人免疫缺陷病毒跨膜蛋白gp41在大肠杆菌中的表达

为研究廉价的HIV血清学诊断系统,实现检测试剂国产化,用原核pET系统表达HIV跨膜蛋白gp41.研究发现,全长及缺失C端1／3的gp41在E.coli中均不能有效表达,仅保留N端1／3的gp41(包含gp41主要抗原决定簇,594－613氨基酸）有一定量的表达;通过金属螯合层析,产物得到部分纯化,Western blot显示产物有很好的反应原性,推测gp41在E.coli中较低表达量可能与mRNA的二级结构有关。     

原核表达HIV-1抗原蛋白的ELISA活性检测

研究了HIV-1 gp120s和gp41在大肠杆菌中表达产物的抗原特异性.首先构建表达质粒p120s和pT41,并转化到BL21和M15中.宿主细胞经IPTG诱导表达重组蛋白,SDS-PAGE和Western blot表征分析蛋白产物.总蛋白中gp120s占17.9%,为可溶性表达;gp41占24.6%,为包涵体形式.亲和层析法检测蛋白纯度分别为63.1%和89%.酶联免疫吸附(ELISA)实验说明重组蛋白有良好的抗原特异性.     

抗HIV-1gp41合成多肽gp41-5单克隆抗体的制备及初步鉴定

制备抗HIV-1 gp41合成多肽gp41-5的单克隆抗体（mAb），为筛选抗HIV-1多肽及分析gp41的抗原表位提供有用工具。常规动物免疫、细胞融合、克隆化制备抗gp41-5多肽mAb，并用ELISA法对其特异性、抗原识别表位及相对亲和力等做了初步鉴定。获得了4株抗gp41-5多肽的mAb，这4株mAb均特异识别gp41-5多肽，但不与gp41的N36或C34多肽片段反应。得到的4株mAb能特异结合gp41核心结构的空间构象。     

HIV-1外膜蛋白gp120在酵母Pichia pastoris中的表达

目的 用巴斯德毕赤酵母系统表达HIV外膜糖蛋白gp120.方法 将从HIV-1型国际标准株pHXB2-gp160的基因序列中扩增出的gp 120分子的长片段基因(1 419 bp)和短片段基因(417bp)分别克隆入真核表达载体pPICZαA与pPICZB中,以电穿孔法转化酵母GS 115.用YPDS-zeo平板筛选重组子、PCR方法检测整合到酵母菌GS 115基因组中的gp 120片段基因,经甲醇诱导表达后,SDS-PAGE和免疫印迹分析表达产物.结果 诱导后gp 120短片段多肽在GS 115中少量表达,在诱导表达24 h重组蛋白表达量及抗原性达到最高,表达产物被降解,具有良好的抗原特异性.诱导后gp 120长片段多肽未被GS 115所表达.结论 在巴斯德毕赤酵母中成功表达gp 120分子长片段需要优化gp 120基因,为制备HIV-1的诊断抗原和基因工程重组疫苗打下基础.     

不同癌细胞系中gp96的唾液酸化程度研究

以自行合成的叠氮标记的甘露糖进行细胞代谢,代谢后以叠氮化的唾液酸替代天然的唾液酸于细胞糖蛋白的糖链中呈现,依赖于点击化学方法,建立分离唾液酸化糖蛋白的方法,并研究gp96蛋白在不同癌细胞系中的唾液酸化程度.经酶联免疫吸附反应检测各细胞系均含有gp96蛋白,且含量无显著差异.而分析gp96的生物素化和唾液酸修饰程度,发现...     

禽网状内皮组织增生病毒gp90全长蛋白的原核表达、表达形式鉴定与纯化

该研究将禽网状内皮组织增生病毒gp90基因克隆至原核表达载体pET-30a(+),转化至大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导,并通过SDS-PAGE和Western blotting鉴定蛋白表达情况和蛋白表达.结果发现,将gp90基因连接表达载体转化宿主菌后,成功在大肠杆菌以包涵体形式表达.纯化的蛋白纯度超过90%.     

