Preparation method and application of Vibro harveyi and Vibrio parahaemolyticus bigeminy DNA vaccine
A technology of Vibrio harveii and DNA vaccine, applied in the fields of genetic engineering and immunology, can solve the problems of cytotoxicity and low immune protection rate, and achieve the effects of high repetition rate, strong experimental operability and simple preparation method
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Embodiment 1
[0052] Example 1: Preparation of tdh2-oppA fusion gene:
[0053] 1. Preparation of gene tdh2 and gene oppA:
[0054] 1. Preparation of gene tdh2:
[0055] (1) According to the sequence of the thermostable hemolysin subunit gene tdh2 of Vibrio parahaemolyticus included in GeneBank, a pair of primers a for amplifying the gene tdh2 was designed, and its sequence is:
[0056] 5’CGCCTCGAGATGAAGTACCGATATTTTGCA 3’
[0057] 5'CGCGAATTCTTGTTGATGTTTACATTCAAAA 3'
[0058] (2) take the genomic DNA of Vibrio parahaemolyticus as a template, carry out PCR reaction, obtain the PCR product of 570bp;
[0059] The reaction conditions were as follows: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 1 minute; this reaction was performed for 30 cycles; and then extension at 72°C for 10 minutes.
[0060] The reaction system is: 5μl Buffer, 3μl MgCl 2 , 0.5 μl of dNTP, 0.5 μl of Taq enzyme, 2 μl of template DNA,...
Embodiment 2
[0081] Example 2: Construction of tdh2-oppA fusion gene eukaryotic expression vector pEGFP-N1-tdh2-oppA:
[0082] 1. prepare the gene tdh2 carrier DNA that has XhoI and EcoRI double restriction site by the method for embodiment 1;
[0083] 2. Digest the eukaryotic expression vector pEGFP-N1 and the gene tdh2 vector DNA with XhoI and EcoRI, and separate to obtain the linear vector pEGFP-N1 and the linear gene tdh2;
[0084] 3. Take 4.5 μl of the linear gene tdh2, add 0.5 μl of the linear vector pEGFP-N1, mix, then add 5 μl of DNA ligase, and react at 16°C for 16 hours to obtain the gene tdh2 ligation product;
[0085] 4. Transform the gene tdh2 ligation product into Escherichia coli DH5α for culture, pick the colony and extract the carrier DNA, after PCR reaction and XhoI, EcoRI double enzyme digestion detection, screen to obtain the engineering vector pEGFP-N1-tdh2;
[0086] 5. Prepare the gene oppA carrier DNA with EcoRI and BamHI double restriction sites by the method of ex...
Embodiment 3
[0092] Example 3: Preparation and use of double DNA subunit vaccine suspension
[0093] Transform the pEGFP-N1-tdh2-oppA engineering vector obtained in Example 2 above into Escherichia coli DH5α, and after culturing in LB liquid medium containing kanamycin for 12-18 hours, extract the engineering vector pEGFP-N1-tdh2 -oppA, after the endotoxin removal treatment was carried out on the carrier, the carrier was dissolved in 0.05mol / L phosphate buffer saline PBS to a concentration of 200 μg / ml, and the double DNA subunit of Vibrio harveyi and Vibrio parahaemolyticus was obtained Unit vaccine suspension.
[0094] The above-mentioned vaccine suspension can be directly immunized to fish. Taking the cultured flounder as an example, the immunization method is as follows: buy the same batch of healthy flounder two weeks before the immunization experiment, raise them in the water tank at room temperature, ventilate and change the water and feed normally; use the prepared engineering car...
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