GB2604692A

GB2604692A - Method for preparing recombinant subunit vaccine against novel coronavirus

Info

Publication number: GB2604692A
Application number: GB2115860.5A
Authority: GB
Inventors: Bi Shengli; Hou Yunde; Mao Jiangsen; Gao Fu; Wu Guizhen; Qin Chuan; Xu Wenbo; Mao Zi'an; Su Qiudong; Yi Yao; Gao Meng; Chen Gang; Zhuang Fangcheng; Lv Hangjun; Lu Shaohong
Original assignee: Beijing Shangwei Biotechnology Dev Co Ltd
Current assignee: Beijing Shangwei Biotechnology Dev Co Ltd
Priority date: 2020-11-04
Filing date: 2021-11-04
Publication date: 2022-09-14
Anticipated expiration: 2041-11-04
Also published as: GB2604692B; CN112089831B; CN112089831A

Abstract

The method includes: transfecting a plasmid into E. coli inserted with a nucleic acid fragment corresponding to the novel coronavirus receptor binding domain fused to a tetanus toxin sequence (RBD-TT); culturing the E.coli to express the RBD-TT fusion protein; lysing the bacterial cells and separating an inclusion body crude extract of the RBD-TT recombinant protein; dissolving the crude extract in a denaturing buffer solution containing 7.5-8.5 M urea, arginine, reduced glutathione and glycerol at pH 9-10, and purifying by anion exchange chromatography; diluting the crude sample of RBD-TT recombinant protein with a diluent; filtering, renaturing with renaturation solutions comprising lower arginine and glutathione concentrations than the denaturing solution to obtain a renatured protein; purifying the recombinant renatured protein by anion exchange chromatography.

Description

METHOD FOR PREPARING RECOMBINANT SUBUNIT VACCINE

AGAINST NOVEL CORONAVIRUS

TECHNICAL FIELD

1011 The present disclosure relates to the field of vaccine preparation, in particular to a method for preparing a recombinant subunit vaccine against novel coronavirus.

BACKGROUND ART

1021 Novel coronavirus (SARS-CoV-2) is a newly discovered coronavirus in humans, it can cause acute infectious pneumonia and, in severe cases, acute respiratory distress syndrome or septic shock, or even death. At present, there are no clinically specific drugs that have been proved to be effective in the treatment of novel coronavirus infections. Preventive vaccination is the most economical and effective means for preventing and controlling the virus. Therefore, it is necessary to conduct a safe and effective vaccine study on novel coronavirus.

1031 According to the genomic characteristics, SARS-CoV-2 virus belongs to the Sarbecovirus subgenus in I genus of Coronavints* (COY) subfamily. The genome length of SARS-CoV-2 virus is about 29.8Kb, and two-thirds of the genes at the 5' end encode enzymes related to virus replication, while the remaining one-third of the genes encode 4 structural proteins including Spike (S), Envelope (E), Membrane protein (M), and Nucleocapsid protein (N), as well as some other accessory proteins. Like other coronaviruses, the S protein of the SARS-CoV-2 virus is exposed on the surface of the virus and consists of two non-covalently bound subunits, Si and S2. The Si subunit contains the receptor-binding domain (RBD) of the cell receptor, which can bind to angiotensin-converting enzyme 2 (ACE2) on host cells to help viruses adsorption and enter host cells. The 52 subunit contains one fusion peptide (FP), one transmembranedomain (TM), and two heptad repeat regions (HR), which play a key role in the fusion process of virus and cell membrane.

1041 Existing studies have shown that the RBD region in the Si subunit is considered to be a viral neutralizing epitope rich region, which can induce the body to produce neutralizing antibodies. Therefore, it is theoretically feasible to conduct subunit vaccine research based on RBD region. However, the immune effect of this recombinant subunit vaccine is not comparable to that of the traditional inactivated vaccine, and other technical means are usually needed to improve its immunogenicity. The use of vaccine adjuvant is one of the ways to improve the immune effect of vaccine. However, in addition to aluminum adjuvants, only few vaccine adjuvants have been approved for marketing among the clinically approved vaccine adjuvants, and they are expensive. Therefore, researchers have carried out research on intramolecular adjuvants to improve the immunogenicity of vaccines by attaching molecular peptides with immune enhancement effect to the ends of antigenic proteins. This kind of intramolecular adjuvant can reduce research and development cost, is beneficial to improve vaccine quality control, and is an effective scheme for novel vaccine design.

1051 With the help of immune enhancement effect of intramolecular adjuvants, it is of great value and significance for the prevention and treatment of interpersonal transmission of novel coronavirus to construct a recombinant subunit vaccine against novel coronavirus with the characteristics of high expression and good immunogenicity. However, intramolecular adjuvants may change the protein structure, resulting in reduced expression efficiency of the recombinant subunit vaccine against novel coronavirus.