盘基网柄菌粘附分子gp150的定位及其与PKA活性关系的研究

细胞粘附分子gp150在盘基网柄菌细胞发育后期起着重要作用.用gp150抗体定位gp150在细胞发育重要阶段的分布,显示在细胞发育的不同时段,gp150的定位有着明显的区别.在聚集前的细胞流时期,gp150还均匀分布在细胞质内,到了细胞丘时期,gp150就移至多细胞聚集体的边缘部位.到了蛞蝓体时期,gp150在前柄细胞的表达量明显大于前孢子细胞,提示了gp150对柄细胞的作用.而当细胞发育至子实体阶段,gp150大量分布在成熟孢子的孢壁上,这是目前还没有报道的.高效液相色谱法检测野生型KAx-3细胞和gp150过表达细胞KAx-3:acct15/lagC的PKA活性,显示在细胞发育的早期(10 h之前),PKA在野生型细胞中的活性比KAx-3:act15/lagC细胞低.但是在细胞发育的后期,也就是野生型细胞中gp150开始快速积累之后,PKA在野生型细胞中的活性比KAx-3:act15/lagC细胞高.这些结果说明,gp150和PKA之间可能存在某种负反馈环的关系共同调节盘基网柄菌的生长发育.     

盘基网柄菌细胞发育中gp150分子与凋亡蛋白作用分析

用Percoll密度梯度技术分离和收集盘基网柄菌前柄和前孢子细胞,Western blot分析gp150分子和胱天蛋白酶在前孢子细胞和前柄细胞两种类型细胞中的表达情况.结果显示：只能在前柄细胞中检测到gp150蛋白条带,并随细胞发育蛋白的量逐渐增加,提示gp150蛋白的表达量与发育时间,前柄细胞分化有密切关系;在前柄细胞中能检测到31.5 kD和37.5 kD分子量大小的凋亡蛋白,且蛋白量也是随发育时间有所增加,在两种类型细胞中都可检测38.2 kD的凋亡蛋白.这些数据表明盘基网柄菌细胞凋亡过程中有类似Caspase-3的蛋白表达,它们的存在与细胞凋亡存在密切关系; gp150分子的表达与胱天蛋白酶的激活可能存在一定关系.     

盘基网柄菌细胞发育中gp150分子与凋亡蛋白作用分析

用Percoll密度梯度技术分离和收集盘基网柄菌前柄和前孢子细胞,Western blot分析gp150分子和胱天蛋白酶在前孢子细胞和前柄细胞两种类型细胞中的表达情况.结果显示：只能在前柄细胞中检测到gp150蛋白条带,并随细胞发育蛋白的量逐渐增加,提示gp150蛋白的表达量与发育时间,前柄细胞分化有密切关系;在前柄细胞中能检测到31.5kD和37.5kD分子量大小的凋亡蛋白,且蛋白量也是随发育时间有所增加,在两种类型细胞中都可检测38.2kD的凋亡蛋白.这些数据表明盘基网柄菌细胞凋亡过程中有类似Caspase-3的蛋白表达,它们的存在与细胞凋亡存在密切关系;gp150分子的表达与胱天蛋白酶的激活可能存在一定关系.     

中国流行株HIV—1gp120及其与IFNα基因共表达核酸疫苗质粒的构建及表达

目的 研究pGP和pGPIFNα在真核细胞中的表达。方法 以表达中国流行株HIV－1gp120基因的核酸疫苗质粒pGP及其表达中国流行株HIV－1gp120基因与IFNα基因与IFNα基因的核酸疫苗质粒pGPIFNα。转染BHK－21细胞，以间接免疫荧光鉴定其表达产物。结果 荧光显微镜下，pGP和pGPIFNα转染细胞可见绿色荧光，而HK－21细胞和空质粒则不见绿色荧光。结论 pGP和pGPIFNα可在真核细胞中表达目的蛋白gp120。     

A conserved structural motif shared by EIAV gp90 and HIV gp120

Both HIV (human immunodeficiency virus) and EIAV (equine infectious anemia virus) belong to the retroviridae family and lentivirus genus. EIAV is similar to HIV not only in its morphology, genomic structure and antigenicity, but also in its replicative and infectious characteristics. Their env genes comprise surface unit (SU) and trans- membrane protein (TM). The large number of disulfide bonds and N-glycosylation sites are prominent structural properties of SU (HIV gp120 and EIAV g…     

An engineered poliovirus chimaera elicits broadly reactive HIV-1 neutralizing antibodies

