CN115521921A - Expression system of coxsackievirus 6 type recombinant virus-like particles, virus-like particles prepared by expression system and hand-foot-and-mouth disease vaccine - Google Patents

Abstract

The invention provides an expression system for expressing coxsackie virus 6 type recombinant virus-like particles, which is characterized in that an exogenous expression cassette is added on the basis of an original endogenous expression cassette, different promoters are adopted to realize different expression levels of the two sets of expression cassettes, the expression capacity of exogenous proteins is greatly improved, P1 precursor protein and 3CD protease of CA6 virus can be expressed in one expression system respectively, self-assembly is completed, and uniform and stable CA6 virus VLPs are formed finally. The expression system has strong expression capability on target foreign proteins and high product purity, can prepare CA6VLPs with excellent immune prototypes in a large scale, and can be further used for industrial production of high-quality CA6 hand-foot-and-mouth disease vaccines; the multivalent hand-foot-and-mouth disease vaccine prepared by combining the virus-like particles and other immunogenic components of enteroviruses can provide broad-spectrum and comprehensive protection for multiple epidemic strains, so that the prevention effect on hand-foot-and-mouth disease is effectively improved.

Description

Expression system of coxsackievirus 6 type recombinant virus-like particles, virus-like particles prepared by expression system and hand-foot-and-mouth disease vaccine

Technical Field

The application belongs to the field of biomedical engineering, and particularly relates to an expression system of coxsackie virus 6 type recombinant virus-like particles, and virus-like particles and vaccines prepared by the expression system.

Background

The human enterovirus belongs to the family of picornaviridae, and comprises poliovirus, coxsackievirus, echovirus and novel enterovirus, wherein the genome is single-stranded positive-strand RNA, only has one open reading frame and encodes a multimeric precursor protein; the polyprotein is hydrolyzed into 3 precursor proteins P1, P2 and P3 in host cells, wherein P1 is a structural protein and forms a virus capsid, and P2 and P3 non-structural proteins are mainly enzymes related to virus replication. When P2 and P3 are hydrolyzed, the P2 and the P3 are respectively hydrolyzed into 2A, 2BC, 3AB and 3CD, and then are further hydrolyzed into 7 terminal mature proteins of 2A, 2B, 2C, 3A, 3B, 3C and 3D, wherein the 3CD has protease activity and can specifically shear precursor protein P1 into VP0, VP1 and VP3 to form a pentamer subunit; VP0 binds viral RNA, encapsulates it within capsid proteins, and hydrolyzes itself into VP2 and VP4 before capsid protein assembly is complete, ultimately forming infectious viral particles. Therefore, how to realize the co-expression of the 3CD protease and the P1 precursor protein when preparing recombinant virus-like particles (VLPs) based on the gene recombination technology is the key to realize the high-efficiency preparation of the enterovirus VLPs.

Hand-foot-and-mouth disease is a common infectious disease prevalent in infants under 5 years old, and has been listed in the third infectious disease list; specifically, the medicine is characterized in that small herpes or small ulcer appears at the parts of stomachache, anorexia, low fever, hands, feet, oral cavity and the like, most children patients can self-heal for about one week, and a few children patients can cause complications such as myocarditis, pulmonary edema, aseptic meningoencephalitis and the like; the individual critically ill children have a fast disease progression and eventually die. Various enteroviruses are known to cause hand-foot-and-mouth disease, including coxsackievirus A group 4, 5, 6, 9, 10, 16 type, B group 2, 5 type, enterovirus 71 type and the like. The development of high titers of relevant vaccines for the prevention of relevant diseases is an important task for the person skilled in the art.

The virus-like particles are particles which are composed of capsid proteins without viral genomes and with complete three-dimensional structures, have no infectivity, retain antigen epitopes on the capsid proteins, and have stable structures, so the virus-like particles are safer and more effective compared with the traditional attenuated or inactivated vaccines. Currently, in such cases where two proteins need to be co-expressed, there are a method of co-expressing a single vector and a method of separately preparing two recombinant plasmid vectors and introducing the vectors into the same host for expression. However, in any way, only the P1 precursor protein and the 3CD protease are finally expressed, i.e., only the process product and not the final product are expressed, and whether VLPs which are uniform, stable, have excellent immunogenicity and can be industrially produced can be finally formed cannot be determined, so that the co-expression of the 3CD protease and the P1 precursor protein cannot be ensured.

Disclosure of Invention

In order to solve the technical problems, the invention provides an expression system of coxsackie virus 6 type virus-like particles, and the virus-like particles and vaccines prepared by the expression system.

The technical scheme of the invention is as follows:

the invention provides an expression system for expressing a coxsackie virus 6 type recombinant virus-like particle, which comprises a host cell and a recombinant plasmid vector introduced into the host cell, wherein the recombinant plasmid vector can simultaneously express a P1 precursor protein and 3CD protease of the coxsackie virus 6 type, and the expression levels of the P1 precursor protein and the 3CD protease of the recombinant plasmid vector are different.

When coxsackie virus type 6 (CA 6) is assembled in virus-like particles, the precursor protein P1 needs to be decomposed under the action of 3CD protease so as to assemble VLPs (virus-like particles) of the CA6 virus, so that the P1 protein and the 3CD protease need to be simultaneously expressed so as to assemble the VLPs.

Further, the recombinant plasmid vector is provided with an initial MOX expression cassette for expressing the P1 precursor protein and an exogenous expression cassette for expressing the 3CD protease, and the promoter of the exogenous expression cassette is selected from any promoter except the MOX promoter.

By adopting the expression system, the co-expression of the 3CD protease and the P1 precursor protein can be realized, and the efficient self-assembly of the 3CD protease and the P1 precursor protein can be effectively realized, so that VLPs with high expression quantity can be obtained. The special expression vector (namely the recombinant plasmid vector) constructed by the invention can overcome the uncertainty of the expression result, realize the high expression quantity of the target protein 3, and can obtain the CA6VLPs which are uniform, stable, have outstanding immunogenicity and can realize industrial production

Further, the promoter of the exogenous expression cassette is a CYC1 promoter, a GAPDH promoter, a TEF1 promoter, or a DAS promoter.

Further, the promoter of the exogenous expression cassette is a CYC1 promoter.

The promoter is used as an exogenous promoter, so that the recombinant plasmid vector is more suitable for expression of the coxsackie virus 6 type related protein; preferably, the exogenous promoter is CYC1 promoter, and the expression rate of VLP protein is obviously higher than that of other promoters.

Further, the host cell is preferably Hansenula polymorpha, in particular is preferably auxotrophic for at least one substance, and the recombinant plasmid vector has at least one auxotrophic marker corresponding to the substance.

By constructing auxotrophic host cells, the obtained expression system can be screened and cultured by a corresponding nutrient substance deficient culture medium, so that the expression system required by the application can be obtained.

Further, the substance is uracil, leucine, methanol, pep protease, prc protease, or kex protease.

Further, the host cell is leucine auxotroph, and the recombinant plasmid vector has an auxotrophic marker Leu2.

Through screening and verification of a large number of experiments, the Leu2 auxotrophy marker is relatively more suitable for the expression system, and efficient screening is facilitated.

Further, the recombinant plasmid vector also comprises at least one reporter gene expression cassette, preferably any one of a Ura3 expression cassette, a Zeocin expression cassette and a G418 expression cassette.

Further, the recombinant plasmid vector also has an autonomously replicating sequence thereon.

By carrying out optimization screening on an exogenous promoter and introducing an autonomous replication sequence, a strain with high copy and high expression quantity can be obtained, the copy number of exogenous polynucleotide reaches 13, and the expression level of coxsackie virus 6 type recombinant virus-like particles can reach more than 23.0 mu g/ml.

According to another aspect of the present invention, there is provided a recombinant Coxsackie virus 6 virus-like particle expressed by any one of the expression systems described above.

In a third aspect, the invention provides an immune composition for preventing or treating hand-foot-and-mouth disease, which specifically comprises the coxsackie virus 6 type recombinant virus-like particles.

In the immune composition obtained by the method, the purity of VLPs can reach 99 percent, so that a vaccine prepared by using the VLPs has excellent immunogenicity.

Further, in the immune composition with a single administration dose, the content of the coxsackie virus 6 type recombinant virus-like particles is 5-60 mu g.

Specifically, the dosage of CA6VLPs in a single administration dose may be equal to or about equal to 5 μ g, 10 μ g, 15 μ g, 20 μ g, 25 μ g, 30 μ g, 35 μ g, 40 μ g, 45 μ g, 50 μ g, 55 μ g, or 60 μ g, or may be any value between any two adjacent numbers.

