CN106692963B - Combined vaccine for preventing staphylococcus aureus infection and tetanus - Google Patents
Combined vaccine for preventing staphylococcus aureus infection and tetanus Download PDFInfo
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Abstract
The invention discloses a bivalent immunogen combined vaccine for preventing staphylococcus aureus infection and tetanus. The components of the combined vaccine are recombinant staphylococcus aureus surface protein A (SasA) and tetanus neurotoxin C fragment (TeNT-Hc). The antibody response induced by the combined immunization of the SasA and the TeNT-Hc in mice is larger than that of a single component, the combined immunization has a synergistic effect, and the combined vaccine shows higher protection in a mouse staphylococcus aureus and tetanus toxin challenge model.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and immunology, and particularly relates to a bivalent immunogen composition containing Staphylococcus aureus surface protein A (SasA) and tetanus neurotoxin C fragment (C fragment of tetanus neurotoxin, TeNT-Hc) and application thereof in resisting infection of Staphylococcus aureus and tetanus.
Background
Staphylococcus aureus and tetanus are widely distributed in nature and any trauma patient with an open wound has the potential to infect both pathogens. Staphylococcus aureus can cause skin and soft tissue infections, as well as fatal sepsis and invasive complications. Tetanus is caused by the obligate anaerobic bacterium tetanus, and the produced neurotoxin can invade the nervous system, causing systemic muscle spasm and even asphyxiation death. Although the incidence of tetanus is low in developed countries, the mortality rate for tetanus is 6-72% worldwide. In addition, neonates and parturients are also susceptible to staphylococcus aureus infections and tetanus during childbirth in developing countries with limited health care settings.
Combination vaccines are a common strategy for simultaneously controlling infection by two or more pathogens, which can simplify vaccination protocols, increase user compliance, and reduce vaccination costs. However, no research on combination vaccines against staphylococcus aureus infection and tetanus has been reported. The combined vaccine for resisting staphylococcus aureus infection and tetanus is suitable for vulnerable people including athletes, soldiers and policemen, and women of childbearing age in developing countries.
Staphylococcus aureus surface protein A (SasA), a cell wall anchoring protein of Staphylococcus aureus, has 2,271 amino acids. The SasA recombinant protein immunized mice can protect lethal toxicity attack of staphylococcus aureus. In addition, the SasA gene is ubiquitous in clinical strains, and the SasA protein is also expressed in vivo during Staphylococcus aureus infection. Tetanus toxin has a molecular weight of 150kDa and has A, B, C three domains, an N-terminal endopeptidase domain, a translocation heavy chain domain and a C-terminal receptor-binding heavy chain structure (tetanus neurotoxin C-fragment, TeNT-Hc). It has been shown that recombinant TeNT-Hc, which has ganglioside binding activity, can replace the existing tetanus toxoid vaccine (TT). The TeNT-Hc is superior to the tetanus toxoid vaccine in safety, production convenience and uniformity.
These preliminary studies indicate that the recombinant proteins SasA and TeNT-Hc can serve as candidate vaccine components for Staphylococcus aureus and tetanus. In view of simplifying the vaccination scheme, increasing user compliance, reducing vaccination cost and increasing vaccine protection rate, there is a need in the art for a combination vaccine that can effectively prevent staphylococcus aureus infection and tetanus at the same time, but no combination vaccine with significant synergistic effects has been found at present. The combination vaccine does not equal the random mixing of any vaccine, and the following factors need to be considered for preparing the combination vaccine: (1) whether the immunogenic response effects of various antigens are affected; (2) whether there is incompatibility or interference between different antigens; (3) whether antagonistic effects between different antigenic components will occur; (4) whether the mixing ratio of other components of the vaccine (such as adjuvant and the like) and various antigens is proper or not. In addition, the dosage form of the combination vaccine, such as a liquid or lyophilized powder, and the duration of time after mixing, can affect the stability of the vaccine.
