CN102559615B - EV71 vaccine preparation method and the vaccine prepared by the method - Google Patents

Abstract

EV71 vaccine preparation method and vaccine prepared by the method. The invention relates to a method for preparing a virus-like particle vaccine with immunogenicity by using Pichia pastoris as an expression system, co-expressing codon-optimized P1 and 3CD proteins of EV71 using different promoters. It also relates to vaccines prepared by this method.

Description

Preparation method of EV71 vaccine and vaccine prepared by the method

technical field

The invention relates to a method for preparing a virus-like particle vaccine with immunogenicity by using Pichia pastoris as an expression system, co-expressing codon-optimized P1 and 3CD proteins of EV71 using different promoters. It also relates to vaccines prepared by this method.

Background technique

Enterovirus 71 (EV71) belongs to the genus Enterovirus in the Picornaviridae family, and mainly causes central nervous system diseases such as hand, foot and mouth disease, aseptic meningitis, brainstem encephalitis, and polio-like paralysis, causing disability and death It is a neurotropic enterovirus second only to poliovirus in terms of damage. EV71 takes humans as the only host, and the main route of transmission is fecal-oral transmission. The population is generally susceptible to EV71, but its main targets are infants and young children under 5 years old. Since it was first reported in 1974, EV71 has caused more than a dozen large-scale outbreaks around the world. In China alone, it once caused tens of thousands of infections in Taiwan and Shandong in 1998 and 2007-2008. According to the research report, the incidence rate of EV71 in some areas of China is as high as 10%.

EV71 virus particles are non-enveloped icosahedral symmetrical spheres, composed of 60 identical subunits. Its viral genome is a single-stranded positive-strand RNA, about 7400bp long, with only one reading frame and non-coding regions located at 5' and 3', encoding a polyprotein, including three precursor proteins P1-P3, and P1 encodes VP1- VP4 has four capsid proteins, and P2 and P3 encode seven nonstructural proteins, including 2A, 2B, 2C, 3A, 3B, 3C, and 3D. Among them, 3CD is a specific proteolytic enzyme, which recognizes the Gln-Gly site, decomposes the P1 precursor protein, and makes it release the VP1-VP4 that constitutes the viral capsid subunit, and finally completes the assembly of the virus particle.

According to the difference in gene sequence homology, EV71 can be divided into three genotypes, A, B, and C, and types B and C can be further subdivided into subtypes B1-B5 and C1-C4. The prevalence trends of different genotypes showed certain regional differences, and the EV71 genotypes that were prevalent in the same area at different times were different. However, since 1997, the popularity of EV71 in China has been relatively stable, mainly C4 type.

Since the pathogenic mechanism and immune mechanism of EV71 need to be further revealed, so far, there are still no effective antiviral drugs and vaccines to prevent viral infection. In recent years, many studies have shown that some progress has been made in EV71 vaccine research, including inactivated vaccines, subunit vaccines, DNA vaccines, peptide vaccines and live attenuated vaccines. Among them, the most efficient and safe vaccine research direction is recombinant virus-like particle (VLPs) vaccine.

Virus-like particles are particle structures composed of capsid proteins with a complete three-dimensional structure without a viral genome. Inactivated vaccines are more reliable and safe, and compared with other recombinant vaccines, their immunogenicity is also very obvious. Since the hepatitis B virus (HBV) surface antigen (Surface Antigen, HBsAg) particle was found to be used as a hepatitis B vaccine in 1985, VLPs technology has developed rapidly in the research of new virus vaccines. A typical example of successfully using virus-like particles as a vaccine is the human papillomavirus (HPV) virus-like particle. Many research results have shown that many expression systems including Escherichia coli, yeast and baculovirus can express recombinant HPV Late protein L1 and L2, L1/L2-VLPs or L1-VLPs with virus particle structure can be obtained. After these virus-like particles are immunized with the host together with aluminum adjuvant, they can induce the body to produce high titers of neutralizing antibodies and achieve good results. protective effect.