The fourth aspect of the invention provides a hand-foot-and-mouth disease vaccine which comprises the immune composition.

Further, the vaccine also comprises an adjuvant, wherein the adjuvant comprises at least one of aluminum hydroxide, aluminum phosphate, aluminum sulfate, ammonium alum, potassium alum, 3D-MPL, squalene, tween, tocopherol, cpG, poly (I: C) and QS 21.

Further, the adjuvant comprises aluminum hydroxide, and the dosage of the aluminum hydroxide in the hand-foot-and-mouth disease vaccine is 1-2 mg/ml.

Further, the adjuvant comprises the following components:

squalene 40-50 mg/ml (preferably 42.16 mg/ml), alpha-tocopherol 40-50 mg/ml (preferably 47.44 mg/ml), and tween 80-25 mg/ml (preferably 19.44 mg/ml).

Further, the adjuvant comprises the following components:

35 to 45mg/ml (preferably 39 mg/ml) of squalene, 85.5 to 7.5mg/ml (preferably 4.7 mg/ml) of span and 2.5 to 7.5mg/ml (preferably 4.7 mg/ml) of tween.

In a fifth aspect the present invention provides the use of an immunogenic composition as described above in the preparation of a multivalent hand-foot-and-mouth disease vaccine, wherein said immunogenic composition is used in combination with one or more further immunogenic components of an enterovirus.

Further, the enterovirus includes at least one of coxsackievirus 10, coxsackievirus 16 or enterovirus 71, and the immunogenic component is a virus-like particle or an inactivated virus.

The invention has the following beneficial effects:

the invention provides an expression system capable of expressing two proteins simultaneously, which respectively expresses P1 precursor protein and 3CD protease of CA6 virus through two groups of expression cassettes, and can complete self-assembly of the P1 precursor protein and the 3CD protein in one expression system to finally form uniform and stable CA6 VLPs. The expression system has strong expression capacity on target foreign proteins and high product purity, can prepare CA6VLPs with excellent immunogenicity on a large scale, and can be further used for industrial production of high-quality CA6 hand-foot-and-mouth disease vaccines; the multivalent hand-foot-and-mouth disease vaccine prepared by combining the virus-like particles and other immunogenic components of enteroviruses can provide broad-spectrum and comprehensive protection for multiple epidemic strains, so that the prevention effect on hand-foot-and-mouth disease is effectively improved.

Drawings

FIG. 1 is a spectrum of the starting plasmid vector pHP3.0 in example 1;

FIG. 2 is a map of the recombinant plasmid vector of pHP7.0-MOX-MOX in example 3, wherein the promoter of the original MOX expression cassette is the MOX promoter and the promoter of the exogenous MOX expression cassette is the MOX promoter;

FIG. 3 is a map of the recombinant plasmid vector of pHP7.0-MOX-CYC1 in example 3, wherein the promoter of the original MOX expression cassette is the MOX promoter and the exogenous promoter of the exogenous MOX expression cassette is the CYC1 promoter;

FIG. 4 shows the Western Blot detection results of the VLP vaccine of recombinant CA16 of example 7;

FIG. 5 is an electron microscopic image of the recombinant virus-like particles of CA16 type in example 7.

Detailed Description

The present application will be described in further detail with reference to the drawings and examples. In the present application, the nomenclature of each plasmid vector is merely for convenience of description, and has no special meaning. In addition, the P1 precursor protein coding gene and the 3CD protease coding gene adopted in the embodiment are both derived from an epidemic strain of Coxsackie virus type 6 in China, preferably ALB72952.1, the sequence of the P1 protein is SEQ ID NO.1, and the sequence of the 3CD protease is SEQ ID NO.2.

EXAMPLE 1 construction of recombinant plasmid vector

In the examples, the plasmid vector was constructed from a starting plasmid vector pHP3.0 having the gene sequence SEQ ID No.3, which itself contained the Ura3 expression cassette, the Zeocin expression cassette, the G418 expression cassette, and a starting MOX expression cassette for expression of the foreign gene, the profile of which is shown in FIG. 1. The specific method for constructing the recombinant plasmid vector by using the plasmid vector pHP3.0 is as follows:

1. synthesizing primers TT-F-NotI and TT-R of an MOX promoter and primers MOX-F and MOX-R-KpnI of an MOX terminator, wherein the gene sequences of the primers are respectively shown as SEQ ID No. 4-7; two sections of PCR are respectively amplified: and connecting the two sections by PCR through an exogenous MOX promoter and an exogenous MOX terminator, and carrying out enzyme digestion and recovery by using NotI + KpnI after glue is recovered.

2. Construction of plasmid vector pHP4.0: and (3) carrying out NotI + KpnI enzyme digestion on pHP3.0, then connecting the pHP3.0 with an exogenous MOX expression cassette, carrying out selective cloning, and obtaining a plasmid vector pHP4.0 after the enzyme digestion identification is correct.

3. Construction of recombinant plasmid vector

3.1 carrying out double enzyme digestion on a plasmid vector pHP4.0 and a CA6 type P1 precursor protein coding gene (the gene sequence is shown as SEQ ID No. 8) by EcoVI + EcoRV, then connecting by PCR, after transformation, selecting a colony of a monoclonal antibody, and identifying correctly to obtain a pHP4.0-P1 plasmid vector; the pHP4.0-P1 plasmid vector and a CA16 type 3CD protease coding gene (the gene sequence is shown as SEQ ID No. 9) are subjected to double enzyme digestion by SpeI and SalI and then are connected by PCR, and after transformation, a colony of a monoclonal antibody is picked and correctly identified to obtain a recombinant plasmid vector pHP4.0-P1-M3CD. In this case, the exogenous promoter of the recombinant plasmid vector is a MOX promoter.

The recombinant plasmid vector is identified by adopting the following steps: introducing 1 μ L of recombinant plasmid vector into Escherichia coli DH5 α competent cell, ice-cooling the competent cell for 30min, placing in 42 deg.C water bath, heat shocking for 90s, and standing in ice bath for 3min; then adding 850 +/-100 mu L LB culture medium into Escherichia coli DH5 alpha competent cells, shaking-culturing for 45min at 37 ℃ and 160 +/-10 rpm, directly taking 100 mu L culture solution to coat a flat plate, carrying out inverted culture at 37 ℃ overnight, finally taking colony of the monoclonal antibody and identifying correctly to obtain the recombinant plasmid vector.

3.2 carrying out KpnI + SalI double enzyme digestion on the recombinant plasmid vector pHP4.0-P1-M3CD to remove the exogenous MOX promoter in the exogenous MOX expression cassette, then connecting with a CYC1 promoter (the primer sequence is shown as SEQ ID No. 10-11), finally realizing replacement of the MOX promoter for expressing 3CD protease with the CYC1 promoter, obtaining the recombinant plasmid vector pHP4-P1-C3CD, and finally identifying the recombinant plasmid vector by adopting the method in the step 3.1.

EXAMPLE 2 construction of recombinant plasmid vector containing autonomously replicating sequence

The recombinant plasmid vectors pHP4.0-P1-M3CD and pHP4.0-P1-C3CD obtained in example 1 were digested with NotI + SacI, and then ligated to the autonomously replicating sequence HARS1 gene fragment ((the primer sequences are shown in SEQ ID Nos. 12 to 13)) to construct recombinant plasmid vectors pHP5.0-P1-M3CD and pHP5.0-P1-C3CD, respectively.

EXAMPLE 3 construction of recombinant plasmid vector containing autonomously replicating sequence and auxotrophic marker Leu2

The recombinant plasmid vectors pHP5.0-P1-M3CD and pHP5.0-P1-C3CD obtained in example 2 were inserted with Leu2 expression cassettes (the yeast leucine promoter gene sequence of the expression cassettes was synthesized by Beijing Okagaku Biotechnology Co., ltd.), respectively, as follows:

the leucine promoter gene fragment is cut by BamHI/BglII double enzyme digestion, the recombinant plasmid vectors pHP5.0-P1-M3CD and pHP5.0-P1-C3CD obtained in the embodiment 2 are respectively cut by BglII enzyme digestion, then dephosphorylated by CIAP, the leucine promoter gene fragment is connected with the dephosphorylated recombinant plasmid vector, colonies of the monoclonal antibody are respectively selected and identified by PCR, the recombinant plasmid vectors pHP7.0-P1-M3CD and pHP7.0-P1-C3CD are obtained after the sequencing verification is correct, and the specific spectrogram is shown in figures 2-3.

