Chemically modified tetanus toxin C fragment as well as preparation and application thereof

Abstract

The invention provides a chemically modified tetanus toxin C fragment as well as preparation and application thereof. The method for chemically modifying the tetanus toxin C fragment comprises the following steps: 1) reacting the tetanus toxin C fragment with a reducing agent to reduce a disulfide bond in the tetanus toxin C fragment; and 2) carrying out a reaction on the reduced tetanus toxin C fragment and an alkylation reagent, and carrying out modification. The recombinant tetanus toxin C fragment prepared by the method disclosed by the invention not only meets the quality standard required by pharmacopeia, but also has the advantages of stable conformation, high yield, easiness in amplification and low cost, and has a wide application prospect in the preparation of human vaccines, veterinary vaccines and antibody drugs and the development process of in-vitro diagnostic reagents.

Description

Technical Field

[0001] The invention belongs to the field of gene engineering technology and pharmaceutical technology, and relates to the production of recombinant protein drugs using gene engineering technology, and specifically relates to a chemically modified tetanus toxin C fragment and its preparation and application. Background Art

[0002] Tetanus, also known as ankylosing spondylosis and commonly known as lockjaw, is an acute toxic zoonosis caused by Clostridium tetani through wound infection. The main clinical symptoms are clenched jaws and paroxysmal or tonic spasms of local or systemic muscles.

[0003] After Clostridium tetani infects a wound, it rapidly multiplies and produces an exotoxin, tetanus neurotoxin (tetanus toxin for short). Tetanus toxin (TT) is one of the most toxic toxins known. It is a protein composed of 1315 amino acids and has a molecular weight of about 150KDa. The TT initially expressed by Clostridium tetani in vivo is an inactive single polypeptide chain. During the secretion process, TT is cleaved by proteases into two heavy chains and light chains connected by a single disulfide bond. The light chain (A fragment) has a molecular size of 50KDa and is the active part of the toxin. The heavy chain contains two domains of 50KDa each. The C-terminus of the heavy chain (C fragment) is the ganglioside binding region, which recognizes and binds to the gangliosides on the outer cytoplasmic membrane of the motor neuron at the neuromuscular junction, prompting the toxin to enter the synaptic vesicles formed by the cell membrane in the cell. Synaptic vesicles move retrogradely from peripheral nerve endings along nerve axons to the cell bodies of motor neurons. Through trans-synaptic movement, vesicles enter the afferent nerve endings from motor neurons and finally enter the central nervous system. The N-terminus (B fragment) of the heavy chain is a migration domain, which forms an ion channel in the lipid bilayer and transports the catalytic structure (light chain) to the cytosol of neurons. The active light chain interacts with the SNARE protein complex in the cytosol of neurons, inhibiting the release of acetylcholine, thereby blocking nerve conduction, causing an imbalance between the excitation and inhibition of muscle activity, and causing tonic spasms.

[0004] From the mechanism of action of TT, it can be seen that TT binds to receptors on the surface of neurons through the C fragment (TTc for short), and this binding is a prerequisite for the tetanus toxin fragments A and B to exert their toxic effects. A large number of studies have shown that the immune activity of TTc is equivalent to that of the whole fragment of TT, and it is significantly superior to traditional tetanus toxoids in terms of preparation process, safety, and yield, making TTc a key candidate antigen for new tetanus vaccines or combined vaccines. It is also used in the development and research of polysaccharide conjugate vaccines in the form of high-quality carrier proteins.

[0005] So far, a large number of studies have successfully used E. coli expression and elution to achieve recombinant expression of TTc in a soluble form. However, since the TTc protein contains four cysteines, it is very easy to form intra-chain or inter-chain disulfide bonds during protein expression, freezing and thawing, and storage, leading to the formation of aggregates, which poses a great challenge to protein quality control. Summary of the invention

[0006] The purpose of the present invention is to provide a chemically modified tetanus toxin C fragment and its preparation and application, so as to solve the problems of poor stability and low yield of TTc protein in the prior art. Based on the problem of TTc conformational stability, the present invention intends to adopt a chemical modification method to develop a purification process for the preparation of TTc protamine, and evaluate the stability and immunogenicity of the modified TTc protein, laying the foundation for the carrier protein used in the later stage for tetanus subunit vaccine, tetanus multi-valent vaccine and polysaccharide conjugate vaccine.

[0007] In order to solve the above problems, the first aspect of the present invention provides a method for chemically modifying the tetanus toxin C fragment, characterized in that the method comprises the following steps:

[0008] 1) reacting the tetanus toxin C fragment with a reducing agent to reduce the disulfide bonds in the tetanus toxin C fragment; and,

[0009] 2) The reduced tetanus toxin C fragment is reacted with an alkylating agent for modification.

[0010] In some embodiments, the reducing agent in step 1) is selected from one or more of DTT (dithiothreitol), β-mercaptoethanol, TCEP (tris(2-carboxyethyl)phosphine), DTE (dithioerythritol) and GSH (reduced glutathione).

[0011] In some embodiments, the alkylating agent in step 2) is selected from one or more of iodoacetamide, chloroacetamide, N-ethylmaleimide and iodoacetic acid.

[0012] In some embodiments, the final concentration of the reducing agent is 1-1000 mM; preferably 5-20 mM.

[0013] The final concentration of the reducing agent refers to the concentration when the reducing agent reacts with the tetanus toxin C fragment.

[0014] In some embodiments, the final concentration of the alkylating agent is 1-1000 mM; preferably 5-30 mM.

[0015] The final concentration of the alkylating agent refers to the concentration of the alkylating agent when reacting with the reduced tetanus toxin C fragment.

[0016] In some embodiments, the recombinant tetanus toxin C fragment has an amino acid sequence as shown in SEQ ID NO:2.

[0017] In some embodiments, the recombinant tetanus toxin C fragment is encoded by the nucleic acid shown in SEQ ID NO:1.

[0018] In some embodiments, the method further comprises one or more of the steps of constructing an expression strain, fermentation, and crude purification.

[0019] A second aspect of the present invention provides a method for purifying a chemically modified tetanus toxin C fragment, the method comprising the following steps:

[0020] a) modifying the tetanus toxin C fragment using the method provided in the first aspect of the present invention; and,

[0021] b) subjecting the modified tetanus toxin C fragment obtained in step a) to chromatography purification, wherein the chromatography purification preferably comprises the steps of column equilibration, sample loading, washing and elution.

