HIV-1囊膜糖蛋白gp41核心结构域基因的表达及其单克隆抗体的制备
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摘要
艾滋病(AIDS)是由人类免疫缺陷病毒(HIV-1和HIV-2，主要为HIV-1)感染引起的一种后天性人类免疫缺陷综合征。目前用于抑制HIV-1的治疗药物主要针对病毒反转录酶和蛋白酶。尽管这些药物可以减缓HIV-1感染者向AIDS患者发展的进程，但长期使用存在副作用大、价格昂贵、易产生耐药性等缺点。因此，寻找有效而安全的治疗AIDS的药物成为当务之急。
随着对HIV-1进攻靶细胞过程的深入了解，人们不断发现新的极具吸引力的药物靶点，并为研制有效疫苗提供了新的策略。HIV-1属于囊膜病毒，其囊膜蛋白复合物控制着病毒进入靶细胞的关键步骤，即决定着病毒趋向性和促进病毒基因组进入靶细胞以及感染细胞与邻近的非感染的细胞之间的膜融合过程。
介导病毒膜与靶细胞膜融合的关键分子是HIV-1囊膜糖蛋白
第四军医大学硕士学位论文
gp41，其结构相对保守，针对gP41的药物可能避免耐药性的形成，
是抑制病毒进入靶细胞的理想靶位。gp41的晶体结构已经明确，
其融合活性构象的核心结构是一个由三个发卡结构紧密排列而成
的六聚体螺旋束，gP41的N端螺旋形成中心的三聚体，而C端螺
旋以反平行方式堆积在N端螺旋形成的疏水槽表面，组成外层的
三聚体螺旋。该空间构象己被证实是促使病毒与靶细胞膜融合的
关键构象，结构十分稳定。
Nsl(L6)e46和N36(L6)e34均为模拟gp41融合态核心结构
空f司构象的合成肤。Nsl(L6)C46是在N36(L6)C34基础上在C末
端增加了一个含有中和表位ELDKWA序列的12月太序列。它们的
一级序列与具有抑制活性的C多肤重叠。Jiang等针对
N36(L6)C34合成肤研制了单克隆抗体(MAb)NC一1。该MAb
能特异识别Hlv一1 gp41六股。一螺旋束核心结构空间构型，但未显
示中和活性。
本研究首先运用原核表达系统表达了N51(L6)C46多肤，并
用能特异识别Hlv一1 gP41三聚体特征性的六股。一螺旋束核心结
构空间构型的MAb NC一l进行Western blot鉴定，证实Nsl(L6)C46
多肤单体(14KDa)及其三聚体(36KDa)与MAb NC一1反应，
提示该多肤可形成gp41核心结构空间构型特征，结构稳定，SDS、
二琉基乙醇的存在并不影响其空间构型的形成，可作为制备针对
gP41核心结构空间构型MAb的免疫原。
采用Nsl(L6)e46多肤免疫小鼠制备了针对N51(L6)e46多肤
的4株MAb，其中MAb 1 AZ、IA4和IBg针对多肤单体，MAb 3AI
针对多肤三聚体。利用ELISA间接夹心法进一步鉴定了MAb的
特异性，包被兔抗N36(L6) C34抗体，特异吸附多肤后再检测
杂交瘤细胞培养上清，结果显示这4株抗体都不同程度地与兔抗
N36(L6) C34捕获的抗原反应，证实它们能特异识别表达多肤，
但这4株MAb不与单独的N36或C34多肤片段反应，提示它们
3
第四军医大学硕士学位论文
是抗多肤空间构象的MAb。将这4株MAb与MAb NC一1的抗体
相对亲和力作比较，其高低依次为NC一1>IB9>IA2>3AI>IA4。位
点相加和竞争ELISA法分析其抗原表位，发现这4株MAb与MAb
NC一1识别的抗原表位近似，但也存在一些差异。其中识别多肤单
体的MAb 1 AZ、1 A4、1 Bg和MAb NC一l识别的抗原表位较接近，
而识别多肤三聚体的MAb 3AI与其它4株抗体识别表位差异均相
对较大。
上述4株MAb有可能成为筛选抗Hlv多肤及分析gp41表位
免疫原特性的有效工具。
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 envelope