It is unknown whether the combination of SasA and TeNT-Hc has antagonism, i.e., whether the antibody response and protection produced in vivo interfere with each other. The invention aims to provide a bivalent combined vaccine for preventing staphylococcus aureus infection and tetanus and a preparation method thereof, wherein the bivalent combined vaccine has obvious synergistic effect and higher immune protection rate.
Disclosure of Invention
In accordance with the above objects, the present invention provides a bivalent immunogen combination vaccine for protecting a host against diseases caused by staphylococcus aureus and/or tetanus, the vaccine comprising staphylococcus aureus surface protein a and tetanus neurotoxin C fragment.
In a preferred technical scheme, the staphylococcus aureus surface protein A and the tetanus neurotoxin C fragment are both gene recombinant proteins.
In a more preferred technical scheme, the Staphylococcus Aureus surface protein A (SasA) is a truncated protein, and the protein is a sequence of a target fragment which is designed by amplifying 142-999bp according to a reported sequence of Staphylococcus Aureus MASA252 strain (GenBank: BX571856.1), and is an amino acid segment from 48 th to 333 th positions of the full-length protein. The nucleotide sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.
More preferably, the amino acid sequence of the tetanus neurotoxin C fragment (TeNT-Hc) is shown in SEQ ID NO.4, and the nucleotide sequence of the tetanus neurotoxin C fragment is analyzed and optimized according to the sequencing result (GeneBank sequence number: AF154828) of the domestic Clostridium tetani C.tetani virulent strain 64008 strain, and the optimized sequence is shown in SEQ ID NO. 3.
Preferably also, in the combination vaccine, the staphylococcus aureus surface protein a and tetanus neurotoxin C fragment are present as separate components.
In a preferred technical scheme, the weight ratio of the staphylococcus aureus surface protein A to the tetanus neurotoxin C fragment is 1: 1.
In a more preferred embodiment, both the S.aureus surface protein A and the tetanus neurotoxin C fragment are adsorbed on aluminium hydroxide.
In a further preferred embodiment, the weight ratio of the aluminum hydroxide to the staphylococcus aureus surface protein a and the tetanus neurotoxin C fragment is 75:1: 1.
In a further preferred embodiment, the vaccine is prepared as a capsule, a lyophilisate or an injection.
In a preferred technical scheme, the staphylococcus aureus surface protein A and the tetanus neurotoxin C fragment recombinant protein are respectively prepared into capsules, and each capsule comprises the components of the recombinant protein, N-acryloxysuccinimide (NAS), acrylamide (AAm) and methylene bisacrylamide (Bis) according to the combination ratio of 1: 20: 3000: 400, after which each capsule was adsorbed again by aluminium hydroxide.
The invention also provides a method for preparing the combined vaccine, which comprises the following steps: and mixing the staphylococcus aureus surface protein A and the tetanus neurotoxin C fragment.
The combined vaccine immunization of the SasA and the TeNT-Hc provided by the invention induces antibody response in mice more than a single component. Combined immunization (SasA + TeNT-Hc) induced SasA IgG titers at weeks 2, 4 and 6 of mice receiving immunization that were 3.083-fold (p ═ 0.0210), 3.013-fold (p ═ 0.0053) and 3.365-fold (p ═ 0.0077), respectively, that of SasA monocomponent immunization. The titer of TeNT-Hc IgG induced by the combined immunization (SasA + TeNT-Hc) is 2.183 times (p is 0.0342), 2.594 times (p is 0.0124) and 2.377 times (p is 0.0089) that of TeNT-Hc single-component immunization, respectively, which shows that the antibody response induced by the combined immunization of SasA and TeNT-Hc has synergistic effect. The combination vaccine also showed higher protection in mouse s.aureus and tetanus toxin challenge models. The combined immunization of SasA single component (p ═ 0.0467) and TeNT-Hc + SasA (p ═ 0.0001) has protection for mice, and the protection effect of the combined immunization is higher than that of the SasA single component (p ═ 0.0417), indicating that the combined immunization of TeNT-Hc + SasA has synergistic effect on the protection of Staphylococcus aureus infection. In addition, the combination immunity of the TeNT-Hc single component (p <0.0001) and the TeNT-Hc + SasA (p <0.0001) has protection on mice, and the protection effect of the combination immunity is higher than that of the TeNT-Hc single component, which indicates that the combination immunity of the TeNT-Hc + SasA also has synergistic effect on the protection of tetanus. Moreover, the SasA and TeNT-Hc combined vaccine provided by the invention is modified by the nanocapsule, so that the heat stability is further improved, and the protective titer is also remarkably improved.
