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。     

HIV-1CRF07-BC gp41 HR2高突变区氨基酸结构和功能分析

HIV-1包膜蛋白ENV介导病毒进入靶细胞,这个过程可以作为设计抗艾滋病药物的一个重要靶点,进入抑制剂能够抑制HIV进入靶细胞,从而在感染的最初环节抑制病毒的传播.为了更好地筛选针对中国株CRF07-BC的进入抑制剂,建立了细胞-细胞融合系统.通过比较CRF07-BC gp41和B亚型gp41的序列同源性和融合能力,gp41 HR2存在着一个7个氨基酸的高突变区.通过结构生物学对gp41 HR2高突变区进行分析发现,高突变区内氨基酸可以在功能上互补,保持了gp41的融合能力.这种功能互补性的研究可以为HIV gp41进化提供理论参考,为HIV-1进入抑制剂的设计提供更准确的靶点.     

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.     

Biological function of HIV-1 transmembrane protein gp41──A study on a putative cellular receptor of gp41

Since 1992, the study of biological functions of HIV-1 gp41 has made great progress. Experimental evidence from several research groups demonstrated that gp41 has a putative cellular receptor. A recombinant soluble gp41 (aa539-684) and gp41 immunosuppressive peptide (aa583-599) could bind to human B lymphocytes and monocytes, but weakly bind to T lymphocytes. It was found that gp41 contains two cellular binding sites (aa583-599 and 641-675). GP41 could selectively inhibit cell proliferation of human T, B lymphocytes and monocytes, enhance human MHC class Ⅰ, Ⅱ and ICAM-1 molecule expression on cell surface. Gp41 binding proteins and a monoclonal antibody against the first binding site could inhibit this modulation effect. Amino acid sequence homology exists between gp41 and human type Ⅰ interferons, and the homologous region is located in the first binding site on gp41 and in the receptor binding site on type Ⅰ interferons. Studies in other groups indicate that both binding sites in gp41 may be associated with HIV infection of cells. Peptides containing two binding sites could respectively inhibit HIV infection of cells. A monoclonal antibody recognizing the second binding site could neutralize lab-strains and recently separated strains of HIV-1. Besides, antibodies against two regions (homologous with gp41 binding sites) of SIV transmembrane protein gp32 could protect macaques from SIV infection. These results suggest that the study of gp41 binding sites and cellular receptor could contribute to understanding the mechanism of HIV infection and to developing HIV vaccine and anti-HIV drugs.     

影响HIV-GP41N端1/2在E.coli中表达的两段氨基酸序列界定

GP41蛋白二级结构预测显示 ,前 1 /2片段的 N端 ( 4～ 2 6位氨基酸 )和 C端 ( 1 67～ 1 89位氨基酸 )各有一个富含疏水氨基酸的穿膜螺旋 (可能性 >0 .9) ,分别从 N1 (前 1 /2片段无 C端穿膜螺旋 )的 N端和 N6(前1 /2片段无 N端穿膜螺旋 )的 C端进行缺失构建一系列缺失突变体 ,只有 p ET-N2 ,p ET-N3 ,p ET-N4,p ET-N5在大肠杆菌中获得高效表达 ,其表达量占菌体总蛋白的 2 0 % ,Western blot显示表达蛋白与 HIV阳性血清有良好的反应性 .而 p ET-N1 ,p ET-N6在大肠杆菌中表达量很低或不表达 .从而证明 1～ 6位 ,1 84～ 2 0 1位氨基酸是影响 gp41基因 N端 1 /2片段表达的主要因素     

Monoclonal Antibodies Recognizing HIV-1 gp41 Could Inhibit Env-Mediated Syncytium Formation

Some monoclonal antibodies （mAbs） could inhibit infection by HIV-1. In this study, four mAbs against HIV-1 gp41 were prepared in mice. All four mAbs could bind to the recombinant soluble gp41 and recognize the native envelope glycoprotein gp160 expressed on the HIV-Env^＋ CHO-WT cell in flow cytometry analysis. Interestingly, the results show that all four mAbs purified by affinity chromatography could inhibit HIV-1 Env-mediated membrane fusion （syncytium formation） by 40%-60% at 10 μg/mL, which implies potential inhibitory activities against HIV-1.     

含抗HIV广谱中和抗体2F5、4E10靶基因载体的构建及鉴定

目的:构建含HIV gp120,gp41序列中广谱中和抗体2F5,4E10作用靶基因的载体并进行鉴定,为后期重组载体表达产物诱导产生中和抗体及抗HIV亚单位疫苗的研究奠定基础.方法:根据NCBI中HIV gp120,gp41基因序列中可与2F5、4E10结合的区域设计引物并进行PCR反应,将PCR得到的目的片段插入到载体pET28a中,对重组载体进行PCR鉴定、酶切鉴定及DNA测序.结果:PCR鉴定、酶切鉴定及DNA测序结果证实重组载体构建成功.结论:成功构建了含HIV gp120,gp41序列中广谱中和抗体2F5,4E10作用靶基因的载体.     

