A kind of preparation method of oral vaccine of porcine epidemic diarrhea virus
A porcine epidemic diarrhea and oral vaccine technology, which is applied in the field of preparation of new oral vaccines for effective prevention and control of porcine epidemic diarrhea virus, to achieve the effects of reducing stress, avoiding blindness, and stimulating mucosal immune response
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Embodiment 1
[0042] Example 1 Preparation of a novel oral vaccine for the effective prevention and control of porcine epidemic diarrhea virus in the present invention
[0043] Includes the following actions:
[0044] 1. Freeze-drying of virus: The attenuated strain of porcine epidemic diarrhea virus (PEDV) cultured in African green monkey kidney (Vero) cells was filtered with 4 layers of sterile gauze and mixed with an equal volume of sterile freeze-dried protective agent (containing 10% skimmed milk powder, 5% sucrose), stir and mix well. Next, turn on the freeze dryer for pre-freezing, and when the plate temperature drops to -50 °C, put the mixed virus solution into the freeze dryer tray, and keep it at -50 °C for 2 hours after entering the box; then turn on the rear box to cool down and pump Vacuum, open the big butterfly valve, turn on the Roots pump; reduce the vacuum degree of the box to 20pa and maintain it for 1h; after 1h, heat the sample for 200min to raise the temperature of th...
Embodiment 2
[0048] Example 2 Detection of the function of the vaccine prepared by the method of the present invention
[0049] 2.1 In vitro acid resistance test:
[0050] Weigh 2g of NaCl and 3.2g of pepsin (12000U) and fully dissolve them in 900ml of distilled water to prepare artificial gastric juice, then add concentrated HCL to adjust the pH to 1.2, and dilute to 1L. The prepared new PEDV oral vaccine was placed in the small cup of the intelligent dissolution tester by the small cup method (paddle method), and then 100 mL of artificial gastric juice was added, and stirred at 37 °C and 100 r / m for 2 h. After 2 hours, the artificial gastric juice was poured out, washed with pure water and transferred to pH 6.8 DMEM cell culture medium for dissolution and live virus titer determination. The results are shown in Figure 4 , in which PEDV freeze-dried powder has not been treated with artificial gastric acid TCID 50 =10 6.5 , TCID treated with artificial gastric acid 50 =0; TCID of PEDV...
Embodiment 3
[0072] Refer to the preparation method of the novel oral vaccine for the prevention and control of porcine epidemic diarrhea virus in Example 1, the difference is: in step 2, the virus freeze-dried powder is fully mixed with cornstarch, lactose, dextrin, and mannitol of equal quality, and the other steps The operation is the same as the first embodiment. Observe the state of each freeze-dried powder mixture and subsequent process conditions.
[0073] In the solution of the present invention, the freeze-dried powder is easy to absorb moisture and become a block because it contains sucrose milk powder, and the corn starch is very fine and has strong hygroscopicity. Mixing with the freeze-dried powder can dilute and absorb moisture and keep the freeze-dried powder flowing. state, and in the enteric phase, starch can act as a disintegrant to accelerate the release of viruses in the intestine. And the yield of corn starch is high, and powders such as lactose, dextrin, mannitol and...
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