SUMMARY OF THE INVENTION

1061 The objective of the present disclosure is to provide a method for preparing a recombinant subunit vaccine against novel coronavirus, comprising the steps of: 1071 a) transfecting a plasmid containing a nucleic acid fragment set forth in SEQ ID NO: 2 into Li coli, wherein the nucleic acid fragment is capable of expressing an RBD-TT fusion protein; 1081 b) culturing the E. colt to express the RBD-TT fusion protein, breaking bacterial cells and separating a crude extract of the inclusion body of the RBD-TT recombinant protein; 1091 c) dissolving the crude extract of the inclusion body in a denaturing solution containing urea, and purifying the inclusion body by anion exchange chromatography to obtain a crude sample of RBD-TT recombinant protein; 1101 d) diluting the crude sample of RBD-TT recombinant protein with a diluent; 1111 whrerin, the diluent contains 450-510 g of urea, 70-86 g of arginine, 0.3-0.7 g of reduced glutathione, 15-25 g of glycerol, and a buffer substance per liter, with a pH value of 9 -10; 1121 e) renaturing the diluted RBD-TT recombinant protein through dialysis replacement or tangential flow ultrafiltration, using a renaturation solution 1, a renaturation solution 2 and a renaturation solution 3 in sequence; 1131 whrerin, the renaturation solution 1 contains 70-86 g of arginine, 0.3-0.7 g of reduced glutathione, 15-258 of glycerol, 1-3 g of sodium carbonate and 3-68 of sodium bicarbonate per liter, with a pH value of 9-10; [14] whrerin, the renaturation solution 2 contains 0.3-0.7 g of reduced glutathione, 15-25 g of glycerol, 1-3 g of sodium carbonate, 3-6 g of sodium bicarbonate per liter, with a pH value of 9-10; 1151 whrerin, the renaturation solution 3 contains 0.1-0.3 g of reduced glutathione, 0.5-1 g of sodium carbonate, 1-2 g of sodium bicarbonate per liter, with a pH value of 9-10; 1161 0 purifying the protein after renaturation by anion exchange chromatography.

1171 The present disclosure also relates to an isolated nucleic acid fragment corresponding to the RBD-TT recombinant protein expressed in E. coil, the sequence of the nucleic acid fragment is set forth in SEQ ID NO: 2.

1181 The nucleic acid fragment is codon-optimized and is able to be efficiently expressed in an E. coh expression system.

1191 The present disclosure has the following beneficial effects: 1201 A method for preparing a recombinant novel coronavirus subunit vaccine with the characteristics of strong immunogenicity, low cost and high yield is provided. The obtained recombinant subunit vaccine novel coronavirus is able to efficiently induce mice to produce protective neutralizing antibodies and specific cell immune responses.

BRIEF DESCRIPTION OF THE DRAWINGS

1211 In order to more clearly describe the specific embodiments of the present disclosure or the technical scheme in the prior art, the following will briefly introduce the drawings required to be used in detailed description or the description of the prior art, it is obvious that the drawings in the following description are some embodiments of the present disclosure, and other drawings may be obtained according to these drawings by those skilled in the art without making creative effort.

1221 FIG. 1 is an electrophoresis pattern of RBD-TT recombinant protein expression in an example of the present disclosure; where M representes a protein molecular weight mark; 1, 2 represent the electrophoresis results before induction of expression; 3, 4 represent the electrophoresis results after inducing expression for 4 hours; 1231 FIG. 2 is a bar chart showing the effect of different diluent formulas on antigen

titer in an example of the present disclosure;

1241 FIG 3 is a bar chart showing the effect of different renaturation conditions on antigen titer in an example of the present disclosure; 1251 FIG. 4 is an electrophoresis pattern of purified RBD-TT recombinant protein in an example of the present disclosure; wherein M represents a protein molecular weight mark; 1 represents a sample before purification; 2 represents a sample after purification; 3 represents a vaccine bulk sample; 1261 FIG. 5 is a bar chart showing a comparison of the results of the geometric mean of protective neutralizing antibodies induced by recombinant subunit vaccine against novel coronavirus in mice and the RBD group in an example of the present disclosure 1271 FIG. 6 is a bar chart showing a comparison of the results of the expression of specific interferon gamma (INF-y) and interleukin 4 (IL-4) in the lymphocytes of the immunized mice in the RBD-TT test group and the RBD group in an example of the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

1281 Reference will now be made in detail to embodiments of the present disclosure, one or more examples of which are describe below. Each example is provided as an explanation of rather than a limitation to the present disclosure. In fact, it is obvious to those skilled in the art that various modifications and changes can be made to the present disclosure without departing from the scope or spirit of the present disclosure. For example, features illustrated or described as part of one embodiment may be used in another embodiment to obtain still further embodiments.

