Brucella genetic engineering subunit vaccine and preparation method and application thereof
A Brucella and subunit vaccine technology, applied in the field of Brucella genetically engineered subunit vaccine and its preparation, can solve the problems of infectivity, poor safety, and inability to inoculate pregnant animals in humans and animals, and achieve prevention Effect of Brucella infection, good immunogenicity
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Embodiment 1
[0039] The construction of embodiment 1 recombinant expression vector
[0040] Bioinformatic analysis was carried out on the amino acid sequences of the outer membrane proteins OMP2, OMP25, OMP31, OMP28, and OMP22 proteins of the cattle, sheep, sheep testis, pigs, and canis Brucella from Genebank. The following antigenic epitopes were screened from Bacillus outer membrane protein: 22-51, 144-158 of goat Omp2a protein (GenBank: AMM72580.1); 233-245 of goat Omp2b protein (GenBank: AMM72579.1) Position, 314-336; 4-29 position of bovine Omp2b protein (GenBank: AAX94577.1); 41-53 position of pig Omp2b protein (GenBank: AAX94574.1); bovine Omp25 protein (GenBank: AFJ79953.1 ) 4-46, 187-197, 143-154; canine Omp25 protein (GenBank: CDL76104.1) 87-94; pig Omp25 protein (GenBank: AHN46339.1) 120-137 ; 10-27 positions of sheep testis Omp25 protein (GenBank: ABU93464.1) 19-27 positions of sheep Omp31 protein (GenBank: ACS50328.1); 4-14 positions of dog Omp31 protein (GenBank: AAL27296.1)...
Embodiment 2
[0046] Example 2 Plasmid Stability Identification
[0047] Escherichia coli B121 cells were selected as host bacteria, and the recombinant pET28a-MutiE expression vector constructed in Example 1 was transformed into Escherichia coli B121 competent cells by heat shock method, and then genetically engineered strains with stable and high expression of MutiE protein were screened. The specific steps are as follows: ①Thaw 100 μL of the prepared competent E. coli Bl21 cells on ice, and then add 1.5 μg of the prepared pET28a-MutiE vector; ②After mixing, place on ice for 30 minutes, and bathe in water at 45°C for 90 seconds; ③On ice Leave it for 3 minutes, add 700 μL LB medium, and activate at 37°C for 1 hour; ④Spread the activated bacterial solution on an LB solid plate with a kanamycin concentration of 100 μg / mL, and culture it upside down at 37°C overnight; ⑤Randomly select plump After numbering, they were inoculated in 5 mL LB liquid medium with a kanamycin concentration of 100 μg...
Embodiment 3
[0048] Example 3 Seed Bank Establishment
[0049] Establishment of the original seed bank: Take the strains prepared in Example 2 and insert them into 1.5mL LB medium with a kanamycin concentration of 100 μg / mL at a subsequent amount of 1%, and carry out shaking culture at 37°C to OD 600 Reached around 0.8. Then the culture solution was inoculated on LB solid plates with a kanamycin concentration of 100 μg / mL by streaking method, and cultured upside down at 37° C. overnight. Randomly select plump single colonies, number them and inoculate them in 5 mL of LB medium with a kanamycin concentration of 100 μg / mL, and culture with shaking at 37°C. Mix the bacterial solution cultivated to the logarithmic phase with the lyoprotectant at a ratio of 1:1, then take 3mL in a sterile 10mL vial under aseptic conditions, freeze-dry, and name it as the Brucella Genetic Engineering Subunit The original seed of the vaccine, the PCR identification result is as follows figure 1 shown.
[0050...
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