In the early 1990s, David C. Ansardi et al. successfully expressed in Hela cells the VLPs of polioviruses belonging to the Picornaviridae family, which are very similar in structure to EV71 virus. The recombinant vector of P1 precursor protein and P3 precursor protein gene of inflammatory virus, the result proves that the 3CD protease complex in the P3 precursor protein exerts the function of specific proteolytic enzyme, and decomposes the co-expressed P1 precursor protein into VP1, VP0 and VP3 protein, and finally obtained virus-like particles with uniform morphology in electron microscope observation. In 2003, Yuchen Hu et al. also obtained EV71 virus-like particles after co-transfection of recombinant baculovirus carrying EV71P1 and 3CD genes in insect cells. However, the main disadvantage of the insect cell expression system is transient expression. Virus infection will eventually lead to the death of host expression cells, so the infection must be restarted, and each round of protein production must reinfect newly cultured cells, which is expensive and has a long production cycle. Moreover, the expression level of EV71 virus-like particles using insect cells reported in the literature is about 10 mg/10 ⁹ cells, and the expression level is low, which has certain problems in industrial production.

Contents of the invention

In the present invention, the P1 precursor protein coding gene and the 3CD protease coding gene are cloned into the expression vector of Pichia pastoris, and virus-like particles with uniform shape, stability and immunogenicity are successfully obtained after double expression. This conclusion also proves the uniqueness of EV71 virus particles Assembly requires only the P1 precursor protein and 3CD protease.

In order to meet the industrialized production of EV71 vaccine research and development, the present invention selects the Pichia pastoris expression system, which is also the first research to use this system to express and obtain EV71 virus-like particles. The Pichia pastoris expression system has the advantages of simple operation, high expression level, low cost, and short growth cycle, and is suitable for large-scale production of recombinant proteins. The coding DNA sequences of P1 and 3CD proteins are more suitable for expression in Pichia pastoris after codon optimization, which effectively guarantees the high expression level of the recombinant virus-like particle protein.

In order to simulate the translation and expression of P1 and 3CD proteins in the host at the late stage of virus infection, the codon-optimized P1 and 3CD genes were cloned into vectors respectively, and then the two vectors were co-transformed into Pichia pastoris. In addition, 3CD is operated by the weak promoter pYPT1, and the expression level is relatively low, while P1 uses the strong promoter pAOX1, and the expression level is relatively high. P1 and 3CD genes use two vectors to integrate them into the Pichia pastoris genome to control different copies The number is convenient to simultaneously regulate the expression of the two recombinant proteins to achieve the purpose of expressing the recombinant virus-like particle protein more effectively.

The first aspect of the present invention discloses a method for preparing EV71 recombinant virus-like particles. In the method, the gene encoding the P1 precursor protein and the gene encoding the 3CD protease are cloned into the expression vector of Pichia pastoris. Immunogenic virus-like particles.

In a preferred embodiment, the sequences of the gene encoding the P1 precursor protein and the gene encoding the 3CD protease are codon-optimized sequences of Pichia pastoris. In a more preferred embodiment, the sequence of the gene encoding the P1 precursor protein is SEQ ID NO: 5, and the sequence of the gene encoding the 3CD protease is SEQ ID NO: 6.

In another preferred embodiment, the gene encoding the P1 precursor protein and the gene encoding the 3CD protease are cloned into the expression vector of Pichia pastoris through different vectors.

In yet another preferred embodiment, said different expression vectors comprise promoters of different strengths. In a more preferred embodiment, the expression vector of the gene encoding the P1 precursor protein is a recombinant vector consisting of inserting SEQ ID NO: 5 into the BstBI/KpnI site of the pPICZ αB vector, and the expression vector of the gene encoding the 3CD protease The expression vector is a recombinant vector composed of inserting SEQ ID NO: 6 at the BstBI/KpnI site of the pPICZαB vector and replacing the pAOX1 promoter with the YPT1 gene promoter (named pYPT1 promoter herein, SEQ ID NO: 13).

The second aspect of the present invention discloses EV71 recombinant virus-like particles prepared by the method of the present invention.

A third aspect of the present invention discloses a vaccine comprising the EV71 recombinant virus-like particle of the present invention. In a preferred embodiment, the vaccine further comprises an aluminum hydroxide adjuvant.

The fourth aspect of the present invention discloses the use of the EV71 recombinant virus-like particle of the present invention as a vaccine.

The fifth aspect of the present invention discloses a DNA molecule whose nucleotide sequence is the nucleotide sequence encoding the P1 precursor protein or 3CD protein of EV71 virus codon-optimized by Pichia pastoris. In a preferred embodiment, the nucleotide sequence of the DNA molecule is SEQ ID NO:5 or SEQ ID NO:6.

