CN115057944A - Multi-epitope subunit vaccine for all GBS serotypes - Google Patents

Abstract

The invention belongs to the field of medicine, and in particular relates to a multi-epitope subunit vaccine against all GBS serotypes. The present invention first provides a multi-epitope chimeric protein MVSA against all GBS serotypes, and the amino acid sequence of the multi-epitope chimeric protein MVSA is shown in SEQ ID No: 1. The active immune protection test in mice showed that MVSA could provide 100% protection rate against lethal GBS infection in mice. At the same time, the passive immune protection test in mice showed that the injection of MVSA antibody in advance could significantly reduce the bacterial load in the organs and the level of pro-inflammatory cytokines in the organs of the mice after GBS challenge, and provided 100% of the immune system for the mice to resist lethal GBS infection. % protection rate. In vitro, MVSA antibodies can significantly inhibit the growth of GBS of different serotypes. Therefore, as a multi-epitope vaccine, MVSA can stimulate the body to produce protective antibodies against GBS infection, and has a good application prospect.

Description

A multi-epitope subunit vaccine against all GBS serotypes

technical field

The invention belongs to the field of medicine, and in particular relates to a multi-epitope subunit vaccine against all GBS serotypes.

Background technique

Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a Gram-positive bacterium that infects a variety of species, including fish, reptiles, amphibians, birds, and mammals. In addition to animals, GBS can colonize the vagina and gastrointestinal tract of up to 30% of healthy women. Meanwhile, GBS is a major life-threatening pathogen in neonates, causing meningitis, sepsis, and pneumonia. Vaccines to prevent GBS infection are critical in humans and animals. Current GBS vaccines target the bacterial polysaccharide capsule, but lack cross-protection between different serotypes. Therefore, there is an urgent need to develop a broad-spectrum, multivalent novel vaccine that simultaneously provides broad protection against all 10 serotypes of GBS. In recent years, well-established genomics, bioinformatics and proteomic technologies have made it possible to identify and execute widely distributed anti-pathogen conserved immunogenic proteins. Surface or secreted proteins and virulence factors of some pathogens are considered as candidate protective antigens, such as surface immune protein Sip, cell wall surface anchoring family proteins, CAMP factors, C5a peptidase, serine-rich repeat glycoprotein Srr, ornithine Carbamyltransferase OTC, etc. However, a single protein cannot induce an effective immune response that provides protection for the host.

Multiepitope fusion antigen (MEFA) technology is a new vaccine construction technology and platform based on the combination of computer biology and structural biology. The neutralizing (blocking) epitopes of virulence factors are integrated into an antigen using computer biology and structural biology techniques, and the immunogenicity of the epitope itself is simulated to the greatest extent, aiming to construct a broad protective effect of novel multivalent epitope vaccines. The epitope vaccine designed based on the MEFA technology platform can integrate multiple epitope antigens from different sources, so that the vaccine has broad protection against heterologous pathogens, which makes the traditional epitope vaccine concept take a new look.

SUMMARY OF THE INVENTION

In view of the deficiencies of the existing problems, the purpose of the present invention is to provide a multi-epitope subunit vaccine against all GBS serotypes.

The technical scheme adopted by the present invention to solve the technical problem is:

In the first aspect, the present invention protects a multi-epitope chimeric protein MVSA against all GBS serotypes, and the amino acid sequence of the multi-epitope chimeric protein MVSA is as shown in SEQ ID No: 1.

The present invention also protects the gene encoding the previously described multi-epitopic chimeric protein MVSA against all GBS serotypes.

The present invention also protects expression cassettes, expression vectors, transgenic cell lines or recombinant engineered bacteria containing the aforementioned genes.

In a second aspect, the present invention protects a vaccine against all GBS serotypes comprising the multi-epitope chimeric protein MVSA as described above.

The global coverage of MVSA was predicted to be 88.91% based on the major histocompatibility antigen allele (HLA) in humans. In GBS-endemic regions of the United States (24.7%), Australia (23.8%), and Canada (20.5%), the MVSA vaccine covered 100%, 92.79%, and 99.2% of the population, respectively.

As a preferred technical solution of the present application, the vaccine is a subunit vaccine.

Serological tests showed that MVSA antibodies were produced after vaccination with the vaccine of the present invention. The in vitro antibacterial test shows that the MVSA antibody provided by the present invention can significantly inhibit the growth of 6 different serotype GBS strains, and the six popular serotypes (Ia, Ib, II, III, V, VI) cover 98.4% of GBS clinical infection case.

Preferably, the vaccine further comprises a subunit vaccine adjuvant.

More preferably, the subunit vaccine adjuvant is seppic ISA206.

