Production of monoclonal antibodies in eggs of transgenic avians

转基因鸡已被提出作为生产重组蛋白的转基因生物反应器。迄今为止，尽管已经报道了各种产生转基因家禽的程序，但尚未达到商业上可行水平的转基因表达。我们使用一种复制缺陷型泛嗜性逆转录病毒载体，将带有水泡性口炎病毒G蛋白（VSV-G）的Moloney小鼠白血病病毒（MoMLV）引入禽胚。为了使转基因鸡产生单克隆抗体，我们首先构建了逆转录病毒载体质粒，用于表达单链抗体片段（scFv），该片段来源于抗朊病毒蛋白单克隆抗体，与人IgG1的Fc区融合，作为模型抗体。我们将浓缩的逆转录病毒载体注射到发育中的胚胎的心脏中，经过55小时的孵育，然后进行胚胎培养孵化，从而稳定地产生转基因鸡（GO）。结果表明，在整个繁殖期内，转基因产物在鸡的血清和鸡蛋中表达水平较高（约5.6 mg/ml）。其在蛋白中的表达水平几乎相当于蛋白中主要蛋白卵清蛋白的十分之一，与溶菌酶的表达水平相当。此外，我们还可以从GO鸡中获得G1和G2转基因后代。转基因后代在其血清和卵子中也表达了转基因产物。接下来，我们构建了一个逆转录病毒载体，用于生产嵌合抗cd2抗体，其中抗体的h链和l链基因的双链表达由源自脑心肌炎病毒（EMCV）的内核糖体进入位点（IRES）序列介导。将该病毒载体注射到鸡胚中，孵育55 h后可获得氧化石墨烯转基因鸡。孵出的鸡血清和蛋中均表达抗cd2抗体。该系统证明了转基因鸡生物反应器用于单克隆抗体商业化生产的可行性。少

Transgenic chickens have been proposed as a transgenic bioreactor for the production of recombinant proteins. To date, although various procedures for generating transgenic avians have been reported, the expression of a transgene at a commercially feasible level has not been attained. We used a replication-defective pantropic retroviral vector based on Moloney murine leukemia virus(MoMLV) pseudotyped with vesicular stomatitis virus G protein(VSV-G) for gene introduction to avian embryos. For the generation of transgenic chickens producing monoclonal antibodies, we firstly constructed a retroviral vector plasmid for the expression of a single-chain antibody fragment(scFv), derived from an anti-prion protein monoclonal antibody, fused with the Fc region of human IgG1, as a model antibody. We steadily generated transgenic chickens (GO) by injecting a concentrated retroviral vector into the heart of developing embryos after 55 h incubation and followed by embryo culture to hatch. The GO ch … More ickens expressed the transgene product in their serum and eggs at high levels (~5.6 mg/ml) throughout the breeding period. The expression level in egg white corresponds to almost one tenth that of ovalbumin, a major protein in egg white, and more than equivalent to that of lysozyme. Furthermore, we could obtain G1 and G2 transgenic progeny derived from a GO chicken. The transgenic progeny also expressed the transgene product in their serum and eggs. Next, we constructed a retroviral vector for the production of a chimeric anti-CD2 antibody, in which the bicistronic expression of H-and L-chain genes of the antibody was mediated by an internal ribosomal entry site(IRES) sequence derived from encephalomyocarditis virus(EMCV). The viral vector was injected to chicken embryos after 55 h incubation to generate GO transgenic chickens. The chickens hatched expressed anti-CD2 antibody in the serum and eggs. This system demonstrates the reality of a transgenic chicken bioreactor for the commercial production of monoclonal antibodies. Less