| Summary: | 碩士 === 國立東華大學 === 生物技術研究所 === 92 === The progress in genetic engineering and plant tissue culture in recent years has made kinds of biopharmaceutical proteins being successfully produced in plants, including antibodies, vaccines, hormone, cytokines and growth factors etc. Biopharmaceutical proteins can be produced in plants with a large quantity but at a relatively low cost and, more importantly, these proteins contain no contamination with known or unknown human viruses. The objective of this sdudy is to establish plants as a bioreactor for producing human interleukin-7 (IL-7). IL-7 is a cytokine important for B cell and T cell development, and also plays a critical role in T cell homeostasis. To make edible IL-7, we made two constructs, IL7-1 and IL7-2. IL7-1 contains a signal sequence on the N-terminus of the mature IL-7 gene. The signal sequence comes from the gene encoding thaumatin-like protein (TLP), a pathogenesis-related protein isolated from oats. IL7-2 contains a fusion sequence of the mature cholera toxin B subunit (CTB) and the mature IL-7. CTB serves as a carrier molecule for uptake of biopharmaceutical proteins in the intestine. The IL7-1 and IL7-2 were introduced into Nicotiana tabacum cv. Xanthi and cv. Samsun via Agrobacterium-mediated transformation. After selection and regeneration in culture media with hygromycin, a total of 31 putative transgenic tobacco were obtained, including 4 Xanthi with the IL7-1, 7 Xanthi with the IL7-2, 19 Samsun with the IL7-1, and 1 Samsun with the IL7-2. The results of PCR analyses revealed that 23 transgenic plants contain the IL7-1 or the IL7-2 transgene. The Southern blot analysis showed that there are 1 to 4 copies of transgene in the genome. The expression level of the IL-7 transcript in the 23 transgenic tobacco are variable with higher expression level for the plants with single copy of transgene than those with more than one copy, suggesting that gene silencing might be involved in the plants with more than one copy of transgene. The production and biological function of IL-7 in transgenic tobacco will be determined in near future.
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