Construction of brucellosis A19 molecular marker vaccine strain and determination of virulence and immunogenicity
A molecular marker vaccine, A19 vaccine technology, applied in the field of microbial bacterial genetic engineering, can solve the problems of maintaining immunogenicity and biological characteristics, weakening virulence, etc., to improve vaccine function, weak virulence, and good immunogenicity Effect
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Embodiment 1
[0035] Construction of recombinant suicide plasmid
[0036] Materials and Methods
[0037] Strains, plasmids, vectors
[0038]The Brucella A19 strain was purchased from the China Veterinary Drug Administration, and the suicide plasmid pBK-CMV-sacB was preserved by the Veterinary Research Department of the Xinjiang Academy of Animal Sciences; the cloned pGEM-T vector was purchased from Promega, a commercial reagent.
[0039] Reagent
[0040] Restriction enzymes were purchased from Fermentas; plasmid DNA extraction kit and DNA gel recovery kit were purchased from Promega; T4DNA ligase, DNA Marker, and DH5α were purchased from Beijing Zhuangmeng Biological Company; sequence determination was provided by Shanghai Invitrogen Trading Co., Ltd. Ltd. completed.
[0041] Primers were designed according to the GenBankAF141604 sequence, as shown in Table 1:
[0042] Table 1 Primer Sequence
[0043]
[0044] PCR amplification
[0045] Amplification of Homology Arm Fragment Upstr...
Embodiment 2
[0069] Described is the determination of the virulence and immunogenicity of the brucellosis A19 molecular marker vaccine strain.
[0070] Characteristics of A19 molecular marker strain:
[0071] The experimental animals were 4-6 weeks old BALB / C female mice purchased from Xinjiang Experimental Animal Research Center, license number: SCXK (new) 2003-0002.
[0072] Safety test: Dilute A19-ΔVirB12 viable bacteria with normal saline to contain 1 billion viable bacteria per 1ml, subcutaneously inject 5 BABL / C mice with a body weight of 18-20g, 0.25ml each, and all live healthy within 10 days.
[0073] Genetic Stability:
[0074] Passage stability: The frozen A19-ΔVirB12 molecular marker strain was streaked on a tryptone soybean broth medium plate at a temperature of 37°C containing 5% CO 2 Cultured in the incubator for 72 hours, the grown colony was the first-class seed, and the first-class seed was continuously passed on the medium for 50 generations, and the colonies of the pa...
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