Novel carrier protein and application thereof

Abstract

The invention relates to the technical field of immunity, and discloses a novel carrier protein and application thereof. A novel carrier protein is designed, the amino acid sequence of the novel carrier protein is shown as SEQ ID NO: 1, the novel carrier protein is obtained by coding nucleotide shown as SEQ ID NO: 2, the ACYW135 group meningococcus polysaccharide conjugate vaccine prepared by using the carrier protein has the characteristics of simplicity and high efficiency, and the prepared ACYW135 group meningococcus polysaccharide conjugate vaccine has a good effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology, in particular to a novel carrier protein and application thereof. BACKGROUND

[0002] Epidemic cerebrospinal meningitis (meningitis) caused by Neisseria meningitidis (Nm) is a global acute respiratory infectious disease. The current meningitis vaccines mainly include capsular polysaccharide vaccine and polysaccharide-protein conjugate vaccine. The capsular polysaccharide vaccine has several defects, first, the polysaccharide with repetitive structure is a T cell independent type 2 immunogen, without the participation of T cells, it cannot induce immune memory effect, and the antibodies produced by the body are mainly IgM and IgG2, which cannot effectively activate the complement system; second, more importantly, this vaccine cannot induce immune response in infants less than 2 years old to prevent the disease, and this population is a high-risk group for infectious diseases due to the incomplete development of the immune system. In contrast, the polysaccharide-protein conjugate vaccine formed by the covalent combination of polysaccharide and protein molecules can convert non-T cell dependent polysaccharide antigens into T cell dependent antigens, effectively stimulate immune memory and enhance immune response, and is suitable for all age groups.

[0003] At present, the polysaccharide-protein conjugate vaccines marketed at home and abroad mostly use CRM197 (non-toxic diphtheria variant strain toxoid), TT (tetanus toxoid) or DT (diphtheria toxoid) as carrier proteins. However, these carriers have certain limitations: for example, although CRM197 is a mutant of diphtheria toxin, it is difficult to express in Escherichia coli, and the related patents cover a wide range, limiting its application. The production process of TT and DT is complex and costly; and toxicity reversal or contamination may occur during toxoid treatment.

[0004] At present, polysaccharide-protein conjugate vaccines are developing rapidly, while the development of carrier proteins used is relatively slow, which to some extent restricts the development of conjugate vaccines. The main carrier protein used at present is tetanus toxoid (TT), which has a large molecular weight and obvious steric hindrance, resulting in low conjugation efficiency of polysaccharide and TT, and the yield of conjugate is only 10% to 20%.

[0005] Therefore, it is necessary to design a novel carrier protein. SUMMARY

[0006] To solve the above problems, the present application first provides a novel carrier protein, and the amino acid sequence of the carrier protein is shown in SEQ ID NO: 1.

[0007] Preferably, the carrier protein is obtained by encoding the nucleotide shown in SEQ ID NO: 2.

[0008] The second object of the present application is to provide an application of the new carrier protein in preparing ACYW135 group meningococcal polysaccharide conjugate vaccine.

[0009] Compared with the prior art, the present application has the following beneficial effects: (1) The present application obtains a new protein which can be used for preparing ACYW135 group meningococcal polysaccharide conjugate vaccine.

[0010] (2) The present application adopts E. coli expression system, and the culture conditions of the strain of the new carrier protein are simple, without the need of special factory building and the introduction of formaldehyde and other substances for detoxification, thereby reducing the introduction of exogenous toxic substances and the production cost of the vaccine.

[0011] (3) The present application adopts E. coli expression system constructed by high expression plasmid, such as pET30a-BL21 DE3 expression system, so that the protein expression amount is increased, the molecular structure is clear, and the storage is stable.

[0012] (4) The present application can show good immune effect by adopting the new carrier protein to prepare ACYW135 group meningococcal polysaccharide conjugate vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The electropherogram and Western Blot diagram of the new carrier protein.

[0014] Figure 2 The SEC-MALLS diagram of polysaccharide protein conjugate.