The sixth aspect of the present invention discloses a recombinant vector comprising the nucleotide sequence of the fifth aspect of the present invention.

In one embodiment, the recombinant vector is pPICZαB.

In a preferred embodiment, the pAOX1 promoter of pPICZαB is replaced by a weak promoter. In a more preferred embodiment, the weak promoter is the pYPT1 promoter.

The seventh aspect of the present invention discloses a host cell comprising the recombinant vector of the sixth aspect of the present invention. In a preferred embodiment, the host cell is a Pichia cell.

Description of drawings

Figure 1 shows the agarose electrophoresis detection of the double enzyme digestion (HindIII+KpnI) identification of recombinant P1-pYPT1. 1: 250bp ladder DNA marker; 2: Recombinant P1-pPICZαB plasmid (undigested); 3: Recombinant P1-pPICZαB plasmid (HindIII+KpnI).

Figure 2 shows the agarose electrophoresis detection of the double enzyme digestion (HindIII+KpnI) identification of recombinant 3CD-pPICZαB. 1: 250bp ladder DNA marker; 2: Recombinant 3CD-pPICZαB plasmid (undigested); 3: Recombinant 3CD-pPICZαB plasmid (HindIII+KpnI).

Figure 3 shows agarose electrophoresis detection of PCR identification of recombinant 3CD-pYPT1. 1: DL2000Marker; 2: pYPT1 PCR product with recombinant 3CD-pYPT1 plasmid as template; 3: pYPT1 PCR product with ddH ₂ O as template.

Figure 4 shows the Western-blot identification of the induced expression of P13CD-pYPT1-X33. 1-2. P13CD-pYPT1-X33 induced bacterium supernatant for 72 hours; 3-4. P1-pPICZ-X33 induced bacterium supernatant for 72 hours; 5-6. X-33 induced 72 hours 7. EV71 virus; 8. PageRuler Prestained ProteinLadder.

Figure 5 shows the electron microscope observation of recombinant EV71 virus-like particles obtained by expressing P13CD-pYPT1 (a. 10000 times; b. 40000 times).

detailed description

In the present invention, the expression system of Pichia pastoris is used for the first time to obtain EV71 virus-like particles with uniform and stable morphology, immunogenicity and immunoprotection. And it has the following advantages: (1) The Pichia pastoris expression system is easy to operate, low in cost, high in yield, and has uniform and stable product properties, which is conducive to large-scale production; (2) The expression of 3CD protease ensures that the co-expressed P1 protein is decomposed into components that can be assembled into The VP1-VP4 proteins of the viral capsid; (3) P1 and 3CD genes are inserted into different vectors to control the copy number of the two genes respectively, which is convenient for simultaneously regulating the expression of the two recombinant proteins; (4) P1 and 3CD respectively use strong promoters pAOX1 and the weak promoter pYPT1 make the expression of P1 relatively high in the host, simulating the situation that the capsid protein is mainly expressed in the late stage of virus infection; (5) P1 and 3CD genes are adapted to the Pichia expression system after codon optimization, reaching purpose of efficient expression.

The following examples illustrate the present invention by way of example, but are not intended to limit the scope of the present invention.

Example

1. Gene selection and codon optimization design

Referring to the C4 type Chinese strain BJ08-Z020-1 (GenBank: FJ606449.1), DNA sequences encoding EV71P1 and 3CD were synthesized by techniques known in the art. The DNA sequences of BJ08-Z020-1 wild-type P1 and 3CD are SEQ ID NO: 1 and SEQ ID NO: 2, and the amino acid sequences encoded by them are SEQ ID NO: 3 and SEQ ID NO: 4. The DNA sequences of wild-type P1 and 3CD were modified, and codons with high frequency in Pichia pastoris were used as codons to obtain optimized sequences SEQ ID NO: 5 and SEQ ID NO: 6.

2. Construction of P13CD-pPEXZ recombinant expression vector

Firstly, the P1 and 3CD genes are obtained by means of gene synthesis known in the art according to the above synthesis method, and then the P1-pPICZaB and 3CD-pPICZaB recombinant vectors are constructed. Then, the AOX1 promoter of 3CD-pPICZaB was replaced with the pYPT1 promoter to obtain 3CD-pYPT1. The specific implementation steps are as follows:

2.1 Construction of P1-pPICZαB

The synthesized P1 sequence of SEQ ID NO: 5 was cloned into pPICZαB vector (Invitrogen) by the following method.