[0022] In some embodiments, the chromatographic purification conditions in step b) are selected from one or more of the following:

[0023] Ⅰ. The balancing solution used for the column balancing, or the washing, or the elution is PB with a pH of 6-8, such as 7.5, or Tris buffer with a pH of 6-10, such as 8.5; the balancing solution is preferably 20-50 mmol / L Tris with a pH of 8.5, containing 1 mmol / L EDTA and 0.6 mol / L (NH4)2SO4; or 20-50 mmol / L Tris with a pH of 8.5, containing 1 mmol / L EDTA and 0.46 mol / L (NH4)2SO4; or 20-50 mmol / L Tris with a pH of 8.5, containing 1 mmol / L EDTA and 3.5 mol / L (NH4)2SO4;

[0024] Ⅱ. The column chromatography procedure is equilibration for 3CV, sample loading, elution for 3CV, elution for 2-5CV, water washing for 3CV, alkaline washing for 3CV, and equilibration for 3CV again;

[0025] III. The sample loading amount is 3-10 mg of target protein / mL of filler; and,

[0026] IV. The chromatographic medium is selected from a hydrophobic chromatographic filler with phenyl as a ligand, a hydrophobic chromatographic filler with butyl as a ligand or a hydrophobic chromatographic filler with octyl as a ligand, preferably a hydrophobic chromatographic filler with butyl as a ligand, such as Polar MC30 HICButyl.

[0027] In some embodiments, an equilibration solution is required in the column equilibration step; in some embodiments, an equilibration solution is required in the washing step; and in some embodiments, an equilibration solution is required in the elution step.

[0028] In some embodiments, the method further comprises one or more of the following:

[0029] 1) Purifying the tetanus toxin C fragment before step a), for example, by chromatography purification; preferably, performing two chromatography purifications;

[0030] 2) the purity of the tetanus toxin C fragment in step a) is greater than 60%, such as greater than 80%, preferably greater than 90%; and,

[0031] 3) A step of concentrating and replacing the eluate of step b).

[0032] The inventors found that when the purity of the tetanus toxin C fragment is greater than a specific value, or the purity of the tetanus toxin C fragment is first improved and then alkylated, the non-specific binding with the target protein can be reduced, the specificity of the modification can be improved, the interference of impurities on the modification reaction can be reduced, the modification efficiency can be improved, and the effect of downstream applications, such as immunological experiments, structural analysis, etc., can be improved. The method of improving the purity of the tetanus toxin C fragment is not limited, and any method that can improve the purity of the tetanus toxin C fragment can be used.

[0033] In some embodiments, the Tetanus Toxin C fragment is greater than 65%, 70%, 75%, 80%, 85%, 90% or 95% pure.

[0034] In some embodiments, the tetanus toxin C fragment is purified by chromatography before step a), and the chromatography purification conditions are selected from one or more of the following:

[0035] Ⅰ. The balancing solution used for the column balancing, or the washing, or the elution is PB with a pH of 6-8, such as 7.5, or Tris buffer with a pH of 6-10, such as 8.5; the balancing solution is preferably 20-50 mmol / L Tris with a pH of 8.5, containing 1 mmol / L EDTA and 0.6 mol / L (NH4)2SO4; or 20-50 mmol / L Tris with a pH of 8.5, containing 1 mmol / L EDTA and 0.46 mol / L (NH4)2SO4; or 20-50 mmol / L Tris with a pH of 8.5, containing 1 mmol / L EDTA and 3.5 mol / L (NH4)2SO4;

[0036] Ⅱ. The column chromatography procedure is equilibration for 3CV, sample loading, elution for 3CV, elution for 2-5CV, water washing for 3CV, alkaline washing for 3CV, and equilibration for 3CV again;

[0037] III. The sample loading amount is 3-10 mg of target protein / mL of filler; and,

[0038] IV. The chromatographic medium is selected from anion exchange fillers with phenyl as diethylaminoethyl as ligand and / or hydrophobic chromatographic fillers with phenyl as ligand, such as DEAE Sepharose FF, DEAE Bestarose FF, Phenyl Sepharose HP or Phenyl Bestarose HP.

[0039] In some embodiments, the tetanus toxin C fragment is subjected to two chromatography purifications before step a), the chromatography medium for the first purification is an anion exchange filler with diethylaminoethyl as a ligand, preferably DEAE Sepharose FF or DEAE Bestarose FF; and / or, the chromatography medium for the second purification is a hydrophobic chromatography filler with phenyl as a ligand, preferably Phenyl Sepharose HP or Phenyl Bestarose HP.

[0040] In some embodiments, ultrafiltration membranes are used for concentration and replacement; and / or, the buffer used for replacement contains boric acid-sodium tetraborate and NaCl.

[0041] In some embodiments, the pore size of the ultrafiltration membrane is 3-10 KDa, preferably 3 KDa.

[0042] In some embodiments, the method comprises the following steps:

[0043] (1) a first step of purification, wherein the crude sample of the tetanus toxin C fragment obtained by fermentation and crude treatment is subjected to the first step of purification, and a first step of purification eluate sample is collected, wherein the chromatography medium of the first step of purification is an anion exchange filler with a diethylaminoethyl group as a ligand;

[0044] (2) subjecting the eluate sample obtained in step (1) to a second purification step, and collecting the second purified eluate sample, wherein the chromatography medium for the second purification step is a hydrophobic chromatography filler having phenyl as a ligand;

[0045] (3) alkylating the eluate sample obtained in step (2) to obtain an alkylated sample of the second-step purified eluate, wherein the alkylating agent is an olefin, a halogenated hydrocarbon, or the like;

[0046] (4) performing a third step purification on the alkylation-modified sample of the second step purified eluate obtained in step (3), and collecting the third step purified eluate sample, wherein the chromatography medium of the third step purification is a hydrophobic chromatography filler with butyl as a ligand; and,

[0047] (5) Concentrating and replacing the third-step purified eluate obtained in step (4) to harvest protamine.

[0048] In some embodiments, the chromatography medium used in the first step of purification is DEAE Sepharose FF or DEAE Bestarose FF.

[0049] In some specific embodiments, the chromatography conditions of the first step of purification are selected from one or more of the following:

[0050] Equilibration buffer: 20-50 mmol / L Tris at pH 8.5, containing 1 mmol / L EDTA, 5 mmol / L DTT and 27 mmol / L NaCl;

[0051] Elution buffer: 20-50 mmol / L Tris at pH 8.5, containing 1 mmol / L EDTA, 5 mmol / L DTT and 68 mmol / L NaCl;

[0052] Salt wash buffer: 1.5 mol / L NaCl solution;

[0053] Alkaline washing buffer: 0.5 mol / L NaOH solution;

[0054] Column chromatography procedure: balance 3CV, sample loading, elution 3CV, elution 2-4CV, salt wash 3CV, regeneration alkaline wash 3CV, re-balance 5CV; and,

[0055] Sample loading amount: 3-10 mg target protein / mL filler.

[0056] In some embodiments, the chromatography medium for the second purification step is Phenyl Sepharose HP or Phenyl Bestarose HP.

[0057] In some specific embodiments, the chromatography conditions of the second step of purification are selected from one or more of the following:

[0058] Equilibrium buffer: 20-50 mmol / L Tris at pH 8.5, containing 1 mmol / L EDTA, 5 mmol / L DTT and 0.6 mol / L (NH4)2SO4;

[0059] Sample conditioning solution: 20-50mmol / L Tris at pH 8.5, containing 1mmol / L EDTA, 5mmol / L DTT and 3.5mol / L (NH4)2SO4;

[0060] Elution buffer: 20-50 mmol / L Tris at pH 8.5, containing 1 mmol / L EDTA, 5 mmol / L DTT and 0.3 mol / L (NH4)2SO4;

[0061] Washing buffer: water for injection;

[0062] Alkaline washing buffer: 0.5 mol / L NaOH solution;

[0063] Column chromatography procedure: balance 3CV, sample loading, rinse 3CV, elution 2-4CV, water wash 3CV, alkaline wash 3CV regeneration, and balance 3CV; and,

[0064] Sample loading amount: 3-10 mg target protein / ml filler.