In the third aspect, the present invention protects a method for preparing a multi-epitope subunit vaccine for all GBS serotypes, comprising the steps of:

(1) Computer prediction of GBS antigenic epitopes: determine candidate proteins covering 10 different GBS serotypes; perform T/B cell epitope prediction, and then multiple sequence alignment and antigenicity prediction screen out T/B cell epitopes , 11 polypeptide epitopes of B cell epitopes;

(2) Construction of MVSA protein: add a Linker fragment "LRMKLPKS" to the N-terminus of each epitope, and insert a second spacer GGPPG between each pair of Linker epitope sequences; connect the epitopes of step (1) in series and combine Construction of polypeptide chimera MVSA;

(3) construct recombinant plasmid: take amplification product as template, amplify and obtain MVSA protein gene, described MVSA protein gene is connected to pET-28a (+) plasmid to obtain recombinant plasmid;

(4) MVSA protein gene expression: The constructed recombinant plasmid was transformed into BL21 competent cells, and the MVSA protein was obtained through culture, extraction and purification.

As a preferred technical solution of the present application, the epitopes in the step (1) are respectively derived from GBS self-protein NT5, BKD-E2, PK, GAPDH, PGK, Srr1, FbsA, Sip, AP1-2b, Beta C protein, BibA .

As a preferred technical solution of the present application, the preparation method further includes the step of further preparing the MVSA into a commercial vaccine.

The present invention also protects the multi-epitope subunit vaccine obtained by the aforementioned preparation method of a multi-epitope subunit vaccine against all GBS serotypes.

The in vitro antibacterial test shows that the MVSA antibody of the present invention can significantly inhibit the growth of six serotype GBS strains.

The present invention also protects any of the following applications:

(1) the application of the aforementioned multi-epitope chimeric protein as or in the preparation of subunit vaccines against all GBS serotypes;

(2) the application of the multi-epitope chimeric protein described above as or in the preparation of a drug for the prevention of all GBS serotypes;

(3) Application of the aforementioned gene, aforementioned expression cassette, expression vector, transgenic cell line or recombinant engineering bacteria in the aforementioned preparation of the aforementioned multi-epitope chimeric protein.

In the third aspect, the present invention also protects a vaccine or drug against all GBS serotypes, the active ingredient of which is the aforementioned multi-epitope chimeric protein.

beneficial effect

A multi-epitope subunit vaccine for all GBS serotypes provided by the invention has the following beneficial effects compared with the prior art:

(1) The subunit vaccine of the present invention is formed by connecting the protective epitopes of multiple antigens in series, which can not only eliminate immune irrelevant components or immune tolerance components in the entire protein molecule, induce a more effective immune protective response, but also It can overcome the capacity limitation of chimeric expression of various antigens and antibodies of expression vectors.

(2) The distribution of NT5, BKD-E2, PK, GAPDH, PGK, Srr1, FbsA, Sip, AP1-2b, Beta Cprotein, BibA selected in the present invention covers almost all clinical isolates of GBS; Fusion MVSA, predicted by computer human allele HLA, can cover about 88.91% of the global population. Therefore, MVSA is one of the ideal target antigens for the development of GBS vaccines.

(4) The present invention detects the serum antibody production level and the immune protection rate after the challenge of mice immunized with MVSA by indirect ELISA method, WB method and active immune protection test. The results found that MVSA can stimulate the body to produce different levels of specific antibodies. The immune protection rate of MVSA to immunized mice was 100%. (3) In vitro, MVSA antibodies induced by MVSA can inhibit the growth of GBS of different serotypes. In mice, pre-injection of MVSA hyperimmune serum can significantly reduce the bacterial load in the organs and the levels of pro-inflammatory cytokines in the organs of the mice after GBS challenge. The passive immune protection test of mice showed that the survival rate of mice immunized with MVSA hyperimmune serum reached 100% when challenged with lethal dose of GBS. Therefore, the protective antibodies produced by the body stimulated by MVSA can resist GBS infection and have a good application prospect.

Description of drawings

Fig. 1 is a kind of design route of the multi-epitope subunit vaccine for multiple GBS serotypes of the present invention;

Fig. 2 is the computer analysis of predicted epitope;

Figure 3 is a schematic diagram of the tandem protein MVSA;

Figure 4 shows the PCR identification of pET-28a-MVSA-BL21; wherein, A: T7 universal primer identification; B: MVSA target gene primer identification; M: 2000DL Marker; 1: pET-28a-MVSA-BL21-1; 2: pET -28a-MVSA-BL21-2; 3: pET-28a-MVSA; 4: ddH ₂ O;

Figure 5 is the SDS-PAGE analysis of MVSA-induced expression and purification; wherein, M: protein size and molecular mass 180kDa; 1: MVSA;

Fig. 6 is the immunoblotting result of recombinant protein;

Fig. 7 Survival curve of immunized ICR mice to BAA-611 infection;

Fig. 8 is the mouse survival curve of immune protection test;

Fig. 9 is the result of in vitro antibacterial test;

Figure 10 shows the organ distribution of passively immunized mice;

Figure 11 shows the transcriptional levels of mouse organ cytokines.

