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Construction of Bordetella pertussis strains with enhanced production of genetically-inactivated Pertussis Toxin and Pertactin by unmarked allelic exchange
Wasin Buasri, Attawut Impoolsup, Chuenchit Boonchird, Anocha Luengchaichawange, Pannipa Prompiboon, Jean Petre, Watanalai Panbangred
BMC Microbiology , 2012, DOI: 10.1186/1471-2180-12-61
Abstract: Three 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.We 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.Pertussis or whooping cough is a severe respiratory disease resulting from colonisation of the upper respiratory tract by the causative organism Bordetella pertussis [1]. Vaccines have been available for decades, comprising killed whole cells of B. pertussis that are chemically detoxified and formulated with Diphtheria and Tetanus antigens. They are administered as a trivalent Diphtheria-Tetanus-Pertussis combination, or in newer combinations with HBV and Hib, providing additional immunity against Hepatitis B and Haemophilus influenzae type b invasive disease, respectively [2]. The use of whole-cell Pe
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