Using the P1 sequence of SEQ ID NO: 5 as a template, forward primer: 5'CCAAGCTC TTCGAA ACGATGGGTTTCCAAGTCT 3' (SEQ ID NO: 7); reverse primer: 5'AGC GGTACC CTATTATAAAGTAGTA 3' (SEQ ID NO: 8). The P1 DNA fragments with BstBI and KpnI at both ends were amplified by PCR. All the primers mentioned in the present invention are self-designed and entrusted to Shanghai Yingjun Biotechnology Co., Ltd. to synthesize. PCR program: cycle 30 times at 94°C for 5 minutes, 94°C for 30 seconds, 57°C for 30 seconds, 72°C for 2 minutes and 15 seconds, 72°C for 10 minutes, 10°C for 10 minutes, and the operation ends. The PCR product was identified by agarose gel electrophoresis and a band at about 2600 bp was recovered (Qiagen Gel Recovery Kit). The recovered fragment and pPICZαB were digested with BstBI and KpnI (restriction enzymes involved in the present invention were all purchased from NEB Company), identified by agarose gel electrophoresis and recovered about 2600bp and about 3300bp fragments respectively. After recovery, the P1 fragment and pPICZαB were ligated at a molar ratio of 5:1 with T4 ligase (Takara) overnight at 16°C, and the ligation product was transformed into E.coli DH5α (purchased from Beijing Dingguo Biotechnology Co., Ltd.) the next day. Spread it on a low-salt LB (1% tryptone, 0.5% yeast powder, 0.5% NaCl) plate (containing 25 μg/ml Zeocin), and culture overnight at 37°C. Part of the transformed clones were picked to extract plasmids, identified by double enzyme digestion (HindIII+KpnI), and detected by agarose electrophoresis (Figure 1). The identified positive recombinant clones were verified to be correct by DNA sequencing and stored. The recombinant vector was named P1-pPICZαB.

2.23 Construction of CD-pPICZαB

The synthesized 3CD sequence of SEQ ID NO: 6 was cloned into pPICZαB vector by the following method.

Using the 3CD sequence of SEQ ID NO: 6 as a template, use the forward primer: 5'TTTAGTTC TTCGAAGCTAGC ATGGGTCCATCTCTGG 3' (SEQ ID NO: 9) and the reverse primer: 5' GGC GGTACC CTATTATTGTTCTGAA3' (SEQ ID NO: 10) by PCR The 3CD DNA fragments with BstBI and KpnI restriction sites at both ends were amplified by the method. PCR program: 94°C for 5 minutes, 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 50 seconds, 72°C for 10 minutes, 10°C for 10 minutes, and the operation ends. The PCR product was identified by agarose gel electrophoresis and the band at about 600bp was recovered (Qiagen gel extraction kit). The recovered fragments were digested with pPICZαB in combination with BstBI and KpnI, identified by agarose gel electrophoresis, and about 600bp and about 3300bp fragments were recovered respectively. After recovery, the 3CD fragment and pPICZαB were ligated at a molar ratio of 5:1 with T4 ligase (Takara) overnight at 16°C, and the ligation product was transformed into E.coli DH5α the next day, and spread on a low-salt LB plate (containing 25 μg/ ml Zeocin), cultured overnight at 37°C. Part of the transformed clones were picked to extract plasmids, identified by double enzyme digestion (HindIII+KpnI), and detected by agarose electrophoresis (Figure 2). The identified positive recombinant clones were verified by DNA sequencing and then stored. The recombinant vector was named 3CD-pPICZαB.

