CN111253494A

CN111253494A - Fusion protein of pig foot-and-mouth disease virus and pig rotavirus, viroid particle, vaccine and preparation method

Info

Publication number: CN111253494A
Application number: CN202010164198.2A
Authority: CN
Inventors: 贺笋; 闫鹏先; 张国庆; 潘毅平; 郭苗苗; 肖升东; 赵毅; 李延涛
Original assignee: Tecon Biological Co ltd; Tiankang Biology (shanghai) Co Ltd
Current assignee: Tiankang Pharmaceutical Co ltd
Priority date: 2020-03-10
Filing date: 2020-03-10
Publication date: 2020-06-09
Anticipated expiration: 2040-03-10
Also published as: CN111253494B

Abstract

The invention relates to the field of molecular biology, and particularly provides a fusion protein of a swine foot-and-mouth disease virus and a swine rotavirus, a viroid, a vaccine and a preparation method thereof. The fusion protein provided by the invention comprises an epitope of the porcine foot-and-mouth disease virus and VP6 protein of the porcine rotavirus, and the fusion protein can be automatically assembled into viroid particles. The invention uses VP6 protein of pig rotavirus as a carrier, obtains fusion protein after embedding the antigenic epitope of the pig foot-and-mouth disease virus, the fusion protein can be assembled into viroid particles by self, so that the antigenic epitope of the pig foot-and-mouth disease virus can be displayed on the surfaces of the viroid particles, and the obtained viroid particles have good immunogenicity to the pig foot-and-mouth disease virus and the pig rotavirus. The vaccine prepared from the virus particles is a bivalent vaccine, so that animals can simultaneously obtain the immunogenicity of the swine foot-and-mouth disease virus and the swine rotavirus, and the immune effect is good.

Description

Fusion protein of pig foot-and-mouth disease virus and pig rotavirus, viroid particle, vaccine and preparation method

Technical Field

The invention relates to the field of molecular biology, in particular to a fusion protein of a swine foot-and-mouth disease virus and a swine rotavirus, a viroid, a vaccine and a preparation method thereof.

Background

Foot-and-mouth disease (FMD), an acute, hot, highly contagious infectious disease of artiodactyl caused by foot-and-mouth disease virus. It mainly attacks artiodactyl animals, such as pig, cattle, sheep, etc., and occasionally humans and other animals. Clinical diagnosis is characterized by blisters on the oral mucosa, hooves and breast skin. The disease has multiple transmission ways and high speed, and has been epidemic in many times in the world, which causes huge political and economic losses. In view of this, the world animal health Organization (OIE) has listed it as the first infectious disease of class A. At present, FMD is prevalent in two-thirds of OIE member countries, and is a constant threat to livestock safety and livestock product trade in FMD-free countries and regions.

The porcine Rotavirus disease is an infectious zoonosis characterized by diarrhea caused by Rotavirus infection, Rotavirus (RV) widely exists in a pig group, has obvious seasonal epidemics, is mostly sent to piglets of 7-14 days old, has the morbidity of about 50-80 percent and the mortality of about 15 percent, and is clinically characterized by the symptoms of withering, anorexia, vomiting, diarrhea, dehydration and weight loss. Rotavirus diseases are distributed worldwide and are extremely difficult to purify, causing serious economic losses.

The foot-and-mouth disease vaccine in the current market mainly comprises an O/A bivalent inactivated vaccine and an O/A bivalent synthetic peptide vaccine, and the rotavirus is not a commercial vaccine. Among various vaccines for preventing foot and mouth disease, the subunit vaccine prepared by using the genetic engineering technology is a more successful direction in recent decades, and the viroid particle Vaccine (VLP) is the genetic engineering vaccine with the strongest immunogenicity, the most outstanding effect and the wide application prospect in the subunit vaccine. Although there are many technical routes for preparing foot-and-mouth disease virus particles, the poor thermal stability and acid stability of foot-and-mouth disease virus particles limit the application of foot-and-mouth disease VLP vaccines in the aspects of amplification, production and the like.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide a fusion protein of swine foot-and-mouth disease virus and swine rotavirus, a preparation method thereof and a related biological material thereof.

The second object of the present invention is to provide the use of the fusion protein or the biomaterial.

The third purpose of the invention is to provide viroid particles of the porcine foot-and-mouth disease virus and the porcine rotavirus and a preparation method thereof.

The fourth purpose of the invention is to provide a viroid particle vaccine for resisting the foot-and-mouth disease and the rotavirus disease of the pigs.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a fusion protein of swine foot-and-mouth disease virus and swine rotavirus comprises an epitope of the swine foot-and-mouth disease virus and VP6 protein of the swine rotavirus, and the fusion protein can be self-assembled into virus-like particles.

Further, the epitope comprises VP1 protein epitope 129-166aa and VP1 protein epitope 196-213 aa;

preferably, the epitope is selected from VP1 protein epitope 129-166aa of O type foot-and-mouth disease virus and VP1 protein epitope 196-213aa of O type foot-and-mouth disease virus;

the amino acid sequence of VP1 protein epitope 129-166aa of the O-type foot-and-mouth disease virus is SEQ ID NO.1, and the amino acid sequence of VP1 protein epitope 196-213aa of the O-type foot-and-mouth disease virus is SEQ ID NO. 2;

preferably, the amino acid sequence of the VP6 protein is SEQ ID NO. 3;

preferably, the nucleotide sequence of the VP6 protein is SEQ ID NO. 4;

preferably, the amino acid sequence of the fusion protein is SEQ ID No. 5;

preferably, the nucleotide sequence encoding the fusion protein is SEQ ID NO. 6.

The preparation method of the fusion protein comprises the steps of embedding the antigen epitope gene of the porcine foot-and-mouth disease virus into the VP6 protein gene of the porcine rotavirus to obtain a fused gene recombinant fragment, and expressing the recombinant fragment by an expression system to obtain the fusion protein.

