人类免疫缺陷病毒I型包膜糖蛋白gp41的基因重组表达

目的 基因重组表达（ＨＩＶ－１ ｇｐ４１）抗原,并研制一种快速,简便,灵敏性高,特异性强的国产ＨＩＶ－１免疫检测试剂。方法 选用ＨＩＶ－１型ＢＨ１０毒株的包膜糖蛋白ｇｐ４１的部分基因（６９７７－７４９７）,重组在ＰＢＶ２２１表达载体上。表达产物通过１５％ＳＤＳ－聚丙烯酰胺凝胶电泳初步分离纯化,根据ＲＦ值,切下含特异蛋白的胶带,以Ｗｅｓｔｅｒｎ ｂｌｏｔ法转移在硝酸纤维素膜上;     

HIV-1囊膜糖蛋白gp41三聚体结构域基因的表达及其单克隆抗体的制备

目的：表达HIV-1囊膜糖蛋白gp41三聚体结构域基因N51(L6)C46融合蛋白，并制备针对该融合蛋白的单克隆抗体(mAb)，为筛选抗HIV多肽及分析gp41表位的免疫原提供抗体工具。方法：在B121(DE3)中诱导表达NSl(L6)C46融合蛋白，经mAb NC-1鉴定后，采用小鼠腹股沟皮下NC膜免疫的方法免疫小鼠，并进行细胞融合、克隆化制备抗N51(L6)C46 mAb，用ELISA法初步鉴定其特异性位点。结果：获得了高表达的N51(L6)CA6融合蛋白，并能与识别特异性空间构象的mAb NC-1反应。用该融合蛋白免疫小鼠后，获得了6株抗N51(D5)C46的mAb，这6株mAb均特异识别兔抗N36(L6)C34抗体捕获的融合蛋白，但不与N36或C34多肽片段反应。结论：得到高效表达融合蛋白N51(L6)CA6，并呈现gp41核心结构空间构象。用此蛋白免疫小鼠得到6株特异结合gp41核心结构空间构象的单抗。     

HIV—I包膜蛋白gp41单克隆抗体的研究进展

HIV－1gp41在病毒侵入早期,发挥重要作用。针对gp41的单克隆抗体作为一种良好的工具广泛地应用于以gp41为靶点的抗HIV－1药物和疫苗研究工作中。     

HIV 外膜糖蛋白 gp120 和 gp41 在昆虫细胞中表达及其免疫学特性的评价

将编码完整ｇｐ１２０和完整ｇｐ４１的基因分别克隆到杆状病毒转移质粒中。使用重组转移质粒与野生杆状病毒（ＡｃＮＰＶ）ＤＮＡ共转染Ｓｆ９昆虫细胞，经挑选获得分别带有编码ｇｐ１２０和ｇｐ４１基因的重组杆状病毒。重组杆状病毒感染Ｓｆ９细胞后在细胞中分别表达了ＨＩＶ外膜糖蛋白ｇｐ１２０和ｇｐ４１。其重组蛋白的分子量分别为１２０×１０３和４１×１０３。此重组糖蛋白在免疫荧光、免疫印染和酶联免疫实验中都能被ＨＩＶ阳性血清所识别。动物免疫实验表明此重组糖蛋白能诱导很强的特异性抗体产生。     

HIV-1gp41核心结构模拟位的筛选与鉴定

目的：筛选并鉴定HIV-1 gp4l核心表位。方法：用识别HIV-1 gp41的构象特异性单克隆抗体NC-1筛选噬菌体12肽库，通过夹心ELISA、NC-1特异性阻断实验、竞争抑制实验鉴定阳性噬菌体克隆，DNA序列分析阳性克隆。结果：经3轮筛选，随机挑取24个噬菌体克隆，ELISA鉴定表明有lO个克隆可与NC-1结合，DNA序列分析并推导氨基酸序列，共5种序列：HDVHHRWVYLLS、ITVNEWLYTSEQ、HGRSHGMFKPKR、MGPIARPHWHLN、DMYRSPRPKPDT。其中gp41N肽和C肽所形成的复合物可特异性阻断表达HDVHHRWVYLLS，VNEWLYTSEQ和MGPIARPHWHLN的克隆与NC-1的结合。结论：所得序列HDVHHRWVYLLS，VNEWLYTSEQ及MGPIARPHWHLN模拟HIV-1 gp41六螺旋束核心表位。     