2.33 CD-pYPT1 Construction

Using X-33 Pichia pastoris genomic DNA as a template, use forward primer: 5'TTTAGATCTCCATATGATGAGTCACAAT 3' (SEQ ID NO: 11); reverse primer: 5'AAAGCTAGCCTATTATCTCTGTGTGTAT 3' (SEQ ID NO: 12) to amplify by PCR A DNA fragment (SEQ ID NO: 13) of the pYPT1 promoter (GenBank number: AF027960) with BglII and NheI at both ends was obtained, and named as pYPT1 promoter. PCR program: 94°C for 5 minutes, 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 50 seconds, 72°C for 10 minutes, 10°C for 10 minutes, and the operation ends.

pYPT1 and 3CD-pPICZαB were digested with BglII and NheI, identified and recovered by agarose gel electrophoresis about 900bp of pYPT1 and about 3900bp of 3CD-pPICZαB. After recovery, the pYPT1 fragment and 3CD-pPICZαB were ligated at a molar ratio of 5:1 with T4 ligase (Takara) overnight at 16°C, and the ligation product was transformed into E.coli DH5α the next day, and spread on a low-salt LB plate (containing 25 μg/ml Zeocin), cultured overnight at 37°C. Part of the transformed clones were picked to extract the plasmids, the pYPT1 fragment in the recombinant plasmids was identified by PCR, and detected by agarose electrophoresis (Figure 3). The identified positive recombinant clones were verified to be correct by DNA sequencing and then stored. The recombinant vector was named 3CD-pYPT1.

3. Construction and expression of P13CD-pYPT1 recombinant expression strain

The host bacteria used in the present invention is Pichia pastoris X-33 strain (purchased from Invitrogen). The specific construction steps of P13CD-pYPT1 Pichia pastoris expression strain are as follows:

The P1-pPICZαB and 3CD-pYPT1 vectors were linearized with SacI under conventional experimental conditions in the field, co-electroporated to Pichia pastoris X-33, and coated on YPDS (1% yeast powder, 2% peptone, 2% glucose, 1M sorbitol, 2% agar powder) plates (containing 200 μg/ml Zeocin), cultivated at 30°C for 3 days, and hundreds of clones were obtained. Pick dozens of clones and inoculate them on YPD (1% yeast powder, 2% peptone, 2% glucose, 2% agar powder) plates (containing 1500 μg/ml Zeocin), screen high-copy plasmid strains, and cultivate them at 30°C for 2 days. Pick several clones and inoculate them in 4ml YPD liquid medium, replace BMMY (1% yeast powder, 2% peptone, 100mM phosphate buffer pH6.0, 1.34% YNB, 0.5% methanol, 0.00004% biotin) after 24 hours Culture medium, 72 hours after induction with 0.5% methanol, cells were collected. After the cells were broken by glass beads, the supernatant obtained by centrifugation was identified by Western-blot (Figure 3, only the results of two picked clones (1-2) are shown, and the results of other picked clones are not shown). The primary antibody used was a rabbit polyclonal antibody against VP1 protein made by techniques known in the art, and the secondary antibody was goat anti-rabbit IgG-HRP (purchased from Calbiochem). The expression results of P1-pPICZαB-X33 and X-33 under the same conditions were negative controls (Figure 4_3-4 and 5-6), and EV71 virus particles were positive controls (Figure 4_7). Due to the lack of 3CD protease in P1-pPICZ αB-X33, after its induction, the whole bacterial protein has no hybridization band near 34kD of VP1 (Fig. Only expressing P1 in yeast cannot obtain the VP1-VP4 protein with virus particle assembly function; in contrast, P13CD-pYPT1-X33 has obvious hybridization bands around 34kD (Figure 4_1-2), which hybridizes with the natural EV71 virus particles of the positive control The obtained bands were the same (Fig. 4_7), which proved that the 3CD protease co-expressed by P1-pPICZαB and 3CD-pYPT1 played the function of proteolytic enzyme and released VP1 from P1. Take a P13CD-pYPT1-X33 strain (but discard it if the VP1 expression level is found to be abnormally low compared with other strains), such as the strain shown in Figure 4_1 or 2, and freeze it at -80°C as a working seed for fermenter cultivation .

