發展環境友善型食品級乳酸鏈球菌之腸病毒71型口服疫苗並評估對BALB/c小鼠之免疫效果

林惠婷

doi:10.6845/NCHU.2015.00294

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發展環境友善型食品級乳酸鏈球菌之腸病毒71型口服疫苗並評估對BALB/c小鼠之免疫效果

Development of environment-friendly food grade Lactococcus lactis system as oral vaccine against EV71 and evaluation on the vaccination effect by BALB/c mice.

林惠婷 , Masters　　Advisor：葉娟美　　

繁體中文 DOI： 10.6845/NCHU.2015.00294

腸病毒71型；革蘭陽性加強介質(gram-positive enhancer matrix ； GEM) ；重組VP1e anchor motif融合蛋白質 (rVP1e-anchor) ；重組 VP1e蛋白質 (rVP1e) ；乳酸菌表現系統；口服疫苗； Enterovirus 71 ； Gram-positive enhancer matrix (GEM) ； Recombinant VP1e anchor motif ； recombinant VP1e (rVP1e) ； L. lactis expression system ； Oral immunization

Abstract │ Reference (117) │ Times Cited (1) │ Altmetrics

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The Human enterovirus 71 causes hand, foot and mouth disease, especially in infants and young children. These outbreaks happened currently in the Asia-Pacific regions. So far, the effective EV71 vaccine is highly recommended. Human enterovirus 71 belongs to a genus of positive-sense single strand RNA virus. Capsid proteins of EV71 consist of VP1, VP2, VP3 and VP4. In the previous research, the VP1 capsid protein harbors the main neutralizing epitopes of EV71.
The use of a food grade organism that is noninvasive and nonpathogenic as antigen delivery vehicle is a promising vaccine strategy. Lactococcus lactis has long been used in food fermentation, and regarded as generally recognized as safe (GRAS) and food grade microorganisms. Killed L.lactis is suggested to be gram positive enhancer matrix (GEM) which enhances the vaccination as adjuvant. 
	In this study, the optimal GEM preparation conditions were established. Results showed that boiled the stationary phase L.lactis MG1363 in 0.125M lactic acid for 1 min obtained GEM. The recombinant VP1e-anchor motif (rVP1e-anchor) was expressed, purified and bound to GEM. The optimal binding condition was 400 μg recombinant protein to GEM under pH 2 buffer which achieved 4.17*1010 molecules/GEM cell.
The storage stabilities of rVP1e-anchor bound GEM were evaluated, and confirmed by microscopy analysis. The rVP1e-anchor bound GEM (rVP1e-GEM) was stable under 4 °C or -80°C storage. 
For comparison, the recombinant VP1e-His tag protein (rVP1e-His tag) and recombinant VP1e (rVP1e) were expressed by L. lactis constitutive expression system. The secreted level of VP1e-His tag protein was much lower than rVP1e. 
The vaccination effects of rVP1e-GEM by oral route and rVP1e by injection route were evaluated in animal trials.The specific antibody IgG 1, IgG 2a and cytokine IFN-γ, IL-4 were examined. As the results shown, the specific antibody and cytokine were induced after administered to BLAB/c mice. There are no significant difference between oral immunization groups and IP groups in the specific antibody, IgA. Results suggested that the rVP1e-anchor bound GEM can be seen as potent oral vaccine.

Reference ( 117 ) 〈TOP〉

2013 Oral immunization with Lactococcus lactis secreting attenuated recombinant staphylococcal enterotoxin B induces a protective immune response in a murine model. Microb Cell Fact 12:32.
連結：
Avall-Jaaskelainen, S., et al.
連結：
2002 Surface display of foreign epitopes on the Lactobacillus brevis S-layer. Appl Environ Microbiol 68(12):5943-51.
連結：
2012 Lactococcus lactis-based vaccines from laboratory bench to human use: an overview. Vaccine 30(4):685-90.
連結：
2008 Nisin inducible production of listeriolysin O in Lactococcus lactis NZ9000. Microb Cell Fact 7:24.
連結：

Times Cited (1) 〈TOP〉

林翰陞(2017)。發展食品級孢子表層展示之腸病毒71型口服疫苗並評估對BALB/c小鼠之免疫效果。中興大學食品暨應用生物科技學系所學位論文。2017。1-109。

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