HIV—1gp120基因及其与IFN—α基因共表达产物的构建和 …

目的 研究细胞因子ＩＦＮ－α在机体免疫应答过程中的免疫佐剂效应。方法 以表达中国流行株ＨＩＶ－１ｇｐ１２０基因的核酸疫苗质粒ｐＧＰ及共表达中国流行株ＨＩＶ－１ｇｐ１２０基因与ＩＦＮ－α基因的核酸疫苗质粒ｐＧＰＩＦＮ－α免疫Ｂａｌｂ／ｃ鼠，用流式细胞仪测定１００００个免疫鼠脾淋巴细胞中ＣＤ４＾＋，ＣＤ８＾＋Ｔ细胞数及ＣＤ４＾＋／ＣＤ８＾＋比值。结果 ｐＧ－ＰＩＦＮ－α与ｐＧＰ比较，ｐＧＰＩＦＮ－α免     

人类免疫缺陷病毒Ⅰ型包膜糖蛋白gp41的基因重组表达

目的基因重组表达（ＨＩＶ１ｇｐ４１）抗原，并研制一种快速、简便、灵敏性高、特异性强的国产ＨＩＶ１免疫检测试剂。方法选用ＨＩＶ１型ＢＨ１０毒株的包膜糖蛋白ｇｐ４１的部分基因（６９７７７４９７），重组在ＰＢＶ２２１表达载体上。表达产物通过１５％ＳＤＳ聚丙烯酰胺凝胶电泳初步分离纯化，根据ＲＦ值，切下含特异蛋白的胶带，以Ｗｅｓｔｅｒｎｂｌｏｔ法转移在硝酸纤维素膜上；免疫血清法检测合格者制备的抗原检测条带，经国家标准参比血清检测。结果获得１株含ＨＩＶ１ｇｐ４１基因的重组质粒，其蛋白的特异性表达为８％，经ＨＩＶ１阳性血清检测和国家标准参比血清检定，该表达蛋白灵敏性、特异性均为１００％。结论（１）可以选用单一的ｇｐ４１作为ＨＩＶ１感染初筛试剂的抗原。（２）表达载体ＰＢＶ２２１其表达的目的蛋白为非融合蛋白，作为试剂中的抗原，可降低检测中的非特异性。（３）该试剂是一种简便、特异、灵敏性高的试剂。     

HIV-1 膜蛋白基因片段杆状病毒转移载体克隆及重组体的构建

目的：构建编码HIV－1外膜糖蛋白的gp120和gp41基因杆状病毒转移载体，并在昆虫细胞中表达gp120和gp41重组蛋白。方法：从HIV－1基因克隆PNL4－3（NY5／LAV）中应用聚合链反应扩增目的基因gp120和gp41，克隆到PGEM－T载体中，限制性内切酶酶切、DNA序列分析鉴定目的基因，再经EcoRI和BamHI双酶切后定向克隆到杆状病毒转移载体PAC－SecG2T中，再次测序鉴定。通过在sf9昆虫细胞中同源重组、空斑筛选、病毒鉴定、SDS－PAGE、Western-blot对重组病毒和重组蛋白进行分析。结果：限制性内切酶酶切和DNA序列分析表明，gp120和gp41正确克隆到杆状病毒转移载体PACSecG2T中；SDS－PAGE和Eestern-blot结果表明在昆虫细胞中成功表达了HIV外膜糖蛋白gp120和gp41。结论：成功构建了PACSecG2T-gp120和PACSecG25-gp41杆状病毒转移载体，并在杆状病毒－昆虫细胞表达系统中表达了HIV－1 gp120和gp41重组蛋白，Western-blot证明具有较好的免疫原性。     