Further, the chimeric site is a loop region on the surface of VP6 protein of the porcine rotavirus;

preferably, the sequence of the gene recombination fragment is shown as SEQ ID NO. 6;

preferably, the expression system comprises E.coli, yeast, insect cells, plant or mammalian cells, preferably yeast;

preferably, the recombinant fragments are introduced into the same cell of an expression system through different expression vectors, and are expressed to obtain the fusion protein.

The biological material related to the fusion protein is any one of the following materials:

(a) a nucleic acid molecule encoding a fusion protein of the invention;

(b) an expression cassette comprising the nucleic acid molecule of (a);

(c) a recombinant vector comprising the nucleic acid molecule of (a) or the expression cassette of (b);

(d) a recombinant eukaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c);

(e) a recombinant prokaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c).

The fusion protein or the biological material is applied to preparing viroid particles or vaccines.

A viroid particle of pig foot-and-mouth disease virus and pig rotavirus comprises a carrier and a carrier surface display protein, wherein the carrier comprises VP6 protein of the pig rotavirus, and the carrier surface display protein comprises an epitope of the pig foot-and-mouth disease virus.

preferably, the epitope is selected from VP1 protein epitope 129-166aa of O-type foot-and-mouth disease virus, the amino acid sequence of which is SEQ ID NO.1, and VP1 protein epitope 196-213aa of O-type foot-and-mouth disease virus, the amino acid sequence of which is SEQ ID NO. 2;

preferably, the amino acid sequence of the VP6 protein is SEQ ID NO. 3;

preferably, the nucleotide sequence of the VP6 protein is SEQ ID NO. 4.

The preparation method of the viroid comprises the steps of expressing the recombinant fragment for encoding the fusion protein of the invention in an expression system, and automatically assembling the obtained fusion protein into the viroid.

Further, the nucleotide sequence of the recombinant fragment is SEQ ID NO. 6;

preferably, the recombinant fragments are introduced into the same cell of an expression system through different expression vectors, expressed and self-assembled into viroid particles;

preferably, the buffer in which the fusion protein self-assembles into viroid particles comprises 40-60mM sodium citrate and 250-350mM sodium chloride, pH 4.5-5.

A viroid particle vaccine for resisting swine foot-and-mouth disease and swine rotavirus disease comprises pharmaceutically or veterinarily acceptable auxiliary materials and viroid particles;

preferably, the adjuvant comprises an adjuvant for injection preparations and an adjuvant for oral preparations;

preferably, the adjuvant for injectable formulations comprises an adjuvant;

preferably, the adjuvant comprises an aluminium salt adjuvant, an oil-in-water emulsion, a water-in-oil-in-water adjuvant, or a liposome, preferably ISA206 adjuvant.

Compared with the prior art, the invention has the beneficial effects that:

the fusion protein provided by the invention comprises an epitope of the porcine foot-and-mouth disease virus and VP6 protein of the porcine rotavirus, and the fusion protein can be automatically assembled into viroid particles. The invention uses VP6 protein of pig rotavirus as a carrier, obtains fusion protein after embedding the antigenic epitope of the pig foot-and-mouth disease virus, the fusion protein can be assembled into viroid particles by self, so that the antigenic epitope of the pig foot-and-mouth disease virus can be displayed on the surfaces of the viroid particles, and the obtained viroid particles have good immunogenicity to the pig foot-and-mouth disease virus and the pig rotavirus. The vaccine prepared from the virus particles is a bivalent vaccine, so that animals can simultaneously obtain the immunogenicity of the swine foot-and-mouth disease virus and the swine rotavirus, the immune effect is good, the disease prevention and control range is expanded, the labor force is saved, and the working efficiency is improved. In addition, the virus particle vaccine overcomes the defects of the foot-and-mouth disease virus particle vaccine in the prior art, fills the blank that no commercial rotavirus vaccine exists in the prior art, and is suitable for industrial production and popularization and use.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows SDS-PAGE detection of fusion proteins in example 1 of the present invention;

FIG. 2 shows the Western blot detection result of the fusion protein in example 1 of the present invention;

FIG. 3 shows the result of the electron microscopic examination of the viroid particles in example 2 of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

The invention protects the fusion protein of the foot-and-mouth disease virus and the porcine rotavirus, which comprises the epitope of the foot-and-mouth disease virus and the VP6 protein of the porcine rotavirus, and the fusion protein can be automatically assembled into virus-like particles.

The invention is based on the current situation of the vaccine for resisting the pig foot-and-mouth disease virus and the vaccine for resisting the pig rotavirus, creatively combines the main targets of the vaccines of the pig foot-and-mouth disease virus and the pig rotavirus, takes the VP6 protein of the pig rotavirus as a carrier, obtains the fusion protein after being embedded with the epitope of the pig foot-and-mouth disease virus, and the fusion protein can be automatically assembled into viroid particles, so that the epitope of the pig foot-and-mouth disease virus can be displayed on the surfaces of the viroid particles, and the obtained viroid particles have good immunogenicity to the pig foot-and-mouth disease virus and the pig rotavirus. It should be noted that the fusion protein is combined into viroid, and the reasons that the viroid has two immunogenicity of swine foot-and-mouth disease virus and swine rotavirus are at least: the antigenic epitope fragment of the porcine foot-and-mouth disease virus does not hinder the folding of the VP6 protein of the porcine rotavirus, or the VP6 protein fragment of the porcine rotavirus does not hinder the folding of the antigenic epitope fragment of the porcine foot-and-mouth disease virus, or both the two reasons exist.

In a preferred embodiment, the epitopes comprise the G-H loop of the VP1 protein (129-166aa) and the epitope 196-213aa of the VP1 protein. Further preferably selected from a VP1 protein G-H loop (129-166aa) of the type O foot-and-mouth disease virus, the amino acid sequence of which is SEQ ID NO.1, and a VP1 protein epitope 196-213aa of the type O foot-and-mouth disease virus, the amino acid sequence of which is SEQ ID NO. 2.