Drawings
FIG. 1 is a histogram of the levels of IgG antibodies induced in mice by the combination of SasA and TeNT-Hc + SasA vaccines;
FIG. 2 is a histogram of IgG antibody levels induced in mice by the combination vaccine of TeNT-Hc and TeNT-Hc + SasA;
FIG. 3 is a graph of the protective survival of the combination SasA and TeNT-Hc + SasA vaccine against a mouse model of S.aureus infection;
FIG. 4 is a graph of the protective survival of a combination vaccine of TeNT-Hc and TeNT-Hc + SasA in a mouse tetanus model;
FIG. 5 is a histogram of the antibody level of SasAIgG produced by TeNT-Hc + SasA and nanocapsule combination vaccine induction;
FIG. 6 is a histogram of the level of TeNT-Hc antibody induced by TeNT-Hc + SasA and nanocapsule combination vaccine
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.
Example 1 preparation of recombinant S.aureus truncated SasA protein
For the acquisition of the truncated SasA protein from recombinant staphylococcus aureus see chinese patent application CN102268444A, the disclosure of CN102268444A is incorporated by reference into the present specification, and the preparation method is briefly described below.
1. According to the reported sequence of Staphyloccus Aureus MASA252 strain (GenBank: BX571856.1), the primer is designed to amplify the sequence of a target fragment of 142-999bp, and the amino acid sequence of the target fragment is shown as SEQ ID NO.2 and is the 48 th to 333 th amino acid segment of the full-length protein. NdeI and XhoI restriction sites were added to both ends of the SasA sequence, respectively, and the stop codon was removed, and a sequence encoding 6 histidines was introduced to the 3' end.
2. The amplified SasA gene was digested with NdeI and XhoI, ligated to expression vector pET21a (+) digested with NdeI and XhoI, transformed into E.coli competent cell DH5 α, and cultured overnight at 37 ℃. The next day, a single clone was picked up and cultured in 5mL of LB (Amp +) medium at 37 ℃ and 220rpm for 12 hours, and the plasmid was extracted and sequenced correctly and named pET21 a-SasA.
3. Escherichia coli expression and Western-blot identification of staphylococcus aureus truncated SasA
Transformation of E.coli competent cell BL with the vector pET21a (+) correctly ligated to the SasA Gene21(DE3) Selecting a single clone, culturing in 5mL LB (Amp +) liquid medium at 37 deg.C and 220rpm to OD600nm ≡ 0.6, adding IPTG with final concentration of 1mM, and culturing at 28 deg.C and 220rpm for 6 h. The cells were collected at 5,000g by centrifugation at 4 ℃ and resuspended in PBS and then sonicated to fragment the cells. And centrifuging at 12,000g and 4 ℃, taking supernatant, performing SDS-PAGE electrophoresis, identifying the expression of the SasA protein with the size of 40kDa, and verifying that the protein can be specifically combined with the mouse anti-His tag monoclonal antibody by a Western-felt result.
4. 1L of the seed solution expressing the SasA protein was transferred to a 30L fermentor, and cultured until OD600nm ≡ 0.6, then IPTG with a final concentration of 1mM was added, and the culture was continued at 37 ℃ and 300rpm for 6 h. The cells were collected by centrifugation at 4 ℃ at 10,000g, resuspended in 20mM Tris-HCl buffer (pH8.5), and then disrupted by homogenization. The supernatant was collected by centrifugation at 4 ℃ at 20,000 g. The supernatant is passed through QFF column for anion exchange, and the effluent is treated with 20mM NaH2PO40.5M NaCl (pH7.4) was diluted 3-fold and purified by a nickel column using 20mM NaH2PO4Continuous gradient elution was performed with 0.5M NaCl, 0.5M imidazole (pH 7.4). After two-step purification, the target protein SasA is well purified, the purity can reach more than 85%, and the target protein is finally stored in a PBS buffer solution for later use in immune experiments.