[0065] In some embodiments, the alkylating modifying reagent is iodoacetamide.

[0066] In some specific embodiments, the alkylation modification conditions are selected from one or more of the following:

[0067] Reducing agent: 5-15 mmol / L DTT;

[0068] Alkylating blocking agent: 5-30 mmol / L iodoacetamide; and,

[0069] Alkylation modification step: add 5-15mmol / L DTT solution to the second step purified eluate, stir evenly, and react at room temperature with a closed mouth for 10-180min; then add 5-30mmol / L iodoacetamide solution, stir evenly, and react at room temperature with a closed mouth for 6-15h in the dark.

[0070] In some embodiments, the chromatography medium for the third step of purification is Polar MC30 HIC Butyl.

[0071] In some specific embodiments, the chromatography conditions of the third step of purification are selected from one or more of the following:

[0072] Equilibrium buffer: 20-50 mmol / L Tris at pH 8.5, containing 1 mmol / L EDTA and 0.6 mol / L (NH4)2SO4;

[0073] Sample conditioning solution: 20-50mmol / L Tris at pH 8.5, containing 1mmol / L EDTA and 3.5mol / L (NH4)2SO4;

[0074] Elution buffer: 20-50 mmol / L Tris at pH 8.5, containing 1 mmol / L EDTA and 0.46 mol / L (NH4)2SO4;

[0075] Washing buffer: water for injection;

[0076] Alkaline washing buffer: 0.5 mol / L NaOH solution;

[0077] Column chromatography procedure: balance 3CV, sample loading, elution 3CV, elution 2-5CV, water wash 3CV, alkaline wash 3CV, balance 3CV again; and,

[0078] Sample loading amount: 3-10 mg target protein / ml filler.

[0079] In some embodiments, the third step of concentrating and replacing the purified eluate is performed by using an ultrafiltration membrane package.

[0080] In some specific embodiments, the third step of purification, eluent concentration, replacement conditions are selected from one or two of the following:

[0081] Membrane pore size: 3~10KDa; and,

[0082] Replacement buffer: 0.2 mol / mol boric acid-sodium tetraborate, containing 0.9% NaCl.

[0083] In some embodiments, the recombinant tetanus toxin C fragment has an amino acid sequence as shown in SEQ ID NO:2.

[0084] In some embodiments, the recombinant tetanus toxin C fragment is encoded by the nucleic acid shown in SEQ ID NO:1.

[0085] In some embodiments, the method further comprises one or more of the steps of constructing an expression strain, fermentation, and crude purification.

[0086] In some embodiments, the construction of the expression strain comprises one or more of the following steps:

[0087] (1) performing codon optimization of tetanus toxin C; the codon optimization is performed according to the preference of Escherichia coli; preferably, the nucleotide sequence after codon optimization is as shown in SEQ ID NO: 1;

[0088] (2) synthesizing the nucleic acid shown in SEQ ID NO:1, wherein the nucleotide sequence of the nucleic acid encodes the amino acid sequence shown in SEQ ID NO:2;

[0089] (3) inserting the nucleic acid shown in SEQ ID NO: 1 into an expression vector through the restriction site to obtain a recombinant plasmid; preferably, the expression vector is pET30a;

[0090] (4) transforming the recombinant plasmid containing the nucleic acid fragment as shown in SEQ ID NO: 1 into a host strain, and screening to obtain transformants; preferably, the host strain is BL21 (DE3) or a mutant strain of BL21 (DE3); further preferably, the host strain is BL21 (DE3); and,

[0091] (5) Inoculate the transformant containing the nucleic acid fragment shown in SEQ ID NO: 1 into the culture medium for expansion to obtain a glycerol bacteria strain.

[0092] In some embodiments, the fermentation comprises one or more of the following steps:

[0093] (1) inoculating the glycerol bacteria into a shake flask medium and culturing overnight to obtain a primary seed solution; preferably, the shake flask culture conditions are 25-32° C. and 120-220 rpm overnight;

[0094] (2) inoculating the primary seed liquid into the basal medium, and starting the fermentation and feeding procedures; preferably, the fermentation parameters are 35-37° C., 100-600 rpm, aeration 15-25 L / h, and tank pressure 0.05 MPa; when OD 600nm When the value reaches 1 or above, batch feeding begins, once every hour, in stages;

[0095] (3) Cooling and inducing the bacteria in the logarithmic growth phase; preferably, the OD corresponding to the logarithmic growth phase is 600nm The value is 8 to 12, the temperature after cooling is 25 to 32°C, the inducer is isopropylthiogalactoside (IPTG) mother solution, and the final concentration of IPTG is 0.01 to 0.1 mmol / L; and,

[0096] (4) stopping fermentation and discharging the bacteria at the plateau stage; preferably, the OD corresponding to the plateau stage is 600nm The value is 15 to 35.

[0097] In some embodiments, the crude purification comprises one or more of the following steps:

[0098] (1) crushing the fermentation liquid to obtain a crushed sample; preferably, the crushing method is homogenous crushing, ultrasonic crushing or osmotic shock crushing; further preferably, the crushing is homogenous crushing, and the crushing condition is 800-1000 bar crushing 3 times under low temperature conditions;

[0099] (2) clarifying the obtained crushed sample to obtain a clarified flow-through sample; preferably, the clarification is performed using an ultrafiltration membrane package, and the pore size of the membrane package is 0.45 μm, 0.2 μm or 500 KDa; more preferably, the pore size of the membrane package is 500 KDa; and,

[0100] (3) Concentrating and replacing the obtained clarified flow-through sample to obtain a concentrated replacement collected liquid sample; the concentration and replacement is preferably carried out using an ultrafiltration membrane package, and the pore size of the membrane package is 10KDa, 8KDa, 5KDa or 3KDa; more preferably, the pore size of the membrane package is 3KDa.

[0101] The present invention also provides an application of a chromatography column combination in purifying a recombinant tetanus toxin C fragment, the chromatography column combination comprising:

[0102] (Ⅰ) DEAE Sepharose FF or DEAE Bestarose FF;

[0103] (II) Phenyl Sepharose HP or Phenyl Bestarose HP; and

[0104] (III) Polar MC30 HIC Butyl.

[0105] In some specific embodiments, the application includes an alkylation modification step, which is performed before using the chromatography column (I), or between using the chromatography columns (I) and (II), or between (II) and (III); preferably, the alkylation modification step is performed between using the chromatography columns (II) and (III).

[0106] The third aspect of the present invention provides the use of a kit for chemically modifying a tetanus toxin C fragment or purifying a chemically modified tetanus toxin C fragment; the kit comprises a reducing agent and an alkylating agent.