YSGTSKYSASQNRRGDLGPLAARLAAQLPASFNFGAI(SEQ ID NO.1)。

SQDRHKQKIIAPAKQ(SEQ ID NO.2)。

In a preferred embodiment, the amino acid sequence of the VP6 protein is SEQ ID NO.3, and the nucleotide sequence encoding VP6 protein is preferably SEQ ID NO. 4.

MEVLYSLSKTLKDARDKIVEGTLYSNVSDLIQQFNQMIVTMNGNDFQTGGIGNLPIRNWTFDFGLLGTTLLNLDANYVENARTTIEYFIDFIDNVCMDEIARESQRNGIAPQSEALRKLSGIKFKRINFDNSSDYIENWNLQNRRQRTGFVFHKPNILPYSASFTLNRSQPAHDNLMGTMWINAGSEIQVAGFDYSCAFNAPANIQQFEHVVPLRRALTTATITLLPDAERFGFPRVINSAGGTTTWYFNPVILRPSNVEVEFLLNGQIINTYQARFGTIIARNFDTIRLSFQLVRPPNMTPAVANLFPQAPPFIFHATVGLTLRTESAVCESVLADASETLLANVTAVRQEYAIPVGPVFPPGMNWTELVTNYSPSREDNLQRVFTVASIRSMLIK(SEQID NO.3)。

ATGGAAGTTTTGTACTCTTTGTCTAAGACTTTGAAGGACGCTAGAGACAAGATCGTTGAAGGTACTTTGTACTCTAACGTTTCTGACTTGATCCAACAATTCAACCAAATGATCGTTACTATGAACGGTAACGACTTCCAAACTGGTGGTATCGGTAACTTGCCAATCAGAAACTGGACTTTCGACTTCGGTTTGTTGGGTACTACTTTGTTGAACTTGGACGCTAACTACGTTGAAAACGCTAGAACTACTATCGAATACTTCATCGACTTCATCGACAACGTTTGTATGGACGAAATCGCTAGAGAATCTCAAAGAAACGGTATCGCTCCACAATCTGAAGCTTTGAGAAAGTTGTCTGGTATCAAGTTCAAGAGAATCAACTTCGACAACTCTTCTGACTACATCGAAAACTGGAACTTGCAAAACAGAAGACAAAGAACTGGTTTCGTTTTCCACAAGCCAAACATCTTGCCATACTCTGCTTCTTTCACTTTGAACAGATCTCAACCAGCTCACGACAACTTGATGGGTACTATGTGGATCAACGCTGGTTCTGAAATCCAAGTTGCTGGTTTCGACTACTCTTGTGCTTTCAACGCTCCAGCTAACATCCAACAATTCGAACACGTTGTTCCATTGAGAAGAGCTTTGACTACTGCTACTATCACTTTGTTGCCAGACGCTGAAAGATTCGGTTTCCCAAGAGTTATCAACTCTGCTGGTGGTACTACTACTTGGTACTTCAACCCAGTTATCTTGAGACCATCTAACGTTGAAGTTGAATTCTTGTTGAACGGTCAAATCATCAACACTTACCAAGCTAGATTCGGTACTATCATCGCTAGAAACTTCGACACTATCAGATTGTCTTTCCAATTGGTTAGACCACCAAACATGACTCCAGCTGTTGCTAACTTGTTCCCACAAGCTCCACCATTCATCTTCCACGCTACTGTTGGTTTGACTTTGAGAACTGAATCTGCTGTTTGTGAATCTGTTTTGGCTGACGCTTCTGAAACTTTGTTGGCTAACGTTACTGCTGTTAGACAAGAATACGCTATCCCAGTTGGTCCAGTTTTCCCACCAGGTATGAACTGGACTGAATTGGTTACTAACTACTCTCCATCTAGAGAAGACAACTTGCAAAGAGTTTTCACTGTTGCTTCTATCAGATCTATGTTGATCAAG(SEQ ID NO.4)。

Through specific epitope selection, the fusion protein in the invention is easier to assemble into viroid particles by self, and has the immunogenicity of swine foot-and-mouth disease virus and swine rotavirus. In a more preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO. 5.

MEVLYSLSKTLKDARDKIVEGTLYSNVSDLIQQFNQMIVTMNGNDFQTGGIGNLPIRNWTFDFGLLGTTLLNLDANYVENARTTIEYFIDFIDNVCMDEIARESQRNGIAPQSEALRKLSGIKFKRINFDNSSDYIENWNLQNRRQRTGFVFHKPNILPYSASFTLNSQDRHKQKIIAPAKQGTMWINAGSEIQVAGFDYSCAFNAPANIQQFEHVVPLRRALTTATITLLPDAERFGFPRVINSAGGTTTWYFNPVILRPSNVEVEFLLNGQIINTYQARFGTIIARNFDTIRLSFQLYSGTSKYSASQNRRGDLGPLAARLAAQLPASFNFGAIHATVGLTLRTESAVCESVLADASETLLANVTAVRQEYAIPVGPVFPPGMNWTELVTNYSPSREDNLQRVFTVASIRSMLIK(SEQ ID NO.5)。

The nucleotide sequence encoding the fusion protein is preferably as follows:

ATGGAAGTTTTGTACTCTTTGTCTAAGACTTTGAAGGACGCTAGAGACAAGATCGTTGAAGGTACTTTGTACTCTAACGTTTCTGACTTGATCCAACAATTCAACCAAATGATCGTTACTATGAACGGTAACGACTTCCAAACTGGTGGTATCGGTAACTTGCCAATCAGAAACTGGACTTTCGACTTCGGTTTGTTGGGTACTACTTTGTTGAACTTGGACGCTAACTACGTTGAAAACGCTAGAACTACTATCGAATACTTCATCGACTTCATCGACAACGTTTGTATGGACGAAATCGCTAGAGAATCTCAAAGAAACGGTATCGCTCCACAATCTGAAGCTTTGAGAAAGTTGTCTGGTATCAAGTTCAAGAGAATCAACTTCGACAACTCTTCTGACTACATCGAAAACTGGAACTTGCAAAACAGAAGACAAAGAACTGGTTTCGTTTTCCACAAGCCAAACATCTTGCCATACTCTGCTTCTTTCACTTTGAACTCTCAAGACAGACACAAGCAAAAGATCATCGCTCCAGCTAAGCAAGGTACTATGTGGATCAACGCTGGTTCTGAAATCCAAGTTGCTGGTTTCGACTACTCTTGTGCTTTCAACGCTCCAGCTAACATCCAACAATTCGAACACGTTGTTCCATTGAGAAGAGCTTTGACTACTGCTACTATCACTTTGTTGCCAGACGCTGAAAGATTCGGTTTCCCAAGAGTTATCAACTCTGCTGGTGGTACTACTACTTGGTACTTCAACCCAGTTATCTTGAGACCATCTAACGTTGAAGTTGAATTCTTGTTGAACGGTCAAATCATCAACACTTACCAAGCTAGATTCGGTACTATCATCGCTAGAAACTTCGACACTATCAGATTGTCTTTCCAATTGTACTCTGGTACTTCTAAGTACTCTGCTTCTCAAAACAGAAGAGGTGACTTGGGTCCATTGGCTGCTAGATTGGCTGCTCAATTGCCAGCTTCTTTCAACTTCGGTGCTATCCACGCTACTGTTGGTTTGACTTTGAGAACTGAATCTGCTGTTTGTGAATCTGTTTTGGCTGACGCTTCTGAAACTTTGTTGGCTAACGTTACTGCTGTTAGACAAGAATACGCTATCCCAGTTGGTCCAGTTTTCCCACCAGGTATGAACTGGACTGAATTGGTTACTAACTACTCTCCATCTAGAGAAGACAACTTGCAAAGAGTTTTCACTGTTGCTTCTATCAGATCTATGTTGATCAAG(SEQ ID NO.6)。

the invention also protects a preparation method of the fusion protein, and specifically, the epitope gene of the porcine foot-and-mouth disease virus is embedded into the VP6 protein gene of the porcine rotavirus to obtain a recombinant fragment, and the recombinant fragment is expressed by an expression system to obtain the fusion protein. The method ensures the antigen activity of the foot-and-mouth disease virus and the rotavirus of the pig, and the obtained fusion protein can be assembled into viroid particles. The chimeric site is preferably a loop region on the surface of VP6 protein of porcine rotavirus. The antigenic epitope gene of the porcine foot-and-mouth disease virus is embedded in the loop region on the surface of the VP6 gene of the porcine rotavirus, so that the antigenic activity of the two viruses is not influenced, the folding of protein fragments is not influenced mutually, and the viroid particles can be assembled smoothly. The recombinant fragment is preferably the nucleotide sequence shown in SEQ ID NO. 6.

In a preferred embodiment, the recombinant fragment is expressed in an expression system, such as E.coli, yeast, insect cells, plant or mammalian cells, and the like, to produce a fusion protein. The yeast is preferred, and the yeast cell is used as an expression system, so that the expression level of the target protein can be obviously improved, and the production cost can be reduced. In addition, in order to further improve the protein expression level of the expression system, the protein expression level can be improved by a mode of multiple electric transfer, specifically, on the basis of obtaining high expression cells through first screening, other expression vectors containing target recombinant fragments are integrated again, and the expression level of the target protein is improved.

(a) a nucleic acid molecule encoding a fusion protein of the invention;

(b) an expression cassette comprising the nucleic acid molecule of (a);

The invention also protects the application of the fusion protein or the biological material in preparing viroid particles or vaccines.

The invention also protects viroid particles of the porcine foot-and-mouth disease virus and the porcine rotavirus, which comprise a carrier and carrier surface display protein, wherein the carrier comprises VP6 protein of the porcine rotavirus, and the carrier surface display protein comprises an epitope of the porcine foot-and-mouth disease virus. The virus particles lead the antigen to be highly concentrated, and the foot-and-mouth disease epitope has a certain spatial structure and a better immune effect.

In a preferred embodiment, the epitopes comprise epitope 129-166aa of the VP1 protein and epitope 196-213aa of the VP1 protein. Preferably selected from VP1 protein epitope 129-166aa of O-type foot-and-mouth disease virus, the amino acid sequence of which is SEQ ID NO.1, and VP1 protein epitope 196-213aa of O-type foot-and-mouth disease virus, the amino acid sequence of which is SEQ ID NO. 2.

In a preferred embodiment, the amino acid sequence of the VP6 protein is SEQ ID No. 3; the nucleotide sequence of the VP6 protein is SEQ ID NO. 4.

The invention also provides a preparation method of the viroid, which expresses the recombinant fragment for encoding the fusion protein in an expression system, and the obtained fusion protein is automatically assembled into the viroid.

In a preferred embodiment, the nucleotide sequence of the recombinant fragment is SEQ ID NO. 6; expression systems are, for example, E.coli, yeast, insect cells, plant or mammalian cells. Furthermore, the fusion protein can self-assemble into viroid particles in the following buffers: 40-60mM sodium citrate and 250-350mM sodium chloride, pH 4.5-5; preferably 50mM sodium citrate, 300mM NaCl, pH 4.85; the temperature at which the fusion protein self-assembles is preferably 2 to 8 ℃ and more preferably 4 ℃.

The invention finally protects the viroid vaccine for resisting the pig foot-and-mouth disease and the pig rotavirus disease, which comprises pharmaceutically or veterinarily acceptable auxiliary materials and the viroid. The vaccine prepared from the virus particles is a bivalent vaccine, so that animals can simultaneously obtain the immunogenicity of the swine foot-and-mouth disease virus and the swine rotavirus, the immune effect is good, the disease prevention and control range is expanded, the labor force is saved, and the working efficiency is improved. In addition, the virus particle vaccine overcomes the defects of the foot-and-mouth disease virus particle vaccine in the prior art, fills the blank that no commercial rotavirus vaccine exists in the prior art, and is suitable for industrial production and popularization and use.