Example 2 preparation of recombinant tetanus Neurotoxin C fragment (TeNT-Hc)
The recombinant TeNT-Hc protein is obtained in Chinese patent CN101880675B, the disclosure of CN101880675B is incorporated into the specification of the invention in a cited way, and the preparation method is briefly described below.
1. According to the sequencing result (GeneBank sequence number: AF154828) of the domestic clostridium tetani C.tetani virulent strain 64008 strain, the Tet-Hc sequence of 451Aa is analyzed and optimized, the optimized sequence is shown as SEQ ID NO.3, and the whole gene synthesis is carried out.
2. Construction of tetanus toxin subunit vaccine Hc expression vector
After the optimized and synthesized Tet-Hc gene is cut by EcoRI and XhoI, the gene is connected to an expression vector pET32a (+) cut by EcoRI and XhoI, and then transformed into an escherichia coli competent cell DH5 alpha, and cultured overnight at 37 ℃. The next day, the single clone is picked up and cultured in 5mLLB (Amp +) culture medium for 12h at 37 ℃ and 220rpm, plasmids are extracted, EcoRI and XhoI are subjected to double enzyme digestion to identify the insertion of the target gene, and the sequence is sent. The plasmid with the correct sequencing was designated as pET32 a-Tet-Hc.
3. Escherichia coli expression and Western-blot identification of tetanus toxin recombinant subunit vaccine Hc
The pET32a (+) vector of the Tet-Hc gene is correctly connected and transformed into the escherichia coli competent cell BL21(DE3) Selecting single clone, culturing in 5mLLB (Amp +) liquid medium at 37 deg.C and 220rpm to OD600nm ≡ 0.6, adding IPTG with final concentration of 0.2mM, and culturing at 28 deg.C and 220rpm for 6 hr. The cells were collected at 5,000g by centrifugation at 4 ℃ and resuspended in PBS and then sonicated to fragment the cells. The supernatant was centrifuged at 4 ℃ at 12,000g, and SDS-PAGE was performed to identify the expression of Hc protein with a size of 50 KD. Western-blot results prove that the protein can be specifically combined with a murine anti-tetanus monoclonal antibody.
4. Escherichia coli fermentation and purification of tetanus toxin subunit vaccine Hc
1L of the seed solution expressing Hc protein was transferred to a 30L fermentor, and when the seed solution was cultured to OD600nm ≡ 0.6, IPTG was added to the final concentration of 0.2mM, and the culture was continued at 28 ℃ and 300rpm for 6 hours. 10,000g of a total of 10,000g,the cells were collected by centrifugation at 4 ℃ and resuspended in 20mM Tris-HCl buffer (pH8.5) to homogenize the cells. The supernatant was collected by centrifugation at 4 ℃ at 20,000 g. The supernatant was anion-exchanged by QFF column, equilibrated with 20mM Tris-HCl buffer (pH8.5), and eluted with a gradient of 0-0.5M NaCl in Tris-HCl buffer. The eluate containing the target protein was added with (NH) at a final concentration of 0.5M4)2SO4Then, the product was purified further by a phenyl hydrophobic column. The target protein is (NH) with concentration of 0.5-0M4)2SO4The gradient elution is carried out by using Tris-HCl buffer solution. The eluate containing the target protein was subjected to cation exchange with SP column after replacing the buffer with 20mM NaAc (pH4.0) by desalting column, and the target protein was subjected to gradient elution with 20mM NaAc (pH4.0) buffer containing 0-0.5M NaCl. After three-step purification, the target protein Hc without the label is well purified, the purity can reach more than 95%, the yield is more than 300mg/L, and the target protein is finally stored in PBS buffer solution for later use in the following immune experiment.
