Preparation and application for porcine epidemic diarrhea subunit vaccine
A vaccine composition, amino acid technology, applied in medical preparations containing active ingredients, microorganism-based methods, biochemical equipment and methods, etc., can solve problems such as infection and death of newborn piglets, and achieve large-scale production convenience and immunity Good, the effect of preventing diarrheal diseases
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Embodiment 1
[0025] Example 1: Preparation, identification and content determination of fusion protein S1-RBD-Fc.
[0026] Preparation and Identification of Porcine Epidemic Diarrhea Virus Fusion Protein
[0027] 1.1.1 Construction of recombinant vector
[0028] According to the nucleic acid sequence described in the sequence table SEQ ID NO.2, it was synthesized by Wuhan Jinkairui Biotechnology Co., Ltd. by artificial synthesis, and the total length of the synthesized gene was 1223bp. Using the synthetic gene as a template, design a pair of specific primers, the sequence is as follows: Fla-S-his6-F1: 5'-CGGGAATTCAACATTAACAACAACTTGC-3';
[0029] Fla-S-his6-R1: 5'-CGGCTCGAGCTTACCTGGAGACAAAGAC-3'.
[0030] Fragments were amplified by PCR. The PCR conditions were: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 15 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, 30 cycles, and extension at 72°C for 5 min.
[0031] The PCR product was electrophoresed on 1% agarose ge...
Embodiment 2
[0044] Embodiment 2: preparation and purity test of vaccine composition
[0045] 2.1 Adjuvant preparation
[0046] Preparation of MF59 adjuvant (containing 5% squalane, 1% oleic acid, 1% Tween 60, 93% 0.005M sodium citrate): Stir and mix the prescribed amount of Tween 80 and sodium citrate buffer, and then Add the prescribed amount of oleic acid and squalane, and use a high-pressure homogenizer at 1200 bar for 5 cycles, then collect the feed liquid and filter it through a 0.22 μm PTFE membrane to sterilize;
[0047] 2.2 Vaccine ratio
[0048] The fusion protein was administered in doses of 10, 20, 50, 100 μg and the adjuvant at a ratio of 1:1 in a sterile environment, stirred and mixed evenly, marked and grouped.
[0049] Table 2 Animal group record form
[0050] group
Embodiment 3
[0051] Embodiment 3: Animal evaluation experiment of vaccine composition
[0052] 3.1 Animal safety test of vaccine composition
[0053] 3.1.1 Character test The appearance of the vaccine composition is pink latex, without layering.
[0054] 3.1.2 Sterility test The vaccine composition was tested according to the third appendix of the current Veterinary Pharmacopoeia of the People's Republic of China in 2010, and no colonies were observed in T.G, G.P tubes and G.A slant medium.
[0055] 3.1.3 Safety inspection 24 3-day-old pigs that were negative for porcine PEDV virus antibody and antigen were randomly divided into 4 groups, 6 pigs in each group, and 10 pigs were injected intramuscularly with the vaccine. Clinical observation was carried out for 14 days, and all 6 / 6 pigs were healthy. live, no adverse reactions occurred.
[0056] Vaccine Composition Neutralizing Antibody Level Test
[0057] 1) Materials and methods
[0058] Get 30 pregnant sows that were negative for pig ...
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