Hdentification of Small Molecular Anti—HIV—1 Compound that Interferes with Formation of the Fusion—active gp41 Core

The human immunodeficiency virus type 1 (HIV - 1 ) envelope glycoprotein gp41 plays a critical role in the fusion of viral and target cell membranes. The gp41 extracellular domain, which contains fusion peptide (FP), N - and C - terminal hydrophobic heptad repeats (NHR and CHR, respectively). Peptides derived from NHR and CHR regions,designated N- and C- peptides, respectively, can interact with each other to form a six - stranded coiled - coil domain, representing the fusion-active gp41 core. Our previous studies demonstrated that the C- peptides have potent inhibitory activity against HIV- 1 infection.These peptides inhibit HIV- 1 -mediated membrane fusion by binding to NHR regions for preventing the formation of fusion- active gp41 core. One of the C - peptides, T - 20, which is in the phase Ⅲ clinical trails, is expected to become the first peptide HIV fusion inhibitory drug in the near future. However, this peptide HIV fusion inhibitor lacks oral availability and is sensitive to the proteolytic digestion.Therefore, it is essential to develop small molecular non -peptide HIV fusion inhibitors having similar mechanism of action as the C- peptides. We have established an ELISA- based screening assay using a unique monoclonal antibody, NC- 1, which can specifically bind to a conformational epitope on the gp41 core domain. Using this screening assay, we have identified a small molecular anti- HIV- 1 compound,named ADS-Jl, which inhibits HIV- 1- mediated membrane fusion by blocking the interaction between the NHR and CHR regions to form the fusion - active gp41 core. This compound will be used as a lead to design and develop novel HIV fusion inhibitors as new drugs for the treatment of HIV infection and/or AIDS.     

利用gp41蛋白的合成肽抗原检测HIV—1抗体

采用固相多肽合成技术对人免疫缺损病毒-1型(HIV-1)gp41蛋白的一段代表优势抗原表位的22肽进行了化学合成,经反相高效液相层析(HPLC)和多肽序列测定表明,合成肽成品均质性良好,其氨基酸序列与设计相符。利用该合成肽作为包被抗原,以间接 ELISA 法检测 HIV-1血清抗体,其特异性、灵敏性和重复性均达到与国外生产的 gp41合成肽抗原相同的水平.     

我国HIV-1流行株gp41抗原表位筛选与串联表达

目的探讨HIV-1gp41抗原表位串联表达蛋白用于HIV抗体检测的可行性。方法用HIV-1gp41蛋白亲和层析柱制备HIV-1感染者血清中的多克隆抗体，用噬菌体展示随机十二肽库进行生物淘洗，反向吸附非特异性噬菌体。经ELISA鉴定阳性克隆，DNA测序，确定优势表位。将优势表位与文献报道的另一个优势表位串联，克隆人pQE30载体进行蛋白表达。结果成功筛选到位于HIV-1 gp41蛋白上的优势抗原表位(YGPKDAETTAIW)，串联表位(YGPKDAETTAIW-GGGS-SC-SAKFTCTTQI)在pQE30载体中实现可溶性表达。重组蛋白具有良好的抗原性，能与不同的HIV-1抗体阳性血清呈特异反应。结论HIV-1 gp41抗原表位串联表达设计足可行的，串联表位重组蛋白可用于HIV-1抗体检测，但检测灵敏性低于常规方法。     

