Ovalbumin locus BACs for expression of biotherapeutics

描述（由申请人提供）：鸡卵白蛋白基因位点具有每个鸡蛋每天2克蛋白质的生产能力，有可能将大量生物制药蛋白质合成到转基因母鸡的鸡蛋中。该一期SBIR项目将测试基于完整的200千碱基鸡卵白蛋白基因位点使用大表达构建的可行性，以驱动包括人源化单克隆抗体在内的生物制药蛋白的高效表达。该项目的长期目标是将这些结构用于转基因母鸡的培育，在第二阶段，将在蛋清中产生mg/ml浓度的生物制药蛋白。在这个第一阶段的提案中，我们计划利用reca辅助限制性内切酶（RARE）切割技术，从两条重叠的细菌人工染色体（BAC）中创建一个完整的200千碱基卵清蛋白基因。然后，我们将使用类似的技术将报告基因和生物制药基因插入卵清蛋白基因转录区域的不同位置。最后，我们将通过体外和体内实验来检测这些构建体的蛋白表达。

DESCRIPTION (provided by applicant): With a daily production capacity of two grams of protein per egg, the chicken ovalbumin gene locus has the potential to synthesize large amounts of biopharmaceutical proteins into the eggs of transgenic hens. This phase I SBIR project will test the feasibility of using large expression constructs, based on the complete 200 kilobase chicken ovalbumin gene locus, to drive efficient expression of biopharmaceutical proteins including humanized monoclonal antibodies. The longer-term objectives of the program are to use these constructs in the creation of transgenic hens, in phase II that will produce biopharmaceutical proteins at mg/ml concentration in egg white. In this phase I proposal, we plan to create a complete 200 kilobase ovalbumin gene from two overlapping bacterial artificial chromosomes (BAC) by utilizing recA-assisted restriction endonuclease (RARE) cleavage techniques. Using similar techniques, we will then insert reporter and biopharmaceutical genes into various locations within the transcribed region of the ovalbumin gene. Finally, we will assay protein expression from these constructs using in vitro and in vivo assays.