protein 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. Then
the 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
随着对HIV-1进攻靶细胞过程的深入了解，人们不断发现新的极具吸引力的药物靶点，并为研制有效疫苗提供了新的策略。HIV-1属于囊膜病毒，其囊膜蛋白复合物控制着病毒进入靶细胞的关键步骤，即决定着病毒趋向性和促进病毒基因组进入靶细胞以及感染细胞与邻近的非感染的细胞之间的膜融合过程。
介导病毒膜与靶细胞膜融合的关键分子是HIV-1囊膜糖蛋白
第四军医大学硕士学位论文
gp41，其结构相对保守，针对gP41的药物可能避免耐药性的形成，
是抑制病毒进入靶细胞的理想靶位。gp41的晶体结构已经明确，
其融合活性构象的核心结构是一个由三个发卡结构紧密排列而成
的六聚体螺旋束，gP41的N端螺旋形成中心的三聚体，而C端螺
旋以反平行方式堆积在N端螺旋形成的疏水槽表面，组成外层的
三聚体螺旋。该空间构象己被证实是促使病毒与靶细胞膜融合的
关键构象，结构十分稳定。
Nsl(L6)e46和N36(L6)e34均为模拟gp41融合态核心结构
空f司构象的合成肤。Nsl(L6)C46是在N36(L6)C34基础上在C末
端增加了一个含有中和表位ELDKWA序列的12月太序列。它们的
一级序列与具有抑制活性的C多肤重叠。Jiang等针对
N36(L6)C34合成肤研制了单克隆抗体(MAb)NC一1。该MAb
能特异识别Hlv一1 gp41六股。一螺旋束核心结构空间构型，但未显
示中和活性。
本研究首先运用原核表达系统表达了N51(L6)C46多肤，并
用能特异识别Hlv一1 gP41三聚体特征性的六股。一螺旋束核心结
构空间构型的MAb NC一l进行Western blot鉴定，证实Nsl(L6)C46
多肤单体(14KDa)及其三聚体(36KDa)与MAb NC一1反应，
提示该多肤可形成gp41核心结构空间构型特征，结构稳定，SDS、
二琉基乙醇的存在并不影响其空间构型的形成，可作为制备针对
gP41核心结构空间构型MAb的免疫原。
采用Nsl(L6)e46多肤免疫小鼠制备了针对N51(L6)e46多肤
的4株MAb，其中MAb 1 AZ、IA4和IBg针对多肤单体，MAb 3AI
针对多肤三聚体。利用ELISA间接夹心法进一步鉴定了MAb的
特异性，包被兔抗N36(L6) C34抗体，特异吸附多肤后再检测
杂交瘤细胞培养上清，结果显示这4株抗体都不同程度地与兔抗
N36(L6) C34捕获的抗原反应，证实它们能特异识别表达多肤，
但这4株MAb不与单独的N36或C34多肤片段反应，提示它们
3
第四军医大学硕士学位论文
是抗多肤空间构象的MAb。将这4株MAb与MAb NC一1的抗体
相对亲和力作比较，其高低依次为NC一1>IB9>IA2>3AI>IA4。位
点相加和竞争ELISA法分析其抗原表位，发现这4株MAb与MAb
NC一1识别的抗原表位近似，但也存在一些差异。其中识别多肤单
体的MAb 1 AZ、1 A4、1 Bg和MAb NC一l识别的抗原表位较接近，
而识别多肤三聚体的MAb 3AI与其它4株抗体识别表位差异均相
对较大。
上述4株MAb有可能成为筛选抗Hlv多肤及分析gp41表位
免疫原特性的有效工具。
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 envelope
protein 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. Then
the 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
引文
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