, 利用免疫荧光染色方法进行染色, 显微镜观察发现重组酵母细胞表面有绿色荧光, 流式细胞仪结果进一步证实gp41正确折叠展示于酵母细胞表面。采用不同浓度的葡萄糖培养基进行表达优化。当葡萄糖浓度为1%时, 82.46%的酵母细胞表达了gp41抗原; 随着葡萄糖浓度升高, 蛋白表达受到抑制。     

HIV-1 gp41的酵母表面展示及表达优化

以His标签检测蛋白的表达, 利用酿酒酵母表面展示系统, 成功地将HIV-1 gp41片段锚定在酵母表面, 并检测到gp41的活性。以pMD18T-gp41为模板, 通过PCR技术克隆了gp41基因, 将gp41基因通过双酶切连接到载体pICAS-His上,构建了gp41酵母表面展示载体, 并将其转化至酿酒酵母(Saccharomyces cerevisiae)MT8-1中。重组菌经培养, 利用免疫荧光染色方法进行染色, 显微镜观察发现重组酵母细胞表面有绿色荧光, 流式细胞仪结果进一步证实gp41正确折叠展示于酵母细胞表面。采用不同浓度的葡萄糖培养基进行表达优化。当葡萄糖浓度为1%时, 82.46%的酵母细胞表达了gp41抗原; 随着葡萄糖浓度升高, 蛋白表达受到抑制。     

HIV-1跨膜蛋白gp41的截短及表达

将HIV-1跨膜蛋白gp41进行截短,在大肠杆菌中进行表达并纯化。PCR扩增gp41的部分编码基因,回收的PCR产物纯化后克隆到连接载体pGEM-T上,然后用EcoRI和Sal I切下目的基因,并构建到表达载体pGEX-4T3上,导入宿主细胞BL21(DE3),用IPTG诱导表达,表达产物用亲和层析进行纯化并作相应鉴定。截短的HIV-1跨膜蛋白gp41能直接在大肠杆菌内进行表达,利用亲和层析能方便地将目的蛋白进行纯化,为跨膜蛋白的进一步应用打下基础。     

HIV-1包膜蛋白gp41优势抗原的构建及可溶性表达

目的:筛选1型人免疫缺陷病毒(HIV-1)中国流行株中包膜蛋白gp41的优势抗原片段,构建具有区域流行代表性的HIV-1 gp41重组抗原,为改进现有HIV-1初筛试剂盒中使用的同类抗原奠定基础。方法:利用免疫斑点杂交和生物信息学方法,从收集自重庆、广州、上海的区域代表性150份HIV-1感染者血清标本中筛选gp41抗原性强的候选样本,利用RT-PCR及巢式PCR方法扩增包含重要抗原表位决定蔟的gp41基因片段,与原核表达载体pQE30连接,转化大肠杆菌M15构建gp41重组抗原表达菌株,表达后经亲和层析纯化、SDS-PAGE和Western印迹鉴定。结果:兔源HRP标记的gp41多抗能识别标本中gp41抗原性差异,得到候选样本,扩增包含gp41主要抗原表位片段;构建了包含gp41抗原表达簇的重组原核表达质粒,表达、纯化后经His标签抗体Western印迹鉴定为阳性。结论:高纯度的重组优势gp41抗原的构建和鉴定,为进一步改进现有HIV初筛诊断奠定了基础。     

双抗体夹心ELISA检测HIV-1 gp41抗原方法的建立

目的:建立检测HIV-1gp41抗原的双抗体夹心ELISA,并探讨其临床应用的可行性。方法:用饱和硫酸铵(SAS)纯化抗HIV-1gp41-5单克隆抗体(mAb),用HRP标记后建立双抗体夹心ELISA法,对其灵敏度及特异性进行检测,并用该方法对40份HIV-1阳性血清进行了检测。结果:用mAbE12(5μg/mL)为包被抗体,2H6为酶标记抗体(1∶900)建立了双抗体夹心ELISA法,检测gp41-5多肽的灵敏度是100pg/mL。对HIV-1阳性血清中gp41抗原的检出率为67.5%(27/40)。结论:建立了特异性强、灵敏度良好的检测HIV-1gp41抗原的双抗体夹心ELISA法。     

人类免疫缺陷病毒1型gp41结构与功能的研究进展

人类免疫缺陷病毒1型(HIV-1)通过其包膜糖蛋白(Env)介导侵入靶细胞.Env由受体特异性结合单位gp120和膜融合单位gp41组成.HIV-1的gp41分为3个功能区:膜外区、跨膜区和膜内区.膜外区是病毒感染时膜融合的主要结构基础;跨膜区通过疏水残基使Env锚定在脂质膜上;膜内区则表现多重功能,参与病毒的感染、复...     