Detailed ways

The present invention will be described in further detail below in conjunction with the embodiments. If the reagents or equipment used are not marked with the manufacturer, they are regarded as conventional products that can be purchased through the market.

Materials: Streptococcus agalactiae BAA-611 was purchased and preserved by ATCC. The mice (SPF, ICR, female, 4 weeks old) required for this experiment were purchased from the Comparative Medicine Center of Yangzhou University.

Example 1: Selection and retrieval of proteins

Search PubMed (https://www.ncbi.nlm.nih.gov/pubmed/) for keywords such as "Protein Vaccines and Streptococcus", "Antigens and Streptococcus", and "Protein Antigens and Streptococcus Immunity" to sort out Protein antigens that have demonstrated protection have been tested and downloaded and stored (Table 1). In particular, 6 proteins (NT5, OTC, BKD-E2, PK, GAPDH, PGK) were identified as strongly immunoreactive proteins in our previous immunoproteomic study (Table 1).

Table 1 Streptococcus agalactiae has proven immunogenic vaccine candidate proteins

Example 2 Prediction of Affinity Epitopes with B and T Lymphocytes

The protein sequences screened in the previous step were used for B cell and T cell epitope prediction. B cell epitope prediction is performed using two programs, BCPred (http://ailab.ist.psu.edu/bcpred/predict.html) and ABCPred (http://crdd.osdd.net/raghava/abcpred/), Contiguous epitopes between 10 and 20 amino acids in length are recognized with greater than 90% specificity selection. T Cell Epitope Prediction The prediction of peptides binding to MHC class I and II molecules was performed using the IEDB TEPITOP (http://tools.iedb.org/tepitool/) website. MHC class II HLA alleles selected as human hosts DRB1*01:01, DRB1*03.:01, DRB1*04:01, DRB1*07:01, DRB1*11:01, DRB1*13:01, DRB1*15 :01 There are seven in total, and the predicted T cell epitope results of the proteins are sorted.

Example 3 Epitope Alignment, Antigenicity Analysis and Global Coverage Analysis

The sorted B/T cell epitopes were uploaded to the Prabi (https://npsa-prabi.ibcp.fr/) website for multiple sequence alignment. Epitopes recognized by B/T cells were selected and antigenic analysis was performed through the website VaxiJen (VaxiJen (ddg-pharmfac.net)). Polypeptide epitopes with antigenicity greater than 0.4 were selected, and long-chain epitopes with lower antigenicity were removed, so that the selected epitopes were derived from 11 different candidate proteins. In order to determine the coverage of the world population by the constructed polyepitopic vaccine, the antigenic epitopes were analyzed through the database provided by the IEDB website (http://tools.iedb.org/population/).

Example 4 Conservation analysis of antigenic epitopes in 10 serotypes of GBS

The distribution of predicted epitopes in different GBS was analyzed to verify the conservation of selected epitopes in GBS. According to the 10 capsular genotypes of GBS, we downloaded the complete genomes of 139 GBS strains (including Ia, Ib, II-VII) and 21 GBS Scaffold genomes (including types VIII and IX) published on NCBI, And a genome database of 160 GBS strains of 10 serotypes was established. TBLASTN was performed on the predicted epitopes based on the established GBS database, and the distribution of epitopes in each GBS strain was counted and analyzed. At the same time, based on the capsular gene cluster of 160 GBS strains, an evolutionary tree was established by NJ method, combined with the distribution of epitopes in different GBS strains, and the conservation of its epitopes in different serotypes was analyzed.

Example 5 Tandem epitope vaccine design

The determined peptide epitopes were uploaded to EXPASY (https://web.expasy.org/protparam/) for physicochemical property analysis. The average hydrophilicity was less than 0 for hydrophilic proteins, and greater than 0 for hydrophobic proteins.

A Linkey fragment "LRMKLPKS" was added to the N-terminus of each epitope, and a second spacer GPGPG was inserted between each pair of Linkey epitope sequences to connect the above-screened epitopes. The connection sequence is based on hydrophilicity, the middle is a hydrophobic polypeptide epitope, and the two ends are polypeptide epitopes with strong hydrophilicity. Linked into a protein, named MVSA ( M ulti-epitopesubunit v accine for S treptococcus a galactiae). MVSA was uploaded to the website VaxiJen and AllergenFP (http://www.ddg-pharmfac.net/AllergenFP/) for antigenicity and allergen prediction. The ligated sequence is connected to the protein expression vector to prepare for expressing the protein, preparing the vaccine and protecting the mice from the virus.

