以胡蘿蔔生產禽流感口服疫苗，家禽抗病免打預防針

家禽流行性感冒(Avian influenza)造成家禽養殖業嚴重的經濟損失。目前禽流感的疫苗生產主要是以雞胚胎增殖病毒，再製成死毒疫苗，生產過程耗時，且受到蛋源供應的影響。若利用植物細胞作為疫苗工廠，可降低疫苗生產成本、降低污染風險、口服使用等。本研究依據Codon Usage Database中胡蘿蔔(Daucus carota)同義密碼子的使用偏好，優化作為免疫佐劑之大腸桿菌忌熱毒素(heat-labile enterotoxin B subunit, LTB)基因及禽流感病毒血球凝集素(Hemagglutinin, HA)抗原基因的密碼子，再透過農桿菌轉殖法(Agrobacterium-mediated transformation)轉殖至胡蘿蔔細胞核中。利用聚合酶連鎖反應(polymerase chain reaction)與南方氏雜交分析(Southern blot analysis)已確認轉殖胡蘿蔔的外源基因完整性與拷貝數。透過酵素連結免疫吸附法(enzyme-linked immunosorbent assay)與西方轉漬分析(Western blot analysis)，偵測到LTB-HA蛋白質於胡蘿蔔細胞中產量達0.0108%總可溶蛋白質(total soluble protein)。

Su Y.-C., P.-L. Huang, Y.-Y. Do. 2023. Genetic transformation and expression of hemagglutinin gene from avian influenza virus in carrot (Daucus carota). Journal of Plant Biochemistry and Biotechnology 32:550-559. (SCI)

轉殖胡蘿蔔各部位均表達報導基因表示轉殖成功

禽流感疫苗蛋白質於胡蘿蔔細胞中產量達0.01%總可溶蛋白質

西方轉漬免疫分析偵測到轉殖胡蘿蔔具有禽流感疫苗蛋白質

研究全文：https://doi.org/10.1007/s13562-023-00840-6

Genetic Transformation and Expression of Hemagglutinin Gene from Avian Influenza Virus in Carrot (Daucus carota)

Avian influenza is a disease of poultry that causes economic losses to poultry farms. Conventional vaccine production requires chicken embryos for virus proliferation with time-consuming and also affected by egg supply during the avian influenza virus outbreak. There are several advantages of plant cells as bio-factories for vaccine production, including low cost, low risk of contamination, and even edible. In this study, we chose carrot (Daucus carota) as a bioreactor to produce hemagglutinin (HA), an avian influenza virus antigen protein. HA gene linked with heat-labile enterotoxin B subunit (LTB) gene whose product plays the role of adjuvant protein was codon optimized with codon usage bias of carrot for high-level expression. The native and codon-optimized LTB-HA were transformed into carrots by Agrobacterium-mediated transformation. T-DNA insertion of two version constructs was confirmed by polymerase chain reaction. Integration of codon-optimized LTB-HA gene in all transgenic carrot lines was identified as a single copy by Southern blot analysis. LTB-HA protein expressed in carrot cells was determined through enzyme-linked immunoassay. Codon-optimized LTB-HA gene showed a better expression level than the native LTB-HA gene in transgenic carrots. The highest expression level of LTB-HA in codon-optimized LTB-HA-transformed carrot taproot reached up to 0.0108% of total soluble protein. This study is the first report about expression of HA antigen of H5N2 subtype AIV in a horticultural crop. Our transgenic carrots have successfully produced HA protein which shows great potential to become an edible vaccine against avian influenza virus.

Su Y.-C., P.-L. Huang, Y.-Y. Do. 2023. Genetic transformation and expression of hemagglutinin gene from avian influenza virus in carrot (Daucus carota). Journal of Plant Biochemistry and Biotechnology 32:550-559. (SCI)

Histochemical detection of GUS activity in transgenic carrots transformed with pGKUgfpgus-optLHA

Comparison of LTB-HA protein accumulation level between non-codon-optimized and codon-optimized transgenic lines

Western blot analysis of LTB-HA protein expression in transgenic carrot leaves transformed with pGKUgfpgus-optLHA

More information: https://doi.org/10.1007/s13562-023-00840-6