Example 4 construction of the expression System for the type 6 CAV-like particles and expression of VLPs

A Hansenula polymorpha original strain ATCC26012 was purchased from ATCC, and Leu2 gene knockout was performed by the Beijing institute of microorganisms to obtain a leucine auxotrophic strain HP15. The recombinant plasmid vectors pHP7.0-P1-M3CD and pHP7.0-P1-C3CD obtained in example 3 were respectively and electrically transformed into HP15 Hansenula polymorpha strain to obtain corresponding CA6 type virus-like particle expression systems. The specific method comprises the following steps:

1. expanding preparation of recombinant plasmid vector pHP7.0

mu.L of the recombinant plasmid vector pHP7.0 was added to E.coli DH 5. Alpha. Competent cells and ice-cooled for 30min. Placing the escherichia coli DH5 alpha competent cells in a water bath at 42 ℃ for 90s after ice bath, and then standing for 3min in the ice bath; then, 850. + -.100. Mu.L of LB liquid medium was added to E.coli DH 5. Alpha. Competent cells, followed by shaking culture at 37 ℃ and 160. + -.10 rpm for 45min. Then further scale-up culture was performed. After the amplification culture, the Plasmid was extracted using E.Z.N.A Plasmid Mini Kit (from Omega Bio-Tek) and subjected to single cleavage with Bgl II, followed by recovery with E.Z.N.A Gel Extraction Kit (from Omega Bio-Tek), and then eluted with 50. Mu.L of sterile water preheated to 55 ℃ by OD measurement _260nm DNA quantification was performed and the linearized fragments were diluted to 100 ng/. Mu.L, and mostThen storing in a refrigerator at the temperature of 20 ℃ below zero for later use.

2. Treatment of Hansenula polymorpha cells

Selecting a single colony of HP15 Hansenula polymorpha, inoculating the single colony into a test tube containing 5ml of YPD liquid culture medium, and culturing at 30 ℃ for 12h; taking 5ml of bacterial liquid, transferring the bacterial liquid into 200ml of YPD culture medium, and culturing for 4-6 h at 30 ℃ until OD is reached _600nm About 1.3, the cells were resuspended in 200ml of 0.1mol/L phosphate buffer (25 mmol/L, pH 7.5), mixed well, incubated at 30 ℃ for 30min, and centrifuged at 5000rpm for 10min, the supernatant was discarded, and the pellet (i.e., cells) was retained. With precooled STM solution (i.e. sucrose-Tris-MgCl) ₂ Solution) 200mL, the thalli are evenly blown and sucked, the thalli are centrifuged for 3min at the temperature of 4 ℃ and the rpm of 5000, the supernatant is discarded, and the precipitate (namely the thalli) is left. The cells were resuspended in 100mL of an ice-cold STM solution, centrifuged at 5000rpm at 4 ℃ for 3min, and the supernatant was discarded to leave a pellet (i.e., cells). And (3) resuspending the bacteria by using 50-200 mu L of ice-cold STM solution according to the bacterial amount, transferring the bacteria solution to a centrifugal tube with high pressure, and carrying out ice bath to obtain the bacteria solution to be electrically transformed.

3. Electrotransformation transfer recombinant plasmid vector pHP7.0

Adding 15 mu L of recombinant plasmid vector and 30 mu L of bacterial liquid according to the volume ratio of the recombinant plasmid vector to the bacterial liquid = 1: 2, fully and uniformly blowing and sucking to obtain a recombinant plasmid vector-bacterial liquid mixed solution, and placing the mixed solution in an ice bath for conversion. Taking out the electric revolving cup which is soaked in alcohol in advance, refrigerated at the temperature of minus 20 ℃ after ultraviolet irradiation, and adding the recombinant plasmid vector-bacterial liquid mixed solution. Shock recombinant plasmid vector-bacteria liquid mixture according to voltage 2500V, resistance 150 omega, electric capacity 50 uF conditions, after shock rapidly add 1mL had already balanced to room temperature YPD solution, gently mix, transfer to EP tube. Placing the recombinant plasmid vector-bacterial liquid mixed solution after the electric transformation in a water bath at 30 ℃ for 2h, and slightly reversing for 3 times every 15 min; then, centrifuging the recombinant plasmid vector-bacterial liquid mixed solution incubated for 2h for 10min at 5000rpm, and discarding the supernatant; finally, 200 mu L of YPD solution is used for resuspending the thalli, 100 mu L of the thalli are coated on a YPD plate containing 0.5mg/mL Zeocin and not added with leucine, a single colony grows out after inverted culture is carried out for 3-7 days at the temperature of 30 ℃, and the result shows that the recombinant plasmid vector is successfully introduced, and the recombinant hansenula polymorpha single strain which can overcome the defect of leucine and can grow is obtained.

4. Passage and stabilization of recombinant Hansenula

And (4) picking a single colony of the recombinant strain grown in the step (3), inoculating the single colony into 5mL of YPD liquid culture medium containing 0.5mg/mL of Zeocin, carrying out shake culture at 30 ℃ and 200rpm (24-48 h) until the OD value reaches 50, transferring the single colony into 5mL of YPD liquid culture medium containing 0.5mg/mL of Zeocin according to the content ratio of 1:1000, culturing until the OD value reaches 50, transferring into 5mL of YPD liquid culture medium containing 0.5mg/mL of Zeocin according to the ratio of 1:1000, and continuing for 10 times by the same method, and preserving the strain. The storage system is bacterial liquid and 60% glycerol (v/v) = 1:1, and the bacterial strain is stored according to the required amount, and is usually 500 μ L bacterial liquid and 500 μ L60% glycerol. Inoculating the recombinant strain which is transferred for 10 times into 5mL YPD liquid culture medium without Zeocin resistance, carrying out shake culture at 30 ℃ and 200rpm until the OD value reaches 50, and then transferring the strain into 5mL YPD liquid culture medium according to the volume ratio of 1: 1000; by analogy, and 5 times in succession in YPD liquid medium without Zeocin resistance. The stabilized bacterial solution was spread on YPD plates containing 16mg/mLG418, and cultured in an inverted state at 30 ℃ for 3 days.

Picking single colonies of a plurality of recombinant strains from YPD plates containing 16mg/mL G418, inoculating the single colonies into a test tube containing 5mL of YPG liquid culture medium, and culturing at 30 ℃ for 24h; transferring the bacterial liquid into a 100mL triangular flask containing 30mL YPM induction medium with initial density of OD _600nm =1, shake-induced for 72h, and methanol solution with final concentration of 0.5% (v/v,%) was added to the bacterial solution every 12 h.

Transferring 10mL of induced bacterial liquid into a 50mL centrifuge tube, centrifuging at 10000rpm for 10min, and removing supernatant; resuspending the thallus precipitate with 50mL of cell lysate, mixing well, and performing thallus crushing in an ultrasonic instrument according to an ultrasonic crushing program with the power of 20%, the total ultrasonic time of 20min, the opening time of 2s and the closing time of 3s, wherein an ice bath is required to be kept in the crushing process; and centrifuging the ultrasonically-crushed bacterial liquid at 10000rpm for 10min, collecting the supernatant, and detecting.

Example 5 preparation of a vaccine for the CA6 type hand-foot-and-mouth disease

The preparation of the recombinant vaccine comprises the fermentation process by using the recombinant hansenula polymorpha strain and the separation and purification process of CA6VLPs, and specifically comprises the following steps:

(1) Fermentation of recombinant Hansenula polymorpha expression strains

Seed culture: the recombinant Hansenula polymorpha strain obtained in example 4 (obtained by electrotransformation of the recombinant plasmid vector pHP7.0-P1-C3CD to the HP15 Hansenula polymorpha strain obtained in example 3) was seed-cultured in the following manner:

fermenting the seed liquid: thawing 1 frozen glycerol strain (HP-1 #/pRMHP2.1-58 HP), inoculating 50 μ L into 5mL YPD medium, shake culturing at 30 deg.C and 200rpm (20-24 hr) to optical density of bacterial thallus A _600nm About 2-5, sucking 1mL of the qualified bacteria, inoculating into 2 bottles of 500mL YPD medium, shake culturing at 30 deg.C and 200rpm for 20-24h until the optical density A of the bacteria _600nm About 15-20, and using the qualified product as fermentation seed liquid for later use.