Example 6 Synthesis and identification of tandem proteins

The pET-28a-MVSA plasmid dry powder synthesized by the company was dissolved in sterile water and transformed into E.coli competent cells BL21 (DE3). The steps of transformation are as follows: 1) Take commercial DH5α competent cells (containing 100 μL of competent cells) and place them on ice to thaw, add 5 μL of pET-28a-MVSA plasmid to the competent cells, mix well and then ice bath for 30 min; 2 ) After 30min, put the EP tube into a 42°C water bath for heat shock for 45s-90s; 3) Continue to ice-bath the EP tube for 3-5min; 4) Add 1 mL of LB liquid medium to the EP tube, place at 37°C, 180rpm Shaker for 1 h; 5) Centrifuge at 5000 rpm for 5 min, discard 1 mL of supernatant, resuspend bacteria with the remaining 100 μL of supernatant, spread on Kan+ resistant LB plates, and incubate overnight at 37 °C. Use a sterile pipette tip to pick single clones on the plate, place them in 1 mL of LB liquid medium containing Kan (50 μg/mL), and cultivate at 37° C. in a constant temperature shaker at 180 rpm for 6 h. The clones were identified by colony PCR.

The primer sequences are:

MVSA upstream primer-P1: GGGCCAGGTCCCGGATTAAGGA;

MVSA downstream primer-P2: TTCTTTGGTCTGACCATCTCT;

P1 primer and P2 primer can amplify MVSA epitope tandem gene tandemly connected to pET-28a plasmid PCR reaction system and reaction conditions: PCR 20μL reaction system: 1μL upstream primer, 1μL downstream primer, 10μL 2×Rapid Taq Mix, template 1 μL and 7 μL ddH ₂ O. PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; 30 cycles (95°C, 30s, 55°C, 30s, 72°C for 20min); extension at 72°C for 10 min. The PCR product was identified by 1% agarose gel electrophoresis, and the target band was excised, and the gel was recovered with a TaKaRa gel recovery kit, and the recovered product was sent to Nanjing Jinweizhi Company for sequencing.

Example 7 Prokaryotic expression of tandem proteins

1 Expression and purification

The fresh bacterial solution was inoculated into 200 mL of LB liquid medium containing the corresponding antibiotics at a ratio of 1:100, and incubated at 37 °C with shaking at 180 rpm for 3 to 4 h. When the OD ₆₀₀ reached 0.4 to 0.6, induced, collected the bacterial cells, and washed with PBS for two times. Repeatedly, resuspend the bacteria with 20 mL of supernatant lysis buffer, and sonicate on ice. The working time is 5 s, the interval time is 10 s, and the working frequency is 80 times. The supernatant was filtered through 0.45 μm and 0.22 μm filters in turn. Purify by His-tag Ni-NTA affinity chromatography as follows:

Washing: Wash the affinity chromatography column with 5 times the bed volume of ddH ₂ O (5 mL);

Equilibration: Equilibrate the affinity column with 5 mL of supernatant dissolution buffer;

Loading: slowly inject the supernatant solution into the affinity chromatography column with a syringe, and collect the effluent;

Washing: Rinse the column with 10 times the column volume of supernatant dissolution buffer;

Elution: The target protein was eluted with 10 mL of upper washing buffer, and each 1 mL was collected in an EP tube, and the collected solution was subjected to SDS-PAGE electrophoresis to analyze the purity of the protein and quantify the protein concentration by BCA method.

Washing: first wash the column with ddH ₂ O of 5 times the column bed volume, then wash it with 3 column bed volumes of 20% ethanol, and store it at 4°C for later use.

The collected proteins were analyzed by SDS-PAGE electrophoresis.

2 Determination of protein concentration

The protein concentration was measured using the BCA ProteinKit kit. According to the instructions, the BSA standard protein was serially diluted (1000, 500, 250, 125, 62.5, 0 μg/mL), and the working solution of reagent A and reagent B was prepared at a ratio of 50:1. The protein gradient dilution solution and working solution 1:8 (20 μL protein, 200 μL working solution) were mixed in a 96-well plate by pipetting, incubated at 37° C. for 30 min, and the absorbance at OD ₅₆₂ was measured. Taking the protein concentration as the abscissa and the corresponding OD ₅₆₂ absorbance value as the ordinate, draw a standard curve, Y is the protein content (μg) per 20 μL, and X is the absorbance value. Calculate the purified ultrafiltered protein concentration based on the standard curve.

Example 8 Mouse immunization experiment of tandem protein MVSA

1 Preparation of inactivated vaccine

The BAA-611 strain was inoculated on the Columbia blood plate at 37°C for 16 hours, and single colonies were picked for expansion and culture, and the turbidity and viable bacteria were counted. Incubate in a 37°C incubator for 24 hours, during which time it is shaken several times to prepare an inactivated bacterial solution. Centrifuge and rinse with sterilized normal saline (0.9%) for 3 times, dilute or concentrate it to 5×10 ⁹ CFU/mL according to the counting result, and inoculate the inactivated bacterial solution on Columbia blood plate, incubate at 37°C for 16 hours, and observe whether there is bacterial growth. .