[0015] Figure 3 The antibody titer of mice immunized by polysaccharide-new carrier protein conjugate and polysaccharide-TT conjugate; Figure 4 The SBA of mice immunized by the ACYW135 group meningococcal polysaccharide conjugate vaccine prepared by the present application and the marketed ACYW135 group meningococcal polysaccharide conjugate vaccine. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0017] Example 1 The plasmid for expressing the new carrier protein is constructed, and the specific steps are as follows: (1) The gene sequence (SEQ ID NO: 2) encoding the carrier protein of the application was synthesized by the Bioengineering (Shanghai) Co., Ltd. and connected to the pET30a (+) vector to directly obtain the pET30a (+)-TTHc-Cys869Asp plasmid.

[0018] (2) The pET30a (+)-TTHc-Cys869Asp plasmid was transformed into E. coli DH5α competent cells and coated on an LB plate containing 50 μg / ml of kanamycin (Kanamycin). Positive colonies were picked and inoculated in LB-Kan culture solution for amplification. The pET30a (+)-TTHc-Cys869Asp plasmid was extracted and the gene sequence (SEQ ID NO: 2) was verified by sequencing.

[0019] Example 2 The expression and purification of the novel protein specifically included the following steps: (1) The pET30a (+)-TTHc-Cys869Asp plasmid prepared in Example 1 was transformed into BL21 DE3 competent cells and inoculated into a 50 μg / ml kanamycin LB culture dish and incubated at 37°C in a CO2 incubator until colonies grew.

[0020] (2) The colonies were scraped and inoculated into a 40 liter LB culture solution containing 50 μg / ml kanamycin in a fermenter at an initial OD 600 of about 0.05 for further incubation. When the OD 600 reached about 20, 0.2 mmol / L IPTG (isopropyl-β-D-thiogalactoside) was added to induce protein expression. The induction was stopped when the OD 600 was greater than 35. The bacterial cells were collected by centrifugation at 14000 rpm using a tubular centrifuge. When the bacteria were harvested, 0.1 ml of bacterial solution was centrifuged at 12000 rpm for 1 minute. The precipitate was suspended in 1 ml of 10 mmol / L Tris-HCl. Protein expression electrophoresis and Western Blot were performed, and the results are shown in Figure 1 .

[0021] (3) The bacteria were suspended in 10 mmol / L Tris-HCl, 1 mmol / L EDTA (pH 8.0) solution. The bacteria were broken by a high-pressure homogenizer (15,000 psi, about 100 MPa). The supernatant was collected by centrifugation at 14000 g and 4°C for 25 minutes.

[0022] (4) 1 mol / L Tris-HCl pH 8.0 was added to the supernatant obtained in step (3) to adjust the pH of the supernatant to about 8.2. 20 mmol / L Tris-HCl pH 8.0 was added to dilute the conductivity to 2.5 mS / cm.

[0023] NaMic Gel 50Q elution conductivity test: A solution is 20 mmol / L Tris-HCl pH 8.0, B solution is 20 mmol / L Tris-HCl pH 8.0 + 0.5 mol / L NaCl, when 7% B solution, conductivity is 4.7 mS / cm; when 8% B solution, conductivity is 5.2 mS / cm.

[0024] NaMic Gel 80Q chromatography: flow rate is 50 mL / min, PreC pressure limit is 0.3 MPa, after equilibration for 5 column volumes, sample loading is performed, after sample loading, elution is performed for 5 column volumes, and then 8% B solution is used to elute the target protein. In the harvested eluate, 1.34 mol / L (NH4)2SO4 is added.

[0025] Elution conductivity test by hydrophobic column UniHR PhenyL-30L: A solution is 20 mmol / L Tris-HCl pH 8.0 + 0.67 mol / L (NH4)2SO4, B solution is 20 mmol / L Tris-HCl pH 8.0, when 32.5% B solution, conductivity is 75 mS / cm; when 62% B solution, conductivity is 45 mS / cm. The final protein purification solution is harvested.