In a preferred embodiment, the adjuvants include adjuvants for injectable preparations and adjuvants for oral preparations. The adjuvant for the injection preparation comprises an adjuvant, wherein the adjuvant can be an aluminum salt adjuvant, an oil-in-water emulsion, a water-in-oil-in-water adjuvant or a liposome, and is preferably an ISA206 adjuvant.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Example 1 expression of fusion proteins by Pichia expression System

Through means of structural biology, the porcine rotavirus VP6 protein structure is analyzed, an amino acid sequence suitable for integration and substitution of porcine foot-and-mouth disease virus VP1 protein antigen sites is found out for substitution, the substituted sequence is named as VP6-VP1 fusion protein, and the specific sequence is shown as SEQ ID NO. 5;

carrying out codon optimization on a Pichia pastoris expression system on the replaced VP6-VP1 fusion protein, synthesizing a gene (recombinant fragment shown by SEQ ID NO. 6) for coding the VP6-VP1 fusion protein by means of genetic engineering, inserting a target gene into a pPICZ α A vector through Bst BI and Xba I, carrying out enzyme digestion identification and sequencing, screening out a positive recombinant vector, transforming the positive recombinant vector into an E.Coli DH5 α competent cell for amplification, and extracting a large number of plasmids, namely pPICZ α A-VP 6-FMDVP 1.

The plasmid pPICZ α A-RVP6-FMDVP1 was digested with Pme I restriction enzyme in a linearized manner, the linearized expression plasmid was recovered and the purity and concentration of the plasmid were determined.

Preparing a pichia pastoris X33 strain, electrically converting competent cells, mixing and placing a linearized pPICZ α A-RVP6-FMDVP1 carrier and the competent cells, and then electrically converting the carrier, wherein the electrically converted bacterial liquid is directly coated on a high-concentration Zeocin antibiotic plate for screening;

and (3) carrying out shake culture and amplification expression on the monoclonal strains growing on the flat plate, carrying out crushing, SDS-PAGE detection and Western blot detection on the fermented thalli, and obtaining the strains with positive detection results, namely the pichia pastoris X33 engineering bacteria capable of expressing the VP6-VP1 fusion protein. The SDS-PAGE detection result is shown in figure 1, and the Western blot detection result is shown in figure 2, wherein C is a control group, and 1 is an experimental group.

Taking the engineering bacteria to culture in a BMGY culture medium at 30 ℃ for 24h for activation; taking activated culture bacteria liquid according to the proportion of 1: inoculating the mixture in a BMGY culture medium according to the proportion of 400 to ferment, culturing at 30 ℃ for 24h, then changing the BMGY culture medium to a BMMY culture medium to continue induction expression, supplementing methanol every 24h to enable the final concentration of the methanol to be 0.5%, and harvesting the strain after induction expression for 72 h.

Example 2 purification of VP6-VP1 fusion protein and Assembly of VLPs

Adding the mixture into the thalli according to the mass-volume ratio of 1: 10, buffer A (20mM HEPES, 300mM NaCl, pH 7.3) is added, and the thalli are crushed by a high-pressure crushing method;

and (3) centrifuging the crushed cells at 4 ℃ and 12000rpm for 60min, precipitating the centrifugal supernatant for 1h at 4 ℃ by using 30% ammonium sulfate, and then re-suspending the precipitate by using buffer A for coarse purification.

The purified target protein was purified twice by gel filtration chromatography, the target protein was collected, the target protein was transferred to buffer B (50mM sodium citrate, 300mM NaCl, pH 4.85), and then left at 4 ℃ for assembly, and the particle size and uniformity thereof were examined by a microscope, and the results are shown in FIG. 3.

EXAMPLE 3 preparation of the vaccine

The fusion protein is diluted into different concentrations by PBS solution, the diluted fusion protein solution and SEPPIC ISA206 adjuvant are mixed together according to the proportion of 1:1 of antigen components and adjuvant, and finally different antigen gradient vaccines of 25 mug/head, 50 mug/head, 100 mug/head and 150 mug/head are prepared. Stirring at 8000r/min for 8-10min, adding 0.01% (volume ratio) thimerosal solution before stopping stirring, shaking thoroughly, mixing, performing aseptic inspection, viscosity measurement and stability measurement according to the requirement of appendix of Chinese veterinary pharmacopoeia (current edition), and standing at 4 deg.C for use.

Example 4 recombinant VLP vaccine safety and antibody detection

4.1 method

4.1.1 animals and immunization

The 21-day-old newborn piglets are all negative by the detection of pig foot-and-mouth disease, pig vesicular stomatitis and pig vesicular inflammation virus antibody kits, and the detection of the PCR shows that the pig rotavirus is negative. Example 3 the resulting vaccine was prepared. 10 newborn piglets are divided into 5 groups, 1 group is a normal saline control group, and 2-5 groups of newborn piglets are injected with vaccines of 25 mug/head part, 50 mug/head part, 100 mug/head part and 150 mug/head part through muscle respectively. Blood was taken prior to immunization. And carrying out secondary immunization 21 days after the immunization of the newborn piglet is completed. Blood was taken 14 days after the secondary immunization and FMDV and rotavirus antibody levels were measured. The weight change of each group of pigs was observed before and after immunization to see if there were fever, anorexia, etc.

4.1.2 serum antibody detection

The antibody detection method is respectively carried out according to a foot-and-mouth disease virus O-type antibody liquid phase blocking ELISA detection kit produced by Lanzhou veterinary research institute of Chinese agricultural academy of sciences and a self-made pig rotavirus detection method. Two weeks after the second immunization, blood was collected weekly, and serum was separated for antibody detection.