1291 Therefore, it is intended that the present disclosure cover such modifications and changes that fall within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the present disclosure are disclosed in or are obvious from the following detailed description. It should be understood by those of ordinary skill in the art that this discussion is only a description of exemplary embodiments and is not intended to limit the broader aspects of the present disclosure pol The present disclosure relates to a method for preparing a recombinant novel coronavirus subunit vaccine, comprising the steps of 1311 a) transfecting a plasmid containing a nucleic acid fragment set forth in SEQ ID NO: 2 into E. coli, wherein the nucleic acid fragment is capable of expressing an RBD-TT fusion protein; 1321 b) culturing the E. colt, to express the RBD-TT fusion protein, breaking bacterial cells and separating an inclusion body crude extract of the RBD-TT recombinant protein; 1331 c) dissolving the inclusion body crude extract in a denaturing solution containing urea, and purifying the inclusion body by anion exchange chromatography to obtain a crude sample of RBD-TT recombinant protein; 1341 d) diluting the crude pure sample of RBD-TT recombinant protein with a diluent 1351 whrerin, the diluent contains 450-510 g of urea, 70-86 g of arginine, 0.3-0.7 g of reduced glutathione, 15-25 g of glycerol, and a buffer substance per liter, with a pH value of 9 -10; 1361 e) renaturing the diluted RBD-TT recombinant protein through dialysis replacement or tangential flow ultrafiltration, using a renaturation solution 1, a renaturation solution 2 and a renaturation solution 3 in sequence; [37] whrerin, the renaturation solution 1 contains 70-86 g of arginine, 0.3-0.7 g of reduced glutathione, 15-25 g of glycerol, 1-3 g of sodium carbonate and 3-6 g of sodium bicarbonate per liter, with a pH value of 9-10; [38] whrerin, the renaturation solution 2 contains 0. 3-0.7 g of reduced glutathione, 15-25 g of glycerol, 1-3 g of sodium carbonate, 3-6 g of sodium bicarbonate per liter, with a pH value of 9-10; [39] whrerin, the renaturation solution 3 contain 0.1-0.3 g of reduced glutathione, 0.5-1 g of sodium carbonate, 1-2 g of sodium bicarbonate per liter, with a pH value of 9-10; [40] 0 purifying the protein after renaturation by anion exchange chromatography.

[41] In some embodiments, the dialysis displacement volume ratio of the renaturation solution to protein solution used for each dialysis displacement is independently selected from 1:(50-1000), such as 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, and 1:900, time for each replacement is 4-6 hours.

[42] In some embodiments, the ultrafiltration displacement volume ratio of the renaturation solution to the protein solution used for each tangential flow ultrafiltration renaturation is independently selected from 1:(2-5), such as 1:3, 1:4, and the feed pressure for each tangential flow ultrafiltration is 20 bar to 30 bar.

[43] In some embodiments, the pore size of the dialysis bag used in the dialysis replacement is in a range of 5 kd to 30 kd. Optionally, the pore size of the dialysis bag used in the dialysis replacement is selected from 8 kd, 10kd, 12kd, 15kd, 20kd and 25kd.

[44] In some embodiments, the pore size of the tangential flow ultrafiltration membrane package used for the tangential flow ultrafiltration renaturation is in a range of 5 kd to 30 kd. Optionally, the pore size of the tangential flow ultrafiltration membrane package used for the tangential flow ultrafiltration renaturation is selected from 8kb, 10kd, 12kd, 15kd, 20kd and 25kd.

[45] In some embodiments, in steps c) and 0, the exchange groups of the anion exchange chromatography include a diethylaminoethyl group or a quaternary ammonium group.

1461 In some embodiments, in step d), the dilution is performed in a volume ratio of 1:(1-10) of the crude pure sample of RED-TI recombinant protein to the diluent.

[47] In some embodiments, the diluent includes 470-490 g of urea, 73-83 g of arginine, 0.4-0.6 g of reduced glutathione, 17-23 g of glycerol, and a buffer substance per liter.

[48] The term "plasmid" refers to a nucleic acid transfer vector into which a polynucleotide can be inserted. When a plasmid enables expression of a protein encoded by an inserted polynucleotide, the plasmid is also referred to as an expression vector. The plasmid can be introduced into the host cells through transformation, transduction or transfection so that the genetic material elements carried by it are expressed in the host cells. Vectors are well known to those skilled in the art, in some embodiments, the plasmids of the present disclosure comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and polyU sequences, etc.).

[49] In some embodiments, the plasmid is a pET28a E coil expression plasmid.

[50] In some embodiments, the denaturing solution contains 17-23 g of sodium chloride, 450-510 g of urea, and a buff substance per liter, with a pH of 7.2 to 7.8.

[51] In some embodiments, the denaturing solution contains 18-22 g of sodium chloride, 470-490 g of urea, and a buff substance per liter, with a pH of 7.3 to 7.7.

[52] The term "buffer substance" as used herein refers to an aqueous solution or composition containing the buffer substance, and the aqueous solution or composition resists the changes in pH when the aqueous solution or composition is added in an acid or base. This resistance to pH change is due to the buffering properties of such substances. The buffering substance generally does not have an unlimited ability to maintain the pH of the solution or composition. Instead, they are generally capable of maintaining a pH within a specific range, e.g., pH 7-pH 9. Generally, the buffer is capable of maintaining pH within the previous logarithm and the next logarithm of its pKa (see, for example, Mohan, Buffers, A guide for the preparation and use of buffers inbiologica1 systems, CALBIOCTIEM, 1999). The buffer and buffer solution are generally prepared from buffer salts, or preferably nonionic buffer components such as IRIS and HEPES. The buffer that may be used in the method of the present disclosure is preferably selected from phosphate buffer, phosphate buffered saline buffer (PBS), 2-amino-2 -hydroxymethy1-1,3-propanediol (TRIS) buffer, TRIS buffered saline (TBS) and TRIS/EDTA(TE). In some specific embodiments, the buffer substance is tri s(hydroxymethyl)aminomethane.

[53] In some embodiments, the method for separating the crude extract of inclusion body of the RBD-TT recombinant protein in step b) is centrifugation [54] In some embodiments, the purification method by anion exchange chromatography in step c) comprises: equilibrating the column with 5-fold of column volumes of denatured liquid and then loading the samples onto the chromatography column, and collecting the samples flowing through the column to obtain a crude sample of RBD-TT recombinant protein.