[0026] Example 3 ACYW135 group meningococcal polysaccharide is prepared according to the following specific steps: (1) Take 1 branch of working seed batch of A group, C group, Y group or W135 group Neisseria meningitidis (purchased from China Institute for Food and Drug Control, A group strain preservation number is 29201, C group strain preservation number is 29205, Y group strain preservation number is 29028, and W135 group strain preservation number is 29037) from the biosafety cabinet, resuspend with EF1 solution, and uniformly coat on the surface of GC solid culture medium (BD company, 228950), and invert in a CO2 constant temperature incubator at 37.0°C, 5% CO2 for 16 hours to recover the strain.

[0027] Table 1 EF-A solution formula After the reagent is fully dissolved with water for injection at room temperature, 1 mol / L NaOH is used to adjust the pH to 7.4, and high-pressure sterilization is performed at 121°C for 15 min, and then it is cooled for standby use.

[0028] Table 2 EF1-B solution formula Yeast extract was prepared into 200 g / L, ultrafiltrated with 3 kDa ultrafiltration membrane, after ultrafiltration, CTAB (cetyltrimethylammonium bromide) was added to the yeast ultrafiltrate to a concentration of 1%, and whether a precipitate was observed; after it was determined that no precipitate was produced, the absorbance value of the yeast ultrafiltrate was detected at 405 nm using a spectrophotometer, according to the detection results, using the principle that the concentration of yeast liquid is linear with the absorbance value at 405 nm, a standard curve was established, the absorbance value of the yeast ultrafiltrate at 405 nm was substituted into the standard curve to obtain the corresponding concentration. Then after adding glucose and MgSO4·7H2O, water for injection was used to make up the volume, 0.22 µm filter membrane was used to filter and sterilize, and it was divided and stored for standby use at 2-8 °C.

[0029] Table 3 EF1 solution formula When preparing the EF1 medium, 1 volume of EF1-B solution was added to 25 volumes of high-pressure sterilized EF-A solution.

[0030] (2) The recovered strain was inoculated into 200 mL of EF1 liquid medium at an initial OD 600 = 0.07, and cultured at 37 °C, 160 rpm on a shaker for 9 hours for primary amplification; after the primary amplification was completed, it was inoculated into 4 bottles of 1 L of EF1 liquid medium at an initial OD 600 = 0.07, and continued to be cultured at 37 °C, 160 rpm on a shaker for 9 hours to complete secondary amplification.

[0031] (3) After completing the secondary amplification, the strain was inoculated into 36 L of EF1 liquid medium at a ratio of 1:10 for primary fermentation, the fermentation temperature was set to 36.5 °C, the pH was 7.0, the rotation speed was 100 rpm, the dissolved oxygen was 30%, and the ventilation volume was adjusted according to the dissolved oxygen. Primary fermentation was carried out for 4 hours; after the primary fermentation was completed, the fermentation liquid was inoculated into 160 L of EF1 liquid medium at a ratio of 1:4 for secondary fermentation, and the fermentation conditions were the same as above. For W135 group, 2.5 g / L of glucose was added at the 3rd and 4th hours of fermentation, and for Y group, 2.5 g / L of glucose was added at the 5th and 7th hours of fermentation. The sugar production was used as an index at the end of secondary fermentation: the fermentation liquid was centrifuged to remove bacteria, and the supernatant was taken, 10% cetyltrimethylammonium bromide solution was added to the supernatant, the amount of cetyltrimethylammonium bromide solution added was 1% of the volume of the supernatant, and the absorbance OD 405 value was measured, and a value greater than 1.0 was the harvest standard.

[0032] (4) When the sugar production reaches the harvest standard, sample the fermentation broth for pure bacteria inspection and gram staining microscopic examination. After passing the inspection, add 1.0% (v / w) formaldehyde solution to the fermentation broth, stir for 30 minutes, sterilize at 36.5°C and 100 rpm for 30 minutes, centrifuge the sterilized culture solution at low temperature (2-8°C) by a disc centrifuge, collect the supernatant, filter it with a 0.45 μm PES filter core, and collect the filtrate.

[0033] (5) Add 10% cetyltrimethylammonium bromide solution to the collected supernatant, with the addition amount of the cetyltrimethylammonium bromide solution being 1% of the volume of the supernatant. After stirring at 4°C and 70 rpm for 30 minutes, stand still at 2-8°C for 10 hours, and centrifuge the precipitate collected at 2-8°C by a tubular centrifuge.

