Production of monoclonal antibodies in eggs of transgenic avians

转基因鸡被认为是一种生产重组蛋白的转基因生物反应器。到目前为止，虽然已经报道了产生转基因禽类的各种方法，但还没有达到商业上可行的水平表达转基因。以水疱性口炎病毒G蛋白(VSV-G)为假型的Moloney鼠白血病病毒(MoMLV)为基础，构建了复制缺陷型泛嗜性逆转录病毒载体，用于鸡胚胎的基因导入。为了获得产生单抗的转基因鸡，我们首先构建了逆转录病毒载体表达单链抗体片段(ScFv)，该单链抗体片段与人IgG1的Fc区融合，作为模型抗体。我们通过将浓缩的逆转录病毒载体注射到发育中的胚胎心脏，55h孵化，然后进行胚胎培养孵化，稳定地获得了转基因鸡(GO)。The Go ch…在整个繁殖期，更多的KENS在它们的血清和鸡蛋中表达了高水平的转基因产物(~5.6 mg/ml)。蛋清中的表达水平几乎相当于卵清蛋白的十分之一，而卵清蛋白是蛋清中的一种主要蛋白质，与溶菌酶的表达水平相当。此外，我们还可以获得来自围棋鸡的G1和G2转基因后代。转基因后代还在其血清和卵子中表达了转基因产物。接下来，我们构建了抗CD2嵌合抗体的逆转录病毒载体，其中抗体的H链和L链基因的双顺反子表达是由脑心肌炎病毒的内部核糖体进入位点(IRES)序列介导的。55h后将病毒载体注射到鸡胚中，获得转GO基因的鸡。孵化的雏鸡在血清和鸡蛋中都表达了抗CD2抗体。该系统展示了转基因鸡生物反应器用于商业生产单抗的现实。较少

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