| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 95 === Hyperlipidemia can cause the heart disease and the brain stroke. Effective suppression of the amount of cholesterol in blood will help to reduce their potential risks. The bioactive peptides─Val-Val-Tyr-Pro (VVYP) is one of the well known effective materials to suppress the triglyceride absorption of intestinal tract, and to reduce the blood cholesterol. In this study, we engineered transgenic soybean plants to carry bioactive peptides VVYP. The strategy included the construction of soybean storage protein Glycinin Gy5 and Gy1 genes containing VVYP and using intestine protease to release the bioactive peptides when ingested. The constructs of Gy5 containing 5 copies of VVYP or 10 copies of VVYP were complete. In principle, the soybean may contain the fixed amount of storage protein in seeds. In order to increase the engineered VVYP-containing recombinant proteins in transgenic soybeans, using RNA interference approach to suppress the expression of endogenous soybean storage protein can be effective. The RNAi vectors had been constructed and agrobacterium-mediated transformation is ongoing. In addition, this study also prepares antibodies against Gy5 and Gy1 proteins respectively. The western hybridization results revealed that Gy5 and Gy1 antibodies can differentially recognize acidic and basic chains of different groups of glycinin, providing an useful tool to identify the engineered proteins in transgenic soybeans.
Comparing to current microbial and mammalian cell culture systems, plants have many advantages as bioreactors for the production of pharmaceuticals, antibodies and vaccines, such as the less cost of production and no risk of contamination by animal pathogens. The L-homophenylalanine (L-HPA) is a chiral and unnatural amino acid. It was used in the synthesis of angiotensin converting enzyme inhibitors and many pharmaceuticals. In this study, we engineered transgenic rice plants to produce the pharmaceutical enzyme N-acylamino acid racemase (NAAAR) in seeds. Seven homozygous transgenic rice plants (three 705-NAAAR, two 35S-NAAAR, and two Act-NAAAR) were obtained and confirmed by using hygromycin selection, PCR analysis, Southern blot and western blot analysis. NAAAR combining with L-aminoacylase (LAA) can convert N-acetyl-D-homophenylalanine to L-HPA. The activity assay were performed with high performance liquid chromatography (HPLC) using a chirobiotic T column. The optimal separation condition of NAc-D-HPA and NAc-L-HPA was methanol/ 0.01 M ammonium acetate=70/30 as mobile phase at a flow-rate of 0.5 mL/min. The reaction time and temperature optima of the NAAAR were 2 hrs and 50℃, respectively. This study confirmed that the rice-based-production of NAAAR combining with LAA can be used for the production of L-HPA.
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