Bordetella bronchiseptica gene deleted vaccine and application
A gene deletion vaccine, Bordetella blood technology, applied in the direction of bacteria, antibacterial drugs, bacterial antigen components, etc., can solve the problem of no product coming out, achieve the effect of reduced virulence, broad market application prospects, and protection against attacks
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[0033] 1. aroA primer design and PCR amplification
[0034] According to the reported whole genome sequence of Bordetella bronchiseptica RB50 (refer to the gene sequence of GenBank accession number AF315119), two pairs of primers were designed (all primers were synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd.) to amplify the upstream of aroA gene respectively. Homology arm (SEQ NO.3) and downstream homology arm (SEQ NO.1), the amplified fragment sizes are 863bp and 900bp respectively, KpnI and BamHI restriction sites are designed at both ends of the upstream arm, and respectively designed at both ends of the downstream arm BamHI and SacI restriction sites. The primer sequences are as follows:
[0035] C1:5`-TGC GGTACC TAGGTTGGAATCCATTTGGC-3`(Kpn I)
[0036] C2: 5`-TAA GGATCC CCATGGCTATTTCCGCATCC-3`(BamH I)
[0037] Primers C1 and C2 amplify the upstream homology arm 863bp
[0038] T1: 5`-TGT GGATCC TGGAATCCTTGCGCCAATTG-3`(BamH I)
[0039] T2: 5`-TA...
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