[0034] (6) Weigh the CTAB precipitate, grind it into granules, add 3L of 0.2 mol / L calcium chloride solution (1 kg of CTAB precipitate) pre-cooled at 2-8°C, and stir at 700 rpm for 5 hours to dissociate the polysaccharide from the CTAB. After dissociation, centrifuge at 4°C and 13600g for 45 minutes to remove the precipitate, collect the supernatant, add 2-8°C pre-cooled anhydrous ethanol to the supernatant to a final concentration of 250 ml / L, stand still at 2-8°C for 18 hours, and centrifuge twice (4°C, 13600g, 45 minutes) to collect the supernatant.

[0035] (7) Add 2-8°C pre-cooled anhydrous ethanol to the supernatant obtained in step (6) to a final concentration of 75%, mix thoroughly, stand still at 2-8°C for 30 minutes, centrifuge at 4°C and 500g for 10 minutes, discard the supernatant, mix the precipitate with 2-8°C pre-cooled anhydrous ethanol thoroughly, centrifuge again, repeat twice, collect the precipitate, wash and centrifuge the precipitate with pre-cooled acetone again, repeat twice, obtain the precipitate, scrape the precipitate into a mortar, grind it into powder, store it in a vacuum dryer at -20°C for more than 48 hours to obtain the crude polysaccharide product.

[0036] (8) Dissolve the polysaccharide crude product in 2-8°C water for injection to 5 mg / mL, add 1% (W / V) sodium deoxycholate dry powder, stir thoroughly for 4 hours, centrifuge at 4°C, 13600g for 45 minutes, collect the supernatant, use 100 kDa membrane ultrafiltration to concentrate to 1 / 4 of the original volume, add water for injection to the original volume, ultrafiltrate to a conductivity of ≤1 μS / cm, collect the concentrated solution, dilute the concentrated solution to 5 mg / mL, add (NH4)2SO4 dry powder (final concentration 31.7 g / L), mix, add one quarter of the volume of No. II extraction solution (Triton X-114 / ammonium sulfate solution), stir at 2-8°C for 45 minutes, 18°C water bath for 30 minutes, 20°C, 15000g centrifuge for 45 minutes, stand for 10 minutes to separate the layers, draw the supernatant, repeat this step 4 times. Finally, the supernatant is ultrafiltrated with a 100 kDa membrane to remove residual (NH4)2SO4, and repeatedly supplemented with water and ultrafiltrated to a conductivity of ≤1 μS / cm to obtain a concentrated solution. The concentrated solution is added with NaCl dry powder (final concentration 29.25 g / L), and pre-cooled anhydrous ethanol is added to a final concentration of 75%, mixed, and stood at 2-8°C for 1 hour. Centrifuge at 4°C, 500g for 10 minutes to collect the polysaccharide precipitate, wash with ethanol and acetone, and centrifuge twice, grind the precipitate into a vacuum dryer, vacuumize, and store at -20°C for 48 hours or more to obtain the polysaccharide refined product, which is required to be stored below -20°C, and the storage period is not more than 12 months.

[0037] Table 4 No. II extraction solution formula Dissolve with pyrogen-free water for injection, and then dilute to 1000 ml. Store at 2-8°C.

[0038] (9) Dissolve the polysaccharide refined product with water for injection (10 mg / mL), filter sterilize, and obtain the monovalent polysaccharide stock solution.

[0039] Example 4 Preparation of ACYW135 group meningococcal polysaccharide conjugate vaccine with a novel carrier protein (1) Use a microfluidic device to lyse the monovalent polysaccharide solution prepared in the example, control the parameters to reduce the average molecular weight of the polysaccharide to about 100 kDa, and after the lysis is completed, the polysaccharide solution is concentrated by an ultrafiltration device (such as a 100 kDa cutting membrane) to obtain a polysaccharide lysate with a target molecular weight range.

