期刊
BIOTECHNOLOGY AND BIOENGINEERING
卷 112, 期 10, 页码 2005-2015出版社
WILEY-BLACKWELL
DOI: 10.1002/bit.25625
关键词
baculovirus; EV71; VLP; vaccine; hand-foot-and-mouth disease; protease
资金
- Ministry of Science and Technology [MOST 103-2622-E-007-024-CC1, NSC 102-2622-E-007-022-CC1, NSC 101-2628-E-007-009-MY3, NSC 100-2622-E-007-003-CC2]
- Ministry of Health and Welfare [MOHW103-TDU-PB-211-122021, MOHW102-TDU-PB-111-NSC108, DOH99-TD-I-111-TM022, DOH98-DC-1013]
Enterovirus 71 (EV71) is responsible for the outbreaks of hand-foot-and-mouth disease in the Asia-Pacific region. To produce the virus-like particle (VLP) vaccine, we previously constructed recombinant baculoviruses to co-express EV71 P1 polypeptide and 3CD protease using the Bac-to-Bac((R)) vector system. The recombinant baculoviruses resulted in P1 cleavage by 3CD and subsequent VLP assembly in infected insect cells, but caused either low VLP yield or excessive VLP degradation. To tackle the problems, here we explored various expression cassette designs and flashBAC GOLD vector system which was deficient in v-cath and chiA genes. We found that the recombinant baculovirus constructed using the flashBAC GOLD system was insufficient to improve the EV71 VLP yield. Nonetheless, BacF-P1-C3CD, a recombinant baculovirus constructed using the flashBAC GOLD(TM) system to express P1 under the polh promoter and 3CD under the CMV promoter, dramatically improved the VLP yield while alleviating the VLP degradation. Infection of High Five(TM) cells with BacF-P1-C3CD enhanced the total and extracellular VLP yield to approximate to 268 and approximate to 171mg/L, respectively, which enabled the release of abundant VLP into the supernatant and simplified the downstream purification. Intramuscular immunization of mice with 5g purified VLP induced cross-protective humoral responses and conferred protection against lethal virus challenge. Given the significantly improved extracellular VLP yield (approximate to 171mg/L) and the potent immunogenicity conferred by 5g VLP, one liter High Five(TM) culture produced approximate to 12,000 doses of purified vaccine, thus rendering the EV71 VLP vaccine economically viable and able to compete with inactivated virus vaccines. Biotechnol. Bioeng. 2015;112: 2005-2015. (c) 2015 Wiley Periodicals, Inc.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据