[55] In some embodiments, the method for purifying the recombinant protein renaturation protein in step 0 by anion exchange chromatography comprises: equilibrating the column with 5-fold of column volumes of denaturant liquid and then loading the samples onto the column, and collecting the samples flowing through the column.

[56] In some embodiments, the culturing is carried out in a shaker and/or a fermentor.

[57] In some embodiments, breaking of the bacterial cells is carried out by an ultrasonic method.

[58] In some embodiments, no strong solvents (e.g., guanidine hydrochloride, detergents such as sodium lauryl sulfate and thiocyanate) are used in the process of breaking the bacteria cells.

[59] In some embodiments, buffer used in breaking of the cells contains 2.0-2.8 g of tris(hydroxymethyl)aminomethane and 15-25 g of sodium chloride per liter of, with a pH value of 7.7-8.3.

[60] In some embodiments, in step b), the E. coif is cultured to a logarithmic growth phase, and 0.7 mM to 1.3 m1VI of isopropyl-f3-D-thioga1actoside inducer is used to induce the E. colt to express the fusion protein RBD-TT at a temperature of 25°C to 37°C for 4 to24 hours.

1611 In some embodiments, the following step is further included after step 0: filtering and sterilizing the renatured recombinant protein and using the renatured recombinant protein in combination with an adjuvant.

1621 Tn some embodiments, the adjuvant is selected from one or more of squalene, muramyl dipeptide, MFS9, AS03, monophospholipid lipid A, flagellin, CpG-ODN, poly (I:C), and small molecules of aluminum or calcium salt. These adjuvants are well known in the art and are available through several commercial channels.

1631 Preferred adjuvants that can be used with the RBD-TT recombinant protein of the present disclosure include aluminum or calcium salts (e.g., hydroxides or phosphates). A preferred adjuvant that is particularly suitable for use in the present disclosure is an aluminum hydroxide gel, such as Alhydrogelml The aluminum hydroxide gel is obtained by mixing the chimeric protein with the adjuvant, so that each dose contains about 50 to about 800 gg of aluminum, preferably about 400 to about 600 lig of aluminum. Another particularly preferred adjuvant is aluminum phosphate available from SuperfosBiosector of Denmark under the trademark of Adju-PhosTm. The primary aluminum phosphate particles have a plate-like morphology with a diameter of about 50 to about 100nm, and a final particle size in the product is of about 0.5 to about 10gm. Calcium phosphate nanoparticle (CAP) is an adjuvant developed by Biosante, Inc(Lincolnshire, IL), 1641 In some embodiments, the adjuvant includes an aluminum phosphate adjuvant and/or an aluminum hydroxide adjuvant.

1651 In some embodiments, the vaccine is a water-in-oil emulsion having an aqueous phase and an oil phase.

166] In some embodiments, the vaccine is an oil-in-water emulsion having an aqueous phase and an oil phase.

1671 The present disclosure also relates to an isolated nucleic acid fragment corresponding to the RBD-TT recombinant protein expressed in E. colt, the sequence of the nucleic acid fragment is set forth in SEQ ID NO: 2.

1681 The embodiments of the present disclosure will be described in detail below with reference to examples.

1691 Example 1 Optimization of diluent [70] (1) A nucleotide sequence SEQ ID NO: 2 for expressing an RBD-TT recombinant protein was designed by using an amino acid sequence set forth in SEQ ID NO: 1 as a template according to the codon merger principle and the use frequency of codons by E. coil, and the synthesized genes were inserted into the pET28a E. col/ expression plasmid by a seamless cloning method to obtain a recombinant expression plasmid. The obtained recombinant expression plasmid was transferred into a competent cell of E. coil BL21(DE3) by a thermal activation method at 42°C, and the obtained E. coil was screened by an LB solid culture medium plate for monoclonal screening to obtain a monoclonal colony of the recombinant E. coil expressing the target protein, the monoclonal colony of the recombinant E. coil was picked out and cultured in 5 mL of LB liquid medium at 37°C overnight, a seed culture of the recombinant E. coil expressing the target protein was obtained.

[71] (2) The seed culture of the recombinant E. coil was inoculated in 5 mL of LB liquid medium at a a ratio of 1:100 at 37°C overnight for activation. The culture after overnight culturing was inoculated into fresh LB liquid medium in a ratio of 1 100 and cultured at 37°C until OD 600 was 0.7 followed by induction with an inducer (IPTG) (final concentration of the inducer: t mmol) at 37°C for four consecutive hours. The samples before and after induction were analyzed by SDS-PAGE protein electrophoresis (FIG. 1). The expression level of RBD-TT recombinant protein after induction reached 30% of the protein in the cells.

[72] (3) The colony after induction expression was centrifuged for 20 minutes by using a centrifuge under the condition of 6000 revolutions per minute to collect the colony precipitate after induction expression, the obtained colony precipitate was re-suspended by using a bacteria breaking buffer in a mass-volume ratio of 1:20, and the cell wall of the colonies were ultrasonically broken by using a sonicator, the bacteria suspension after ultrasonic breaking was centrifuged for 30 minutes by using the centrifuge at 12000 revolutions per minute and the broken bacteria precipitate were collected.