[0107] In some embodiments, the reducing agent is selected from one or more of DTT, β-mercaptoethanol, TCEP, DTE, and GSH.

[0108] In some embodiments, the alkylating agent is selected from one or more of iodoacetamide, chloroacetamide, N-ethylmaleimide, and iodoacetic acid.

[0109] The fourth aspect of the present invention provides a chemically modified tetanus toxin C fragment, wherein the tetanus toxin C fragment is modified by alkylation.

[0110] In some embodiments, the tetanus toxin C fragment is prepared by the method provided in the first aspect of the present invention, or by the method provided in the second aspect of the present invention.

[0111] The fifth aspect of the present invention provides a polysaccharide-carrier protein conjugate, which comprises the tetanus toxin C fragment provided in the fourth aspect of the present invention and a polysaccharide, wherein the tetanus toxin C fragment is coupled to the polysaccharide; the polysaccharide is, for example, a capsular polysaccharide.

[0112] In some embodiments, the capsular polysaccharide is a pneumococcal capsular polysaccharide, and the pneumococcal capsular polysaccharide is selected from one or more of type 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F polysaccharides.

[0113] In some embodiments, the pneumococcal capsular polysaccharide is selected from type 12F and / or 19A polysaccharides.

[0114] The sixth aspect of the present invention provides a pharmaceutical composition, which comprises the tetanus toxin C fragment provided by the fourth aspect of the present invention or the polysaccharide-carrier protein conjugate provided by the fifth aspect of the present invention, and a pharmaceutically acceptable carrier.

[0115] The seventh aspect of the present invention provides the use of the tetanus toxin C fragment provided in the fourth aspect of the present invention in constructing an antigen-carrier protein conjugate or preparing an immune preparation.

[0116] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0117] The reagents and raw materials used in the present invention are commercially available.

[0118] The positive and progressive effects of the present invention are: the present invention proposes a chemically modified tetanus toxin C fragment and its preparation and application. The present invention optimizes codons and column chromatography to make the TTc protein yield reach 305 mg target protein / L fermentation broth. The purity of the alkylated modified refined protein is more than 99% after SDS-PAGE electrophoresis and capillary electrophoresis detection, and the purity is still more than 95% after repeated freezing and thawing and long-term storage at 2-8°C. There is no significant difference in immunogenicity with unmodified protein, indicating that the alkylated modified TTc of the present invention has broad application prospects in the field of recombinant tetanus vaccines, combined vaccines and vaccine carrier proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 It is the SDS-PAGE electrophoresis pattern of protamine in Examples 2 to 5.

[0120] The protamine-1 spectrum is the protamine prepared in Example 2, and 10 μg was loaded for both the reducing electrophoresis and the non-reducing electrophoresis.

[0121] The spectrum of protamine-2 is the protamine prepared in Example 3, lane 1 is the protamine before ultrafiltration substitution without adding any alkylating agent, lane 2 is the protamine after ultrafiltration substitution without adding any alkylating agent; lane 3 is the protamine before ultrafiltration substitution after alkylation modification by adding chloroacetamide, lane 4 is the protamine after ultrafiltration substitution after alkylation modification by adding chloroacetamide; lane 5 is the protamine before ultrafiltration substitution after alkylation modification by adding iodoacetamide, lane 6 is the protamine after ultrafiltration substitution after alkylation modification by adding iodoacetamide; all samples were subjected to reduction electrophoresis, and 10 μg was loaded.

[0122] The protamine-3 spectrum is the protamine prepared in Example 4, and 10 μg was loaded for both the reducing electrophoresis and the non-reducing electrophoresis.

[0123] The protamine-4 spectrum is the protamine prepared in Example 5, with 5 μg of protamine loaded in lane 1 and 10 μg of protamine loaded in lane 2; all samples were subjected to reduction electrophoresis.

[0124] Figure 2 This is a purity test chart of TTc protamine-3 under reducing electrophoresis conditions and non-reducing electrophoresis conditions.

[0125] Figure 3 This is a stability test chart of protamine-3 (alkylated modification) and protamine-1 (non-alkylated modification) after repeated freeze-thaw at -80°C and storage at 2-8°C for 7 days. DETAILED DESCRIPTION

[0126] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0127] Example 1 Fermentation of TTc recombinant expression strain

[0128] The TTc amino acid sequence published in GenBank AF154828.1 was codon-optimized according to the codon preference of Escherichia coli to obtain the nucleic acid sequence shown in SEQ ID NO: 1. The synthesized nucleotide sequences were connected to the pET30a vector by chemical synthesis to obtain the pET30a-TTc recombinant plasmid, which was transformed into DH5α competent cells. The bacterial liquid plasmid with correct sequencing was extracted using a kit, and the plasmid was then transformed into BL21 (DE3) competent cells. The plasmid was extracted again, and the strain was preserved after correct sequencing. The strain was named BL21 (DE3) pET30a-TTc and stored at -80°C.

[0129] Take 2.0mL of glycerol bacteria and evenly inoculate them into four 1L sterile LB medium for cultivation; add 1mL of 30mg / mL kanamycin sulfate to each bottle to a final concentration of 30μg / ml. Set the shaker temperature to 32℃ and the speed to 140rpm, shake overnight, and the OD 600nm Reach 1 to 3. Add 10L of basal culture medium to the fermenter for sterilization. Before inoculation, add 20mL of sterile 30mg / mL kanamycin sulfate (final concentration 30μg / mL), and sterile inorganic salt solution (K2HPO4 (final concentration 5g / L), MgSO4 (final concentration 0.5g / L)). When the temperature reaches the set temperature of 35±0.5℃, inoculate under flame protection and sterile conditions. The seed liquid inoculation ratio is 1:5 (final volume 20L). The fermentation process parameters are: pH 7.0; speed 200-600rpm; ventilation 15-25L / H; tank pressure 0.05MPa; dissolved oxygen ≥30%. Defoaming agent is added during the process to eliminate foam. Batch feeding begins after 1h of fermentation, and feeding is carried out once every hour in stages. Feeding is completed in the logarithmic phase; A is measured every hour. 600nm , staining and microscopy. 600nm When the value reaches about 10, the temperature is lowered to 28°C, and sterile IPTG is added to a final concentration of 0.1 mmol / L to start induction. After 4 hours of induction, the fermentation is stopped, and the fermentation liquid is harvested.

[0130] About 20L of fermentation liquid was harvested and the bacterial liquid was crushed using a high-pressure homogenizer at a crushing pressure of 900-1000 bar for 3 cycles, and the crushed bacterial liquid was collected. 2 *2) The crushed liquid was clarified and the clarified permeate was collected. A 10 kDa ultrafiltration membrane was used (Cobetter, 0.5 m 2 *2) The clarified permeate was concentrated. When it was concentrated to about 4 L, an equal volume of the concentrated retentate was diafiltered with a buffer containing 50 mM Tris-HCl (pH 8.5), 1 mM EDTA, 5 mM DTT, and 18.3 mM NaCl. Finally, a certain volume of the same buffer was added to rinse the membrane package. This was repeated 3 times to collect the crude sample.