2 Immunization of mice

Immunization of mice To evaluate the immune effect of MVSA multi-epitope tandem vaccine, the purified recombinant protein MVSA and inactivated bacterial solution BAA-611 and ISA 201 adjuvant (SEPPIC, France) were shaken and mixed at a ratio of 1:1. MVSA recombinant vaccine and BAA-611 inactivated vaccine were prepared. 4-week-old SPF-grade female ICR mice were divided into 3 groups. The first group was the recombinant vaccine protection group, immunized with MVSA recombinant vaccine, and the immunization dose was 50 μg/mouse. The second group is the BAA-611 inactivated vaccine protection group, immunized with BAA-611 inactivated vaccine, and the immunization dose is 1×10 ⁸ CFU/mouse; the third group is only immunized with PBS+adjuvant, which is set as the control group, and the mice in each experimental group are immunized The immunization route of subcutaneous multi-point injection was adopted. The immunization schedule was as follows: the second immunization was performed 10 days after the first immunization, and the third immunization was performed 10 days later. The blood of mice in each group was collected by orbital venous blood collection before each immunization and before challenge. After incubating at 37°C for 1 h, the mice were left at 4°C overnight, centrifuged at 1000 rpm and 4°C for 10 min, and serum was collected. Bacteria were aliquoted and stored at -80°C for later use.

Example 9 ELISA specific antibody titer determination

1) Take a 96-well detachable ELISA plate, coat with 0.2 μg/100 μL of purified MVSA protein per well and BAA-611 1×10 ⁷ CFU per well, and coat overnight at 4°C;

2) Wash the plate 3 times with PBST washing solution, drain the water, add 200 μL of 0.5% BSA solution to each well, and coat it at 4°C.

3) Wash the plate 3 times with PBST washing solution and drain the water;

4) The ratio is 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400 and 1 : Serum collected after 204800 dilution immunization;

5) Add the above diluted serum to the ELISA plate that has been coated with the antigen, 100 μL per well, and make duplicate wells;

6) After incubating in a 37°C incubator for 1 h, wash the plate 3 times with PBS-T wash solution and drain the water;

7) Add 100 μL of HRP-goat anti-mouse IgG diluted 1:4,000 to each well, and incubate at 37°C for 1 h;

8) Wash the plate 3 times with PBST washing solution and drain the water;

9) Add 100 μL of TMB chromogenic solution to each well, and incubate at room temperature for 15 min in the dark until the reaction is complete;

10) Add 50 μL of stop solution to each well, and measure the OD value at 450 nm of the microplate reader.

11) Calculate the P/N value with a critical value of 2.1.

Example 10 Detection of WB-specific antibodies

The purified MVSA protein was subjected to SDS-PAGE electrophoresis, and the protein gel was cut according to the target protein, and the protein was transferred to PVDF (Millipore) with a membrane transfer machine. After transfer, the PVDF membrane was immersed in 5% skim milk and blocked at 37°C for 2 hours. . After the blocking was completed, the self-made triple immune serum was added as the primary antibody (1:1,000 dilution) and incubated at 37°C at 70rpm for 2h. After washing 3 times with PBST, HRP-goat anti-mouse IgG diluted 1:4000 was added as the secondary antibody and incubated at 37°C for 45min at 70rpm. After washing 3 times with PBST, the color was developed with ECL luminescent solution in a dark environment, and finally exposed with an exposure meter.

Example 11 Mice challenge experiment

1 strain rejuvenation

Streptococcus agalactiae BAA-611 was inoculated into THB medium, cultured at 37°C at 180 rpm for 6 h, centrifuged at 5,000 rpm to collect bacterial cells, resuspended in PBS, washed 3 times, resuspended in 200 μL PBS and injected into mice intraperitoneally for 6-24 h The morbidity of the mice was observed, and blood was taken from the heart apex of the dying mice and the blood plates were incubated at 37°C overnight. The bacterial suspension was taken to identify the strain by rapid PCR.

2 Challenge experiments

Pick a single colony on the rejuvenated blood plate, take the bacterial suspension after the bacteria shake and use rapid PCR to identify the strain. At about OD ₆₀₀ =0.6, the cells were collected by centrifugation at 5,000 rpm, washed three times with PBS, and the amount of bacteria was adjusted. Seven days after the last immunization, different groups of mice were challenged with 20 x LD ₅₀ of BAA-611 ( ² x 108 CFU/mouse) by intraperitoneal injection.