HIV—1gp41基因的高效表达及表达产物的纯化

为了获得高效表达的人类免疫缺陷病毒（ＨＩＶ１）ｇｐ４１蛋白,从而为ＨＩＶ１基因工程诊断抗原的国产化打下基础,用ＰＣＲ的方法从ＨＩＶ１全基因序列中扩增出编码ｇｐ４１Ｎ端的６９０ｂｐ片段。经酶切后,克隆到ｐＥＴ２８ａ载体中,再将重组质粒转化到表达宿主菌ＢＬ２１（ＤＥ３）中,经ＩＰＴＧ诱导,高效表达出ｇｐ４１蛋白。间接ＥＬＩＳＡ、Ｗｅｓｔｅｒｎｂｌｏｔ、ＳＤＳＰＡＧＥ电泳证实,该表达产物具有良好的抗原性和特异性,且表达量约占总菌体蛋白的４５％。重组蛋白经金属鏊合纯化,纯度达９９％。     

Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan

The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotein gp41 has been made by determining the effect on membrane integrity of a 15-mer gp41-derived peptide library. By monitoring the effect of this peptide library on membrane leakage, we have identified three regions on the gp41 ectodomain with membrane-interacting capabilities: Region 1, which would roughly correspond to the polar sequence which follows the fusion domain and extends to the N-terminal heptad repeat region; Region 2, which would correspond to the immunodominant loop; and Region 3, which would correspond to the pre-transmembrane region of gp41. The identification of these three regions supports their direct role in membrane fusion as well as facilitating the future development of HIV-1 entry inhibitors.     

Characterization of the steric defense of the HIV-1 gp41 N-trimer region

During viral entry, HIV gp41 adopts a transient conformation called the "prehairpin intermediate" in which a highly conserved therapeutic target, the N-trimer, is exposed. Despite extensive discovery efforts, potent and broadly neutralizing antibodies that target the N-trimer are elusive. We previously demonstrated the N-trimer is protected by a steric block that prevents large proteins, such as antibodies, from accessing it. Here we further characterize the steric block and identify its source. To study the N-trimer steric accessibility, we produced two sets of C-peptide inhibitors (a potent inhibitor targeting the N-trimer) fused to cargo proteins of increasing size facing either the virus or cell side of the prehairpin intermediate. Both bulky inhibitor sets show a steric block, but the effect is more pronounced with virus-side cargo. Additionally, both sets maintain their potencies in a modified entry assay that removes possible sources of target cell steric hindrance. These results implicate a viral source, likely gp120, as the primary component of the steric block. In addition, we studied the steric accessibility of the "pocket" region of the N-trimer, a highly attractive drug and vaccine target. We demonstrated a pocket-specific antibody, D5, is more potent as an scFv than as a full-length IgG, suggesting the N-trimer steric restriction extends to the pocket. This characterization will facilitate the design of sterically restricted antigens that mimic the steric environment of the N-trimer in the prehairpin intermediate and are capable of inducing potent and broadly neutralizing antibodies that circumvent the N-trimer steric block.     

Identification of the HIV-1 gp41 core-binding motif--HXXNPF

The HIV-1 gp41 core, a six-helix bundle formed between the N- and C-terminal heptad repeats, plays a critical role in fusion between the viral and target cell membranes. Using N36(L8)C34 as a model of the gp41 core to screen phage display peptide libraries, we identified a common motif, HXXNPF (X is any of the 20 natural amino acid residues). A selected positive phage clone L7.8 specifically bound to N36(L8)C34 and this binding could be blocked by a gp41 core-specific monoclonal antibody (NC-1). JCH-4, a peptide containing HXXNPF motif, effectively inhibited HIV-1 envelope glycoprotein-mediated syncytium-formation. The epitope of JCH-4 was proven to be linear and might locate in the NHR regions of the gp41 core. These data suggest that HXXNPF motif may be a gp41 core-binding sequence and HXXNPF motif-containing molecules can be used as probes for studying the role of the HIV-1 gp41 core in membrane fusion process.