Then, 200mL of the seed solution was inoculated into a 30L fermentor and subjected to fermentation culture. The initial fermentation medium is a complex medium (the specific medium components (wt): potassium sulfate 1.21%, magnesium sulfate heptahydrate 0.99%,85% concentrated phosphoric acid 1.78%, potassium hydroxide 0.28%, peptone 0.33%, yeast powder 0.67%, and glycerol 0.67%), the temperature is set at 30 ℃, the pH is 5.0, and the ventilation and rotation speed are controlled to make the Dissolved Oxygen (DO) not less than 20%. After the carbon source in the initial medium was exhausted, 1.2% (wt,%) PTM (CuSO) was fed in ₄ ·5H ₂ O 6.0g/L，KI 0.088g/L，MnSO ₄ ·H ₂ O 3.0g/L，Na ₂ MoO ₄ ·2H ₂ O 0.2g/L，H ₃ BO ₃ 0.02g/L，CoCl ₂ ·6H ₂ O 0.5g/L，ZnCl ₂ 20.0g/L，FeSO ₄ ·7H ₂ O 65.0g/L，H ₂ SO ₄ 5mL/L, biotin 0.2 g/L), the glycerol feeding speed is related to dissolved oxygen, and the Dissolved Oxygen (DO) is not less than 20% in the feeding process. And after the wet weight of the thalli reaches 150-200g/L, adjusting the pH value to 6.0. After 0.5-1h of starvation, methanol containing 1.2% (wt,%) of PTM is started to flow for induction: during the induction process, the methanol concentration is controlled to be not higher than 15g/L, the Dissolved Oxygen (DO) is not lower than 20%, and the induction time is 40-50h.

(2) Purification of CA6VLPs in fermentation broths

And (3) harvesting thalli: the cells were centrifuged at 6000g for 25min, and the supernatant was discarded to harvest the cells.

And (3) resuspending the thalli: the wet cells were adjusted to 200g/L using Q buffer (50 mM Tris buffer, 5% (v/v,%) glycerol, 2mM EDTA), pH8.5, and the cells were resuspended.

High-pressure homogenization: the cell disruption was carried out 3-5 times using a high pressure homogenizer with a working pressure of 1200 bar.

Removing cell debris: the cell-disrupted bacterial suspension was centrifuged at 12000r/min for 25min, and the supernatant was collected and then passed through a 0.45 μm filter.

And (3) ultrafiltration: the filtrate treated with 0.45 μm filter was ultrafiltered using 750K hollow fibers, passing through a flux of 20LMH and finally reduced to the original volume.

Anion chromatography: column equilibration with 50mM Tris, pH8.5 using Q Sepharose HP packing, followed by addition of the above ultrafiltrated filtrate to an anion chromatography column, followed by gradient elution with 1M NaCl solution, collection of the eluate and collection of UV _280nm Ultraviolet absorption peak of interest.

And (3) ultrafiltration concentration: the sample after anion exchange chromatography was concentrated 10-fold using a 300K membrane pack to obtain a concentrated solution.

Molecular sieve chromatography: the molecular sieve chromatography column uses ephacryl S-300HR filler, and after the column is equilibrated with 50mM PB (pH 6.8-7.2), the concentrate is added to the column to perform molecular sieve chromatography, and the first UV is collected _280nm Ultraviolet absorption peak.

Hydroxyapatite II chromatography: the column was equilibrated with 50mM PB, and the sample after molecular sieve chromatography was loaded and then eluted with a 500mM PB gradient to collect the desired peak of UV absorption at UV280 nm.

Ultrafiltration concentration liquid exchange: samples after hydroxyapatite II chromatography were solution-exchanged with PBS (ph 7.4) using a 300K membrane pack at a permeation flux of 20LMH and concentrated to the appropriate volume.

And (3) terminal sterilization: the VLP vaccine stock solution (containing recombinant virus-like particles of CA16 type) was obtained by terminal sterile filtration using a 0.22 μm sterile filter.

(3) The vaccine stock solution containing CA6VLPs and an aluminum hydroxide adjuvant normal saline solution are adsorbed and prepared according to the proportion of the CA6VLPs to aluminum with the mass of 1.

The prepared recombinant CA6VLP vaccine is then subjected to related detection.

Detection of vaccines

(1) Western Blot detection

The VLP vaccine of the recombinant CA6 prepared in the example 5 is subjected to HPLC and Western Blot detection, wherein the HPLC detection conditions are as follows: the assay was performed on a TSKgel G5000PW gel column using 10mM PB as mobile phase at a flow rate of 1mL/min. The results showed that the recombinant CA6VLPs produced in example 5 had a purity of 99% for the targeted peak at retention time 6.9 min.

The vaccine was subjected to Western Blot assay, with a loading of 25 μ L, and the primary antibody was diluted with sterile deionized water at a volume ratio of 1; the secondary antibody is a commercial goat anti-rabbit antibody, and is diluted by sterile deionized water according to the volume ratio of 1. As a result, clear bands appeared in each lane as shown in FIG. 4. The sample with the band is sent to Yangzhou university for electron microscope detection, and as a result, as shown in FIG. 5, virus-like particles of about 20-30nm can be seen, indicating that VLP particles of CA6 exist in the vaccine.

(2) Immunogenicity testing of recombinant CA6VLP vaccines

50 BALB/c mice (female, 6-8 weeks old, purchased from institute of laboratory animals, national academy of medical sciences) were immunized intraperitoneally with 10 animals per group at intervals of 14 days (0 and 14 days). All mice were immunized in 5 groups: negative control group (0.9 wt% sodium chloride group) and adjuvant control group and high, medium and low three vaccine dose groups. Wherein, the negative control group is inoculated with 0.5mL0.9wt% of sodium chloride per mouse, the adjuvant control group is inoculated with 0.5mL of aluminum hydroxide adjuvant (500 mu g/mouse) per mouse, the high, medium and low vaccine dose groups contain 500 mu g of aluminum hydroxide adjuvant, the CA6VLPs in VLP stock solution are respectively 0.01 mu g, 0.1 mu g and 0.5 mu g, and the inoculation volume is 0.5mL. Blood was collected at 28 days after the first immunization, and the serum was obtained by standing at 37 ℃ for 1h, at 4 ℃ for 0.5h, and centrifuging at 8000rpm for 10 min.

The serum positive conversion rate of the mice immunized with CA6VLPs was determined by ELISA and detected by a commercially available ELISA detection kit. The method comprises the following specific steps: the ELISA plate is pasted with a sealing plate membrane and is placed in a water-proof incubator at 37 ℃ for incubation for 85-90min. The purified CA6VLPs prepared in example 5 were diluted to 1. Mu.g/mL with coating solution, 100. Mu.L of each well of the microplate was added, and the mixture was coated overnight at 4 ℃. Removing the coating liquid and washing the plate. Add 300. Mu.L of blocking solution (5% nonfat dry milk + PBST) per well and incubate at 37 ℃ for 2h. Removing the coating solution, adding the diluted serum sample into an enzyme label plate, wherein each hole is 100 mu L and the two holes are parallel; adding the same volume of sample diluent into the blank control hole, preserving the temperature for 1h at 37 ℃, removing blood serum, and washing the plate. Adding 100 mu L of HRP-labeled goat anti-mouse IgG antibody which is diluted by 5000 times by using an enzyme-labeled diluent into each hole, preserving the heat at 37 ℃ for 0.5 hour, removing the enzyme-labeled solution, and washing the plate; then 100 mul of color development liquid is added into each hole, the incubation is carried out for 10min in the dark at room temperature, 50 mul of stop solution is added into each hole to stop the reaction, and the absorbance value is measured at 450nm of enzyme label.

A Cutoff value of

Negative control group OD value; and (3) judging that the OD value of the serum sample of the vaccine group is greater than the Cutoff, namely the antibody of the vaccine group is positively converted, and judging that the serum sample with the OD value less than the Cutoff is negative. The antibody positive conversion rate was shown in table 1 based on the detection results. The results of the positive conversion rates of the three detection groups are shown in table 1, and the positive conversion rates of the CA6VLPs are 100% when the dosages of the CA6VLPs are 0.01 mu g, 0.1 mu g and 0.5 mu g, which shows that the VLP vaccine of the recombinant CA6 prepared by the application has excellent immunogenicity.

TABLE 1 calculation of the antibody turnover

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

Sequence listing

<110> Jiangsu Rike Biotechnology Ltd

ABZYMO BIOSCIENCES Co.,Ltd.