4.1.3 establishment of porcine rotavirus detection method

The isolated porcine rotavirus was used at 50mMThe enzyme-linked immunosorbent assay plate is coated in a 100 mu L/hole enzyme-linked immunosorbent assay plate after being diluted by phosphate buffer solution with pH 7.6 and is kept at 4 ℃ overnight. The following day the coating solution was discarded and washed 3 times with PBST, 200. mu.L/well. 2% Bovine Serum Albumin (BSA) was added as a blocking solution at 100. mu.L/well for 1h at 37 ℃. The blocking solution was discarded, and after 3 washes, the negative and positive sera were diluted with 1% Bovine Serum Albumin (BSA) in proportion and added to the ELISA plate at 100. mu.L/well for 1h at 37 ℃. The plate was discarded, washed 5 times and added with 1% Bovine Serum Albumin (BSA) at a rate of 1: HRP-labeled goat anti-porcine IgG at a dilution of 2500, 100. mu.L/well, 1h at 37 ℃. Discarding the liquid in the plate, washing for 5 times, adding TMB color development solution, developing at 100 μ L/well in a dark place at 37 deg.C for 15min, and adding 2mol/L H₂SO₄The reaction was stopped, 100. mu.L/well. Reading OD on microplate reader_450nmThe value is obtained.

4.2 results:

4.2.1 FMD antibody detection 50 immunized piglets, blood is collected every week 14 days after the second immunization to separate serum, the serum is used for detecting the FMD ELISA antibody level, the detection results are shown in tables 1-3, and the detection statistical results are shown in table 4.

TABLE 1 detection results of type O foot-and-mouth disease antibody for second immunization day 14 (Unit: Log)₁₀(potency)

	Group A	Group B	Group C	Group D	Group E
							1	1.5	1.5	1.95	1.95	0
2	1.65	1.5	1.8	1.8	0
						3	1.2	1.65	1.8	1.8	0
4	1.5	1.2	2.1	2.1	0
						5	1.35	1.8	1.8	2.1	0
6	1.65	1.65	1.8	2.25	0
						7	1.5	1.5	1.8	1.8	0
8	1.05	1.8	1.8	2.1	0
						9	1.2	1.65	1.95	1.8	0
10	1.2	1.5	1.8	1.8	0
						Mean value	1.38	1.58	1.86	1.95	0

TABLE 2 detection results (unit: Log) of type-O foot-and-mouth disease antibody for hyperimmunization day 21₁₀(potency)

	Group A	Group B	Group C	Group D	Group E
							1	1.2	1.35	1.8	1.8	0
2	1.35	1.65	1.8	2.1	0
						3	1.5	1.65	1.8	2.1	0
4	1.65	1.8	2.1	1.95	0
						5	1.5	1.95	1.95	2.1	0
6	1.5	1.8	2.1	2.1	0
						7	1.2	1.5	1.95	1.8	0
8	1.2	1.8	1.95	1.95	0
						9	1.2	1.8	1.8	2.1	0
10	1.35	1.65	1.8	2.1	0
						Mean value	1.37	1.70	1.91	2.01	0

TABLE 3 detection results of type O foot-and-mouth disease antibody for second immunization day 28 (Unit: Log)₁₀(potency)

TABLE 4 experimental piglet FMD antibody detection

4.2.2 rotavirus antibody detection 14 days after the piglet secondary immunization, all piglet antibodies in the 50 ug/head dose group turned positive on average (S/N value > 2.1). Antibody levels of all piglets in the 100. mu.g/head dose group and the 150. mu.g/head dose group were continuously increased 14 days after the second immunization, which is obviously superior to other low dose groups. The specific test results are shown in Table 5.

TABLE 5 experimental piglet rotavirus antibody detection

The results show that the increase of the antigen content in the vaccine corresponds to the corresponding increase of the antibody titer, which indicates that the antigen immunized piglet can generate good immunogenicity.

4.2.3 No obvious difference exists between the weight of the immunized piglets and the weight of the piglets of a control group after the immunization observation, and no phenomena of fever, anorexia and the like are observed, which shows that the vaccine of the invention is safe and is shown in Table 6.

TABLE 6 clinical observations of post-vaccine immunization safety trials

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

SEQUENCE LISTING

<110> Tiankang biological (Shanghai) Co., Ltd

TECON BIOLOGICAL Co.,Ltd.

Fusion protein of foot-and-mouth disease virus and rotavirus of pig, viroid particle, vaccine and preparation method

