Sola I, Castilla J, Pintado B, Sánchez-Morgado J M, Whitelaw C B, Clark A J, Enjuanes L
Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Department of Molecular and Cell Biology, Campus Universidad Autónoma, Cantoblanco, Madrid, Spain.
J Virol. 1998 May;72(5):3762-72. doi: 10.1128/JVI.72.5.3762-3772.1998.
Ten lines of transgenic mice secreting transmissible gastroenteritis coronavirus (TGEV) neutralizing recombinant monoclonal antibodies (rMAbs) into the milk were generated. The rMAb light- and heavy-chain genes were assembled by fusing the genes encoding the variable modules of the murine MAb 6A.C3, which binds an interspecies conserved coronavirus epitope essential for virus infectivity, and a constant module from a porcine myeloma with the immunoglobulin A (IgA) isotype. The chimeric antibody led to dimer formation in the presence of J chain. The neutralization specific activity of the recombinant antibody produced in transiently or stably transformed cells was 50-fold higher than that of a monomeric rMAb with the IgG1 isotype and an identical binding site. This rMAb had titers of up to 10(4) by radioimmunoassay (RIA) and neutralized virus infectivity up to 10(4)-fold. Of 23 transgenic mice, 17 integrated both light and heavy chains, and at least 10 of them transmitted both genes to the progeny, leading to 100% of animals secreting functional TGEV neutralizing antibody during lactation. Selected mice produced milk with TGEV-specific antibody titers higher than 10(6) as determined by RIA, neutralized virus infectivity by 10(6)-fold, and produced up to 6 mg of antibody per ml. Antibody expression levels were transgene copy number independent and integration site dependent. Comicroinjection of the genomic beta-lactoglobulin gene with rMAb light- and heavy-chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and beta-lactoglobulin genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of beta-lactoglobulin cointegration. This approach may lead to the generation of transgenic animals providing lactogenic immunity to their progeny against enteric pathogens.
培育出了十株能在乳汁中分泌可传播性胃肠炎冠状病毒（TGEV）中和重组单克隆抗体（rMAb）的转基因小鼠。rMAb轻链和重链基因是通过将编码鼠单克隆抗体6A.C3可变模块的基因与猪骨髓瘤恒定模块融合组装而成，鼠单克隆抗体6A.C3可结合对病毒感染至关重要的种间保守冠状病毒表位，猪骨髓瘤恒定模块具有免疫球蛋白A（IgA）同种型。嵌合抗体在J链存在的情况下会形成二聚体。在瞬时或稳定转化细胞中产生的重组抗体的中和比活性比具有IgG1同种型和相同结合位点的单体rMAb高50倍。通过放射免疫分析（RIA），这种rMAb的效价高达10⁴，可中和高达10⁴倍的病毒感染性。在23只转基因小鼠中，17只整合了轻链和重链，其中至少10只将两个基因传递给了后代，导致100%的动物在哺乳期分泌功能性TGEV中和抗体。通过RIA测定，挑选出的小鼠所产乳汁中TGEV特异性抗体效价高于10⁶，可中和10⁶倍的病毒感染性，每毫升乳汁中可产生多达6毫克抗体。抗体表达水平与转基因拷贝数无关，而与整合位点有关。将基因组β-乳球蛋白基因与rMAb轻链和重链基因共显微注射，导致产生了携带这三个转基因的转基因小鼠。整合了抗体和β-乳球蛋白基因的转基因小鼠产生的抗体效价最高，尽管所产生的转基因动物数量不足以就β-乳球蛋白共整合的增强作用得出明确结论。这种方法可能会导致产生能为后代提供针对肠道病原体的泌乳免疫的转基因动物。