[73] (4) The obtained broken bacteria precipitate was dissolved in the denaturant liquid, and the supernatant was collected by centrifuging at 12,000 rpm for 30 minutes. The obtained supernatant was purified by DEAF anion exchange chromatography with a column volume of 5 mL, and the chromatographic conditions were as follows: the denatured liquid with a 5-fold of column volumes was first used to equilibrate the chromatographic columns, and then the samples were loaded onto the chromatographic columns. The samples flowing through the columns were collected to obtain a crude sample of RBD-TT recombinant protein (FIG. 2).

[74] (5) The obtained crude sample of RBD-TT recombinant protein were diluted with a diluent in a volume ratio of 11, and the diluted samples were subjected to dialysis renaturation using a renaturation solution 1, a renaturation solution 2 and a renaturation solution 3 in sequence to obtain a renaturated protein.

[75] The formulas of different diluents used were as follows: [76] Diluent 1: 2.4 g of tris(hydroxymethyl)aminomethane, 480 g of urea, 0.5 g of reduced glutathione, dissolved in water and made up to 1 L, and pH adjusted to 9.5.

[77] Diluent 2: 2.4 g of tris(hydroxymethyl)aminomethane, 480 g of urea, 0.5 g of reduced glutathione, and 20 g of glycerol, dissolved in water and made up to 1 L, and pH adjusted to 9.5.

[78] Diluent 3: 2.4 g of tris(hydroxymethyl)aminomethane, 480 g of urea, 78 g of arginine, 0.5 g of reduced glutathione, dissolved in water and made up to 1 L and pH adjusted to 9.5.

[79] Diluent 4: 2.4 g of tris(hydroxymethyl)aminomethane, 480 g of urea, 78 g of arginine, 0.5 g of reduced glutathione, and 20 g of glycerol, dissolved in water and made up to 1 L, and pH adjusted to 9.5.

180] The dialysis replacement ratio of renaturation solution to protein solution was 1:500 for each dialysis replacement, and time for each replacement was 5 hours. The pore size of the dialysis bag used in the dialysis replacement was 10 kd [81] (6) The obtained renaturated protein was diluted to 0.1mg/mL, and the antigenic activity under different renaturation conditions was detected by ELISA, the method was as follows.

1821 1) Antigen samples to be detected were diluted serially in different degrees of dilution of 1:2, L4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256 by using PBS diluent. Specifically, the dilution method comprised the following steps of:100!IL of antigen protein samples to be detected was added into 900 RI. of PBS diluent, and uniformly mixed to obtain a 1:10 sample; 100 RI-of the 1:10 sample was added into SOO gL of PBS diluent and uniformly mixed to obtain a 1:90 sample; and other dilutions were diluted likewise. The samples with each dilution degree were sequentially added into the wells of an ELISA plate pre-coated with ACE2 receptor protein in an amout of 100 pt/well, with each dilution sample added in two wells were separately. At the same time, two other wells of the ELISA plate were set for negative samples, and two wells were left blank for zero adjustment. The ELISA plate was put into a incubator and cultured at 37°C for 0.5 h, and the plate was washed 4 times with PBS washing solution; 1831 2) HRP-labeled RBD protein polyclonal antibody with a dilution ratio of 1:2000 (100 gL/well) was added into each well of the enzyme-labeled plate, the enzyme-labeled plate was cultured at 37°C for 0.5 h, and the plate was washed 4 times with PBS washing solution; 1841 3) TMB chromogen solution was added in each well of the ELI SA plate in an amount of 100 pt/well, the ELISA plate was put into a wet box and kept the temperature at 37°C for 15 minutes; 1851 4) a stop solution was added into each well of the ELISA plate in an amount of pt/well, the ELISA plate was placed under a wavelength of 450nm to detect the absorbance A value after zero adjustment with respect to the blank well; 1861 5) Determination of results: 1871 Cutoff value = average A value for negative control wells z 2.1=0.1260, (average A value of negative control was greater than 0.05 and calculated based on actual values).

1881 6) the sample was determined as a positive sample according to the formula A value of the sample Cutoff value, and the sample was determined as a negative sample according to the formula A value of the sample < Cutoff value.

[89] The solutions used were as follows: [90] L Coating buffer (0.05N4 of carbonate buffer, pH9.6): L59 g of sodium carbonate, 2.93 g of sodium bicarbonate, distilled water was made up to 1000 mL [91] 2. Phosphate buffer (PBS, pH7.4): 0.2 g of potassium dihydrogen phosphate, 2.9 g of disodium hydrogen phosphate, 8.0 g of sodium chloride, 0.2 g of potassium chloride, and 0.5 mL of Tween-20, distilled water was made up to 1000 mL [92] 3. PBS washing solution: 0.5 mL of Tween 20 was made up to 1000 mL of phosphate buffer.

[93] 4. Blocking solution: 1 g of bovine serum albumin was added into 100 mL of PBS washing solution, and dissolved and mixed evenly.

[94] 5. Stop solution (2N sulfuric acid):10 mL of concentrated sulfuric acid was slowly added into 80 mL of water and cooled for later use.

[95] The results are shown in FIG. 2. It can be seen from FIG. 2 that the effects of different diluent formulas on antigen titer varied significantly, and the sample treated with diluent 4 had the highest antigen titer.

[96] The solutions used were as follows: [97] LB liquid medium:10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride were dissolve in water and water was added to make up to 1 L. [98] LB solid medium:10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar powder were dissolve in water and water was added to made up to 1 L. [99] Bacteria breaking buffer: 2.4 g of tris(hydroxymethyl)aminomethane and 29.22 g of sodium chloride were dissolved in water and water was added to made up to 1 L, and pH was adjusted to 8.0.