Example 3 immunogenicity Studies of SasA and TeNT-Hc combination vaccines
The following antigens were adsorbed separately with 0.75mg of aluminium hydroxide adjuvant: 10 μ grSasA, 10 μ g TeNT-Hc, 10 μ grSasA +10 μ g TeNT-Hc. 0.75mg of aluminum hydroxide adjuvant without adsorbed antigen was used as a negative control. At weeks 0, 2 and 4, the four groups of antigens were used to immunize female BALB/c mice (age 6-8 weeks) with 10 individuals per group by intraperitoneal injection. Blood samples were taken and serum was extracted at tail vein at weeks 2, 4 and 6.
The serum antibody titers of the antigen-specific IgG were analyzed by enzyme-linked immunosorbent assay (ELISA). Mu.g/ml of rSasA or TeNT-Hc were coated onto the 96-well plate overnight at 4 ℃ in 50mM carbonate buffer (pH 9.6). Blocking was performed with PBS containing 2% (w/v) bovine serum albumin at 37 ℃ for 1 hour. The gradient diluted serum was added to the enzyme-linked plate, incubated at 37 ℃ for 1 hour, followed by 3 washes with PBST (PBS containing 0.05% Tween 20). Horseradish peroxidase-labeled goat anti-mouse IgG antibody was added, incubated at 37 ℃ for 1 hour, and PBST washed 3 times. TMB (3,3',5,5' -tetramethylbenzidine dihydrochloride) substrate was added and incubated for 10 minutes in the dark. The color reaction was stopped with 2M sulfuric acid and read in a microplate readerTaking the light absorption value at 450nm (A)450). Antibody Positive reaction set to A450Greater than twice the mean value of the non-immune sera. ELISA antibody titers were expressed as the highest serum dilution showing a positive reaction. Statistical analysis used a two-sided unpaired t-test.
At weeks 2, 4 and 6, combined immunizations (SasA + TeNT-Hc) induced SasA igg titers that were 3.083-fold (p 0.0210), 3.013-fold (p 0.0053) and 3.365-fold (p 0.0077), respectively, that of SasA monocomponent immunizations (fig. 1). Combined immunization (SasA + TeNT-Hc) induced TeNT-Hc IgG titers that were 2.183-fold (p 0.0342), 2.594-fold (p 0.0124) and 2.377-fold (p 0.0089) that of TeNT-Hc monocomponent immunization, respectively (fig. 2). The results show that the antibody response induced by the combined immunization of SasA and TeNT-Hc has synergistic effect.
Example 4 protective Studies of a combination TeNT-Hc + SasA vaccine in a mouse model of S.aureus infection
Staphylococcus aureus strain USA300 was cultured overnight, diluted 1:100 in TSB medium, and cultured to mid-log phase at 37 ℃. Mice immunized 3 times in the abdominal cavity were challenged 6 weeks after the initial immunization at a dose of 3X 109USA300 of CFU. Survival of mice was observed over a period of 5 days and survival curves were analyzed using the log rank Mantel-Cox test. As a result, all mice in the control group died, and both the SasA monocomponent (p ═ 0.0467) and the combination immunization of TeNT-Hc + SasA (p ═ 0.0001) were protective to the mice, and the protective effect of the combination immunization was higher than that of the SasA monocomponent (p ═ 0.0417) (fig. 3). The result shows that the combined immunity of TeNT-Hc + SasA has synergistic effect on the protection of staphylococcus aureus infection.
Example 5 protective Studies of a combination TeNT-Hc + SasA vaccine in a mouse tetanus model
Subcutaneous injection of 2X 10 6 weeks after primary immunization3LD50Tetanus neurotoxin of (2) mice immunized 3 times. Survival of mice was observed over a period of 5 days and survival curves were analyzed using the log rank Mantel-Cox test. The result is that the control mice all died, TeNT-Hc monocomponent (p)<0.0001) and TeNT-Hc + SasA (p)<0.0001) protective against mice (FIG. 4), and combined protective effects of immunizationHigher than that of the TeNT-Hc single component, but the survival rate of the mice is higher than 90 percent, and the mice have no difference statistically. The results show that the combined immunization of TeNT-Hc + SasA has synergistic effect on the protection of tetanus.