Expression system of <120> coxsackie virus 6 type recombinant virus-like particle, virus-like particle prepared by expression system and hand-foot-and-mouth disease vaccine

<150> CN202210057187.3

<151> 2022-01-19

<150> CN202110707967.3

<151> 2021-06-24

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 870

<212> PRT

<213> Coxsackie virus group A (Coxsackie virus A)

<400> 1

Met Gly Ala Gln Val Ser Thr Glu Lys Ser Gly Ser His Glu Thr Lys

1 5 10 15

Asn Val Ala Thr Glu Gly Ser Thr Ile Asn Phe Thr Asn Ile Asn Tyr

20 25 30

Tyr Lys Asp Ser Tyr Ala Ala Ser Ala Ser Arg Gln Asp Phe Ala Gln

35 40 45

Asp Pro Ala Lys Phe Thr Arg Pro Val Leu Asp Thr Ile Arg Glu Val

50 55 60

Ala Ala Pro Leu Gln Ser Pro Ser Val Glu Ala Cys Gly Tyr Ser Asp

65 70 75 80

Arg Val Ala Gln Leu Thr Val Gly Asn Ser Thr Ile Thr Thr Gln Glu

85 90 95

Ala Ala Asn Ile Val Leu Ser Tyr Gly Glu Trp Pro Glu Tyr Cys Pro

100 105 110

Ser Thr Asp Ala Thr Ala Val Asp Lys Pro Thr Arg Pro Asp Val Ser

115 120 125

Val Asn Arg Phe Tyr Thr Leu Ser Thr Lys Ser Trp Lys Thr Glu Ser

130 135 140

Thr Gly Trp Tyr Trp Lys Phe Pro Asp Val Leu Asn Asp Thr Gly Val

145 150 155 160

Phe Gly Gln Asn Ala Gln Phe His Tyr Leu Tyr Arg Ser Gly Phe Cys

165 170 175

Met His Val Gln Cys Asn Ala Ser Lys Phe His Gln Gly Ala Leu Leu

180 185 190

Val Ala Ala Ile Pro Glu Phe Val Val Ala Ala Ser Ser Pro Ala Thr

195 200 205

Lys Pro Asn Gly Gln Gly Leu Tyr Pro Asp Phe Ala His Thr Asn Pro

210 215 220

Gly Lys Asn Gly Gln Glu Phe Arg Asp Pro Tyr Val Leu Asp Ala Gly

225 230 235 240

Val Pro Leu Ser Gln Ala Leu Val Tyr Pro His Gln Trp Ile Asn Leu

245 250 255

Arg Thr Asn Asn Cys Ala Thr Ile Ile Met Pro Tyr Val Asn Ala Leu

260 265 270

Pro Phe Asp Ser Ala Leu Asn His Ser Asn Phe Gly Leu Val Val Ile

275 280 285

Pro Ile Ser Pro Leu Lys Tyr Cys Asn Gly Ala Thr Thr Glu Val Pro

290 295 300

Ile Thr Leu Thr Ile Ala Pro Leu Asn Ser Glu Phe Ser Gly Leu Arg

305 310 315 320

Gln Ala Ile Lys Gln Gly Phe Pro Thr Glu Leu Lys Pro Gly Thr Asn

325 330 335

Gln Phe Leu Thr Thr Asp Asp Gly Thr Ser Pro Pro Ile Leu Pro Gly

340 345 350

Phe Glu Pro Thr Pro Leu Ile His Ile Pro Gly Glu Phe Thr Ser Leu

355 360 365

Leu Asp Leu Cys Gln Ile Glu Thr Ile Leu Glu Val Asn Asn Thr Thr

370 375 380

Gly Thr Thr Gly Val Ser Arg Leu Leu Ile Pro Val Arg Ala Gln Asn

385 390 395 400

Asn Val Asp Gln Leu Cys Ala Ser Phe Gln Val Asp Pro Gly Arg Asn

405 410 415

Gly Pro Trp Gln Ser Thr Met Val Gly Gln Ile Cys Arg Tyr Tyr Thr

420 425 430

Gln Trp Ser Gly Ser Leu Lys Val Thr Phe Met Phe Thr Gly Ser Phe

435 440 445

Met Ala Thr Gly Lys Met Leu Ile Ala Tyr Thr Pro Pro Gly Ser Ala

450 455 460

Gln Pro Ala Thr Arg Glu Ala Ala Met Leu Gly Thr His Ile Val Trp

465 470 475 480

Asp Phe Gly Leu Gln Ser Ser Val Thr Leu Val Ile Pro Trp Ile Ser

485 490 495

Asn Thr His Phe Arg Ala Val Lys Thr Gly Gly Val Tyr Asp Tyr Tyr

500 505 510

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

515 520 525

Pro Asp Thr Pro Thr Glu Ala Asn Ile Ile Ala Leu Gly Ala Ala Gln

530 535 540

Lys Asn Phe Thr Leu Lys Leu Cys Lys Asp Thr Asp Glu Ile Gln Gln

545 550 555 560

Thr Ala Glu Tyr Gln Asn Asp Pro Ile Thr Asn Ala Val Glu Ser Ala

565 570 575

Val Ser Ala Leu Ala Asp Thr Thr Ile Ser Arg Val Thr Ala Ala Asn

580 585 590

Thr Ala Ala Ser Thr His Ser Leu Gly Thr Gly Arg Val Pro Ala Leu

595 600 605

Gln Ala Ala Glu Thr Gly Ala Ser Ser Asn Ala Ser Asp Glu Asn Leu

610 615 620

Ile Glu Thr Arg Cys Val Met Asn Arg Asn Gly Val Asn Glu Ala Ser

625 630 635 640

Val Glu His Phe Tyr Ser Arg Ala Gly Leu Val Gly Val Val Glu Val

645 650 655

Lys Asp Ser Gly Thr Ser Leu Asp Gly Tyr Thr Val Trp Pro Ile Asp

660 665 670

Val Met Gly Phe Val Gln Gln Arg Arg Lys Leu Glu Leu Ser Thr Tyr

675 680 685

Met Arg Phe Asp Ala Glu Phe Thr Phe Val Ser Asn Leu Asn Asp Ser

690 695 700

Thr Thr Pro Gly Met Leu Leu Gln Tyr Met Tyr Val Pro Pro Gly Ala

705 710 715 720

Pro Lys Pro Asp Ser Arg Lys Ser Tyr Gln Trp Gln Thr Ala Thr Asn

725 730 735

Pro Ser Val Phe Ala Lys Leu Ser Asp Pro Pro Pro Gln Val Ser Val

740 745 750

Pro Phe Met Ser Pro Ala Thr Ala Tyr Gln Trp Phe Tyr Asp Gly Tyr

755 760 765

Pro Thr Phe Gly Glu His Lys Gln Ala Thr Asn Leu Gln Tyr Gly Gln

770 775 780

Cys Pro Asn Asn Met Met Gly His Phe Ala Ile Arg Thr Val Ser Glu

785 790 795 800

Ser Thr Thr Gly Lys Asn Val His Val Arg Val Tyr Met Arg Ile Lys

805 810 815

His Val Arg Ala Trp Val Pro Arg Pro Leu Arg Ser Gln Ala Tyr Met

820 825 830

Val Lys Asn Tyr Pro Thr Tyr Ser Gln Thr Ile Thr Asn Thr Ala Thr

835 840 845

Asp Arg Ala Ser Ile Thr Thr Thr Asp Tyr Glu Gly Gly Val Pro Ala

850 855 860

Asn Pro Gln Arg Thr Ser

865 870

<210> 2

<211> 645

<212> PRT

<213> Coxsackie virus group A (Coxsackie virus A)