抗羊布鲁菌核糖体蛋白L7/L12单克隆抗体制备及初步鉴定

制备抗羊布鲁菌核糖体蛋白L7／L12的单克隆抗体（mAb）,为进一步研制布鲁菌检测方法和免疫制剂提供基础。常规动物免疫、细胞融合、克隆化制备抗核糖体蛋白L7／L12的mAb,并用Western blot和ELISA方法对其特异性、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L7／L12蛋白的mAb：1B1,2A2,2H9和2H10,相对亲和力为2A2〉2H10〉2H9〉181,4株mAb识别的抗原表位相近,但仍存在一定的差异。得到的4株稳定的杂交瘤细胞系分泌的mAb能特异结合L7／L12蛋白。     

Monoclonal Anti-CD4 Antibody MT310 Binds HIV-1 gp120 Binding Site on CD4

IntroductionHuman immunodeficiency virus type1 ( HIV- 1 ) ,after binding by the envelope protein gp1 2 0 to itscellular receptor CD4,enters susceptible cellsthrough p H- independent fusion[1,2 ] . The sitewhich HIV- 1 gp1 2 0 binds is located in the firstdomain on the CD4molecule[3 5] .The anti- CD4m Ab Leu3a recognizes the gp1 2 0 binding site onCD4[6] and the anti- CD4m Ab MT31 0 stronglyinhibits HIV- 1 infection of CD4+ T cells,while theanti- CD4 m Ab MT1 5 1 weakly inhibitsinf…     

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 interaction between the membrane-proximal external region and the N-trimer region of HIV-1 gp41: Involvement in viral fusion

The membrane proximal external region (MPER) of gp41 is extremely conserved among diverse HIV-1 variants, implying its important role in viral infection. Interestingly, two of the most broadly neutralizing antibodies, 2F5 and 4E10, specifically recognize this region. Our previous study demonstrated that the antigenicity and immunogenicity of 4E10 epitope are affected by remodeling gp41 fusion core, suggesting that the MPER may be associated with gp41 core and involved in gp41-mediated membrane fusion. Here we measured the binding activity of 4E10 epitope peptide (D4E10P) with various gp41 core-derived peptides and found that the N-trimer region in a construct designated N-trimer-6HB interacted significantly with D4E10P. Using N-trimer-6HB to screen a phage library, we identified a motif (WF) located in 4E10 epitope that may play a certain role in the interaction of gp41 MPER with the N-trimer in gp41 fusion core and, we thus speculated upon the potential involvement of MPER in the fusion process between viral envelope and target cell membrane. Supported by National Key Basic Research and Development Program of China (Grant No. 2007CB914402)     

Biological function of H1V-1 transmembrane protein gp41

Since 1992, the study of biological functions of HIV-1 gp41 has made great progress. Experimental evidence from several research groups demonstrated that gp41 has a putative cellular receptor. A recombinant soluble gp41 (aa539–684) and gp41 immunosuppressive peptide (aa583–599) could bind to human B lymphocytes and monocytes, but weakly bind to T lymphocytes. It was found that gp41 contains two cellular binding sites (aa583–599 and 641–675). GP41 could selectively inhibit cell proliferation of human T, B lymphocytes and monocytes, enhance human MHC class I, II and ICAM-1 molecule expression on cell surface. Gp41 binding proteins and a monoclonal antibody against the first binding site could inhibit this modulation effect. Amino acid sequence homology exists between gp41 and human type I interferons, and the homologous region is located in the first binding site on gp41 and in the receptor binding site on type I interferons. Studies in other groups indicate that both binding sites in gp41 may be associated with HIV infection of cells. Peptides containing two binding sites could respectively inhibit HIV infection of cells. A monoclonal antibody recognizing the second binding site could neutralize lab-strains and recently separated strains of HIV-1. Besides, antibodies against two regions (homologous with gp41 binding sites) of SIV transmembrane protein gp32 could protect macaques from SIV infection. These results suggest that the study of gp41 binding sites and cellular receptor could contribute to understanding the mechanism of HIV infection and to developing HIV vaccine and anti-HIV drugs.     

Induction of multi-epitopespecific antibodies against HIV-1 by multi-epitopevaccines

Some neutralizing antibodies against HIV-1 envelope proteins were highly effective to inhibit the infection of different strainsin vitro, and existed in the infected individuals with very low levels. We suggested multi-epitope-vaccine as a new strategy to increase levels of neutralizing antibodies and the abilities against HIV mutationin vivo. Two candidate multi-epitope-vaccines induced antibodies with predefined multi-epitope-specificity in rhesus macaque. These antibodies recognized corresponding neutralizing epitopes on epitope-peptides, gp41 peptides, V3 loop peptide, rsgp41 and rgp120. Besides, three candidate epitope-vaccines in combination (another kind of multi-epitopevaccines) showed similar potency to induce predefined multiple immune responses in rabbits. These results suggest that multi-epitope-vaccines may be a new strategy to induce multi-antiviral activities against HIV-1 infection and mutations.