Example 6 Heat stability Studies of SasA and TeNT-Hc nanocapsule combination vaccines
Example 5 it has been confirmed that a recombinant subunit combination vaccine of SasA and TeNT-Hc, which can be prepared for use as a solution injection, a lyophilizate, etc., has a good protective effect against staphylococcus aureus infection and tetanus. However, conventional vaccines administered in these dosage forms have poor thermostability and, in the absence of the cold chain, the antigenicity and effectiveness of the vaccine can be greatly affected. The invention tries to use the nanocapsule for preparing the vaccine, and can improve the stability of the vaccine at normal temperature, thereby potentially reducing the transportation and storage cost of the vaccine.
The SasA and TeNT-Hc nanocapsules are respectively called n (SasA) and n (TeNT-Hc), and the preparation method is as follows:
the SasA or TeNT-Hc recombinant proteins were diluted in 50mM borate buffer (pH8.5) and co-incubated with N-acryloyloxysuccinimide (NAS) for 2 hours at room temperature (molar ratio of each recombinant protein to NAS 1: 20). Then adding acrylamide (AAm) and methylene bisacrylamide (Bis) (the molar ratio of each recombinant protein to AAm and Bis is 1: 3000: 400), adding a proper amount of ammonium persulfate and tetramethylethylenediamine to initiate free radical polymerization, reacting at room temperature for 1 hour, and dialyzing and changing the solution to PBS buffer solution.
SasA + TeNT-Hc and n (SasA) + n (TeNT-Hc) mixed in equal proportions are stored at 37 ℃ for 7 days, and then adsorbed with an aluminum hydroxide adjuvant. Female BALB/c mice (age 6-8 weeks) were immunized with the above 2 groups of antigens by intraperitoneal injection at weeks 0 and 2, respectively, with 10 mice per group. Blood was taken and serum was extracted at the tail vein at week 4. Serum antibody titers of antigen-specific IgG were analyzed by enzyme-linked immunosorbent assay (ELISA) as described previously. The SasA igg titer and the TeNT-Hc titer induced by the nanocapsule combination vaccine n (SasA) + n (TeNT-Hc) are respectively 27.86 times (p <0.001) and 32.00 times (p <0.001) of the immunity of the SasA + TeNT-Hc (fig. 5, 1 is SasA + TeNT-Hc in fig. 5, 2 is n (SasA) + n (TeNT-Hc)) and 32.00 times (p <0.001) (fig. 6, 1 is SasA + TeNT-Hc in fig. 6, and 2 is n (SasA) + n (TeNT-Hc)). The result shows that the heat stability of the combined vaccine of the SasA and the TeNT-Hc is obviously improved by the modification of the nano capsule.
The nano capsule is a technology for modifying biological macromolecules by nano materials, and can polymerize a high-molecular protective layer on the surface of recombinant protein. The experimental result of the embodiment 6 proves that the SasA and TeNT-Hc nano-capsule combined vaccine provided by the invention has stronger stability, and the protective titer of the TeNT-Hc + SasA combined vaccine can be further improved.