[100] denaturant liquid: 2.4 g of tris(hydroxymethyl)aminomethane, 29.22 g of sodium chloride, 480g of urea were dissolved in water and water was added to made up to 1 L, and pH was adjusted to 7.5.

[101] Diluent: 2.4 g of tris(hydroxymethyl)aminomethane, 480 g of urea, 78 g of arginine, 0.5 g of reduced glutathione, 20 g of glycerol were dissolved in water and water was added to 1 L, and pH was adjusted to 9.5.

11021 Renaturation solution 1: argin ne of 78 g, reduced glutathione of 0.5 g, glycerol of 20 g, sodium carbonate of 2g, sodium bicarbonate of 4.5 g, pH was 9.5.

11031 Renaturation solution 2: 0.5 g of reduced glutathione, 20 g of glycerol, 2 g of sodium carbonate, 4.5 g of sodium bicarbonate, pH was 9.5.

11041 Renaturation solution 3: 0.2 g of reduced glutathione, 0.75 g of sodium carbonate, 1.5 g of sodium bicarbonate, pH was 9.5.

11051 Eluent: 1.5 g of sodium carbonate, 3 g of sodium bicarbonate, 5 g of glycerol, 58.44 g of were dissolved in water and water was added to made up to 1 L. 11061 Example 2 Screening of recombination conditions 11071 The operation was carried out according to steps (1) to (5) in Example 1 (diluent 4 was selected for dilution, and the renaturation conditions were chosen based on the comparisons by three schemes. Scheme 1: the renaturation solution t and the renaturation solution 3 were used in sequence to perform tangential flow ultrafiltration renaturation; Scheme 2: the renaturation solution 2 and the renaturation solution 3 were used in sequence to perform tangential flow ultrafiltration renaturation; Scheme 3: the renaturation solution 1, the renaturation solution 2 and the renaturation solution 3 were used in sequence to perform tangential flow ultrafiltration renaturation. The ultrafiltration displacement volume ratio of the renaturation solution to the protein solution used for each run of tangential flow ultrafiltration renaturation was 1:3, the feed pressure for each tangential flow ultrafiltration was 25 bar, and the pore size of the tangential flow ultrafiltration membrane package used for the tangential flow ultrafiltration renaturation was 10 kd).

11081 The following step (6) was proceeded: 11091 (6) The obtained sample of RBD-TT recombinant protein renaturationwas diluted to 0.1mg/mL, and the antigen titers under different renaturation conditions were detected by ELISA.

11101 The results are shown in FIG. 3. The antigen obtained in Scheme 3 hahd the highest titer.

11111 Example 3 Preparation of recombinant subunit vaccine against novel coronavirus 11121 The operation was carried out according to steps (1) to (5) in Example 2 (diluent 4 was selected for dilution, and the renaturation condition was: the renaturation solution 1, the renaturation solution 2 and the renaturation solution 3 were used in sequence to perform tangential flow ultrafiltration renaturation).

11131 The following steps (6) and (7) were proceeded: 11141 (6) The obtained sample of RBD-TT recombinant protein renaturation was purified by DEAE anion exchange chromatography under the following chromatographic conditions: the di alysate with 5-fold of column volumes was first used to equilibrate the chromatographic column, and then the samples were loaded onnto the chromatographic column, 20% of the eluent was used for elution to obtain the vaccine bulk (FIG. 4).

11151 (7) The obtained recombinant protein vaccine bulk was filtered and sterilized with a 0.22 pm filter, an aluminum adjuvant with a final concentration of 1 mg/mL was added, and the mixture was packaged to obtain the required recombinant subunit vaccine against novel coronavirus.

11161 Example 4 Induction of the body by recombinant subunit vaccine against novel coronavirus to produce protective neutralizing antibodies 11171 (1) The RBD recombinant protein vaccine was prepared according to the preparation method of Example 2.

11181 (2) 30 female mice aged four weeks were divided into three groups: a RBD-TT test group, a RBD recombinant protein group and a control group, with 10 mice in each group. The RBD-TT recombinant subunit protein vaccine was administered to mice in the RBD-TT test group at the dosage of 40ug/ mouse. The mice in the RBD recombinant protein group was immunized with single RBD recombinant protein vaccine at an immunization dose of 40g/mouse. The control group was treated by intramuscular injection of the same amount of phase-changing solution plus aluminum adjuvant. Immunization was then boosted twice a week, the serum was collected two weeks after the last immunization, and the resulting serum was heat-inactivated at 60°C for 30 minutes.

11191 (3) Mouse serum (1:2-1:32) was diluted with serum-free medium multiple ratio, the diluted serum were mixed with novel coronavirus diluent (1000 median tissue culture infective dose (TC1D 50)/mL) at a volume ratio of 1:1 respectively, and the mixture was incubated at 37°C for 1 hour.

