Mutant bordetella strains and methods for use
A technology of Bordetella and Bacillus, applied in the fields of immunology, microbiology, allergy and medicine, can solve problems such as difficult production, troublesome attenuation, and poor immunogenicity
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example 1
[0065] Example 1 - Construction and characterization of a pertactin-deficient strain of Bordetella pertussis.
[0066] Escherichia coli DH5α, SM10 and Bordetella pertussis BPZE1, BPSM (Menozzi et al., Infect Immun [Infection and Immunity] 1994; 62:769-778) and B1917 (Bart et al. Bulletin] 2014; 2(6)). Bordetella strains were grown at 37°C on Bordet-Gengou agar (BG) supplemented with 1% glycerol and 10% defibrinated sheep blood. After growth, bacteria were harvested by scraping the plates and resuspended in phosphate-buffered saline (PBS) at the desired density. For liquid culture, Bordetella strains were incubated at 37°C in a modified Stainer- Grow in Scholte's medium (Imaizumi et al. Infect Immun [Infection and Immunity] 1983; 41:1138-1143). E. coli strains used in the cloning procedure were grown in LB broth or LB agar plates. Streptomycin (Sm) was used at 100 μg / ml, gentamicin (Gm) at 10 μg / ml, and ampicillin (Amp) at 100 μg / ml as needed.
[0067] To delete the gene p...
example 2
[0070] Example 2 - BPZE1P colonizes mice like BPZE1.
[0071] As previously described (Mielcarek et al., supra), with 10 6 Groups of 18 6-week-old mice were inoculated intranasally with 20 μl of PBS of live bacteria. At the indicated time points (3 hours, 3 days, 7 days, 14 days, 21 days and 28 days), 3 mice per group were sacrificed and lungs were harvested and homogenized to measure the total number of colony forming units (CFU) . Statistical analysis was performed by two-way ANOVA test using post hoc Bonferroni test (95% confidence interval). see figure 2 , BPZE1 and BPZE1P colonize animals equally well. Both strains exhibited a proliferation peak 3 days after vaccination and colonization continued for 4 weeks. No statistically significant differences were observed between these strains in their ability to colonize mouse lungs.
example 3
[0072] Example 3 - BPZE1P is as immunogenic as BPZE1 and protective against virulent B. pertussis challenge.
[0073] BPZE1P-induced immunity compared to BPZE1 was measured by antibody titration of mouse immune sera following nasal vaccination. use 10 5 Groups of 8 mice were inoculated intranasally with live BPZE1 or BPZE1P. After 4 weeks, mice were bled and total IgG titers were measured against total BPSM lysates. The blood was centrifuged at 5,000 xg for 5 minutes to separate the serum from the cells. Antibody titers against B. pertussis were estimated using enzyme-linked immunosorbent assay (ELISA) as previously described (Mielcarek et al., supra), lysed using total B. pertussis BPSM at 1 μg total protein per well thing. Statistical analysis was performed using GraphPad Prism software. Such as image 3 As shown, BPZE1 and BPZE1P vaccinated mice exhibited much higher antibody titers than naive control mice. No significant difference in antibody titers between BPZE1 a...
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