Labeled swine fever virus E2 protein recombinant baculovirus inactivated vaccine
A technology of recombinant baculovirus and swine fever virus, which is applied in the field of vaccines, can solve the problems of contaminating bovine viral diarrhea, affecting the safety of vaccines, and the inability of Erns antibodies to effectively carry out differential diagnosis, and achieve the effect of effective purification.
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Embodiment 1
[0071] Example 1 Gene optimization, mutation and synthesis of classical swine fever virus E2
[0072]The published E2 sequence of classical swine fever virus and the E2 sequence of new strains popular in recent years were downloaded from Genebank as a reference, and optimized on the basis of the E2 gene of the C strain, and the optimized E2 gene was combined with the reference strains of each subgroup After comparison, the results showed that the optimized E2 gene was determined to be group 1.1. At the same time, the optimized E2 gene sequence epitope "TAVSPTTLR" was mutated into "ATVSPTTRL, TASVPTTLR, TAVSPTTRL, TAVSPTTRL, TAPVSTTRL or TASPVTTRL", and the obtained 6 optimized mutated sequences were submitted to the gene company for synthesis.
Embodiment 2
[0073] The construction of embodiment 2 transfer vectors
[0074] For the test method, refer to the Invitrogen operation manual, and the specific operation is as follows.
[0075] prepare Clone reaction mixture, its composition is as follows: 0.5~4μl synthesized E2 nucleotide, 1μl salt solution (1.2mol / LNaCl, 0.06mol / LMgCl2)), 1μl carrier, and double distilled water was added to make the total volume 6 μl. The steps of the cloning reaction are as follows: pat the wall of the centrifuge tube lightly to mix the reaction mixture evenly, let it sit at room temperature (22-23°C) for 15 minutes, and then place the reaction tube on ice. Take 2μl Add the clone reaction solution to the competent cells, pat the tube wall to mix evenly, place it on ice for 30 minutes, then place it in a water bath at 42°C for 30 seconds for heat shock, then immediately put the reaction tube back on ice Then add 250 μl of warmed SOC culture solution into the reaction tube, put it into a constant t...
Embodiment 3
[0077] Embodiment 3 Recombination of transfer vector and linear baculovirus
[0078] With the extracted recombinant vector, measure the nucleic acid concentration of the recombinant vector at a wavelength of 260nm with a spectrophotometer. Then carry out the LR recombination reaction according to the operation manual of "BaculoDirectTM Baculovirμs Expression System" provided by Invitrogen. The specific operation is as follows: 1 μl recombinant transfer vector (100-300ng), 4 μl 5×LR Reaction buffer and 1μl double distilled water were added to 10μl BaculoDirectTM linear DNA (300ng). Take out LR from -70℃ refrigerator Put the enzyme mix on ice for 2 minutes, shake twice, 2 seconds each time, take 4μl LR Add enzyme mix to the above mixture, pat the tube wall several times to make it evenly mixed, do not shake violently to avoid the baculovirus DNA fragmentation. After infecting at 25°C for 18 hours, add 2 μl of proteinase K and place it at 37°C for 10 minutes to stop the LR...
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