The Sabin type 1 vaccine strain of poliovirus is probably the safest and most successful live-attenuated vaccine virus used in humans. Its widespread use since the early 1960s has contributed significantly to the virtual eradication of poliomyelitis in developed countries. We have reported previously the construction of an intertypic antigen chimaera of poliovirus, based on the Sabin 1 strain, and proposed that this virus could be modified to express on its surface antigenic determinants from other pathogens. We describe here the construction and characterization of a poliovirus antigen chimaera containing an epitope from the transmembrane glycoprotein (gp41) of human immunodeficiency virus type 1 (HIV-1). In antibody absorption experiments, the virus chimaera inhibited neutralization of HIV-1 by antipeptide monoclonal antibodies specific for the gp41 epitope and significantly reduced the group specific neutralizing activity of HIV-1-positive human sera. Rabbit antisera raised by subcutaneous injection of the polio/HIV chimaera in adjuvant was shown to be specific for HIV-1 gp41 in peptide-binding assays and by western blotting. Moreover, the antisera neutralized a wide range of American and African HIV-1 isolates and also inhibited virus-induced cell fusion. Monoclonal antibodies against the HIV-1 derived regions of the chimaera also neutralized HIV-1. These results establish the potential of using poliovirus for the presentation of foreign antigens and suggest that Sabin 1 poliovirus/HIV chimaeras could offer an approach to the development of an HIV vaccine.     

Structure of an unliganded simian immunodeficiency virus gp120 core

Envelope glycoproteins of human and simian immunodeficiency virus (HIV and SIV) undergo a series of conformational changes when they interact with receptor (CD4) and co-receptor on the surface of a potential host cell, leading ultimately to fusion of viral and cellular membranes. Structures of fragments of gp120 and gp41 from the envelope protein are known, in conformations corresponding to their post-attachment and postfusion states, respectively. We report the crystal structure, at 4 A resolution, of a fully glycosylated SIV gp120 core, in a conformation representing its prefusion state, before interaction with CD4. Parts of the protein have a markedly different organization than they do in the CD4-bound state. Comparison of the unliganded and CD4-bound structures leads to a model for events that accompany receptor engagement of an envelope glycoprotein trimer. The two conformations of gp120 also present distinct antigenic surfaces. We identify the binding site for a compound that inhibits viral entry.     

Molecular architecture of native HIV-1 gp120 trimers

The envelope glycoproteins (Env) of human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate virus binding to the cell surface receptor CD4 on target cells to initiate infection. Env is a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120), and forms trimers on the surface of the viral membrane. Using cryo-electron tomography combined with three-dimensional image classification and averaging, we report the three-dimensional structures of trimeric Env displayed on native HIV-1 in the unliganded state, in complex with the broadly neutralizing antibody b12 and in a ternary complex with CD4 and the 17b antibody. By fitting the known crystal structures of the monomeric gp120 core in the b12- and CD4/17b-bound conformations into the density maps derived by electron tomography, we derive molecular models for the native HIV-1 gp120 trimer in unliganded and CD4-bound states. We demonstrate that CD4 binding results in a major reorganization of the Env trimer, causing an outward rotation and displacement of each gp120 monomer. This appears to be coupled with a rearrangement of the gp41 region along the central axis of the trimer, leading to closer contact between the viral and target cell membranes. Our findings elucidate the structure and conformational changes of trimeric HIV-1 gp120 relevant to antibody neutralization and attachment to target cells.     

Molecular motions and conformational transition between different conformational states of HIV-1 gp 120 envelope glycoprotein

The HIV-1 gp120 exterior envelope glycoprotein undergoes a series of conformational rearrangements while sequentially interacting with the receptor CD4 and coreceptor CCR5 or CXCR4 on the surface of host cells to initiate virus entry. Both the crystal structures of the HIV-1 gp120 core bound by the CD4 and antigen 17b and the SIV gp120 core pre-bound by CD4 are known. Despite the wealth of knowledge on these static snapshots of molecular conformations,the details of molecular motions involved in conformational transition that are crucial to intervention remain elusive. We presented comprehensive comparative analyses of the dynamics behaviors of the gp120 in its CD4-complexed,CD4-free and CD4-unliganded states based on the homology models with modeled V3 and V4 loops by means of CONCOORD computer simulation to generate ensembles of feasible protein structures that were sub-sequently analysed by essential dynamics analyses to identify preferred concerted motions. The re-vealed collective fluctuations are dominated by complex modes of combinational motions of the rota-tion/twisting,flexing/closure,and shortness/elongation between or within the inner,outer,and bridg-ing-sheet domains,and these modes are related to the CD4 association and HIV neutralization avoid-ance. Further essential subspace overlap analyses were performed to quantitatively distinguish the preference for conformational transitions between the three states,revealing that the unliganded gp120 has a greater potential to translate its conformation into the conformational state adopted by the CD4-complexed gp120 than by the CD4-free gp120,whereas the CD4-free gp120 has a greater potential to translate its conformation into the unliganded state than the CD4-complexed gp120 does. These dynamics data of gp120 in its different conformations are helpful in understanding the relationship between the molecular motion/conformational transition and the function of gp120,and in gp120-structure-based subunit vaccine design.     

Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160.

The envelope glycoprotein of human immunodeficiency virus (HIV) initiates infection by mediating fusion of the viral envelope with the cell membrane. Fusion activity requires proteolytic cleavage of the gp160 protein into gp120 and gp41 at a site containing several arginine and lysine residues. Activation at basic cleavage sites is observed with many membrane proteins of cellular and viral origin. We have recently found that the enzyme activating the haemagglutinin of fowl plague virus (FPV), an avian influenza virus, is furin. Furin, a subtilisin-like eukaryotic endoprotease, has a substrate specificity for the consensus amino-acid sequence Arg-X-Lys/Arg-Arg at the cleavage site. We show here that the glycoprotein of HIV-1, which has the same protease recognition motif as the FPV haemagglutinin, is also activated by furin.     

Predefined GPGRAFY-Epitope-Specific Monoclonal Antibodies with Different Activities for Recognizing Native HIV-1 gp120

A seven-amino acid epitope GPGRAFY at the tip of the V3 loop in HIV-1 gp120 is the principal neutralizing epitope, and a subset of anti-V3 antibodies specific for this epitope shows a broad range of neutralizing activity. GPGRAFY-epitope-specific neutralizing antibodies were produced using predefined GPGRAFY-epitope-specific peptides instead of a natural or recombinant gp120 bearing this epitope. All six monoclonal antibodies (mAbs) could recognize the GPGRAFY-epitope on peptides and two of the antibodies, 9D8 and 2D7, could recognize recombinant gp120 in enzymelinked immunosorkentassy (ELISA) assays. In the flow cytometry analysis, the mAbs 9D8 and 2D7 could bind to HIV-Env CHO-WT cells and the specific bindings could be inhibited by the GPGRAFY-epitope peptide, which suggests that these two mAbs could recognize the native envelope protein gp120 expressed on the cell membrane. However, in syncytium assays, none of the mAbs was capable of inhibiting HIV-Env-mediated cell membrane fusion. The different activities for recognizing native HIV-1 gp120 might be associated with different antibody affinities against the epitopes. The development of conformational mimics of the neutralization epitope in the gp120 V3 loop could elicit neutralizing mAbs with high affinity.     

The envelope glycoprotein of the human immunodeficiency virus binds to the immunoglobulin-like domain of CD4

CD4, a cell-surface glycoprotein expressed on a subset of T-cells and macrophages, serves as the receptor for the human immunodeficiency virus (HIV) (reviewed in ref. 1), binding to the HIV envelope glycoprotein, gp120 with high affinity. Attempts to block infection in vivo by raising antibodies against gp120 have failed, probably because these antibodies have insufficient neutralizing activity. In addition, because of the extensive polymorphism of gp120 in different isolates of HIV, antibodies raised against one HIV isolate are only weakly effective against others. Because interaction with CD4 is essential for infectivity by all isolates of HIV, an agent that could mimic CD4 in its ability to bind to gp120, such as a peptide or monoclonal antibody, might block infection by a wide spectrum of isolates. To aid the identification of such a ligand we have defined regions of CD4 that are required for binding to gp120. Although human CD4 is similar to mouse CD4 in amino-acid sequence (55% identity, ref. 6) and structure, we have found that the murine protein fails to bind detectably to gp120 and have exploited this finding to study binding of gp120 to mouse-human chimaeric CD4 molecules. These studies show that amino-acid residues within the amino-terminal immunoglobulin-like domain of human CD4 are involved in binding to gp120 as well as to many anti-CD4 monoclonal antibodies.