[0040] (2) The protein prepared in Example 2 is dissolved in a buffer (20 mmol / L MES buffer, 0.15 mol / L NaCl, pH 6.5) to obtain a protein solution with a concentration of 5 mg / mL. 5 times the mass of the protein is added with adipohydrazide (ADH) dry powder. Hydrochloric acid (HCl) is used to adjust the pH to 5.6±0.2. Carbon diimide (EDAC) dry powder is slowly added under stirring. The final concentration of EDAC is 10 mg / mL. The reaction is carried out at room temperature for 45 min under the condition of pH 5.6±0.2 (adjusted with HCl at any time). After the reaction, the pH is adjusted to 6.8±0.2 with NaOH solution to terminate the reaction. Free ADH and small molecule impurities are removed by ultrafiltration to obtain an ADH protein derivative for use in the conjugation reaction.

[0041] (3) Conjugation of A group meningococcal polysaccharide with the protein derivative: The polysaccharide lysate obtained in step (1) is dissolved in 100 mmol / L triethanolamine buffer (pH 9.0) to 10 mg / mL at 4°C in an ice bath. CDAP (N-cyano-4-dimethylamino-pyridine tetrafluoroborate) solution is slowly added dropwise under magnetic stirring. The mass ratio of CDAP to polysaccharide is 0.2:1 (for example: 1 mg of polysaccharide plus 0.2 mg of CDAP). Mix quickly and continue stirring for 3 min to allow CDAP to react with the hydroxyl group of the polysaccharide to form an activated intermediate (cyanate). Immediately add 4°C pre-cooled 1 mol / L Tris-HCl buffer to rapidly reduce the pH to 7.1 to terminate the activation. Immediately mix the above "activated polysaccharide solution" with an equal volume of ADH protein derivative solution (5 mg / mL, PBS buffer) and monitor in real time with a pH meter. Slowly add sterile 1 mol / L NaOH solution to adjust the pH to 8.5. React slowly under 4°C rotation or gentle stirring for 45 min. After the reaction, block with 0.1 mol / L glycine for 45 min to terminate the reaction.

[0042] (4) Conjugation of C, Y or W135 group meningococcal polysaccharide with the protein derivative: The purified ADH protein derivative and the polysaccharide lysate obtained in step (1) are mixed in a mass ratio of 1:1 (protein derivative: polysaccharide) and dissolved in a buffer (such as 20 mmol / L MES (2-morpholinoethanesulfonic acid), pH 6.0). Adjust the pH to 5.6±0.2 with HCl. Add EDAC dry powder to a final concentration of 10 mg / mL. Maintain the pH at 5.6±0.2 with HCl during the reaction. React at room temperature for 45 min. After the reaction, adjust the pH to 6.8±0.2 with NaOH solution to terminate the coupling reaction.

[0043] (5) The reaction solution obtained from step (3) and step (4) is subjected to Sepharose 4 Fast Flow gel chromatography, eluted with physiological buffer (such as 0.9% NaCl), and the high-molecular-weight conjugate component is collected to remove unbound polysaccharide and protein and impurities. The collected conjugate can be further concentrated and sterilized by ultrafiltration or sterile filtration, and the final product is the A, C, Y or W135 group meningococcal polysaccharide-new carrier protein conjugate. The SEC-MALS detection spectrum of the prepared polysaccharide conjugate is as follows Figure 2 As shown in the figure, the A, C, Y or W135 group meningococcal polysaccharide-carrier protein conjugate all have a high molecular weight, with a molecular weight range of 7000-10000 kDa and a retention time of 12-15 minutes, which is consistent with the macromolecular structure characteristics of the polysaccharide conjugate.

[0044] (6) The A, C, Y or W135 group meningococcal polysaccharide-carrier protein conjugate prepared in step (5) is prepared into a lyophilized vaccine according to the following formula: A group meningococcal polysaccharide-carrier protein conjugate: 10ug; C group meningococcal polysaccharide-carrier protein conjugate: 10ug; Y group meningococcal polysaccharide-carrier protein conjugate: 5ug; W135 group meningococcal polysaccharide-carrier protein conjugate: 5ug; Sodium chloride: 3mg; Lactose: 15mg. The dilution, aliquotting and lyophilization processes are all carried out using known methods.