<160>6

<170>PatentIn version 3.5

<210>1

<211>37

<212>PRT

<213> Artificial sequence

<400>1

Tyr Ser Gly Thr Ser Lys Tyr Ser Ala Ser Gln Asn Arg Arg Gly Asp

1 5 10 15

Leu Gly Pro Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro Ala Ser Phe

20 25 30

Asn Phe Gly Ala Ile

35

<210>2

<211>15

<212>PRT

<213> Artificial sequence

<400>2

Ser Gln Asp Arg His Lys Gln Lys Ile Ile Ala Pro Ala Lys Gln

1 5 10 15

<210>3

<211>397

<212>PRT

<213> Artificial sequence

<400>3

Met Glu Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp

1 5 10 15

Lys Ile Val Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu Ile Gln

20 25 30

Gln Phe Asn Gln Met Ile Val Thr Met Asn Gly Asn Asp Phe Gln Thr

35 40 45

Gly Gly Ile Gly Asn Leu Pro Ile Arg Asn Trp Thr Phe Asp Phe Gly

50 55 60

Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val Glu Asn

65 70 75 80

Ala Arg Thr Thr Ile Glu Tyr Phe Ile Asp Phe Ile Asp Asn Val Cys

85 90 95

Met Asp Glu Ile Ala Arg Glu Ser Gln Arg Asn Gly Ile Ala Pro Gln

100 105 110

Ser Glu Ala Leu Arg Lys Leu Ser Gly Ile Lys Phe Lys Arg Ile Asn

115 120 125

Phe Asp Asn Ser Ser Asp Tyr Ile Glu Asn Trp Asn Leu Gln Asn Arg

130 135 140

Arg Gln Arg Thr Gly Phe Val Phe His Lys Pro Asn Ile Leu Pro Tyr

145 150 155 160

Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Ala His Asp Asn Leu

165 170 175

Met Gly Thr Met Trp Ile Asn Ala Gly Ser Glu Ile Gln Val Ala Gly

180 185 190

Phe Asp Tyr Ser Cys Ala Phe Asn Ala Pro Ala Asn Ile Gln Gln Phe

195 200 205

Glu His Val Val Pro Leu Arg Arg Ala Leu Thr Thr Ala Thr Ile Thr

210 215 220

Leu Leu Pro Asp Ala Glu Arg Phe Gly Phe Pro Arg Val Ile Asn Ser

225 230 235 240

Ala Gly Gly Thr Thr Thr Trp Tyr Phe Asn Pro Val Ile Leu Arg Pro

245 250 255

Ser Asn Val Glu Val Glu Phe Leu Leu Asn Gly Gln Ile Ile Asn Thr

260 265 270

Tyr Gln Ala Arg Phe Gly Thr Ile Ile Ala Arg Asn Phe Asp Thr Ile

275 280 285

Arg Leu Ser Phe Gln Leu Val Arg Pro Pro Asn Met Thr Pro Ala Val

290 295 300

Ala Asn Leu Phe Pro Gln Ala Pro Pro Phe Ile Phe His Ala Thr Val

305 310 315 320

Gly Leu Thr Leu Arg Thr Glu Ser Ala Val Cys Glu Ser Val Leu Ala

325 330 335

Asp Ala Ser Glu Thr Leu Leu Ala Asn Val Thr Ala Val Arg Gln Glu

340 345 350

Tyr Ala Ile Pro Val Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr

355 360 365

Glu Leu Val Thr Asn Tyr Ser Pro Ser Arg Glu Asp Asn Leu Gln Arg

370 375 380

Val Phe Thr Val Ala Ser Ile Arg Ser Met Leu Ile Lys

385 390 395

<210>4

<211>1191

<212>DNA

<213> Artificial sequence

<400>4

atggaagttt tgtactcttt gtctaagact ttgaaggacg ctagagacaa gatcgttgaa 60

ggtactttgt actctaacgt ttctgacttg atccaacaat tcaaccaaat gatcgttact 120

atgaacggta acgacttcca aactggtggt atcggtaact tgccaatcag aaactggact 180

ttcgacttcg gtttgttggg tactactttg ttgaacttgg acgctaacta cgttgaaaac 240

gctagaacta ctatcgaata cttcatcgac ttcatcgaca acgtttgtat ggacgaaatc 300

gctagagaat ctcaaagaaa cggtatcgct ccacaatctg aagctttgag aaagttgtct 360

ggtatcaagt tcaagagaat caacttcgac aactcttctg actacatcga aaactggaac 420

ttgcaaaaca gaagacaaag aactggtttc gttttccaca agccaaacat cttgccatac 480

tctgcttctt tcactttgaa cagatctcaa ccagctcacg acaacttgat gggtactatg 540

tggatcaacg ctggttctga aatccaagtt gctggtttcg actactcttg tgctttcaac 600

gctccagcta acatccaaca attcgaacac gttgttccat tgagaagagc tttgactact 660

gctactatca ctttgttgcc agacgctgaa agattcggtt tcccaagagt tatcaactct 720

gctggtggta ctactacttg gtacttcaac ccagttatct tgagaccatc taacgttgaa 780

gttgaattct tgttgaacgg tcaaatcatc aacacttacc aagctagatt cggtactatc 840

atcgctagaa acttcgacac tatcagattg tctttccaat tggttagacc accaaacatg 900

actccagctg ttgctaactt gttcccacaa gctccaccat tcatcttcca cgctactgtt 960

ggtttgactt tgagaactga atctgctgtt tgtgaatctg ttttggctga cgcttctgaa 1020

actttgttgg ctaacgttac tgctgttaga caagaatacg ctatcccagt tggtccagtt 1080

ttcccaccag gtatgaactg gactgaattg gttactaact actctccatc tagagaagac 1140

aacttgcaaa gagttttcac tgttgcttct atcagatcta tgttgatcaa g 1191

<210>5

<211>417

<212>PRT

<213> Artificial sequence

<400>5

Met Glu Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp

1 5 10 15

Lys Ile Val Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu Ile Gln

20 25 30

Gln Phe Asn Gln Met Ile Val Thr Met Asn Gly Asn Asp Phe Gln Thr

35 40 45

Gly Gly Ile Gly Asn Leu Pro Ile Arg Asn Trp Thr Phe Asp Phe Gly

50 55 60

Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val Glu Asn

65 70 75 80

Ala Arg Thr Thr Ile Glu Tyr Phe Ile Asp Phe Ile Asp Asn Val Cys

85 90 95

Met Asp Glu Ile Ala Arg Glu Ser Gln Arg Asn Gly Ile Ala Pro Gln

100 105 110

Ser Glu Ala Leu Arg Lys Leu Ser Gly Ile Lys Phe Lys Arg Ile Asn

115 120 125

Phe Asp Asn Ser Ser Asp Tyr Ile Glu Asn Trp Asn Leu Gln Asn Arg

130 135 140

Arg Gln Arg Thr Gly Phe Val Phe His Lys Pro Asn Ile Leu Pro Tyr

145 150 155 160

Ser Ala Ser Phe Thr Leu Asn Ser Gln Asp Arg His Lys Gln Lys Ile

165 170 175

Ile Ala Pro Ala Lys Gln Gly Thr Met Trp Ile Asn Ala Gly Ser Glu

180 185 190

Ile Gln Val Ala Gly Phe Asp Tyr Ser Cys Ala Phe Asn Ala Pro Ala

195 200 205

Asn Ile Gln Gln Phe Glu His Val Val Pro Leu Arg Arg Ala Leu Thr

210 215 220

Thr Ala Thr Ile Thr Leu Leu Pro Asp Ala Glu Arg Phe Gly Phe Pro

225 230 235 240