Example 12 In vitro antibacterial test

To determine the antibacterial activity of MVSA antibodies against GBS, an in vitro antibacterial test was performed. The strains of different serotypes were single colonized in THB liquid medium and cultured overnight. 1:100 was transferred to 5 mL of THB liquid medium, and incubated at 37°C with shaking at 180 rpm to log phase (OD ₆₀₀ =0.6-08). The GBS bacterial solutions of different serotypes were diluted 1:50 in THB solution and added to 100 μL in the microplate. In each different GBS dilution solution, 100 μL of THB liquid was added to dilute 50-fold MVSA, BAA-611 hyperimmune serum and negative serum. repeat three times. After incubation at 37°C for 2h, the different wells were serially diluted and counted on THB solid medium.

Example 13 Passive immune protection test-organ distribution test

To determine the preventive and neutralizing effects of MVSA antibody on GBS in vivo, 4-week-old female ICR mice were intraperitoneally injected with 200 μL of MVSA hyperimmune serum, and control mice were intraperitoneally injected with 200 μL of PBS. 24 hours later, a lethal dose of GBS BAA-611 (2×10 ⁸ CFU) was injected intraperitoneally, and the mice were observed for 7 days and the death was recorded.

Blood from mice was collected by ocular bleed 9 hours after challenge and counted on drop plates on THA after 10-fold constant dilution with PBS buffer. Mice were humanely euthanized simultaneously by CO ₂ sedation followed by cervical dislocation. Before removing the organ, wipe the visceral surface with a sterile cotton swab to check for bacterial translocation. The spleen, liver and brain were further removed and transferred to sterile pre-weighed MP tubes. After homogenization with PBS, both blood and homogenate were diluted 10-fold, then plated and incubated at 37°C for 24 hours and colony counts were measured.

Example 14 Passive Immune Protection Test - Organ Cytokine Determination

In order to further evaluate the effect of MVSA antibody on organ inflammation in mice after GBS challenge, RT-qPCR was performed to analyze the transcriptional levels of cytokines in different isolated organs. First, according to the TRIzol portable method, the total RNA of each organ was taken. Exogenous DNA was removed and RNA was reversed to cDNA using a two-step method using a reverse transcription kit (PrimeScript ^™ RT reagent Kit with gDNA Eraser). The QuantStudio 6Flex real-time PCR instrument and ChamQ UniversalSYBR qPCR master mix were used to perform fluorescence quantitative assay to detect the transcription levels of cytokines IL-1β, IL-6 and TNFα. The housekeeping gene GAPDH was used as an internal reference, the primers used were shown in Table 2, and the 2 ^-ΔΔCT method was used to calculate the transcription difference.

Table 2 RT-qPCR primers

2 Test results

1 Epitope prediction and analysis

The downloaded proteins that have been shown to have protective effects were used for B cell epitope prediction through BCPred and ABCPred and T cell epitope prediction through IEDB TEPITOP, and only the epitopes recognized by both B cells and T cells were selected. The OTC proteins did not generate suitable B cell predicted epitopes. Epitopes with predicted antigenicity greater than 0.4 were selected, and 2 long-chain epitopes with lower antigenicity, such as BP-2b and lrrg, were removed. Finally, 11 epitopes were screened, as shown in Table 2. Its predicted global coverage based on human alleles is 88.91%. The 11 epitopes covered 100%, 92.79% and 99.2% of the population, respectively, in GBS-endemic regions of the United States (24.7%), Australia (23.8%), and Canada (20.5%).

According to the analysis of the physicochemical properties of EXPASY, the linkers "GPGPG" and "LRMKLPKS" were selected to connect 11 epitopes to obtain the sequence of MVSA (SEQ ID No: 1). The predicted antigenicity of MVSA is 1.1909, with no sensitization. The MVSA polyepitope protein contains a total of 430 amino acids, and was sent to the company to synthesize and link to pET-28a(+).

Table 2 Epitopes and antigenicity scores with affinity for B/T cells

2 Identification of recombinant plasmids

The pET-28a-MVSA was sequenced, and the BLAST comparison confirmed that the obtained sequence had 100% homology with the target sequence. The results showed that the MVSA recombinant protein gene was correctly inserted into the multiple cloning site of the prokaryotic expression vector pET-28a. The plasmids were transformed into BL21(DE) competent cells, spread on LB plates containing kan ⁺ , and cultured for 12-16 h. The single colony was cultured and identified by PCR, and the result was positive (Figure 4).

3 Expression and purification of tandem vaccine prokaryotic

The results of SDS-PAGE electrophoresis after the recombinant plasmid genetically engineered bacteria BL21 induced the protein at a small dose showed that the size of the MVSA protein was 55kDa, and the protein existed in the supernatant and precipitate after ultrasonic lysis. The induction conditions were 28°C, 14h, IPTG 0.5mM When the supernatant expressed the most, it was the optimal induction condition. The results of induction and purification SDS-PAGE electrophoresis are shown in Figure 6. The standard curve was determined with BCA protein quantitative detection kit: Y=24.788X-2.7234, and the purified protein concentration was determined to be 0.84 mg/mL.