<400> 2

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

1 5 10 15

Gln Val Gln Thr Asp Gln Gly His Phe Thr Met Leu Gly Val Arg Asp

20 25 30

Arg Leu Ala Val Leu Pro Arg His Ser Gln Pro Gly Lys Thr Ile Trp

35 40 45

Val Glu His Lys Leu Val Asn Ile Leu Asp Ala Val Glu Leu Val Asp

50 55 60

Glu Gln Gly Val Asn Leu Glu Leu Thr Leu Ile Thr Leu Asp Thr Asn

65 70 75 80

Glu Lys Phe Arg Asp Ile Thr Lys Phe Ile Pro Glu Asn Ile Ser Ala

85 90 95

Ala Ser Asp Ala Thr Leu Val Ile Asn Thr Glu His Met Pro Ser Met

100 105 110

Phe Val Pro Val Gly Asp Val Val Gln Tyr Gly Phe Leu Asn Leu Ser

115 120 125

Gly Lys Pro Thr His Arg Thr Met Met Tyr Asn Phe Pro Thr Lys Ala

130 135 140

Gly Gln Cys Gly Gly Val Val Thr Ser Val Gly Lys Val Ile Gly Ile

145 150 155 160

His Ile Gly Gly Asn Gly Arg Gln Gly Phe Cys Ala Gly Leu Lys Arg

165 170 175

Ser Tyr Phe Ala Ser Glu Gln Gly Glu Ile Gln Trp Val Lys Pro Asn

180 185 190

Lys Glu Thr Gly Arg Leu Asn Ile Asn Gly Pro Thr Arg Thr Lys Leu

195 200 205

Glu Pro Ser Val Phe His Asp Ile Phe Glu Gly Asn Lys Glu Pro Ala

210 215 220

Val Leu His Ser Lys Asp Pro Arg Leu Glu Val Asp Phe Glu Gln Ala

225 230 235 240

Leu Phe Ser Lys Tyr Val Gly Asn Thr Ile His Glu Pro Asp Glu Tyr

245 250 255

Ile Lys Glu Ala Ala Leu His Tyr Ala Asn Gln Leu Lys Gln Leu Asn

260 265 270

Ile Asp Thr Ser Gln Met Ser Met Glu Glu Ala Cys Tyr Gly Thr Asp

275 280 285

Asn Leu Glu Ala Ile Asp Leu His Thr Ser Ala Gly Tyr Pro Tyr Ser

290 295 300

Ala Leu Gly Ile Lys Lys Arg Asp Ile Leu Asp Pro Thr Thr Arg Asp

305 310 315 320

Val Ser Lys Met Lys Phe Tyr Met Asp Lys Tyr Gly Leu Asp Leu Pro

325 330 335

Tyr Ser Thr Tyr Val Lys Asp Glu Leu Arg Ser Ile Asp Lys Ile Lys

340 345 350

Lys Gly Lys Ser Arg Leu Ile Glu Ala Ser Ser Leu Asn Asp Ser Val

355 360 365

Tyr Leu Arg Met Ala Phe Gly His Leu Tyr Glu Thr Phe His Ala Asn

370 375 380

Pro Gly Thr Val Thr Gly Ser Ala Val Gly Cys Asn Pro Asp Val Phe

385 390 395 400

Trp Ser Lys Leu Pro Ile Leu Leu Pro Gly Ser Leu Phe Ala Phe Asp

405 410 415

Tyr Ser Gly Tyr Asp Ala Ser Leu Ser Pro Val Trp Phe Arg Ala Leu

420 425 430

Glu Leu Val Leu Arg Glu Ile Gly Tyr Gly Asn Glu Ala Val Ser Leu

435 440 445

Ile Glu Gly Ile Asn His Thr His His Val Tyr Arg Asn Lys Thr Tyr

450 455 460

Cys Val Leu Gly Gly Met Pro Ser Gly Cys Ser Gly Thr Ser Ile Phe

465 470 475 480

Asn Ser Met Ile Asn Asn Ile Ile Ile Arg Ser Leu Leu Ile Lys Thr

485 490 495

Phe Lys Gly Ile Asp Leu Asp Glu Leu Asn Met Val Ala Tyr Gly Asp

500 505 510

Asp Val Leu Ala Ser Tyr Pro Phe Pro Ile Asp Cys Ser Glu Leu Ala

515 520 525

Arg Thr Gly Lys Glu Tyr Gly Leu Thr Met Thr Pro Ala Asp Lys Ser

530 535 540

Pro Cys Phe Asn Glu Val Asn Trp Glu Asn Ala Thr Phe Leu Lys Arg

545 550 555 560

Gly Phe Leu Pro Asp Glu Gln Phe Pro Phe Leu Ile His Pro Thr Met

565 570 575

Pro Met Lys Glu Ile His Glu Ser Ile Arg Trp Thr Lys Asp Ala Arg

580 585 590

Asn Thr Gln Asp His Val Arg Ser Leu Cys Leu Leu Ala Trp His Asn

595 600 605

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

610 615 620

Ile Gly Lys Ala Leu Ala Ile Pro Asn Tyr Glu Asn Leu Arg Arg Asn

625 630 635 640

Trp Leu Glu Leu Phe

645

<210> 3

<211> 6463

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgaacagaag gaagaacgaa ggaaggagca cagacttaga ttggtatata tacggatatg 60