[0045] The A, C, Y or W135 group meningococcal polysaccharide-carrier protein conjugate prepared in the above examples is prepared into a lyophilized vaccine according to the following formula: A group meningococcal polysaccharide-carrier protein conjugate: 10ug; C group meningococcal polysaccharide-carrier protein conjugate: 10ug; Y group meningococcal polysaccharide-carrier protein conjugate: 5ug; W135 group meningococcal polysaccharide-carrier protein conjugate: 5ug; Sodium chloride: 3mg; Sucrose: 10mg; Mannitol: 20mg. The dilution, aliquotting and lyophilization processes are all carried out using known methods.

[0046] Example 5 Comparison of IgG immunogenicity of polysaccharide-carrier protein conjugate vaccine and polysaccharide-TT conjugate vaccine Healthy 6-8 week old BALB / c mice were selected and randomly divided into two groups, 5 mice in each group. Group 1 (new carrier protein vaccine group): each mouse was subcutaneously immunized with 1 / 4 dose of polysaccharide-carrier protein conjugate vaccine (containing 2.5 μg, 2.5 μg, 1.25 μg, 1.25 μg of meningococcal polysaccharide of A, C, Y, W135 group, respectively). Group 2 (TT carrier group): each mouse was subcutaneously immunized with 2.5 μg of polysaccharide-TT conjugate vaccine (containing 2.5 μg, 2.5 μg, 1.25 μg, 1.25 μg of meningococcal polysaccharide of A, C, Y, W135 group, respectively). The immunization program was to immunize once at 0 week and 2 weeks. One week after the second immunization (i.e. at 3 weeks), the mouse orbital blood was collected, and the serum was separated. ELISA method was used to detect the IgG antibody titer of the mouse serum against A, C, Y, W135 group polysaccharide by coating the microplate with the corresponding polysaccharide. The results are as follows Figure 3 As shown in the table, the geometric mean of the serum IgG antibody titer of the mice against each group of polysaccharide was calculated, and the experimental results showed that there was no significant difference in IgG antibody titer between the two groups.

[0047] Example 6 Comparison of SBA immunization effect of carrier protein vaccine and commercially available CRM197 conjugate vaccine Healthy 6-8 week old BALB / c mice were selected and randomly divided into two groups, 5 mice in each group. Group 1 (new carrier protein vaccine group): each mouse was subcutaneously immunized with the ACYW135 group meningococcal polysaccharide-carrier protein conjugate vaccine prepared by the present unit, each dose containing 2.5 μg, 2.5 μg, 1.25 μg, 1.25 μg of polysaccharide of A, C, Y, W135 group. Group 2 (commercial vaccine group): each mouse was subcutaneously immunized with the commercially available ACYW135 group meningococcal polysaccharide conjugate vaccine using CRM197 as the carrier, with the same composition as group 1 (2.5 μg, 2.5 μg, 1.25 μg, 1.25 μg of polysaccharide of A, C, Y, W135 group, respectively). Both groups were immunized once every 2 weeks, for a total of 3 times. One week after the third immunization (week 7), the mouse serum was collected. Using meningococcal strain 29019 of A group, 29026 of C group, 29028 of Y group, and 29055 of W135 group as target bacteria, the serum bactericidal antibody (SBA) test method was used to detect the serum bactericidal antibody titer of each group of mice against each group of meningococcal. The results are as follows Figure 4 As shown in the table, the geometric mean of the serum IgG antibody titer of the mice against each group of polysaccharide was calculated, and the experimental results showed that there was no significant difference in IgG antibody titer between the two groups.

Claims

1. A novel carrier protein characterized in that: The amino acid sequence of the carrier protein is shown as SEQ ID NO:

1.

2. A novel carrier protein as claimed in claim 1, wherein: The novel carrier protein is encoded by the nucleotide shown as SEQ ID NO:

2.

3. Use of the novel carrier protein according to any one of claims 1-2 in the preparation of a meningococcal polysaccharide conjugate vaccine against serogroup ACYW135.

Images