Table 3. Antibody detection enzyme-linked immunosorbent assay in the serum of mice immunized with recombinant protein and inactivated vaccine

4 Assessment of Immunogenicity

To evaluate the immunogenicity of MVSA, antibody detection enzyme-linked immunosorbent assay was performed with the collected recombinant protein and sera from mice immunized with inactivated vaccine. As shown in Table 3, compared with unimmunized mice, the total immunoglobulin content in the serum of MVSA hyperimmune mice was extremely high, with the lowest titer being 1/25600. Table 3 shows that the lowest titer of total immunoglobulin in the serum of inactivated vaccine hyperimmune mice was 1/200 compared to unimmunized mice. The West blot analysis of the MVSA hyperimmune serum showed that there was a specific band around 55kDa as shown in the figure, which confirmed that the MVSA protein had good immunogenicity (Figure 7).

5 Mice challenge protection test

The experimental results showed that when challenged with 20LD ₅₀ of BAA-611 (2×10 ⁸ CFU/mouse), the mice in the MVSA recombinant vaccine group did not die, and had no symptoms for 7 days, and the protection rate was 100%; Compared with the mice in the BAA-611 inactivated vaccine group, the death of the mice in the BAA-611 inactivated vaccine group was reduced, and the protection rate was 58.33% (Fig. 8).

6Immune protection test

Serological tests showed that MVSA antibodies were produced after vaccination with the vaccine of the present invention. When challenged with 20×LD ₅₀ of BAA-611 (2×10 ⁸ CFU/mouse), 100% of the challenged mice survived the observation period in the MVSA antibody-treated group, while all mice in the PBS-treated group survived within 2 days died (Figure 9), indicating that rMVSA provided 100% protection to the mice.

7 In vitro antibacterial test

The results of the in vitro antibacterial test are shown in Figure 10. The MVSA antibody provided by the present invention can significantly inhibit the growth of 6 different serotype GBS strains, and the six popular serotypes (Ia, Ib, II, III, V, VI) cover GBS clinical infection 98.4% cases. At the same time, the hyperimmune serum of the BAA-611 inactivated vaccine can significantly inhibit the growth of BAA-611, but it has no obvious inhibitory effect on the other serotypes (Ib, II, III, VI), indicating that the BAA-611 inactivated vaccine induces The antibodies generated showed limited protection against cross-serotype strains, similar to previous studies.

8. Organ distribution and determination of cytokines

To evaluate the protective effect of MVSA antibody on GBS invasion, mice were injected with MVSA hyperimmune serum in advance, and the bacterial load and cytokine levels in different organs and tissues of mice were measured 9 h after challenge. Bacterial counts on different organs or tissues showed that after 20×LD ₅₀ BAA-611 challenge, the number of bacteria in the blood, liver and spleen of the mice in the MVSA antibody-treated group was less than that in the PBS-treated group (control group), while the two groups No GBS viable bacteria were counted in the brains of the mice (Fig. 11). The transcriptional levels of pro-inflammatory cytokines in different organs or tissues by RT-qPCR showed that the transcriptional levels of IL-1β, IL-6 and TNFα in the spleen of the MVSA antibody-treated group were significantly higher than those of the PBS-treated group. The transcript levels of TNFα in the liver of the MVSA antibody-treated group were significantly increased, except for IL-1β and IL-6. These data demonstrate that MVSA immune serum inhibits GBS invasion and colonization in vivo.

The protection content of the present invention is not limited to the above embodiments. Variations and advantages that can occur to those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the appended claims are the scope of protection.

sequence listing

<110> Nanjing Agricultural University

<120> A multi-epitope subunit vaccine against all GBS serotypes

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Gly Pro Gly Pro Gly Leu Arg Met Lys Leu Pro Lys Ser Asn Ser Thr