[0131] The expression of samples during fermentation and crude purification is shown in Figure 1 As shown, the protein content was about 57.02 at 4h of induction, and the yield was 2.85g / L fermentation liquid. After homogenization, clarification, concentration and substitution, the target protein content in the obtained crude sample was 13.63g, and the yield was 0.68g / L.

[0132] Table 1 Expression of target proteins in samples and crude samples of TTc expression strain induced for 4 h at 20 L fermentation scale

[0133] Sample type Sample volume (L) Target protein content (g) Yield (g / L fermentation liquid) Induction 4h fermentation broth 20 57.02 2.85 Rough sample 4 13.63 0.68

[0134] Note: The target protein content of each sample in Table 1 was estimated based on SDS-PAGE electrophoresis.

[0135] Example 2 Preparation of TTc Protamine by DEAE FF-Phenyl HP-Polar MC30 HIC Butyl Chromatography

[0136] (1) A DEAE Bestarose FF (XK50 / 30, Bogelon) chromatography column was equilibrated for 3CV using an equilibration buffer of 50 mM Tris-HCl (pH 8.5), containing 1 mM EDTA, 5 mM DTT, and 18.3 mM NaCl, and the crude sample obtained in Example 1 was loaded onto the chromatography column, with the loading amount being controlled to be 5 mg TTc / ml gel; then the column was eluted for 3CV using the equilibration buffer, and then the column was dissociated for 3CV using an elution buffer of 50 mM Tris-HCl (pH 8.5), containing 1 mM EDTA, 5 mM DTT, and 68 mM NaCl, and the DEAE eluate was collected; then the column was cleaned and regenerated using a 1.5 M NaCl solution and a 0.5 M NaOH solution; and finally the column was equilibrated for 3CV using the equilibration buffer.

[0137] (2) The pH and conductivity of the DEAE eluate were adjusted with a sample conditioning solution of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 5 mM DTT, and 3.5 M (NH4)2SO4 to be basically consistent with the equilibrium buffer of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 5 mM DTT, and 0.6 M (NH4)2SO4. The Phenyl Bestarose HP (XK50 / 30, Bogelon) chromatography column was equilibrated for 3CV with 50mM Tris-HCl (pH8.5), equilibration buffer containing 1mM EDTA, 5mM DTT, and 0.6M (NH4)2SO4, and the adjusted DEAE eluate was loaded onto the chromatography column, and the loading amount was controlled to be 5mg TTc / ml gel; then it was eluted for 3CV with the equilibration buffer, and then it was dissociated for 3CV with 50mM Tris-HCl (pH8.5), elution buffer containing 1mM EDTA, 5mM DTT, and 0.3M (NH4)2SO4, and the Phenyl eluate was collected; then the chromatography column was cleaned and regenerated with pure water and 0.5M NaOH solution; finally, it was equilibrated for 3CV with the equilibration buffer.

[0138] (3) The pH and conductivity of the phenyl eluate were adjusted with a sample conditioning solution of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 5 mM DTT, and 3.5 M (NH4)2SO4 to be basically consistent with the equilibrium buffer of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 5 mM DTT, and 0.6 M (NH4)2SO4. The Polar MC30 HIC Butyl (XK50 / 30, Saifen Technology) chromatography column was equilibrated for 3CV with 50mM Tris-HCl (pH8.5), 1mM EDTA, 5mM DTT, and 0.6M (NH4)2SO4 equilibration buffer, and the adjusted Phenyl eluate was loaded onto the chromatography column, and the loading amount was controlled to be 5mg TTc / mlgel; then it was eluted for 3CV with equilibration buffer, and then it was dissociated for 3CV with 50mM Tris-HCl (pH8.5), 1mM EDTA, 5mM DTT, and 0.46M (NH4)2SO4 elution buffer, and the Polar eluate was collected; then the chromatography column was cleaned and regenerated with pure water and 0.5M NaOH solution; finally, it was equilibrated for 3CV with equilibration buffer.

[0139] (4) The Polar eluate was packaged with a 10KDa membrane (0.1m 2 *2) Concentrate, then perform diafiltration with an equal volume of 0.2 M boric acid-sodium tetraborate, 5 mM DTT, 0.85% NaCl buffer, and the retentate collected is TTc protamine-1.

[0140] Example 3 Preparation of TTc protamine by DEAE FF-Phenyl HP-Polar MC30 HIC Butyl-alkylation modified chromatography process

[0141] (1) A DEAE Bestarose FF (XK50 / 30, Bogelon) chromatography column was equilibrated for 3CV using an equilibration buffer of 50 mM Tris-HCl (pH 8.5), containing 1 mM EDTA, 5 mM DTT, and 18.3 mM NaCl, and the crude sample obtained in Example 1 was loaded onto the chromatography column, with the loading amount being controlled to be 5 mg TTc / ml gel; then the column was eluted for 3CV using the equilibration buffer, and then the column was dissociated for 3CV using an elution buffer of 50 mM Tris-HCl (pH 8.5), containing 1 mM EDTA, 5 mM DTT, and 68 mM NaCl, and the DEAE eluate was collected; then the column was cleaned and regenerated using a 1.5 M NaCl solution and a 0.5 M NaOH solution; and finally the column was equilibrated for 3CV using the equilibration buffer.

[0142] (2) The pH and conductivity of the DEAE eluate were adjusted with a sample conditioning solution of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 5 mM DTT, and 3.5 M (NH4)2SO4 to be basically consistent with the equilibrium buffer of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 5 mM DTT, and 0.6 M (NH4)2SO4. The Phenyl Bestarose HP (XK50 / 30, Bogelon) chromatography column was equilibrated for 3CV with 50mM Tris-HCl (pH8.5), equilibration buffer containing 1mM EDTA, 5mM DTT, and 0.6M (NH4)2SO4, and the adjusted DEAE eluate was loaded onto the chromatography column, and the loading amount was controlled to be 5mg TTc / ml gel; then it was eluted for 3CV with the equilibration buffer, and then it was dissociated for 3CV with 50mM Tris-HCl (pH8.5), elution buffer containing 1mM EDTA, 5mM DTT, and 0.3M (NH4)2SO4, and the Phenyl eluate was collected; then the chromatography column was cleaned and regenerated with pure water and 0.5M NaOH solution; finally, it was equilibrated for 3CV with the equilibration buffer.

[0143] (3) The Phenyl eluate was adjusted with a sample conditioning solution of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 5 mM DTT, and 3.5 M (NH4)2SO4 to make the pH and conductivity of the DEAE eluate basically consistent with the equilibrium buffer of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 5 mM DTT, and 0.6 M (NH4)2SO4. The Polar MC30 HIC Butyl (XK50 / 30, Saifen Technology) chromatography column was equilibrated for 3CV with 50mM Tris-HCl (pH8.5), 1mM EDTA, 5mM DTT, and 0.6M (NH4)2SO4 equilibration buffer, and the adjusted Phenyl eluate was loaded onto the chromatography column, and the loading amount was controlled to be 5mg TTc / ml gel; then it was eluted for 3CV with equilibration buffer, and then it was dissociated for 3CV with 50mM Tris-HCl (pH8.5), 1mM EDTA, 5mM DTT, and 0.46M (NH4)2SO4 elution buffer, and the Polar eluate was collected; then the chromatography column was cleaned and regenerated with pure water and 0.5M NaOH solution; finally, it was equilibrated for 3CV with equilibration buffer.