tagtgttgaa gaaacatgaa attgcccagt attcttaacc caactgcaca gaacaaaaac 120

ctggaggaaa cgaagataaa tcatgtcgaa agctacatat aaggaacgtg ctgctactca 180

tcctagtcct gttgctgcca agctatttaa tatcatgcac gaaaagcaaa caaacttgtg 240

tgcttcattg gatgttcgta ccaccaagga attactggag ttagttgaag cattaggtcc 300

caaaatttgt ttactaaaaa cacatgtgga catcttgact gatttttcca tggagggcac 360

agttaagccg ctaaaggcat tatccgccaa gtacaatttt ttactcttcg aagacagaaa 420

atttgctgac attggtaata cagtcaaatt gcagtactct gcgggtgtat acagaatagc 480

agaatgggca gacattacga atgcacacgg tgtggtgggc ccaggtattg ttagcggttt 540

gaagcaggcg gcagaagaag taacaaagga acctagaggc cttttgatgt tagcagaatt 600

gtcatgcaag ggctccctat ctactggaga atatactaag ggtactgttg acattgcgaa 660

gagcgacaaa gattttgtta tcggctttat tgctcaaaga gacatgggtg gaagagatga 720

aggttacgat tggttgatta tgacacccgg tgtgggttta gatgacaagg gagacgcatt 780

gggtcaacag tatagaaccg tggatgatgt ggtctctaca ggatctgaca ttattattgt 840

tggaagagga ctatttgcaa agggaaggga tgctaaggta gagggtgaac gttacagaaa 900

agcaggctgg gaagcatatt tgagaagatg cggccagcaa aactaaaaaa ctgtattata 960

agtaaatgca tgtatactaa actcacaaat tagagcttca atttaattat atcagttatt 1020

acccgggaat ctcggtcgta atgattttta taatgacaag cttggaccag tttttctcgc 1080

acattatcaa ttgctcttta gtacaaagat aatatagaaa caatcatatg ctgactgttt 1140

gacaattaat catcggcata gtatatcggc atagtataat acgactcact ataggagggc 1200

caccatgagc catattcaac gggaaacgtc ttgctcgagg ccgcgattaa attccaacat 1260

ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac 1320

aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg 1380

tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat 1440

gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac 1500

tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa 1560

tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg 1620

tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg 1680

tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg 1740

gaaagaaatg cataagcttt tgccattctc accggattca gtcgtcactc atggtgattt 1800

ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg 1860

agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt 1920

ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa 1980

taaattgcag tttcatttga tgctcgatga gtttttctaa gcacggcctc atctacatat 2040

ttacggctta actgattttt atagttaagg agaaaaaaag ttcaacatac gtcattatta 2100

ttgtacgcgc tttcgtgttt caaacttggc tgccatgata aataaatcta ttgttgcttg 2160

ctatgtaaaa attatttgat tacttcttcc atgcactttc tttatttgga ttgtggtccg 2220

agatctcgcg gagaacgatc tcctcgacgc ggagaacgat ctcctcgagc tgctcgcgga 2280

tcagcttgtg gcccggtaat ggaaccaggc cgacggcacg ctccttgcgg accacggtgg 2340

ctggcgagcc cagtttgtga acgaggtcgt ttagaacgtc ctgcgcaaag tccagtgtca 2400

gatgaatgtc ctcctcggac caattcagca tgttctcgag cagccatctg tctttggagt 2460

agaagcgtaa tctctgctcc tcgttactgt accggaagag gtagtttgcc tcgccgccca 2520

taatgaacag gttctctttc tggtggcctg tgagcagcgg ggacgtctgg acggcgtcga 2580

tgaggccctt gaggcgctcg tagtacttgt tcgcgtcgct gtagccggcc gcggtgacga 2640

tacccacata gaggtccttg gccattagtt tgatgaggtg gggcaggatg ggcgactcgg 2700

catcgaaatt tttgccgtcg tcgtacagtg tgatgtcacc atcgaatgta atgagctgca 2760

gcttgcgatc tcggatggtt ttggaatgga agaaccgcga catctccaac agctgggccg 2820

tgttgagaat gagccggacg tcgttgaacg agggggccac aagccggcgt ttgctgatgg 2880

cgcggcgctc gtcctcgatg tagaaggcct tttccagagg cagtctcgtg aagaagctgc 2940

caacgctcgg aaccagctgc acgagccgag acaattcggg ggtgccggct ttggtcattt 3000

caatgttgtc gtcgatgagg agttcgaggt cgtggaagat ttccgcgtag cggcgttttg 3060

cctcagagtt taccatgagg tcgtccactg cagagatgcc gttgctcttc accgcgtaca 3120

ggacgaacgg cgtggccagc aggcccttga tccattctat gaggccatct cgacggtgtt 3180

ccttgagtgc gtactccact ctgtagcgac tggacatctc gagactgggc ttgctgtgct 3240

ggatgcacca attaattgtt gccgcatgca tccttgcacc gcaagttttt aaaacccact 3300

cgctttagcc gtcgcgtaaa acttgtgaat ctggcaactg agggggttct gcagccgcaa 3360

ccgaactttt cgcttcgagg acgcagctgg atggtgtcat gtgaggctct gtttgctggc 3420

gtagcctaca acgtgacctt gcctaaccgg acggcgctac ccactgctgt ctgtgcctgc 3480

taccagaaaa tcaccagagc agcagagggc cgatgtggca actggtgggg tgtcggacag 3540

gctgtttctc cacagtgcaa atgcgggtga accggccaga aagtaaattc ttatgctacc 3600

gtgcagtgac tccgacatcc ccagtttttg ccctacttga tcacagatgg ggtcagcgct 3660

gccgctaagt gtacccaacc gtccccacac ggtccatcta taaatactgc tgccagtgca 3720

cggtggtgac atcaatctaa aggaattcga tatcaaggag acgtggaagg acataccgct 3780

tttgagaagc gtgtttgaaa atagttcttt ttctggttta tatcgtttat gaagtgatga 3840

gatgaaaagc tgaaatagcg agtataggaa aatttaatga aaattaaatt aaatattttc 3900

ttaggctatt agtcaccttc aaaatgccgg ccgcttctaa gaacgttgtc atgatcgaca 3960

actacgactc gtttacctgg aacctgtacg agtacctgtg tcaggaggga gccaatgtcg 4020

aggttttcag gaacgatcag atcaccattc cggagattgg gatcccagag ctctggcggc 4080

cgcggtaccc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg 4140

gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 4200

cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 4260

gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 4320

cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 4380

tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 4440

aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 4500

gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 4560

ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 4620

ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 4680

gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 4740

ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 4800

cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 4860

cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 4920

gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 4980

aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 5040

tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 5100

gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 5160

tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 5220

gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 5280

tgagtaaact tggtctgagg accacaatcc aaataaagaa agtgcatgga agaagtaatc 5340

aaataatttt tacatagcaa gcaacaatag atttatttat catggcagcc aagtttgaaa 5400

cacgaaagcg cgtacaataa taatgacgta tgttgaactt tttttctcct taactataaa 5460

aatcagttaa gccgtaaata tgtagatgag gccgtgctca gtcctgctcc tcggccacga 5520

agtgcacgca gttgccggcc gggtcgcgca gggcgaactc ccgcccccac ggctgctcgc 5580

cgatctcggt catggccggc ccggaggcgt cccggaagtt cgtggacacg acctccgacc 5640

actcggcgta cagctcgtcc aggccgcgca cccacaccca ggccagggtg ttgtccggca 5700

ccacctggtc ctggaccgcg ctgatgaaca gggtcacgtc gtcccggacc acaccggcga 5760

agtcgtcctc cacgaagtcc cgggagaacc cgagccggtc ggtccagaac tcgaccgctc 5820

cggcgacgtc gcgcgcggtg agcaccggaa cggcactggt caacttggcc atattgtttc 5880

tatattatct ttgtactaaa gagcaattga taatgtgcga gaaaaactgg tccttatata 5940

ccgtttgcag cactccctcc cgaactttac gaaaagtcgt gcgccacctg attttcatca 6000

cgccaaaaac ctacacgtat gactactccg ggccagtgtt caccacgagc tatatagtgt 6060

taattaatta ccttattggt tagctctgca tgtaagggtg gtgtgagccg ggaattgggt 6120

ctactctagc gttcagtaag gtgatataaa gctctgtata gccagaagtg gacatcaccc 6180

aacaaggcgt ctccgggact tgcctgtccg tgcaaggttg ttccatggaa gctctaccgc 6240

cggagcggcc caaaggacaa taagaagtgc tacaccacct ccgcagagga cacaggctta 6300

aaaccctctt tctcggtttc gggaccggtt cccggagatt gtctttaccc cacgcaccgt 6360

gctggagcca tagcagttgt tgcaactttg cgagttgtca ccttttcctc cgtggcccgc 6420

ctcttttctg gtgcacggat gtagtctaga ccaacggtaa tct 6463

<210> 4

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aaggaaaaaa ggcggccgcc ccgggctgca gatcga 36

<210> 5

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aaaggtcgac actagtaagg agacgtggaa ggacata 37

<210> 6

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ccttactagt gtcgaccttt agattgatgt caccaccgtg cac 43

<210> 7

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cggggtacca acgatctcct cgagctgct 29

<210> 8

<211> 2611

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gaattcaaaa acaaaatggg ctcgcaggtg tctacccaga gatcgggttc tcacgagaac 60

tccaactcgg cctctgaggg atctaccatc aactacacca ccattaacta ctacaaggac 120

gcttacgccg cttctgccgg tagacaggac atgtcccagg acccaaagaa gttcaccgac 180

cctgtgatgg acgttatcca cgagatggcc ccacctctga agtctccatc tgctgaggcc 240

tgcggatact ctgacagagt ggctcagctg accatcggaa actctaccat taccacccag 300

gaggccgcta acatcgtgat tgcctacggt gaatggccag agtactgtcc tgacaccgac 360

gctaccgccg ttgacaagcc aaccagacct gacgtgtccg ttaacagatt ctttaccctg 420

gacaccaagt cgtgggctaa ggactctaag ggctggtact ggaagtttcc agacgtgctg 480

accgaagtgg gcgttttcgg acagaacgcc cagtttcact acctgtacag atccggcttc 540

tgcgtgcacg ttcagtgtaa cgcttcgaag tttcaccagg gagccctgct ggtggctgtt 600

ctgcctgagt acgtgctggg caccattgcc ggcggaaccg gaaacgagaa ctcgcaccca 660

ccttacgcta ccacccagcc tggtcaagtg ggagctgttc tgacccaccc atacgtgctg 720

gacgctggca tccctctgtc tcagctgacc gtttgcccac accagtggat taacctgaga 780

accaacaact gtgccaccat cattgtgcca tacatgaaca ccgttccttt cgactccgct 840

ctgaaccact gcaactttgg cctgctggtt atcccagtgg ttcctctgga cttcaacacc 900

ggtgctacct cggagatccc aattaccgtg accatcgccc ctatgtgtgc cgagtttgct 960

ggactgagac aggccgttaa gcagggtatt ccaaccgagc tgaagcctgg caccaaccag 1020

ttcctgacca ccgacgacgg agtgtctgct ccaattctgc ctggttttca cccaacccca 1080

cctatccaca ttcctggaga ggttcacaac ctgctggaga tttgcagagt ggagactatt 1140

ctggaggtta acaacctgaa gaccaacgag actaccccaa tgcagagact gtgcttccct 1200

gtgtctgttc agtccaagac cggagagctg tgtgccgctt ttagagccga cccaggaaga 1260

gacggtcctt ggcagtctac catcctgggt cagctgtgta gatactacac ccagtggtct 1320

ggctccctgg aggtgacctt catgtttgct ggttccttca tggccaccgg caagatgctg 1380

attgcctaca ccccacctgg tggcaacgtt cctgctgaca gaatcaccgc catgctgggc 1440

acccacgtga tttgggactt cggactgcag tcgtctgtta ccctggtggt tccatggatc 1500

tccaacaccc actacagagc ccacgccaga gctggttact ttgactacta caccaccggc 1560

atcattacca tctggtatca gaccaactac gtggttccaa tcggcgcccc taccaccgct 1620

tacattgtgg ccctggccgc tgcccaggac aacttcacca tgaagctgtg caaggacacc 1680

gaggacatcg agcagaccgc taacattcag ggcgacccaa tcgccgacat gattgaccag 1740

accgtgaaca accaggttaa cagatcgctg accgctctgc aggtgctgcc taccgctgcc 1800

aacaccgagg cctcctcgca cagactggga accggtgtgg ttccagctct gcaggctgcc 1860

gagactggcg cttcttccaa cgcctcggac aagaacctga tcgagactag atgcgttctg 1920

aaccaccact ctacccagga gactgctatt ggtaacttct tttccagagc cggcctggtg 1980

tcgatcatta ccatgccaac caccggaacc cagaacaccg acggttacgt taactgggac 2040

atcgacctga tgggatacgc ccagctgaga agaaagtgtg agctgttcac ctacatgaga 2100

tttgacgctg agttcacctt tgtggttgcc aagccaaacg gagagctggt gcctcagctg 2160

ctgcagtaca tgtacgttcc acctggcgcc ccaaagccta cctcgagaga ctctttcgct 2220

tggcagaccg ccaccaaccc ttctgtgttt gttaagatga ccgacccacc tgctcaggtg 2280

tccgttccat tcatgtcccc tgcctcggct taccagtggt tctacgacgg ctaccctacc 2340

tttggagagc acctgcaggc caacgacctg gactacggac agtgcccaaa caacatgatg 2400

ggcaccttct cgatcagaac cgtgggcacc gagaagtctc cacactccat taccctgaga 2460

gtgtacatga gaatcaagca cgttagagct tggattccaa gacctctgag aaaccagcca 2520

tacctgttca agaccaaccc taactacaag ggcaacgaca tcaagtgtac ctcgacctct 2580

agagacaaga ttaccaccct gtaatgatat c 2611

<210> 9

<211> 1963

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gaattcaaaa acaaaatggg accatccctg gacttcgccc tgtcgctgct gagaagaaac 60

