Construction of Bordetella pertussis strains with enhanced production of genetically-inactivated Pertussis Toxin and Pertactin by unmarked allelic exchange

BMC Microbiology

Research Article

Jean Petre¹  Anocha Luengchaichawange¹  Pannipa Prompiboon¹  Wasin Buasri²  Chuenchit Boonchird²  Attawut Impoolsup²  Watanalai Panbangred³ 

[1] Bionet-Asia Co. Ltd., Hi-Tech Industrial Estate, 81 Moo 1, Baan-Lane, 13160, Bang Pa-In, Ayutthaya, Thailand;Department of Biotechnology, Mahidol University, 272 Rama 6 Road, Ratchathewi, 10400, Bangkok, Thailand;Department of Biotechnology, Mahidol University, 272 Rama 6 Road, Ratchathewi, 10400, Bangkok, Thailand;Mahidol University-Osaka University Collaborative Research Center for Bioscience and Biotechnology (MU-OU:CRC), Faculty of Science, Mahidol University, 272 Rama 6 Road, Ratchathewi, 10400, Bangkok, Thailand;

关键词: Pertussis;    Pertussis Toxin;    Pertussis Vaccine;    Bordetella Pertussis;    Acellular Pertussis Vaccine;   

DOI  :  10.1186/1471-2180-12-61

 received in 2011-11-29, accepted in 2012-04-23,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundAcellular Pertussis vaccines against whooping cough caused by Bordetella pertussis present a much-improved safety profile compared to the original vaccine of killed whole cells. The principal antigen of acellular Pertussis vaccine, Pertussis Toxin (PT), must be chemically inactivated to obtain the corresponding toxoid (PTd). This process, however, results in extensive denaturation of the antigen. The development of acellular Pertussis vaccines containing PTd or recombinant PT (rPT) with inactivated S1, Filamentous Hemagglutinin (FHA), and Pertactin (PRN) has shown that the yield of PRN was limiting, whereas FHA was overproduced. To improve antigen yields and process economics, we have constructed strains of Bordetella pertussis that produce enhanced levels of both rPT and PRN.ResultsThree recombinant strains of Bordetella pertussis were obtained by homologous recombination using an allelic exchange vector, pSS4245. In the first construct, the segment encoding PT subunit S1 was replaced by two mutations (R9K and E129G) that removed PT toxicity and Bp-WWC strain was obtained. In the second construct, a second copy of the whole cluster of PT structural genes containing the above mutations was inserted elsewhere into the chromosome of Bp-WWC and the Bp-WWD strain was obtained. This strain generated increased amounts of rPT (3.77 ± 0.53 μg/mL) compared to Bp-WWC (2.61 ± 0.16 μg/mL) and wild type strain (2.2 μg/mL). In the third construct, a second copy of the prn gene was inserted into the chromosome of Bp-WWD to obtain Bp-WWE. Strain Bp-WWE produced PRN at 4.18 ± 1.02 μg/mL in the cell extract which was about two-fold higher than Bp-WWC (2.48 ± 0.10 μg/mL) and Bp-WWD (2.31 ± 0.17 μg/mL). Purified PTd from Bp-WWD at 0.8-1.6 μg/well did not show any toxicity against Chinese hamster ovary (CHO) cell whereas purified PT from WT demonstrated a cell clustering endpoint at 2.6 pg/well.ConclusionsWe have constructed Bordetella pertussis strains expressing increased amounts of the antigens, rPT or rPT and PRN. Expression of the third antigen, FHA was unchanged (always in excess). These strains will be useful for the manufacture of affordable acellular Pertussis vaccines.

【 授权许可】

Unknown   
© Buasri et al; licensee BioMed Central Ltd. 2012. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.