African swine fever virus P12 protein nanoparticle and preparation method and application thereof
An African swine fever virus and nanoparticle technology, applied in the field of African swine fever virus P12 protein nanoparticles and their preparation, can solve the problems of low protective efficacy, no protective effect, and inability to resist virulent attacks.
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Embodiment 1
[0042] Embodiment 1 plasmid construction
[0043] This embodiment provides the amino acid sequence of African swine fever virus P12 protein nanoparticle (GSTP1-MT3-P12), as shown in SEQ ID NO:4. Entrust Qingke Biological Company to synthesize the DNA fragment of GSTP1-MT3-P12 with His tag fused at the C-terminus, its nucleic acid sequence is shown in SEQ ID NO: 5 (wherein, the 13th-987th bits encode the DNA fragment shown in SEQ ID NO: 4 sequence, the 988-1005th encoding His tag), and constructed into the plasmid vector pPICZaA, the map of the constructed plasmid is shown in figure 1 As indicated, the plasmid was named pPICZaA-GSTP1-MT3-P12. The plasmid vector pPICZaA contains the α-factor secretion signal peptide sequence before inserting the fragment. The sequenced correct monoclonal strain was cultivated, and the plasmid pPICZaA-GSTP1-MT3-P12 was extracted for use.
[0044] Amino acid sequence of GSTP1-MT3-P12 (SEQ ID NO: 4):
[0045] DPETCPCPSGGSCTCADSCKCEGCKCTSCKKSCCS...
Embodiment 2
[0046] Example 2 Induced expression of protein nanoparticles
[0047] After the plasmid prepared in Example 1 was linearized, it was electroporated into X-33 yeast competent cells for inducible expression. The specific steps are as follows:
[0048] (1) Plasmid linearization
[0049] The plasmid pPICZaA-GSTP1-MT3-P12 prepared in Example 1 was taken and digested with linearase SacI. The restriction system was as follows:
[0050]
[0051] After mixing, centrifuge at 12000 rpm at room temperature for 1 min to shake the liquid to the bottom of the tube, and bathe in water at 37 °C for 4 h.
[0052] After digestion, take 2 μL, and conduct 1% agarose gel electrophoresis with the original plasmid to identify the linearization result. After identification, the plasmid has been digested completely. Use the plasmid kit to recover the digested plasmid and use it in the subsequent test steps. .
[0053] (2) Electroporation (aseptic operation)
[0054] Add 10μL of the linearized pl...
Embodiment 3
[0058] Example 3 SDS-PAGE identification
[0059] Take 1 mL of the culture product prepared in Example 2, centrifuge in a centrifuge at the maximum speed for 2 min at room temperature, take the supernatant, mix the supernatant with Loading buffer to make a loading sample, carry out SDS-PAGE identification, and analyze the expression level. SDS-PAGE identification results see figure 2 As shown in (left), it was identified that the target protein was successfully expressed.
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