Arg Val Ile Asn Ser Ala Gly Gly Thr Thr Thr Trp Tyr Phe Asn Pro

245 250 255

Val Ile Leu Arg Pro Ser Asn Val Glu Val Glu Phe Leu Leu Asn Gly

260 265 270

Gln Ile Ile Asn Thr Tyr Gln Ala Arg Phe Gly Thr Ile Ile Ala Arg

275 280 285

Asn Phe Asp Thr Ile Arg Leu Ser Phe Gln Leu Tyr Ser Gly Thr Ser

290 295 300

Lys Tyr Ser Ala Ser Gln Asn Arg Arg Gly Asp Leu Gly Pro Leu Ala

305 310 315 320

Ala Arg Leu Ala Ala Gln Leu Pro Ala Ser Phe Asn Phe Gly Ala Ile

325 330 335

His Ala Thr Val Gly Leu Thr Leu Arg Thr Glu Ser Ala Val Cys Glu

340 345 350

Ser Val Leu Ala Asp Ala Ser Glu Thr Leu Leu Ala Asn Val Thr Ala

355 360 365

Val Arg Gln Glu Tyr Ala Ile Pro Val Gly Pro Val Phe Pro Pro Gly

370 375 380

Met Asn Trp Thr Glu Leu Val Thr Asn Tyr Ser Pro Ser Arg Glu Asp

385 390 395 400

Asn Leu Gln Arg Val Phe Thr Val Ala Ser Ile Arg Ser Met Leu Ile

405 410 415

Lys

<210>6

<211>1251

<212>DNA

<213> Artificial sequence

<400>6

atggaagttt tgtactcttt gtctaagact ttgaaggacg ctagagacaa gatcgttgaa 60

ggtactttgt actctaacgt ttctgacttg atccaacaat tcaaccaaat gatcgttact 120

atgaacggta acgacttcca aactggtggt atcggtaact tgccaatcag aaactggact 180

ttcgacttcg gtttgttggg tactactttg ttgaacttgg acgctaacta cgttgaaaac 240

gctagaacta ctatcgaata cttcatcgac ttcatcgaca acgtttgtat ggacgaaatc 300

gctagagaat ctcaaagaaa cggtatcgctccacaatctg aagctttgag aaagttgtct 360

ggtatcaagt tcaagagaat caacttcgac aactcttctg actacatcga aaactggaac 420

ttgcaaaaca gaagacaaag aactggtttc gttttccaca agccaaacat cttgccatac 480

tctgcttctt tcactttgaa ctctcaagac agacacaagc aaaagatcat cgctccagct 540

aagcaaggta ctatgtggat caacgctggt tctgaaatcc aagttgctgg tttcgactac 600

tcttgtgctt tcaacgctcc agctaacatc caacaattcg aacacgttgt tccattgaga 660

agagctttga ctactgctac tatcactttg ttgccagacg ctgaaagatt cggtttccca 720

agagttatca actctgctgg tggtactact acttggtact tcaacccagt tatcttgaga 780

ccatctaacg ttgaagttga attcttgttg aacggtcaaa tcatcaacac ttaccaagct 840

agattcggta ctatcatcgc tagaaacttc gacactatca gattgtcttt ccaattgtac 900

tctggtactt ctaagtactc tgcttctcaa aacagaagag gtgacttggg tccattggct 960

gctagattgg ctgctcaatt gccagcttct ttcaacttcg gtgctatcca cgctactgtt 1020

ggtttgactt tgagaactga atctgctgtt tgtgaatctg ttttggctga cgcttctgaa 1080

actttgttgg ctaacgttac tgctgttaga caagaatacg ctatcccagt tggtccagtt 1140

ttcccaccag gtatgaactg gactgaattg gttactaact actctccatc tagagaagac 1200

aacttgcaaa gagttttcac tgttgcttct atcagatcta tgttgatcaa g 1251

Claims

1. The fusion protein of the porcine foot-and-mouth disease virus and the porcine rotavirus is characterized by comprising an epitope of the porcine foot-and-mouth disease virus and VP6 protein of the porcine rotavirus, and the fusion protein can be automatically assembled into viroid particles.

2. The fusion protein of claim 1, wherein the epitope comprises VP1 protein epitope 129-166aa and VP1 protein epitope 196-213 aa;

preferably, the amino acid sequence of the VP6 protein is SEQ ID NO. 3;

preferably, the nucleotide sequence of the VP6 protein is SEQ ID NO. 4;

preferably, the amino acid sequence of the fusion protein is SEQ ID No. 5;

3. The preparation method of the fusion protein as claimed in claim 1 or 2, characterized in that the antigenic epitope gene of the porcine foot-and-mouth disease virus is embedded into the VP6 protein gene of the porcine rotavirus to obtain a fused gene recombinant fragment, and the gene recombinant fragment is expressed by an expression system to obtain the fusion protein;

preferably, the chimeric site is a loop region on the surface of VP6 protein of porcine rotavirus;

4. The biomaterial related to the fusion protein of claim 1 or 2, wherein the biomaterial is any one of:

(a) a nucleic acid molecule encoding the fusion protein of claim 1 or 2;

(b) an expression cassette comprising the nucleic acid molecule of (a);

5. Use of the fusion protein of claim 1 or 2 or the biomaterial of claim 4 in the preparation of a viroid particle or a vaccine.

6. A viroid particle of swine foot-and-mouth disease virus and swine rotavirus is characterized by comprising a carrier and carrier surface display protein, wherein the carrier comprises VP6 protein of swine rotavirus, and the carrier surface display protein comprises an epitope of the swine foot-and-mouth disease virus.

7. The viroid particle according to claim 6, wherein the epitopes comprise VP1 protein epitope 129-166aa and VP1 protein epitope 196-213 aa;

preferably, the amino acid sequence of the VP6 protein is SEQ ID NO. 3;

preferably, the nucleotide sequence of the VP6 protein is SEQ ID NO. 4.

8. The method for producing a viroid according to claim 6 or 7, wherein the recombinant fragment encoding the fusion protein according to claim 1 or 2 is expressed in an expression system, and the resulting fusion protein self-assembles into a viroid.

9. The method of claim 8, wherein the nucleotide sequence of the recombinant fragment is SEQ id No. 6;

10. A viroid-like particle vaccine against porcine foot-and-mouth disease and porcine rotavirus disease, comprising pharmaceutically or veterinarily acceptable auxiliary materials and the viroid-like particle of claim 6 or 7;

preferably, the adjuvant for injectable formulations comprises an adjuvant;