| Summary: | 碩士 === 國立臺灣大學 === 園藝學研究所 === 94 === Antrodia cinnamomea is a well-known traditional Taiwanese herbal medicine which attracted a lot of attention these years due to its curative effects towards various diseases. Although sources of A. cinnamomea are highly limited in nature, overexpressing the bioactive components found in A. cinnamomea might be the answer to the markets’ demands. Immunomodulatory protein ACA1 has been previously isolated from the mycelia of A. cinnamomea by our laboratory. The bioactivities of ACA1 toward murine immune system have been verified and the gene encoding ACA1 has been cloned. To obtain the protein in large quantity, recombinant ACA1 was expressed in both prokaryotic and eukaryotic systems. Recombinant ACA1 expressed in GST fusion system yielded a soluble fusion protein GST-ACA1, which exhibited a molecular weight near 40 KDa. However, the pure rACA1 obtained after GST tag removal was technically too difficult to be collected. rACA1 expressed by pET-32a(+) expression vector obtained a satisfying quantity of both fusion HIS-ACA1 and pure rACA1, which were approximately 10 mg and 4 mg per liter culture respectively. Fusion HIS-ACA1 had a molecular weight of 20 KDa, and rACA1 had a molecular weight of 14 KDa. The isoelectric point of rACA1 was 5.0. In vitro studies revealed that rACA1 could effectively stimulate murine macrophage RAW264.7 to produce nitric oxide and TNF-α at low concentrations, and the responses increased in a dose-dependent manner. rACA1 could also stimulate the proliferation of murine splenocytes. The stability of rACA1 under various environmental stresses was demonstrated, and rACA1 was found to be stable under heat, acid, and dehydration treatments. Four monoclonal antibodies against rACA1 have also been established, and specific recognitions toward rACA1 were verified. In addition to being expressed in two bacterial systems above, recombinant ACA1 was also expressed in the eukaryotic, yeast Sacchromoyces cerevesiae expression system. Both wildtype aca1 gene and yeast-favored aca1 gene inserted in the constitutive expression vector pYEX-S1 had similar expression levels; the expression levels of C-terminal His-tagged ACA1s were found to be significantly higher than that of N-terminal His tagged ACA1s. In conclusion, recombinant ACA1 has been successfully expressed in both E.coli and yeast expression systems, and the purified rACA1 was proven to have immunomodulatory effects similar to that of natural ACA1. With good stability under various simulated conditions mimicking the industrial processing procedures, recombinant ACA1 expressed in the food-grade expression host S. cerevisiae could therefore possibly be utilized directly in animal feed or food in the near future.
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