11201 (4) Vero E6 cells were inoculated into 96-well cell culture plates at the dose of 3/10 'cells/well and cultured to monolayers at 37°C. The culture solution in the cell culture plate was discarded, the culture plate was washed twice with calcium-magnesium-free buffer, and then the virus-antibody mixed solution in step (3) was added in an amount of 100 pL/well, and an equal volume of virus maintenance solution was added, and 8 replicate wells were set for each serum dilution. Meanwhile, virus-free, serum-free and blank control groups were set as the negative controls, and the cells were cultured under the conditions of 37°C and 5%C07 for 3 days, and the pathological condition of cells was observed. The serum titers of antibodies were calculated according to the Reed-Muench double method. As shown in FIG. 5, in RBD-TT test group, the geometric mean titer (GMT) of the neutralizing antibodies which could effectively be induced in the body to fight against novel coronavirus was 256, which was significantly different from the control group. The GMT of neutralizing antibodies in the single RBD recombinant protein group was 64, indicating that TT peptide could effectively increase the immunogenicity of RBD protein, so that the immune effect of the vaccine was guaranteed.

11211 The materials and solutions used in this example were as follows, but were not limited thereto.

11221 The Vero E6 cells used herein were available from the American type culture collection (ATCC).

11231 Calcium-magnesium-free buffer: 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate and 0.24 g of potassium dihydrogen phosphate. The pH value of the solution was adjusted to 7.4 with hydrochloric acid. Water was added for dissolution and to made up to 1 L. 11241 Virus maintenance solution: prepared by adding 1% fetal bovine serum into commercial DMEM cell culture medium.

11251 Example 5 Induction of specific cellular immune response in the body by recombinant subunit vaccine against novel coronavirus 11261 (1) 30 female mice aged four weeks were divided into three groups: a RBD-TT test group, a RBD recombinant protein group and a control group, with 10 mice in each group. The RBD-TT recombinant subunit protein vaccine was administered to the mice in the RBD-TT test group at the dosage of 40g/mouse, The RBD recombinant protein group was immunized with the single RBD recombinant protein vaccine at the immunization dose of 40ug/mouse. The control group was injected the same amount of phase-changing solution plus aluminum adjuvant. Immunization was then boosted twice a week, the serum was collected two weeks after the last immunization, and the resulting serum was heat inactivated at 60°C for 30 minutes.

11271 (2) The mice were sacrificed on the 14th day after the primary immunization. The spleen was taken and lightly crushed under sterile condition to obtain spleen cell suspension. The obtained spleen cell suspension was treated with red blood cell lysates for 5 minutes and then neutralized with PBS. The supernatant was discarded after centrifugation at 1000 rpm, and then the precipitate was washed twice with serum-free RPM-1640 medium. Finally, the cell precipitate was re-suspended in 5% serum RPM-1640 medium to obtain a mouse spleen lymphocyte suspension.

11281 (3) The mouse spleen lymphocyte suspension was diluted to 3x106 cells/mL and the procedures were conducted by following the EL1S A kit instructions.

11291 The results showed that both the RBD-TT test group and the RBD group could induce the expression of specific gamma interferon (INF-y) and interleukin-4(I1-4) in the lymphocytes of immunized mice, and the specific gamma interferon (INF-y) and interleukin-4(IL-4) in the RBD-TT test group were higher than those in the RBD group (see FIG. 6).

11301 Any combination of the technical features of the above-described examples may be made. For purpose of brevity, not all possible combinations of the technical features of the above-described examples are described. However, the combinations of the technical features should be considered to fall within the scope of the specification unless there are contradictions between them.

11311 In the above-mentioned examples, only several embodiments of the present disclosure are described specifically and detailed, but they should not be understood as limiting the scope of the disclosure. It should be noted that for those skilled in the art, several modifications and improvements can be made without departing from the spirit of the disclosure, and these modifications and improvements are all intended to fall within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be subject to the appended claims

SEQUENCE LISTING

<HO> Zhejiang Pukang Biotechnology co,Itd National Institute for Viral DISEASE Control and Prevention, China CDC Hangzhou Medical College Institute of Laboratory Animals Science,CAMS & PUIVIC <120> Method for preparing recombinant subunit vaccine against novel coronavirus <130> GWP202108350 <150> 202011216187.0 <151> 2020-11-04 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 258 <212> PAT <213> Artificial Sequence <220> <223> amino acid sequence as a template <400> 1 Met Val Glu Lys Gly Ile Tyr Gin Thr Ser Asn Phe Arg Val Gin Pro 1 5 10 15 Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe 25 30 Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn 40 45 Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn 55 60 Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys 70 75 80 Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile 90 95 Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile 105 110 Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile 120 125 Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn 135 140 Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg 150 155 160 Asp Ile Ser Thr Glu Ile Tyr Gin Ala Gly Ser Thr Pro Cys Asn Gly 170 175 Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gin Ser Tyr Gly Phe Gin 185 190 Pro Thr Asn Gly Val Gly Tyr Gin Pro Tyr Arg Val Val Val Leu Ser 200 205 Phe Glu Leu Leu His Ma Pro Ala Thr Val Cys Gly Pro Lys Lys Ser 210 215 220 Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu 225 230 235 240 Thr Gly Gly Gly Gin Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile 245 250 255 Phe Glu <210> 2 <211> 777 <212> DNA <213> Artificial Sequence <220> <223> nucleic acid fragment for expressing a RBD-TT recombinant protein <400> 2 atggttgaga aaggtatcta ccagacctct aacttccgtg ttcagccgac cgaatctatc 60 gttcgtttcc cgaacatcac caacctgtgc ccgttcggtg aagtgttcaa cgctacccgt 120 ttcgcttctg tttacgcttg gaaccgtaaa cgtatctcta actgcgttgc tgactactct 180 gttctgtaca actctgctag cttctctacc ttcaaatgct acggtgttag tccgaccaaa 240 ctgaacgacc tgtgcttcac caacgtttac gctgacagct tcgttatccg tggtgacgaa 300 gttcgtcaga tcgctccggg tcagaccggt aagatcgctg actacaacta caaactgccg 360 gacgacttca ccggttgcgt tatcgcttgg aactctaata acctggactc taaagttggt 420 ggtaactaca actacctgta ccgtctgttc cgtaaatcta acctgaaacc gttcgaacgt 480 gacatctcta ccgaaatcta ccaggctggt tctactccgt gcaacggtgt tgaaggtttc 540 aactgctact tcccgctgca gtcttacggt ttccagccga ccaacggtgt tggttaccag 600 ccgtaccgtg ttgtggttct gagctttgaa ctgctgcacg ctccggctac tgtttgcggt 660 ccgaagaaat ctaccaacct ggttaagaac aaatgcgtta acttcaactt caacggtctg 720 actggtggcg gtcagtacat caaagctaac tctaaattca Icggtatctt cgaataa 777

