| Animal mammary gland bioreactor is an in vivo bioreactor which is specialized for the production of recombinant protein,and is a branch of transgenic animal technology. With the development of biotechnology, transgenic technology will gradually shift towards using mammary gland bioreactor for many medicinal proteins of clinical value.Human interleukin-2 is a very important human cell growth factor, which has a wide range of biological activity, and it has broad prospects. In this study, we began with cloning human interleukin-2 and the construction of goatβ-casein gene locus vector,then used liposomes to make reorganization plasmid transfect goat fetal fibroblast cells, preliminary verified positioning integration efficiency, and layed a foundation for the in-depth study of using mammary gland bioreactor to produce human interleukin-2 . The tests carried out in this study were as follows:1. We used human blood as the experimental materials, extracted total RNA from human whole blood and used RT-PCR technology, designed a pair of specific primers for the human interleukin-2 gene coding region. As a result, we successfully expaned a fragments at the length 462bp, which was then cloned to T vector and expressed in E.coli DH5α.2. We used pBluskm as the vector backbone, used the upstream 2.5Kb regulatory sequence of the first exon of goatβ-casein gene as the 5 'homologous short arm,and used the downstream 3.5Kb sequence of the ninth exon as the 3 'homologous long arm. As a result, we using cloning and connecting technology successfully constructed a gene targeting vector which has a 5 'homologous short arm with a length of 2.5Kb, a 3 'homologous long arm with a length of 3.5Kb and a penicillin resistance selection marker gene with a length of 1.4Kb.3. We connected the IL-2 coding region to the targeting vector at the downstream of 5 'regulatory sequence to build goatβ-casein gene locus vector.4. We used the 40d old goat fetuses as experimental material, and used tissue block method to culture goat fetal fibroblast cells. After three passages, we successfully got spindle fibroblasts cells, which was used for follow-up experiments to transfect reorganization plasmid.5. We used liposomes to make goatβ-casein gene locus plasmid transfect fetal fibroblast cells,and positive clones were screened by G418 and identified. As a result,we successfully go thet monoclonal contained the gene of interest. Experimental studies can be used for follow-up. |