50 55 60

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

65 70 75 80

Ile Pro Val Val Arg Leu Lys Val Gly Pro Gly Pro Gly Leu Arg Met

85 90 95

Lys Leu Pro Lys Ser Ile Val Lys Asn Asp Val Leu Ala Ala Met Ser

100 105 110

Pro Gln Ala Ala Met Ser Pro Gln Ala Ala Glu Ala Pro Val Glu Thr

115 120 125

Lys Ala Thr Pro Thr Thr Gly Pro Gly Pro Gly Leu Arg Met Lys Leu

130 135 140

Pro Lys Ser Gly Val Met Asp Ala Ile Val Lys Gln Pro Gly Val Lys

145 150 155 160

Ser Ile Ile Gly Gly Gly Asp Gly Pro Gly Pro Gly Leu Arg Met Lys

165 170 175

Leu Pro Lys Ser Ala Ala Glu Thr Pro Ala Pro Val Ala Lys Val Ala

180 185 190

Pro Val Arg Thr Val Ala Ala Pro Arg Val Ala Gly Pro Gly Pro Gly

195 200 205

Leu Arg Met Lys Leu Pro Lys Ser Ile Val Ile Val Ala Gly Val Pro

210 215 220

Val Gly Thr Gly Gly Thr Asn Thr Met Arg Val Arg Thr Val Lys Gly

225 230 235 240

Pro Gly Pro Gly Leu Arg Met Lys Leu Pro Lys Ser Lys Glu Leu Gln

245 250 255

Ala Lys Asn Val Lys Ala Ile Val Val Leu Ala His Val Pro Ala Thr

260 265 270

Ser Gly Pro Gly Pro Gly Leu Arg Met Lys Leu Pro Lys Ser Ser His

275 280 285

Phe Asn Leu Phe Lys Ala Ile Lys Gly Arg Ala Thr Val Glu Ala Asp

290 295 300

Val Cys Val Gln Asn Ile Glu Gly Pro Gly Pro Gly Leu Arg Met Lys

305 310 315 320

Leu Pro Lys Ser Phe Lys Thr Asn His Phe Ser Leu Phe Ala Ile Lys

325 330 335

Thr Leu Ser Lys Asp Gln Asn Val Thr Gly Pro Gly Pro Gly Leu Arg

340 345 350

Met Lys Leu Pro Lys Ser Ile Asn Gly Phe Gly Arg Ile Gly Arg Leu

355 360 365

Ala Phe Arg Arg Ile Leu Asp Gly Pro His Arg Gly Gly Asp Leu Arg

370 375 380

Arg Ala Arg Ala Gly Ala Ala Asn Gly Pro Gly Pro Gly Leu Arg Met

385 390 395 400

Lys Leu Pro Lys Ser Lys Asp Phe Leu Phe Asn Pro Ser Glu Thr Leu

405 410 415

Gln Gln Glu Asn Phe Pro Leu Arg Asp Gly Gln Thr Lys Glu

420 425 430

Claims (10)

1. A multi-epitope chimeric protein MVSA for all GBS serotypes, wherein the amino acid sequence of the multi-epitope chimeric protein MVSA is shown in SEQ ID No: 1. 2. A gene encoding the multi-epitope chimeric protein MVSA of claim 1 against all GBS serotypes. 3. An expression cassette, expression vector, transgenic cell line or recombinant engineering bacteria containing the gene of claim 2. 4. A vaccine against all GBS serotypes comprising the multi-epitope chimeric protein of claim 1. 5. The vaccine according to claim 4, wherein the vaccine is a subunit vaccine; preferably, the vaccine further comprises a subunit vaccine adjuvant, more preferably, the subunit vaccine adjuvant is seppic ISA206. 6. a preparation method for the multi-epitope subunit vaccine of all GBS serotypes, is characterized in that, comprises the steps: (1) Computer prediction of GBS epitopes: determine candidate proteins covering all 10 serotypes of GBS; perform T/B cell epitope prediction, and then screen out T and B cell epitopes by multiple sequence alignment the polypeptide epitope; (2) Construction of MVSA protein: add a Linker fragment "LRMKLPKS" to the N-terminus of each epitope, and insert a second spacer GPGPG between each pair of Linker epitope sequences; connect the epitopes of step (1) in series and combine Construction of polypeptide chimera MVSA; (3) Construction of a recombinant plasmid: using the amplified product as a template, amplify and obtain the MVSA protein gene, which is connected to the pET-28a(+) plasmid to obtain a recombinant plasmid; (4) MVSA protein gene expression: The constructed recombinant plasmid was transformed into BL21 competent cells, and MVSA protein was obtained after culture, extraction and purification. 7. The method for preparing a multi-epitope subunit vaccine against all GBS serotypes according to claim 6, wherein in the step (1), the epitopes are respectively derived from GBS self-protein NT5, BKD -E2, PK, GAPDH, PGK, Srr1, FbsA, Sip, AP1-2b, Beta C protein, BibA. 8. The multi-epitope subunit vaccine obtained by the method for preparing a multi-epitope subunit vaccine for all GBS serotypes according to claim 6 or 7. 9. Any of the following applications: (1) Application of the multi-epitope chimeric protein of claim 1 as or in the preparation of subunit vaccines against all GBS serotypes; (2) The application of the multi-epitope chimeric protein of claim 1 as or in the preparation of a drug for preventing infection of all GBS serotypes; (3) Use of the gene of claim 2, the expression cassette, expression vector, transgenic cell line or recombinant engineering bacteria of claim 3 in the preparation of the chimeric protein MVSA of claim 1. 10. A vaccine or drug against all GBS serotypes, the active ingredient of which is the multi-epitope chimeric protein MVSA of claim 1.

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