Claims

WHAT IS CLAIMED IS: 1. A method for preparing a recombinant subunit vaccine against SARS-CoV-2, comprising the steps of: a) transfecting a plasmid containing a nucleic acid fragment set forth in SEQ ID NO: 2 into LI coil, wherein the nucleic acid fragment is capable of expressing an RBD-TT fusion protein; b) culturing the E. coli to express the RBD-TT fusion protein, breaking bacterial cells and separating a crude extract of the inclusion body of the RBD-TT recombinant protein; c) dissolving the crude extract of the inclusion body in a denaturing solution containing urea, and purifying the inclusion body by anion exchange chromatography to obtain a crude sample of RBD-TT recombinant protein; d) diluting the crude sample of RBD-TT recombinant protein with a diluent; whrerin, the diluent contains 450 -510 g of urea, 70 -86 g of arginine, 0.3-0.7 g of reduced glutathione, 15-25 g of glycerol, and a buffer substance per liter, with a pH value of 9 -10; e) renaturing the diluted RBD-TT recombinant protein through dialysis replacement or tangential flow ultrafiltration, using a renaturation solution 1, a renaturation solution 2 and a renaturation solution 3 in sequence; whrerin, the renaturation solution 1 contains 70-86 g of arginine, 0.3-0.7 g of reduced glutathione, 15-25 g of glycerol, 1-3 g of sodium carbonate and 3-6 g of sodium bicarbonate per liter, with a pH value of 9-10; whrerin, the renaturation solution 2 contains 0. 3-0.7 g of reduced glutathione, 15-25 g of glycerol, 1-3 g of sodium carbonate, 3-6 g of sodium bicarbonate per liter, with a pH value of 9-10; whrerin, the renaturation solution 3 contains 0.1-0.3 g of reduced glutathione, 0.5-1 g of sodium carbonate, 1-2 g of sodium bicarbonate per liter, with a pH value of 9-10; f) purifying the protein after renaturation by anion exchange chromatography.
2. The method according to claim 1, wherein the dialysis displacement volume ratio of the renaturation solution to protein solution used for each dialysis displacement is independently selected from 1: (50-1000), and time for each displacement time is 4-6 hours.
3. The method according to claim 2, the pore size of the dialysis bag used in the dialysis replacement is in a range of 5 kd to 30 kd.
4. The method according to claim 1, wherein the ultrafiltration displacement volume ratio of the renaturation solution to the protein solution used for each tangential flow ultrafiltration renaturation is independently selected from 1: (2-5), and the feed pressure for each tangential flow ultrafiltration is 20 bar to 30 bar.
5. The method according to claim 4, wherein the pore size of the tangential flow ultrafiltration membrane package used for the tangential flow ultrafiltration renaturation is in a range of 5 kd to 30 kd.
6. The method according to claim 1, wherein the exchange groups of the anion exchange chromatography include a diethylaminoethyl group or quaternary ammonium group.
7. The method according to claim 1, wherein in step d), the dilution is performed in a volume ratio of 1: (1-10) of the crude pure sample of RBD-TT recombinant protein to the diluent.
8. The method e according to claim I, wherein the plasmid is a pET28a E. coil expression pl asmi d.
9. The method according to claim 1, wherein denaturing solution includes 17-23 g of sodium chloride, 450-510 g of urea, and a buff substance per liter, with a pH value of 7.2 to 7.8.
10. The method according to claim 1 or claim 9, wherein the buffer substance is tris(hydroxymethyl)aminomethane.
11. The method according to any one of claims 1-9, wherein in step b), the E. coil is cultured to a logarithmic growth phase, and 0.7 mM to 1.3 mM of isopropyl-p-D-thiogalactoside inducer is used to induce the E. coil to express the fusion protein RBD-TT at a temperature of 25°C to 37°C for 4 to 24 hours.
12. The method according to any one of claims 1-9, wherein the following step is further included after step 1): filtering and sterilizing the renatured recombinant protein and using the renatured recombinant protein in combination with an adjuvant.
13. The according to claim 12, wherein adjuvant comprises an aluminum phosphate adjuvant and/or an aluminum hydroxide adjuvant.