[0144] (4) 1M DTT (Sigma) solution was first added to the Polar eluent to a final concentration of 10 mM, followed by uniform stirring for several minutes, and the mixture was allowed to react at room temperature for 60 min with the mouth closed. The sample was then divided into three portions, one without any reagent, one with 0.5M iodoacetamide (Sigma) solution to a final concentration of 20 mM, and one with 0.5M chloroacetamide (Sigma) solution to a final concentration of 20 mM. The mixture was uniformly stirred for several minutes, and the mixture was allowed to react at room temperature with the mouth closed overnight in the dark to obtain an alkylated sample.

[0145] (5) The alkylated sample was concentrated using a 3KDa ultrafiltration centrifuge tube, and then diafiltration was performed using an equal volume of 0.2M boric acid-sodium tetraborate buffer containing 0.85% NaCl. The retentate finally collected was TTc protamine-2.

[0146] Example 4 Preparation of TTc protamine by DEAE FF-Phenyl HP-alkylation-Polar MC30 HIC Butyl chromatography

[0147] (1) A DEAE Bestarose FF (XK50 / 30, Bogelon) chromatography column was equilibrated for 3CV using an equilibration buffer of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 5 mM DTT, and 18.3 mM NaCl, and the crude sample obtained in Example 1 was loaded onto the chromatography column, with the loading amount being controlled to be 5 mg TTc / ml gel; then the column was eluted for 3CV using the equilibration buffer, and then the column was dissociated for 3CV using an elution buffer of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 5 mM DTT, and 68 mM NaCl, and the DEAE eluate was collected; then the column was cleaned and regenerated using a 1.5 M NaCl solution and a 0.5 M NaOH solution; and finally the column was equilibrated for 3CV using the equilibration buffer.

[0148] (2) The pH and conductivity of the DEAE eluate were adjusted with a sample conditioning solution of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 5 mM DTT, and 3.5 M (NH4)2SO4 to be basically consistent with the equilibrium buffer of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 5 mM DTT, and 0.6 M (NH4)2SO4. The Phenyl Bestarose HP (XK50 / 30, Bogelon) chromatography column was equilibrated for 3CV with an equilibration buffer of 50mM Tris-HCl (pH8.5), containing 1mM EDTA, 5mM DTT, and 0.6M (NH4)2SO4, and the adjusted DEAE eluate was loaded onto the chromatography column, and the loading amount was controlled to be 5mg TTc / ml gel; then the column was rinsed with the equilibration buffer for 3CV, and then the column was dissociated with an elution buffer of 50mM Tris-HCl (pH8.5), containing 1mM EDTA, 5mM DTT, and 0.3M (NH4)2SO4 for 3CV, and the Phenyl eluate was collected; then the chromatography column was cleaned and regenerated with pure water and 0.5M NaOH solution; finally, the column was equilibrated with the equilibration buffer for 3CV.

[0149] (3) First, add 1 M DTT (Sigma) solution to the phenyl eluate to a final concentration of 10 mM, then stir evenly for several minutes, close the mouth, stand at room temperature, and react for 60 minutes; then add 0.5 M iodoacetamide (Sigma) solution to a final concentration of 20 mM, stir evenly for several minutes, close the mouth, stand at room temperature, and react overnight in the dark to obtain an alkylated sample.

[0150] (4) The pH and conductivity of the alkylated sample were adjusted using a sample adjustment solution of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA and 3.5 M (NH4)2SO4 to be basically consistent with the equilibrium buffer solution of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA and 0.6 M (NH4)2SO4. The Polar MC30 HIC Butyl (XK50 / 30, Saifen Technology) chromatography column was equilibrated for 3CV with 50mM Tris-HCl (pH8.5), 1mM EDTA, 0.6M (NH4)2SO4 equilibration buffer, and the adjusted alkylated sample was loaded on the chromatography column, and the loading amount was controlled to be 5mg TTc / ml gel; then it was eluted with equilibration buffer for 3CV, and then it was dissociated with 50mM Tris-HCl (pH8.5), 1mM EDTA, 0.46M (NH4)2SO4 elution buffer for 3CV, and the Polar eluate was collected; then the chromatography column was cleaned and regenerated with pure water and 0.5M NaOH solution; finally, it was equilibrated with equilibration buffer for 3CV.

[0151] (5) The Polar eluate was packaged with a 3KDa membrane (0.1m 2 *4) was concentrated, and then diafiltration was performed with an equal volume of 0.2 M boric acid-sodium tetraborate buffer containing 0.85% NaCl. The retentate collected was TTc protamine-3.

[0152] Example 5 Preparation of TTc protamine by DEAE FF-Phenyl HP-alkylation-Polar MC30 HIC Butyl chromatography

[0153] (1) A DEAE Bestarose FF (XK50 / 30, Bogelon) chromatography column was equilibrated for 3CV using an equilibration buffer of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 5 mM DTT, and 18.3 mM NaCl, and the crude sample obtained in Example 1 was loaded onto the chromatography column, with the loading amount being controlled to be 5 mg TTc / ml gel; then the column was eluted for 3CV using the equilibration buffer, and then the column was dissociated for 3CV using an elution buffer of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 5 mM DTT, and 68 mM NaCl, and the DEAE eluate was collected; then the column was cleaned and regenerated using a 1.5 M NaCl solution and a 0.5 M NaOH solution; and finally the column was equilibrated for 3CV using the equilibration buffer.

[0154] (2) The pH and conductivity of the DEAE eluate were adjusted with a sample conditioning solution of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 5 mM DTT, and 3.5 M (NH4)2SO4 to be basically consistent with the equilibrium buffer of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 5 mM DTT, and 0.6 M (NH4)2SO4. The Phenyl Bestarose HP (XK50 / 30, Bogelon) chromatography column was equilibrated for 3CV with an equilibration buffer of 50mM Tris-HCl (pH8.5), containing 1mM EDTA, 5mM DTT, and 0.6M (NH4)2SO4, and the adjusted DEAE eluate was loaded onto the chromatography column, and the loading amount was controlled to be 5mg TTc / ml gel; then the column was rinsed with the equilibration buffer for 3CV, and then the column was dissociated with an elution buffer of 50mM Tris-HCl (pH8.5), containing 1mM EDTA, 5mM DTT, and 0.3M (NH4)2SO4 for 3CV, and the Phenyl eluate was collected; then the chromatography column was cleaned and regenerated with pure water and 0.5M NaOH solution; finally, the column was equilibrated with the equilibration buffer for 3CV.

