O type foot and mouth disease virus-like particle vaccine as well as preparation method and application thereof

A foot-and-mouth disease virus and particle technology, applied in the fields of genetic engineering and immunology, can solve the problems of poor safety and unsatisfactory use effect, and achieve the effect of good stability, favorable for popularization and use, and good safety.

Inactive Publication Date: 2015-09-02
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
5 Cites 9 Cited by

AI-Extracted Technical Summary

Problems solved by technology

[0004] In order to solve the problems of poor safety and unsatisfactory use effect of existing inactivated foot-and-mouth disease vaccines, the present invention provides a safe...
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Abstract

The invention discloses an O type foot and mouth disease virus-like particle vaccine as well as a preparation method and an application thereof, and belongs to the fields of genetic engineering and immunology, aiming at solving the problems of an existing inactivated foot and mouth disease vaccine which is poor in safety and unsatisfying in use effect. The amino acid sequence of the vaccine is shown as SEQ ID NO: 1 in a sequence list, and a nucleotide sequence for encoding the amino acid sequence is shown as SEQ ID NO: 2 in the sequence list. After being immunized, the vaccine can induce an animal to simultaneously generate an O type foot and mouth disease resistant antibody; the vaccine exists in the form of virus-like particles, and foot and mouth disease virus VP1 epitope locates on the surface of the virus-like particles. According to the invention, the amino acid sequence constituting the virus-like particle vaccine and the nucleotide sequence for encoding the amino acid sequence are provided, and the nucleotide sequence contains all genes of coat protein which can be self-assembled as the virus-like particles in expression. The preparation of the O type foot and mouth disease virus-like particle vaccine disclosed by the invention is good in safety and free from the probability of reversion or toxin dispersion; and the vaccine is long in immunizing validity period and convenient in transportation and storage.

Application Domain

AntiviralsGranular delivery +3

Technology Topic

Genetic engineeringToxin +12

Image

  • O type foot and mouth disease virus-like particle vaccine as well as preparation method and application thereof
  • O type foot and mouth disease virus-like particle vaccine as well as preparation method and application thereof
  • O type foot and mouth disease virus-like particle vaccine as well as preparation method and application thereof

Examples

  • Experimental program(5)

Example Embodiment

[0038] Example 1: Obtaining the VP1 gene of type O foot-and-mouth disease virus antigen
[0039] The VP1 gene of type O foot-and-mouth disease virus antigen was constructed by PCR amplification method. First, synthesize the upstream and downstream primers, the primer sequence is as follows:
[0040] Upstream primer: 5’-GGGTCGAC ATGACCACTTCGACGGGCGAGTCGGCTG- 3’;
[0041] Downstream primer: 5’-CCCTCGAG CAAGGACTGCTTTACAGGCGCCACT- 3’.
[0042] In the above-mentioned upstream and downstream primer sequences, the underlined part is the complementary sequence to the VP1 gene, and the bold and italicized part is the introduced XhoI and SalI restriction sites. The specific fragment amplified by PCR was recovered by 1% agarose gel electrophoresis to obtain the VP1 gene fragment of type O foot-and-mouth disease virus antigen with XhoI and SalI restriction sites. The nucleotide sequence of the VP1 gene fragment is as follows Sequence 2 in the sequence listing is shown (the nucleotide sequence of the VP1 gene, both Xhol and SalI restriction sites contain one base mutation), and the encoded amino acid sequence is shown in sequence 1 in the sequence listing. The result is figure 1 As shown, the size of the generated O-type foot-and-mouth disease virus antigen VP1 gene fragment is 651 bp, which is consistent with the expected size.

Example Embodiment

[0043] Example 2: Construction of recombinant expression vector plasmid pET30-FMD-VP1
[0044] Insert the O-type foot-and-mouth disease virus antigen VP1 gene fragment (that is, the complete reading frame containing the O-type foot-and-mouth disease virus VP1 gene) with the XhoI and SalI restriction sites obtained in Example 1 into the prokaryotic that has been digested with XhoI and SalI. At the polyclonal restriction site of the expression vector pET30a, the ligation product was transformed into E. coli DH5α, and the white clone was picked from the resistant plate for SalI/XholI double restriction digestion and sequencing. The results are as follows figure 2 As shown, it is confirmed by sequencing that the connection is successful, the VP1 virus code frame is correct, and the restriction site used for the connection is mutated, and the clone with the correct sequencing is named the recombinant expression vector plasmid pET30-FMD-VP1.

Example Embodiment

[0045] Example 3: VP1 protein expression, identification and purification
[0046] The recombinant expression vector plasmid pET30-FMD-VP1 with correct sequencing obtained in Example 2 was transformed into E. coli BL21 (DE3) to obtain recombinant genetically engineered bacteria, and the obtained recombinant genetically engineered bacteria was inoculated into LB liquid with kana resistance. In the medium, shake culture to OD600=0.6, add IPTG to a final concentration of 1mmol/L to induce the expression of recombinant genetic engineering bacteria. The induction conditions are: temperature 37℃, rotation speed 180r/min, induction 6h; after induction, centrifuge Collect the bacterial cells, SDS-PAGE electrophoresis to detect the expression of the target protein; the bacterial protein induced by IPTG and the negative control (uninduced empty bacteria) are subjected to SDS-PAGE electrophoresis. The result is image 3 As shown, compared with the uninduced recombinant bacteria, the induced recombinant bacteria expressed a specific protein band at about 25.0 ku, which was consistent with the predicted target protein size.
[0047] After SDS-PAGE electrophoresis detection, the obtained target protein was electro-transferred to nylon membrane, and the pig anti-O-type foot-and-mouth disease serum was used as the primary antibody, and horseradish peroxidase (HRP) coupled mouse anti-pig IgG was used as the primary antibody. The secondary antibody is subjected to Western-blote analysis to confirm whether the target protein has the antigenicity of type O foot-and-mouth disease virus. The result is Figure 4 As shown, the induced recombinant bacteria has a specific band, and the expressed target protein can be recognized by the positive serum of type O foot-and-mouth disease virus, which proves that the target protein has immunological activity.
[0048] Cultivate the correctly expressed target protein in large quantities. IPTG induces the expression of the target protein. The induction conditions are: temperature 37℃, rotation speed 180r/min, induction 6h; after the induction, the bacterial cells are collected by centrifugation, freeze-thaw cycles are repeated 3 times, and about 1 The cells were resuspended in PBS buffer with a volume of /10 cells, the cells were thoroughly disrupted by ultrasound, and the cells were centrifuged at 15000r/min for 15min. The supernatant was taken and purified by a nickel column to obtain O-type foot-and-mouth disease virus-like particle vaccine.
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Description & Claims & Application Information

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