模拟HIV-1 gp41 CHR表位短肽的筛选及研究

目的 利用针对HIV-1跨膜蛋白gp41CHR序列合成肽C34的单克隆抗体1G1筛选噬菌体12肽库，旨在找寻模拟C34肽表位的序列，同时探索该短肽成为HIV-1gp41NHR与CHR结合抑制物的可能性。方法 以1G1为钓饵蛋白对噬菌体12肽库进行亲和筛选，以双夹心ELISA鉴定阳性克隆。结果 经3轮筛选后，随机挑选17个噬菌体克隆，其中6个克隆与1G1显示出较强的结合活性，上述6个阳性克隆经DNA测序，氨基酸序列相同；HYEFWAWNWEAN，其明显的疏水性质类似于G34N末端，特异性鉴定显示这些克隆均能够与HIV-1gp41N多肽结合，结论 该噬菌体克隆展示肽可模拟HIV-1gp41CHR多肽表位，并可与N多肽结合。     

抗HIV-1 gp41 合成多肽C34单抗的制备及生物学活性

目的 制备针对C34和C46单抗，并以此为工具研究gp41表位，进一步了解HIV－1包膜蛋白的作用机理和病毒的感染机制，为寻找新的治疗靶点提供抗体工具。方法 常规动物免疫、细胞融合、克隆化制备抗C34和C46的单克隆抗体，并鉴定其特异性位点，还用ELISA法、MTTI法及荧光分子探针技术对单抗的生物学活性进行研究。结果 获得了3株抗C34单克隆抗体、2株抗C46单抗。此5个单抗均与C34结合，并抑制N36肽与C34肽复合物的形成；其中效价最高的1G1对H9／HIVⅢB细胞的生长有刺激作用，对H9／HIVⅢB细胞和MT－2细胞的融合无明显影响。结论 得到5株可与C34反应，并可抑制C34与N36多肽结合的单抗，其中1G1所识别的表位可能为增强性或刺激性表位。     

Assessment of antibody responses against gp41 in HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M group consensus envelope

Human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein gp41 is targeted by broadly-reactive neutralizing antibodies 2F5 and 4E10, making it an attractive target for vaccine development. To better assess immunogenic properties of gp41, we generated five soluble glutathione S-transferase fusion proteins encompassing C-terminal 30, 64, 100, 142, or 172 (full-length) amino acids of gp41 ectodomain from M group consensus envelope sequence. Antibody responses in HIV-1-infected patients were evaluated using these proteins and overlapping peptides. We found (i) antibody responses against different regions of gp41 varied tremendously among individual patients, (ii) patients with stronger antibody responses against membrane-proximal external region exhibit broader and more potent neutralizing activity, and (iii) several patients mounted antibodies against epitopes that are near, or overlap with, those targeted by 2F5 or 4E10. These soluble gp41 fusion proteins could be an important source of antigens for future vaccine development efforts.     

The envelope glycoprotein of HIV-1 gp120 and human complement protein C1q bind to the same peptides derived from three different regions of gp41, the transmembrane glycoprotein of HIV-1, and share antigenic homology

Gp41, the transmembrane glycoprotein of HIV-1, has been shown to be non-covalently associated with gp120. We have shown that it also binds human C1q. To analyze the interaction site(s) of gp41 with these two molecules, we established an enzyme-linked immunosorbent assay (ELISA) system using recombinant soluble gp41 [amino acids (aa) 539–684] and peptides thereof. In the cell-external part of gp41 three sites (aa 526–538, aa 590–613 and aa 625–655) were found to bind both gp120 and C1q. That gp120 and C1q use the same sites was evidenced by the fact that these proteins competed with each other for the same sites in recombinant soluble gp41 and gp41 peptides. It could be demonstrated by ELISA, that rabbit antibodies against human C1q recognized gp120, and rabbit antibodies against gp120 cross-reacted with C1q. Rabbit anti-gp120, HIV-1-positive human sera and anti-gp120 obtained from such sera agglutinated sensitized sheep erythrocytes with human C1q (EAC1q). These data suggest that in addition to functional homology between C1q and gp120 structural homology between these two molecules exists. This molecular mimicry might become the basis for immunologically relevant autoimmune phenomena.     