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tataccagct acctgtctat caccttcctg cgtgacttct ggggtaaccc gctgcgttac 780
gacaccgaat attacctgat cccggtagct tacagctcta aagacgttca gctgaaaaac 840
atcactgact acatgtacct gaccaacgcg ccgtcctaca ctaacggtaa actgaacatc 900
tactaccgac gtctgtacag cggcctgaaa ttcatcatca aacgctacac tccgaacaac 960
gaaatcgatt ctttcgttcg ctctggtgac ttcatcaaac tgtacgtttc ttacaacaac 1020
aacgaacaca tcgttggtta cccgaaagac ggtaacgctt tcaacaacct ggacagaatc 1080
ctaagagtag gttacaacgc tccgggtatc ccgctgtaca aaaaaatgga agctgttaaa 1140
ctgcgtgacc tgaaaaccta ctctgttcag ctgaaactgt acgacgacaa agatgcttct 1200
ctgggtctgg ttggcaccca caacggtcag atcggtaacg acccgaaccg tgacatcctg 1260
atcgcttcta actggtactt caaccacctg aaagacaaaa ccctgacctg cgactggtac 1320
ttcgttccga ccgatgaagg ttggaccaac gac 1353
<210> 4
<211> 451
<212> PRT
<213> Clostridium tetani
<400> 4
Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile
1 5 10 15
Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile
20 25 30
Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala
35 40 45
Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn
50 55 60
Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn
65 70 75 80
Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys
85 90 95
Val Ser Ala Ser His Leu Glu Gln Tyr Asp Thr Asn Glu Tyr Ser Ile
100 105 110
Ile Ser Ser Met Lys Lys Tyr Ser Leu Ser Ile Gly Ser Gly Trp Ser
115 120 125
Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala
130 135 140
Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Ser Asp Lys Phe Asn
145 150 155 160
Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp Arg
165 170 175
Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser Ala
180 185 190
Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu
195 200 205
Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys
210 215 220
Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu
225 230 235 240
Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn
245 250 255
Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Tyr Ser
260 265 270
Ser Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr
275 280 285
Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg
290 295 300
Leu Tyr Ser Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn
305 310 315 320
Glu Ile Asp Ser Phe Val Arg Ser Gly Asp Phe Ile Lys Leu Tyr Val
325 330 335
Ser Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn
340 345 350
Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro
355 360 365
Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu
370 375 380
Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asp Ala Ser
385 390 395 400
Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn
405 410 415
Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu Lys Asp
420 425 430
Lys Thr Leu Thr Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp
435 440 445
Thr Asn Asp
450
Claims (4)
1. A bivalent immunogen combined vaccine is used for protecting a host against diseases caused by staphylococcus aureus and/or tetanus bacillus, and is characterized by comprising a gene recombinant protein staphylococcus aureus surface protein A and a gene recombinant protein tetanus neurotoxin C fragment, wherein the staphylococcus aureus surface protein A is a truncated protein, the amino acid sequence of the protein is shown as SEQ ID NO.2, the amino acid sequence of the tetanus neurotoxin C fragment is shown as SEQ ID NO.4, the staphylococcus aureus surface protein A and the tetanus neurotoxin C fragment are adsorbed by aluminum hydroxide, and the weight ratio of the aluminum hydroxide to the staphylococcus aureus surface protein A to the tetanus neurotoxin C fragment is 75:1: 1.
2. Combination vaccine according to claim 1, characterized in that the vaccine is prepared as a capsule, a lyophilisate or an injection.
3. The vaccine of claim 2, wherein the staphylococcus aureus surface protein a and tetanus neurotoxin C fragment recombinant proteins are separately prepared as capsules, each capsule consisting of recombinant protein, N-acryloxysuccinimide, acrylamide, methylene bisacrylamide in a combination ratio of 1: 20: 3000: 400, after which each capsule was adsorbed again by aluminium hydroxide.
4. A process for preparing the combination vaccine of claim 1, wherein the S.aureus surface protein A and tetanus neurotoxin C fragment are combined.
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| CN201611236043 | 2016-12-28 | ||
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| CN109651491B (en) * | 2019-01-16 | 2021-02-02 | 上海赛伦生物技术股份有限公司 | Immunization method capable of improving titer of immunoglobulin for resisting tetanus of horses |
| CN114366809A (en) * | 2022-01-12 | 2022-04-19 | 广东粤港澳大湾区国家纳米科技创新研究院 | Aluminum nanocrystal delivery system and combined vaccine antigen molecule self-assembled particle adjuvant vaccine thereof |
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| GB0204387D0 (en) * | 2002-02-26 | 2002-04-10 | Secr Defence | Screening process |
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