attagacagg ttcagaccga ccagggacac tttaccatgc tgggtgtgag agacagactg 120

gccatcctgc caagacactc tcagcctggc aagaccattt gggtggagca caagctgatc 180

accgttctgg acgctgtgga gctggttgac gagcagggcg tgaacctgga gctgaccctg 240

gttaccctgg acaccaacga gaagttcaga gacgttacca agtttattcc tgagactatc 300

accggcgcct ctgacgctac cctggttatc aacaccgagc acatgccatc catgttcgtg 360

cctgttggcg acgtggttca gtacggattt ctgaacctgt ccggcaagcc aacccacaga 420

accatgatgt acaacttccc taccaaggct ggccagtgcg gcggagtggt tacctcggtg 480

ggaaagatca ttggtattca catcggtggc aacggcagac agggattctg tgccggcctg 540

aagagaggat actttgcttc ggagcaggga gagattcagt ggatgaagtc taacaaggag 600

actggcagac tgaacatcaa cggaccaacc agaaccaagc tggagccttc tgccttctac 660

gacgtgtttg agggctcgaa ggagccagct gttctgacct cgaaggaccc tagactggag 720

gtggacttcg agcaggccct gttttccaag tacgttggta acaccctgca cgagccagac 780

gagtacgtga cccaggccgc tctgcactac gctaaccagc tgaagcagct ggacattaac 840

accaacaaga tgtccatgga ggaggcctgc tacggcaccg agtacctgga ggctatcgac 900

ctgcacacct ctgccggtta cccatactcc gctctgggcg tgaagaagag agacattctg 960

gaccctatca ccagagacac caccaagatg aagttctaca tggacaagta cggcctggac 1020

ctgccttact cgacctacgt taaggacgag ctgagatcgc tggacaagat taagaagggc 1080

agatccagac tgatcgaggc ctcgtctctg aacgactcgg tgtacctgag aatgaccttc 1140

ggacacctgt acgagacttt tcacgctaac cctggcaccg tgaccggctc cgctgttgga 1200

tgtaaccctg acgtgttctg gtcgaagctg ccaattctgc tgcctggatc gctgttcgcc 1260

tttgactact ctggttacga cgcttccctg tcgccagttt ggtttagagc cctggaggtg 1320

gttctgagag agatcggcta ctcggaggag gctgtgtctc tgattgaggg aatcaaccac 1380

acccaccacg tgtacagaaa cagaacctac tgcgttctgg gaggtatgcc ttctggttgt 1440

tccggcacct cgattttcaa ctcgatgatc aacaacatca ttatcagaac cctgctgatt 1500

aagaccttta agggtatcga cctggacgag ctgaacatgg tggcctacgg cgacgacgtt 1560

ctggcttctt acccattccc tattgactgc tccgagctgg ccagaaccgg caaggagtac 1620

ggcctgacca tgaccccagc tgacaagtct ccttgtttta acgaggttac ctgggagaac 1680

gccaccttcc tgaagagagg ctttctgcca gaccaccagt tcccttttct gatccaccca 1740

accatgccta tgagagagat tcacgagtcg atcagatgga ccaaggacgc cagaaacacc 1800

caggaccacg tgagatcgct gtgcctgctg gcttggcaca acggaaagga ggagtacgag 1860

aagttcgtgt ctaccattag atccgttcca atcggtagag ccctggctat ccctaacttc 1920

gagaacctga gaagaaactg gctggagctg ttttaatgat atc 1963

<210> 10

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

acgcgtcgac tattaattta gtgtgtgtat ttg 33

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cggggtacca gcgttggtgg atca 24

<210> 12

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gagctcagct tctggtaaac gttgtagta 29

<210> 13

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

aaggaaaaaa gcggccgcca tcgactcccg cgactc 36

Claims (19)

1. An expression system for expressing recombinant virus-like particles of coxsackie virus 6, which comprises a host cell and a recombinant plasmid vector introduced into the host cell, wherein the recombinant plasmid vector can simultaneously express P1 precursor protein and 3CD protease of the coxsackie virus 6, and the expression levels of the P1 precursor protein and the 3CD protease of the recombinant plasmid vector are different.

2. The expression system of claim 1, wherein said recombinant plasmid vector has both an initial MOX expression cassette for expression of said P1 precursor protein and an exogenous expression cassette for expression of said 3CD protease, and wherein the promoter of said exogenous expression cassette is selected from any promoter other than the MOX promoter.

3. The expression system of claim 2, wherein the promoter of the exogenous expression cassette is a CYC1 promoter, a GAPDH promoter, a TEF1 promoter, or a DAS promoter.

4. The expression system of claim 3, wherein the promoter of the exogenous expression cassette is CYC1.

5. The expression system of claim 2, wherein the host cell is auxotrophic for at least one substance and the recombinant plasmid vector has at least one auxotrophic marker corresponding to the substance.

6. The expression system of claim 5, wherein the substance is uracil, leucine, methanol, pep protease, prc protease, or kex protease.

7. The expression system of claim 6, wherein the host cell is leucine auxotrophic and the recombinant plasmid vector has the auxotrophic marker Leu2.

8. The expression system of claim 2, further comprising at least one reporter gene expression cassette on the recombinant plasmid vector.

9. The expression system of claim 7, wherein the recombinant plasmid vector further comprises an autonomously replicating sequence.

10. A recombinant virus-like particle of coxsackievirus type 6 expressed by the expression system according to any one of claims 1 to 9.

11. An immunological composition for preventing or treating hand-foot-and-mouth disease, comprising the coxsackievirus type 6 recombinant virus-like particle of claim 10.

12. The immunogenic composition of claim 11, wherein the coxsackievirus type 6 recombinant virus-like particles are present in an amount of 5 to 60 μ g in a single dose of the immunogenic composition.

13. A hand-foot-and-mouth disease vaccine comprising the immunogenic composition of claim 11 or 12.

14. The hand-foot-and-mouth disease vaccine of claim 13, further comprising an adjuvant comprising at least one of aluminum hydroxide, aluminum phosphate, aluminum sulfate, ammonium alum, potassium alum, 3D-MPL, squalene, tween, tocopherol, cpG, poly (I: C), and QS 21.

15. The hand-foot-and-mouth disease vaccine of claim 14, wherein the adjuvant comprises aluminum hydroxide, and the amount of aluminum hydroxide in the hand-foot-and-mouth disease vaccine is 1-2 mg/ml.

16. The hand-foot-and-mouth disease vaccine of claim 14, characterized in that the adjuvant comprises the following components: 40-50 mg/ml of squalene, 40-50 mg/ml of alpha-tocopherol and 15-25 mg/ml of tween.

17. The hand-foot-and-mouth disease vaccine of claim 14, characterized in that the adjuvant comprises the following components: 35 to 45mg/ml of squalene, 85.5 to 7.5mg/ml of span and 2.5 to 7.5mg/ml of tween 80.

18. Use of the immunogenic composition according to claim 11 or 12 for the preparation of a multivalent hand-foot-and-mouth disease vaccine, characterized in that said immunogenic composition is used in combination with one or more further immunogenic components of enteroviruses.

19. The use of claim 18, wherein the enterovirus comprises at least one of coxsackievirus type 10, coxsackievirus type 16 or enterovirus type 71 and the immunogenic component is a virus-like particle or an inactivated virus.

Images