中国HIV/AIDS患者保守表位的2F5抗体与疾病进展关系

目的 研究中国HIV/AIDS患者gp41保守表位中和抗体2F5与疾病进程的关系.方法 应用合成4(ELDKWA)-C24肽及gp41抗原肽包被96孔酶标板,ELISA方法检测特异性抗体水平.提取正常人外周血单个核细胞(PBMC),应用HIV-1 SF33毒株进行病毒多轮感染PBMC中和试验.结果 缓慢进展者(slow progressor,SP)的2F5样中和抗体水平显著高于典型进展者(typical progressor,TP)(P<0.05);2F5样中和抗体水平与gp41特异性抗体水平正相关(r=0.406,P<0.05).结论 中国HIV.-缓慢进展者体内具有高水平的2F5样抗体,2F5抗体可能是疾病不进展的保护性因素之一.     

HIV-1 gp41 C-螺旋模拟位的筛选和鉴定

目的：筛选可作为小分子先导化合物的HIV-1 gp41 C螺旋模拟位，为开发抗HIV-1早期感染的小分子药物奠定基础。方法：以源于HIV-1 gp41端的肽N36为靶，对噬菌体环七肽库进行亲和筛选。利用ELISA鉴定噬菌体阳性克隆并对其进行DNA序列分析。结果：3轮筛选后随机挑取26个噬菌体克隆，ELISA鉴定表明有16个克隆可与N36结合，将其中10个克隆DNA测序并推导氨基酸序列，每一克隆至少含有2个疏水氨基酸，其中9个克隆均有WW，7个克隆有WWH保守序列。挑选阳性噬菌体克隆No.8进一步鉴定，游离N36肽阻断No.8克隆与固相化N36结合，C34肽竞争抑制N36肽与No.8克隆结合(IC50为12.5μg/ml)。结论：表达WW保守序列的肽模拟HIV-1 gp41C螺旋与N螺旋结合，该结果为设计针对HIV介导融膜的抑制剂提供技术支持。     

Effects of sequence changes in the HIV-1 gp41 fusion peptide on CCR5 inhibitor resistance

A rare pathway of HIV-1 resistance to small molecule CCR5 inhibitors such as Vicriviroc (VCV) involves changes solely in the gp41 fusion peptide (FP). Here, we show that the G516V change is critical to VCV resistance in PBMC and TZM-bl cells, although it must be accompanied by either M518V or F519I to have a substantial impact. Modeling VCV inhibition data from the two cell types indicated that G516V allows both double mutants to use VCV-CCR5 complexes for entry. The model further identified F519I as an independent determinant of preference for the unoccupied, high-VCV affinity form of CCR5. From inhibitor-free reversion cultures, we also identified a substitution in the inner domain of gp120, T244A, which appears to counter the resistance phenotype created by the FP substitutions. Examining the interplay of these changes will enhance our understanding of Env complex interactions that influence both HIV-1 entry and resistance to CCR5 inhibitors.     

Characterization of a trimeric MPER containing HIV-1 gp41 antigen

The membrane-proximal external region (MPER) of gp41 is considered as a prime target for the induction of neutralizing antibodies, since it contains the epitopes for three broadly neutralizing antibodies (2F5, 4E10 and Z13). Here we present a novel gp41 construct (HA-gp41) comprising gp41 HR2 and MPER fused to two triple-stranded coiled-coil domains at both ends. HA-gp41 is trimeric, has a high helical content in solution and forms rod-like structures as revealed by negative staining electron microscopy. Immunization of rabbits with HA-gp41 induced antibodies directed against MPER, which failed to exert significant neutralization capacity against envelopes from primary isolates. Thus trimerisation of MPER regions does not suffice to induce a potent neutralizing antibody response specific for conserved regions within gp41.