Bckström M, Holmgren J, Schödel F, Lebens M
Department of Medical Microbiology and Immunology, Göteborg University, Sweden.
Gene. 1995 Nov 20;165(2):163-71. doi: 10.1016/0378-1119(95)00444-b.
We previously described the construction of novel hybrid proteins based on the B-subunit of cholera toxin (CTB) [Bäckström et al., Gene 149 (1994) 211-217], in which a neutralizing B-cell epitope from the third variable (V3) loop in the envelope glycoprotein gp120 from human immunodeficiency virus type 1 (HIV-1) was inserted within a surface-exposed region between amino acids (aa) 55 and 64. The resulting protein retained properties of native CTB and could induce strong anti-CTB antibody (Ab) responses, but the inserted gp120 epitope was only modestly immunogenic. In this study, the potential use of this internal permissive site in CTB for the insertion of heterologous epitopes has been further investigated. Six additional plasmids were constructed encoding HIV::CTB hybrid proteins with ten to fourteen aa from the V3 loop of gp120 genetically inserted at different positions between aa 52 and 65, with deletions of different CTB aa. Plasmids encoding proteins with peptides inserted between aa 53 and 64 in CTB gave rise to stable proteins which reacted with CTB-specific monoclonal antibodies (mAb) and bound to GM1 gangliosides (GM1), indicating that insertions between these positions do not drastically alter the conformation or the receptor-binding properties of native CTB. Plasmids were also constructed encoding CTB hybrid proteins which had either an 11-aa peptide from hepatitis B virus (HBV) pre-S(2) or one of two peptides related to the heat-stable toxin (STa) of enterotoxigenic Escherichia coli inserted between aa 55 and 64 of CTB. This resulted in the production of HBV::CTB or ST::CTB hybrid proteins and illustrated that the internal permissive site can be used for insertion of peptides of varying aa composition. The reactivity of the inserted epitopes with epitope-specific mAb in GM1-ELISA and immunoblots varied greatly between hybrid proteins and depended on the position in CTB and the aa composition of the inserted peptides. Despite these differences, all the HIV::CTB, ST::CTB and HBV::CTB hybrid proteins could induce low, but significant, levels of serum Ab in mice against gp120, STa or pre-S(2), in addition to strong serum Ab responses against CTB. The Ab response against the internally inserted gp120 peptide was similar to that against the same peptide fused to the N-terminus of CTB, indicating that internally placed peptides had similar immunogenicity to the same peptides added terminally.
我们之前描述了基于霍乱毒素（CTB）B亚基构建新型杂合蛋白的方法[Bäckström等人，《基因》149（1994）211 - 217]，其中来自1型人类免疫缺陷病毒（HIV - 1）包膜糖蛋白gp120第三个可变（V3）环的一个中和性B细胞表位被插入到氨基酸（aa）55和64之间的一个表面暴露区域内。所得蛋白保留了天然CTB的特性，并且能够诱导强烈的抗CTB抗体（Ab）反应，但插入的gp120表位免疫原性仅适度。在本研究中，进一步研究了CTB中这个内部允许位点用于插入异源表位的潜在用途。构建了另外六个质粒，编码HIV::CTB杂合蛋白，其中来自gp120 V3环的10至14个氨基酸被基因插入到aa 52和65之间的不同位置，并缺失了不同的CTB氨基酸。编码在CTB的aa 53和64之间插入肽段的蛋白的质粒产生了稳定的蛋白，这些蛋白与CTB特异性单克隆抗体（mAb）反应并结合GM1神经节苷脂（GM1），表明在这些位置之间的插入不会显著改变天然CTB的构象或受体结合特性。还构建了编码CTB杂合蛋白的质粒，这些杂合蛋白在CTB的aa 55和64之间插入了来自乙型肝炎病毒（HBV）前S(2)的一个11氨基酸肽段或与产肠毒素大肠杆菌热稳定毒素（STa）相关的两个肽段之一。这导致产生了HBV::CTB或ST::CTB杂合蛋白，并表明该内部允许位点可用于插入不同氨基酸组成的肽段。在GM1 - ELISA和免疫印迹中，插入表位与表位特异性mAb的反应性在杂合蛋白之间差异很大，并且取决于其在CTB中的位置以及插入肽段的氨基酸组成。尽管存在这些差异，但所有HIV::CTB、ST::CTB和HBV::CTB杂合蛋白除了能诱导针对CTB的强烈血清Ab反应外，还能在小鼠中诱导针对gp120、STa或前S(2)的低水平但显著的血清Ab。针对内部插入的gp120肽段的Ab反应与针对融合到CTB N端的相同肽段的反应相似，表明内部放置的肽段与末端添加的相同肽段具有相似的免疫原性。