[0155] (3) First, add 1 M DTT (Sigma) solution to the phenyl eluate to a final concentration of 10 mM, then stir evenly for several minutes, close the mouth, stand at room temperature, and react for 60 minutes; then add 0.5 M chloroacetamide (Sigma) solution to a final concentration of 20 mM, stir evenly for several minutes, close the mouth, stand at room temperature, and react overnight in the dark to obtain an alkylated sample.

[0156] (4) The pH and conductivity of the alkylated sample were adjusted using a sample adjustment solution of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA and 3.5 M (NH4)2SO4 to be basically consistent with the equilibrium buffer solution of 50 mM Tris-HCl (pH 8.5) containing 1 mM EDTA and 0.6 M (NH4)2SO4. The Polar MC30 HIC Butyl (XK50 / 30, Saifen Technology) chromatography column was equilibrated for 3CV with 50mM Tris-HCl (pH8.5), 1mM EDTA, 0.6M (NH4)2SO4 equilibration buffer, and the adjusted alkylated sample was loaded on the chromatography column, and the loading amount was controlled to be 5mg TTc / ml gel; then it was eluted with equilibration buffer for 3CV, and then it was dissociated with 50mM Tris-HCl (pH8.5), 1mM EDTA, 0.46M (NH4)2SO4 elution buffer for 3CV, and the Polar eluate was collected; then the chromatography column was cleaned and regenerated with pure water and 0.5M NaOH solution; finally, it was equilibrated with equilibration buffer for 3CV.

[0157] (5) The Polar eluate was packaged with a 3KDa membrane (0.1m 2 *4) was concentrated, and then diafiltration was performed with an equal volume of 0.2 M boric acid-sodium tetraborate buffer containing 0.85% NaCl. The retentate collected was TTc protamine-4.

[0158] Example 6 TTc protamine SDS-PAGE electrophoresis purity analysis

[0159] The protein concentration of protamine-1, protamine-2, protamine-3 and protamine-4 in Examples 2 to 5 was determined by the Lowry method, and then diluted to 1.25 mg / mL with 0.85% NaCl solution. Two 40 μL portions of each diluted protamine sample were taken in 0.6 mL centrifuge tubes, one portion was added with 10 μL of 5x reducing electrophoresis loading buffer, and the other portion was added with 10 μL of 5x non-reducing electrophoresis loading buffer. After vortex mixing, the mixture was heated in a boiling water bath for 5 min, and then loaded on 12% or 15% concentration of (GenScript Biotech Co., Ltd.) precast gel, set the voltage to 120V, and run the equipment for 120min. Use after electrophoresis Protein staining instrument (GenScript Biotech Co., Ltd.) was used for staining and destaining, and then proteinsimple gel imager was used for image scanning and analysis.

[0160] The results are shown in Table 2 and Figure 1As shown, the protamine obtained without alkylation modification in Example 2 had obvious specificity under non-reducing conditions, with a purity of 94.02%, which did not meet the pharmacopoeia standard; the purity of the protamine obtained after the alkylation modification (chloroacetamide or iodoacetamide) was placed on the Polar MC30 HIC Butyl column chromatography in Example 3 was lower than 90%, which did not meet the pharmacopoeia standard; the purity of the protamine obtained after the alkylation modification (chloroacetamide) was placed between the Polar MC30 HIC Butyl column chromatography and the PhenylBestarose HP column chromatography in Example 5 was lower than 90%, which did not meet the pharmacopoeia standard; the purity of the protamine obtained after the alkylation modification (iodoacetamide) was placed between the Polar MC30 HIC Butyl column chromatography and the Phenyl Bestarose HP column chromatography in Example 4 was higher than 95%, which met the pharmacopoeia standard.

[0161] Table 2 SDS-PAGE purity analysis of protamine in Examples 2 to 5

[0162]

[0163] Note: “ / ” in the table means no test was performed.

[0164] Example 7 CE-SDS electrophoresis purity analysis of TTc protamine

[0165] The protamine-3 in Example 4 was desalted with purified water, and then 5 μL of sample, 90 μL of sample buffer (SCIEX, A10663), 2 μL of internal standard (SCIEX, A26487), and 5 μL of β-mercaptoethanol were taken in a 0.2 ml centrifuge tube, mixed and centrifuged at 5200 rpm for 1 min, 70°C water bath for 10 min, cooled for 3 min, and then 100 μL was transferred to a micro sample tube, and then the micro sample bottle was placed in a universal bottle and the bottle cap was covered. The IgG Purity mode in the capillary electrophoresis instrument (Beckman, PA800plus) was selected and the program was started.

[0166] The results are as follows Figure 2 As shown, under reducing electrophoresis conditions, the purity of TTc protamine-3 was 99.857%; under non-reducing electrophoresis conditions, the purity of TTc protamine-3 was 99.605%.

[0167] Example 8 Study on the stability of TTc protamine

[0168] In order to characterize the stability of the protein after alkylation modification, the protamine-1 obtained in Example 2 and the protamine-3 obtained in Example 4 were subjected to repeated freeze-thaw at -80°C and storage stability at 2-8°C, and the reducing and non-reducing SDS-PAGE electrophoresis purity of the protein during the experimental period was investigated.

[0169] The results are shown in Table 3 and Figure 3 As shown, protamine-3 (alkylation modification) was frozen and thawed once at -80℃, repeatedly frozen and thawed 3 times, and stored at 2-8℃ for 7 days, and the purity of reduced and non-reduced electrophoresis was still higher than 95%; protamine-1 (non-alkylation modification) was frozen and thawed once at -80℃ or stored at 2-8℃ for 7 days, and obvious aggregates appeared at the top of the non-reduced electrophoresis, and obvious cracks appeared at the monomer position, with a purity of about 40%. This shows that the TTC protein after alkylation modification has good stability in freezing and thawing and 2-8℃.

[0170] Table 3 TTc protamine stability analysis

[0171]

[0172] Example 9 Study on the immunogenicity of TTc protamine

[0173] In order to further investigate the effect of alkylation modification on the immunoenhancing effect of TTc as a carrier protein, the protamine-1 obtained in Example 2 and the protamine-3 obtained in Example 4 were conjugated with 12F and 19A pneumococcal capsular polysaccharides, respectively (12F and 19A pneumococcal capsular polysaccharides were prepared and conjugated according to the method disclosed in CN113173977B) to prepare Pn12FTTc-1, Pn19ATTc-1, Pn12FTTc-3, and Pn19ATTc-3 conjugate stock solutions, and the protein concentration in the conjugate was determined by the Lowry method, and the total sugar concentration in the conjugate was determined by the orcinol method, and the sugar / protein ratio was calculated according to the protein concentration and the total sugar concentration.

[0174] BALB / c mice aged 6 to 8 weeks were used as animal models, with half males and half females in each group, and 6 mice in each group. The obtained conjugate stock solution was injected into the leg muscles of BALB / c mice with two injections and an interval of 2 weeks. The injection volume was 0.1 mL / mouse. The immunization dose of sugar antigen in the monovalent conjugate stock solution of each group was 2 μg / dose. Blood was collected from the eye sockets on D28 after immunization. After the mouse serum was allowed to stand for a period of time to precipitate, it was centrifuged at 3000 rpm, the serum was collected, and stored at -20°C or below for the detection of specific polysaccharide IgG antibody content in the serum.

