Anti-foot-and-mouth disease vaccine composition, preparation method and applications thereof
A vaccine composition and a technology for foot-and-mouth disease, which can be applied to medical preparations containing active ingredients, antiviral agents, pharmaceutical formulations, etc., can solve the problems of reduced activity, weak immunogenicity, and variable stability of foot-and-mouth disease virus-like particle structure.
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Embodiment 1
[0086] Preparation of Type O Foot-and-Mouth Disease Virus Ⅰ Virus-like Particles (VP4, VP2, VP3, VP1)
[0087] 1. Synthesize the full length of the gene shown in the amino acid sequence of O-type foot-and-mouth disease virus I VP4, VP2, VP3, and VP1 shown in SEQ ID NO.1, 2, 3, and 4 by Sangon Bioengineering (Shanghai) Co., Ltd. The full lengths of the synthesized gene fragments were 255bp, 654bp, 660bp, 639bp, respectively. The foot-and-mouth disease gene template of the present invention is prepared on the basis of the artificially synthesized foot-and-mouth disease gene fragment.
[0088] 2. Construction of foot-and-mouth disease gene expression vector
[0089] For the foot-and-mouth disease gene template synthesized in the previous step, primers were designed respectively (see Table 1), and the O-type foot-and-mouth disease virus I VP4, VP2, VP3, and VP1 genes were amplified.
[0090] Table 1 O-type foot-and-mouth disease virus Ⅰ primer list
[0091]
[0092] Perform ...
Embodiment 2
[0103] Preparation of Type O Foot-and-Mouth Disease Virus Ⅰ Virus-like Particles (VPO, VP3, VP1)
[0104] Referring to the method in Example 1, primers were respectively designed according to the gene sequences of type O foot-and-mouth disease virus I structural proteins VPO, VP3, and VP1 for tandem expression to prepare virus-like particles. The collected bacteria were resuspended according to the ratio of 1 g of bacteria to 10 ml of lysate, and the bacteria were crushed 4 times with a homogenizer at a pressure of 800 bar. Centrifuge at 13500rpm for 40min, save the supernatant, and detect by 15% SDS-PAGE electrophoresis. At this time, the expression level of the three serially expressed proteins in the supernatant is about 20%. Ammonium sulfate fractional precipitation was used for crude protein purification, followed by chromatographic purification. The purified protein was subjected to SDS-PAGE electrophoresis, which showed that the target protein was purified and enriched....
Embodiment 3
[0107] Preparation of Asian Foot-and-Mouth Disease Type 1 Virus-like Particles (VP0, VP3, VP1)
[0108] The genes shown in the amino acid sequences of Asian type 1 foot-and-mouth disease virus VPO, VP3, and VP1 shown in SEQ ID NO.21, 22, and 23 were respectively designed according to the method of Example 1 and expressed in tandem to prepare virus-like particles.
[0109] Negative staining with phosphotungstic acid and electron microscope observation showed that the FMD protein had formed virus-like particles, and the formed virus-like particles were plump, with high assembly efficiency and no aggregation. After placing the foot-and-mouth disease virus-like particles at 4°C for 4 months, negative staining with phosphotungstic acid and electron microscope observation showed that the virus-like particles were still full without aggregation. It shows that the foot-and-mouth disease protein prepared according to the three-stage expression of the screened sequence of the present in...
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