转基因鸡生物反应器载体的构建及其表达特性分析

Construction and expression characterization of transgenic chicken bioreactor vector

摘要:

以增强型绿色荧光蛋白和萤火虫荧光素酶为报告基因，构建了鸡卵清蛋白启动子表达载体和慢病毒载体，以巨细胞病毒 (Cytomegalovirus，CMV)启动子表达载体为对照，转染或感染鸡原代输卵管上皮细胞、鸡胚成纤维细胞、鼠3T3-L1前脂肪细胞和牛乳腺上皮细胞，通过荧光和酶活性检测，旨在筛选出用于实现转基因鸡生物反应器的高效特异性表达载体。结果发现，鸡卵清蛋白启动子表达载体转染以上4种细胞后2种标记基因均有表达，没有表现出明显的细胞特异性，且荧光素酶检测结果表明其在各细胞组中表达活性都低于CMV启动子表达载体100倍以上；慢病毒载体感染以上4种细胞后2种标记基因均有表达，在鸡输卵管上皮细胞组感染单个细胞的病毒颗粒 (Multiplicity of infection，MOI) 为20时绿色荧光蛋白表达量就可以达到CMV启动子表达载体的水平。上述结果表明，基于卵清蛋白基因调控序列构建的表达载体无法实现外源基因的高效、特异性表达，而慢病毒载体在表达活性和广泛性上可以用于进行鸡输卵管生物反应器的研究。

Abstract:

We constructed transgenic chicken bioreactor vector, driven by chicken ovalbumin promoter, lentiviral vector and cytomegalovirus (CMV) promoter control vector encoding green fluorescent protein (GFP) and luciferase (Luc) as reporter genes. The three vectors were used to transfect or infect chicken primary oviduct epithelial cells, embryo fibroblasts cells, mouse 3T3-L1 preadipocytes cells and bovine mammary epithelial cells. High efficient and specific expression vector for transgenic chicken bioreactor was determined by detecting fluorescence and luciferase activity. Reporter gene analysis showed that chicken ovalbumin promoter expression vector was not cell type-specific in these four different cells. Additionally, luciferase reporter analysis illustrated that the chicken ovalbumin promoter activity was over 100 times lower than that of the CMV promoter in four different cells. Both of these two reporter genes were expressed in those four different cells infected by lentiviral expression vectors. Similarly, the GFP reached the similar expression level in cells infected by lentivirus and cells transfected with CMV promoter plasmid vectors when the multiplicity of infection was 20. In conclusion, the transgenic chicken bioreactor vector under the control of chicken ovalbumin promoter was not highly efficient and cell type-specific. However, the efficient expression and extensiveness of lentiviral vector could be used for studying chicken oviduct bioreactor.