| The human immunodeficiency virus (HIV) is the etiologic agents of acquired immunodeficiency syndrome in human. All current therapeutic agents for HIV type 1 (HIV-1) infection are directed at the viral enzymes, including reverse transcriptase and protease. Despite the success of these drugs in reducing the progression of HIV-1 infection to AIDS, there are increasing problems with long-term toxicity, high cost, difficulties adhering to treatment regimens, and emergence of multiple, drug-resistant viral strains. So it is urgent to find new type of effective and safe theraputic drug.Fortunately, rapid advances in understanding how HIV-1 enter cells have lead to the identification of promising new drug targets and have also suggested new strategies for the generation of vaccine candidates. HIV-1 is an enveloped virus, and its envelope protein complex controls the key process of viral entry. This envelope protein determines viral tropism and facilitates the membrane fusion process that allows invasion of the viral genome. The envelopeprotein can also promote the fusion of infected cells with uninfected neighboring cells.The transmembrane glycoprotein gp41 could promote the fusion of viral and cellular membranes. Owing to the conservative structure of gp41, anti-gp41 drugs maybe avoid drug resistance development. So gp41 could be an attractive target for the development of antiviral agents. Crystallographic analysis has demonstrated that the gp41 fusion-active core adopts a six-stranded helical bundle, in which three N-terminal peptides adopt a homo-trimeric helical coiled-coli forming the center of the bundle and three C-terminal peptide helices pack into hydrophobic grooves on the outer surface of the N-peptide core in an antiparallel manner forming a trimer-of-hairpins structure. The trimer-of-hairpins likely resembles the fusion-active conformation since this structural motif brings the N-terminal region of gp41 containing the fusion peptide together with the C-terminal region that is anchored to the viral membrane. This conformation rearrangement brings the viral and target cell membranes together and promots membrane fusion.N51(L6)C46 and N36(L6)C34 are synthetic polypeptide which could simulate the fusogenic core structure of gp41. N51(L6)C46 polypeptide includes neutralizing epitope (ELDKWA), but N36(L6)C34 hasn't it. Their sequences are same as part of the C-terminal of gp41. The monoclonal antibody (MAb) against N36(L6)C34, NC-1, could specific recognition of conformational epitopes on the six-helix core of gp41. But it has no detectable inhibitory activity for HIV-1 infection.In this study we performed the expression of N51(L6)C46 gene of HIV-1 gp41 core structural domain in E.Coli BL(21)DE3. Thenthe expressed polypeptide was detected by Western blot with the conformation-specific monoclonal antibody, designated NC-1, specifically recognizes the fusogenic core structure of gp41. The results showed that the N51(L6)C46 polypeptide, including monomer and trimer, could be recognized by NC-1. It suggested that the structure of the polypeptide resemble that of the proposed fusion-active core stucture of gp41. So the expressed polypeptide could be used as immunogen in following study.Then, we generated four cloned MAbs from mice immunized with the polypeptide N51(L6)C46, three of them (1A2, 1A4, 1B9) against the polypeptide N51(L6)C46 monomer and one against the trimer, named 3A1. We found that the four anti-N51(L6)C46 MAbs could bind N51(L6)C46 or N36(L6)C34, but not N36 or C34. The results suggested the antibodies maybe react with the oligomeric forms of gp41 or a -helical core domain specificly. The relative affinity sequence of the MAbs is NC-1>1B9>1 A2>3A1>1A4. The results of epitope analysis with competitive ELISA and pairwise testing showed that the MAbs could recognize the similar epitopes, whereas the difference lies in that the epitope recognized by 3A1 is comparatively different from those of others.All these MAbs will be effective tools in selecting anti-HIV polypeptide and analysing t... |
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