[0175] In this experiment, the indirect ELISA method was used to determine the content of specific polysaccharide IgG antibodies in the mouse antiserum of each test group. Each group used the corresponding type of capsular polysaccharide as the coating antigen. The coating concentrations of type 12F and type 19A capsular polysaccharides were 20μg / ml, 5μg / ml, and 20μg / ml, respectively. 100μL / well was coated on the ELISA plate and coated at 2-8℃ for 17-18h. The antiserum of each group was diluted 2 times according to the 50-fold starting dilution factor, 100μL per well, incubated at 37℃ for 120min, and washed 5 times; added 4000-fold diluted goat anti-mouse IgG alkaline phosphatase-labeled enzyme-labeled secondary antibody, incubated at 37℃ for 60min, and washed 5 times; added 100μL of color development solution, and developed the color for 90min under light-proof conditions; added 50μL of stop solution. Place the ELISA plate in the ELISA reader and read the OD 405 nm value; the SoftMax Pro software that comes with the microplate reader is used to perform four-parameter fitting on the OD value of the positive serum solution to obtain a standard curve. The calculable results of the test samples are obtained based on the standard curve, multiplied by the corresponding dilution factor, and then the antibody concentration in the test sample is calculated based on this.

[0176] The results are shown in Table 4. Alkylated TTC protamine and unalkylated TTC protamine were coupled with Pn12F and Pn19A, respectively. After two immunizations, there was no significant difference in the immunogenicity of the two carrier proteins to Pn12F and Pn19A polysaccharide antigens, indicating that alkylation modification has no effect on the immune-enhancing effect of TTC as a carrier protein.

[0177] Table 4 Study on the quality and immunogenicity of the conjugates with TTc in carrier protein

[0178]

[0179] Sequence information involved in this application:

[0180] SEQ ID NO:1

[0181]

[0182] SEQ ID NO:2

[0183] KNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYDTNEYSI ISSMKKYSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLSDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKF RIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVAYSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYSGLKFIIKRYTPNNEIDSFVRSGDFIKLYVSY NNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKDASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKTLTCDWYFVPTDEGWTND

[0184] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. A method for chemically modifying a tetanus toxin C fragment, characterized in that: The method comprises the following steps: 1) reacting the tetanus toxin C fragment with a reducing agent to reduce the disulfide bonds in the tetanus toxin C fragment; and, 2) The reduced tetanus toxin C fragment is reacted with an alkylating agent for modification.

2. The method according to claim 1, characterized in that The reducing agent in step 1) is selected from one or more of DTT, β-mercaptoethanol, TCEP, DTE and GSH; And / or, the alkylating agent in step 2) is selected from one or more of iodoacetamide, chloroacetamide, N-ethylmaleimide and iodoacetic acid.

3. The method according to claim 1 or 2, characterized in that The final concentration of the reducing agent is 1-1000 mM; preferably 5-20 mM; And / or, the final concentration of the alkylating agent is 1-1000 mM; preferably 5-30 mM.

4. A method for purifying a chemically modified tetanus toxin C fragment, characterized in that: The method comprises the following steps: a) modifying the tetanus toxin C fragment using the method according to any one of claims 1 to 3; and, b) chromatographically purifying the modified tetanus toxin C fragment obtained in step a), wherein the chromatographic purification preferably comprises the steps of column equilibration, sample loading, washing and elution; Preferably, the chromatographic purification conditions in step b) are selected from one or more of the following: Ⅰ. The balancing solution used for the column balancing, or the washing, or the elution is PB with a pH of 6-8, such as 7.5, or Tris buffer with a pH of 6-10, such as 8.5; the balancing solution is preferably 20-50 mmol / L Tris with a pH of 8.5, containing 1 mmol / LEDTA and 0.6 mol / L (NH4)2SO4; or 20-50 mmol / L Tris with a pH of 8.5, containing 1 mmol / LEDTA and 0.46 mol / L (NH4)2SO4; or 20-50 mmol / L Tris with a pH of 8.5, containing 1 mmol / L EDTA and 3.5 mol / L (NH4)2SO4; Ⅱ. The column chromatography procedure is equilibration for 3CV, sample loading, elution for 3CV, elution for 2-5CV, water washing for 3CV, alkaline washing for 3CV, and equilibration for 3CV again; III. The sample loading amount is 3-10 mg of target protein / mL of filler; and, IV. The chromatographic medium is selected from a hydrophobic chromatographic filler with phenyl as a ligand, a hydrophobic chromatographic filler with butyl as a ligand or a hydrophobic chromatographic filler with octyl as a ligand, preferably a hydrophobic chromatographic filler with butyl as a ligand, such as Polar MC30 HIC Butyl.

5. The method according to claim 4, characterized in that The method may further include one or more of the following: 1) Purifying the tetanus toxin C fragment before step a), for example, by chromatography purification; preferably, performing two chromatography purifications; 2) the purity of the tetanus toxin C fragment in step a) is greater than 60%, such as greater than 80%, preferably greater than 90%; and, 3) a step of concentrating and replacing the eluate of step b); Preferably, an ultrafiltration membrane is used for concentration and replacement; and / or, the buffer used for replacement contains boric acid-sodium tetraborate and NaCl; More preferably, the pore size of the ultrafiltration membrane is 3-10 KDa, preferably 3 KDa.

6. Use of a kit for chemically modifying a tetanus toxin C fragment or purifying a chemically modified tetanus toxin C fragment; the kit comprising a reducing agent and an alkylating agent; Preferably, the reducing agent is selected from one or more of DTT, β-mercaptoethanol, TCEP, DTE and GSH; And / or, the alkylating agent is selected from one or more of iodoacetamide, chloroacetamide, N-ethylmaleimide and iodoacetic acid.

7. A chemically modified tetanus toxin C fragment, characterized in that: The tetanus toxin C fragment is modified by alkylation; Preferably, the tetanus toxin C fragment is prepared by the method according to any one of claims 1-3, or the method according to claim 4 or 5.

8. A polysaccharide-carrier protein conjugate, characterized in that: The polysaccharide-carrier protein conjugate comprises the tetanus toxin C fragment as claimed in claim 7 and a polysaccharide, wherein the tetanus toxin C fragment is coupled to the polysaccharide; the polysaccharide is, for example, a capsular polysaccharide; Preferably, the capsular polysaccharide is a pneumococcal capsular polysaccharide, such as a 12F pneumococcal capsular polysaccharide or a 19A pneumococcal capsular polysaccharide.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the tetanus toxin C fragment according to claim 7 or the polysaccharide-carrier protein conjugate according to claim 8, and a pharmaceutically acceptable carrier.

10. Use of the tetanus toxin C fragment as claimed in claim 7 in constructing an antigen-carrier protein conjugate or preparing an immune preparation.

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