Vibrio cross-protective antigen, and preparation and application thereof
A technology for Vibrio and Vibrio disease, which is applied in the fields of genetic engineering and immunology, and can solve the problems of less research on immunological properties and lack of LamB protein.
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Embodiment 1
[0080] Cloning of embodiment 1 maltose porin gene LamB
[0081] According to the Maltoporin (LamB) gene sequence of Vibrio parahaemolyticus (Vibrio parahaemolyticus) RIMD2210633 (accession number: NP_801154), and other Vibrio LamB gene sequences, degenerate primers were designed and synthesized. The genomic DNAs of eight Vibrio strains were used as templates for PCR. The degenerate primer sequences are as follows:
[0082] LamB-F:5'-ATGAAAAAAGTAAGTSNYATTGCAG-3'
[0083] LamB-R:5'-TTACCACCAAGCTTCNRCTTG-3'
[0084] Among them, S=C / G; N=A / C / G / T; Y=C / T; R=A / G;
[0085] Reaction system (25 μL): 1 μL DNA template, 1 μL forward primer, 1 μL reverse primer, 9.5 μL double distilled water, 12.5 μL 2×Taq PCR Master mix.
[0086] Reaction conditions: Pre-denaturation at 95°C for 5 minutes, followed by 30 cycles at 95°C for 30s, 58°C for 45s, and 72°C for 1min. After the end of the reaction, the overall extension was carried out for 10 minutes.
[0087] The PCR product was recovered ...
Embodiment 2
[0090] Example 2 Construction of Escherichia coli expression vector pET32-LamB
[0091] By the LamB gene cloning sequence of Vibrio alginolyticus obtained in Example 1, design the expression primer that removes the signal peptide, synthesize this primer, and the primer sequence is as follows:
[0092] LamBeF:5'-CGCGGATCCGCAGTGGATTTTAAC-3'
[0093] LamBeR:5'-CCGCTCGAGATTCGGCTTTTACCAC-3'
[0094] Using the LamB recombinant cloning plasmid pMD19-T-LamB obtained in Example 1 as a template, PCR was carried out, and the reaction conditions were as follows:
[0095] Reaction system (25 μL): 1 μL DNA template, 1 μL forward primer, 1 μL reverse primer, 9.5 μL double distilled water, 12.5 μL 2×Taq PCR Master mix.
[0096] Reaction conditions: Pre-denaturation at 95°C for 5 minutes, followed by 30 cycles at 95°C for 30s, 58°C for 45s, and 72°C for 1min. After the end of the reaction, the overall extension was carried out for 10 minutes.
[0097] The PCR product was recovered, and the...
Embodiment 3
[0101] Example 3 Construction of Escherichia coli recombinant bacterium BL21-pET32-LamB
[0102] Take 5 μL of the Escherichia coli expression vector pET32-LamB obtained in Example 2 and add it to 50 μL of BL21 competent cells, place on ice for 30 minutes; then heat shock at 42°C for 90 seconds; quickly transfer the EP tube to the ice bath for 15 minutes; then Add 350 μL of LB medium to each tube, transfer the tube to a shaker at 37°C, shake slowly for 60 minutes to recover the bacteria; take 50 μL of the recovered bacteria solution and spread it on a solid LB plate containing Amp, X-Gal and IPTG; invert Plates were cultured at a constant temperature of 37°C for 24 hours; finally, a single white colony was picked for PCR detection to obtain Escherichia coli recombinant BL21-pET32-LamB.
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