4. Fermenter culture of EV71 recombinant protein

Take 1 bacterial strain glycerol cryopreservation tube from the working seed bank, pipette 100 μL into 5 ml YPD medium after thawing, and incubate at 280 rpm at 30°C for 20 hours. The _OD600 is between 1 and 2. Introduce 1 ml of the microscopically qualified activation solution into 500 ml of YPD medium, culture at 280 rpm, 30°C for 20 hours. _OD600 is between 2 and 6. Microscopic examination showed no bacterial contamination. Use BIOENGINEERINGNLF22 fermenter, use basic salt medium BSM ₁ for fermentation (K ₂ SO ₄ 273g, MgSO ₄ 109g, CaSO ₄ .2H ₂ O 17.6g, H ₃ PO ₄ 400.5ml, KOH 62g, glycerol 600g, PTM 160ml, foam enemy 1ml , deionized water was added to 15L). Inoculate at 1:15. The fermentation temperature is 30.0°C, the initial pH is 5.00, the rotation speed is 300rpm, the ventilation rate is 0.5vvm, DO100%, and PTM ₁ (CuSO ₄ .5H ₂ O 6.0g, NaI0.008g, MnSO ₄ 3.0g, NaMoO ₄ 0.2g, H ₃ BO ₃ 0.02g, ZnSO ₄ 20.0g, CoCl ₂ 0.5g, FeSO ₄ .7H ₂ O 65.0g, biotin 0.2g, H ₂ SO ₄ 5.0ml, deionized water to 1L) trace salts. The initial multiplication stage is about 20 hours, and the dissolved oxygen value is maintained above 30%. When the carbon source is consumed, the wet weight of the bacteria reaches about 100g/L. Add 50% glycerol solution (12 ml PTM1 per liter). The dissolved oxygen level was maintained above 20% by adjusting the stirring speed, air flow, and tank pressure (<0.8 bar). Add for about 8 hours, when the wet weight of the bacteria is about 350g/L. At the same time, adjust the pH control to 6.00, and add methanol (12ml PTM1 per liter) for induction. Maintain the dissolved oxygen value higher than 20%, the temperature is 30°C, and the pH value is controlled at 6.00. Release the fermented liquid when inducing 40 hours of fermentation to finish. After the fermentation broth was centrifuged at 4800rpm for 20 minutes at 4°C, the sludge was collected and frozen at -20°C.

5. EV71 VLP Harvesting

After fermentation induction, P13CD-pYPT1-X33 cells were mixed with washing buffer (100mM PB pH7.0, 0.15M NaCl) at a ratio of 1:3, mixed thoroughly, and centrifuged at 8000rpm for 5 minutes to collect the cells. Repeat the above operation two times all over. The washed cells were mixed with a bacteriostasis buffer at a ratio of 1:5, and after thorough mixing, the above cell suspension was crushed by high pressure, and the operation was repeated to crush 90% of the cells. Centrifuge the bacteriostatic liquid obtained by high-pressure crushing at 9000 rpm for 30 minutes at 10° C., and collect the supernatant after centrifugation. Take 1ml of the supernatant after breaking the bacteria, 40000rpm for 3 hours, ultracentrifuge at 4°C, discard the supernatant, and resuspend the pellet in 100μl of PBS (10mM, pH7.4). The results of transmission electron microscope observation showed that the supernatant of the supernatant after ultracentrifugation showed uniform and stable virus-like particles ( FIG. 5 ), and the diameter of the particles was between 20-30nm.

300 grams of thalline can be harvested per liter of fermentation broth, and after the purification step, pure VLP is obtained. According to the yield of the final VLP and the yield of the purification step, the VLP expression amount of the method of the present invention is calculated to be about 150 mg/liter of fermentation broth. Those skilled in the art can understand that the recombinant virus-like particle protein expression of this order of magnitude can meet the requirements of industrial production, and compared with the prior art, unexpected technological progress has been achieved.

6. Preparation of EV71 virus-like particle vaccine

With reference to the "Recombinant Hepatitis B Vaccine (Saccharomyces cerevisiae)" chapter of "Pharmacopoeia of the People's Republic of China" Part III (2010 Edition), the method on page 126, the purified EV71 virus-like particle protein is adsorbed with aluminum hydroxide adjuvant, Prepared as a candidate vaccine for the prevention of EV71 virus.

7. Determination of immunogenicity and immunoprotection of EV71 virus-like particles

SPF grade BALB/c mice aged 6-8 weeks (Shanghai Xipuer-Bicai Experimental Animal Co., Ltd.) were selected and divided into 2 groups with 8 mice in each group. The mice in group 1 were immunized with 0.5ml buffer solution containing aluminum adjuvant (as a negative control group), and the mice in group 2 were immunized with 0.5ml VLP with a concentration of 20 μg/ml of aluminum adjuvant (as a test group). On days 0 and 14, the rats were immunized subcutaneously twice, and blood was collected two weeks after the second immunization. Place the collected blood at 37°C for 2 hours, centrifuge at 4000g for 10 minutes, and absorb the supernatant to obtain the mouse polyantiserum, store it at -20°C, and detect the antibody titer of the mouse serum and the antibody titer of neutralizing EV71 virus. The specific method is as follows:

The antibody titer determination method is as follows: Dilute the purified EV71 virus VLP to 1 μg/ml with the coating solution, add 0.1 ml to each concave hole of the microtiter plate, and overnight at 4°C. The coating solution was removed, and the wells were washed with 0.3 ml PBST (10 mM PBS+0.05% Tween-20). Incubate at 37°C for 2 hours with 0.3ml of blocking solution (5% skim milk powder + PBST). Add 0.1ml each of the tested serum (dilution gradient from 1:100 to 1:3278800) diluted with dilution buffer (2% skimmed milk powder + PBST) in different gradients to each well, and remove the serum after incubation at 37°C for 1 hour solution, wash the wells with 0.3ml washing solution. Then add 0.1ml of HRP-labeled goat anti-mouse IgG diluted 1:5000 with dilution buffer to each concave well, remove the enzyme standard solution after incubation at 37°C for 0.5 hours, and wash the concave well with 0.3ml washing solution; Add 0.1ml of DAB chromogenic solution to the concave hole, and then add 2M H ₂ SO ₄ 0.05ml of stop solution to stop the reaction after 20 minutes at room temperature in the dark, and measure the OD ₄₅₀ value with an enzyme-labeled colorimeter, and calculate the serum based on the OD ₄₅₀ reading value antibody titer value.

Neutralizing antibody titer assay method is as follows: in 96 wells cell culture plate, serum to be tested is carried out gradient dilution, and dilution multiple is from 1: 8 to 1: 512, each dilution gets the EV71 virus liquid of 0.05ml and 0.05ml ( titer 100CCID ₅₀ /0.05ml) were mixed, and the mixture was incubated at 37°C for 2 hours, then RD cell suspension with a concentration of 2×10 ⁵ cells/ml was added, and then placed in a 37°C CO2 incubator for incubation. Use an inverted microscope to observe CPE (cytopathic effect) every day, and record the virus titration results. After 6-7 days, the reciprocal of the highest dilution of serum that inhibits 50% of cytopathic effects is the final serum neutralizing antibody titer value.

The results of serum antibody titers and neutralizing virus antibody titers are shown in Table 1. From the results in Table 1, it can be seen that mice immunized with EV71 vaccine with virus-like particles have strong immunogenicity and immune protection against viruses in vitro sex.

Table 1 EV71 virus-like particles immunized mice obtained serum antibody titer and neutralizing antibody titer

The "techniques known in the art" mentioned herein refer to the technologies that can be found or obtained by those of ordinary skill in the art from dictionaries, textbooks, patents, papers and other documents published in this field or related fields according to the experimental purpose, for example, in Techniques described in Molecular Cloning.

Claims (6)

1. A method for preparing EV71 recombinant virus-like particles, the method comprising cloning the EV71 virus P1 precursor protein coding gene and the 3CD protease coding gene into Pichia pastoris through different expression vectors, and obtaining uniform and stable morphology with Immunogenic virus-like particles, Wherein the expression vector of the gene encoding the P1 precursor protein is a recombinant vector formed by inserting SEQ ID NO: 5 at the BstBI/KpnI site of the pPICZαB vector, and the expression vector of the gene encoding the 3CD protease is BstBI/KpnI of the pPICZαB vector. The KpnI site was inserted into SEQ ID NO: 6 and the recombinant vector constituted by replacing the pAOX1 promoter with the pYPT1 promoter. 2. The EV71 recombinant virus-like particle prepared by the method of claim 1. 3. A vaccine comprising the EV71 recombinant virus-like particle of claim 2. 4. The vaccine of claim 3, further comprising an aluminum hydroxide adjuvant. 5. The use of the EV71 recombinant virus-like particle of claim 2 for preparing a vaccine. 6. Pichia cells containing two different recombinant vectors, one of which is a recombinant vector formed by inserting SEQ ID NO: 5 at the BstBI/KpnI site of the pPICZαB vector, and the other is the BstBI/KpnI of the pPICZαB vector The recombinant vector constructed by inserting SEQ ID NO: 6 and replacing the pAOX1 promoter with the pYPT1 promoter.