CN115814074B

CN115814074B - Codon optimized mRNA vaccine against novel coronaviruses

Info

Publication number: CN115814074B
Application number: CN202211265196.8A
Authority: CN
Inventors: 陆剑; 钱朝晖; 吴鑫凯; 昝富文; 单科家
Original assignee: Peking University; Institute of Pathogen Biology of CAMS
Current assignee: Peking University; Institute of Pathogen Biology of CAMS
Priority date: 2022-10-17
Filing date: 2022-10-17
Publication date: 2023-08-11
Anticipated expiration: 2042-10-17
Also published as: CN115814074A

Abstract

The present invention discloses codon optimized mRNA vaccines against novel coronaviruses. The invention optimizes CAI level, MFE level and translation efficiency and mRNA stability based on the amino acid sequence of novel coronavirus S protein, thereby obtaining CDS sequence with stable and high expression which is applied to the design of novel coronavirus mRNA vaccine. The codon optimization strategies can improve the effective antigen amount generated after the mRNA vaccine is transduced into a machine body, and provide guidance for further optimizing vaccine production.

Description

Codon optimized mRNA vaccine against novel coronaviruses

Technical Field

The invention belongs to the technical field of biology, relates to preparation of an mRNA vaccine, and in particular relates to an mRNA vaccine which is designed by optimizing codons and aims at a novel coronavirus.

Background

Group or community immunity has been proposed as a strategy to protect vulnerable groups, which can be established by past infection or vaccination. The existing vaccine for the highly pathogenic novel coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) mainly comprises a traditional inactivated vaccine (Sinopharm-BBIBP-CorV, sinovac-CoronaVac, covaxin-BBV 152), which is an inactivated vaccine for the whole virus; also protein vaccines (Novavax-NVX-CoV 2373) and viral vector vaccines (Johnson & Johnson Janssen-ad26.cov2.S, oxford-AstraZeneca-AZD 1222/chaadox 1, spatnik V-Gam-covd-Vac-rAd 26/rAd 5) etc., which are mainly directed against the full length of the viral spike protein or Receptor Binding Domain (RBD); and mRNA vaccine (Pfizer-BioNTech Comirnaty-BNT162b2& Moderna-mRNA-1273), wherein the vaccine is prepared by introducing in vitro transcribed mRNA encoding specific protein or RNA encoding specific gene of a certain pathogen into a body through a certain technical means, translating and expressing the protein in the body, and is used as antigen to activate collective immunity, and the vaccine has a superior immune effect and application prospect. In addition, the existing research data show that, because SARS-CoV-2S protein is distributed on the surface of virus and has high immunogenicity, the above-mentioned neutralizing antibodies generated after vaccination are mainly aimed at S protein, so that the S protein is particularly critical in vaccine production and preparation.

So far, SARS-CoV-2 has undergone multiple mutations, yielding several varieties. The world health organization classifies SARS-CoV-2 variant into two classes, VOC (variants of concern) and VOI (variants of interest). Among the VOCs of interest include Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), omicron (B.1.1.529); the VOIs monitored include Epsilon (B.1.427/B.1.429), zeta (P.2), eta (B.1.525), theta (P.3), iota (B.1.526/B.1.526.1), kappa (B.1.617), delta Plus (B.1.617.2.1), lambda (C.37) and Mu (B.1.621). The advent of SARS-CoV-2 variants has further limited the success and natural immunity of vaccines because they contain genomic alterations, particularly the S protein coding region, which increase their adaptation compared to previously prevalent strains. The SARS-CoV-2S protein is one of the 4 major viral structural proteins, consisting of two subunits, S1 and S2. The N-terminal S1 subunit contains a species-specific receptor binding domain (receptor binding domain, RBD), within which most of the fitness-enhancing amino acid mutations observed in prevalent varieties are located, whose occurrence can affect viral transmission capacity, virulence and re-infection rate by evading natural immunity and vaccine-induced immunity. Thus, the study of vaccines against SARS-CoV-2 variant S protein has helped to update improved vaccine formulations and treatment protocols to suppress viral transmission.

Disclosure of Invention

Aiming at the phenomenon that the protein expression level is not very high in the existing mRNA vaccine technology, the invention adopts different codon optimization strategies to improve the effective antigen amount generated after the mRNA vaccine is transduced into a organism, provides guidance for further optimizing vaccine production, and designs several mRNA vaccines aiming at novel coronavirus SARS-CoV-2 based on the codon optimization strategies.

Codon usage is an important consideration in designing mRNA vaccines because different codon usage affects mRNA translation efficiency and mRNA stability, and thus antigen expression. In the embodiment of the invention, based on the S protein of the novel coronavirus, the codon adaptation index (codon adaptation index, CAI) and the minimum folding free energy (minimum folding energy, MFE) of the CDS are tried to be optimized, and finally, the CDS sequence with stable and high expression is obtained and applied to the design of mRNA vaccine.

Optimization of CAI levels

The improvement in mRNA translation efficiency means that the cell can maintain the original protein expression level by synthesizing less mRNA. The translation elongation rate of a gene is one of the important factors determining the translation efficiency of the gene. Binding of tRNA on A-site is the primary rate limiting step during translational elongation. Because of the different intracellular concentrations of tRNA's corresponding to different codons, there is also a large difference in the time required for decoding of different codons. It is generally believed that genes in organisms tend to undergo a strong translational efficiency selection, so that codons on their coding sequences (CDS) tend to have a co-evolution relationship with trnas in the cell, and that the co-evolution results, i.e. the frequency of use of different codons on the CDS, corresponds to the supply of intracellular trnas. So we can find codons with higher translational efficiency for subsequent optimization design by analyzing the codon usage level.

The relative usage frequency of synonymous codons (relative synonymous codon usage, RSCU) can be used to reflect the relative usage of synonymous codons corresponding to amino acids. The calculation process is as follows:

wherein RSCU is provided with _ij Represents the relative frequency of use, X, of the jth codon of the ith amino acid _ij Represents the number of occurrences of the j-th codon of the i-th amino acid, n _i The number of codons corresponding to the i-th amino acid. Based on RSCU value we can calculate the relative fitness w corresponding to each codon _ij I.e. the ratio of the RSCU value corresponding to the current codon to the codon with the highest frequency of use corresponding to the amino acid:

w _ij a value between 0 and 1, the closer to 1 indicates a higher codon fitness. The mRNA level expression level of the binding gene can be estimated by estimating the codon usage frequency of all mRNA in the cell, and calculating w according to the above process _ij Values. The specific calculation is as follows:

wherein Codon _ln Representing the number of occurrences of a codon l on the nth gene, TPM _n Represents the mRNA expression level of the gene in host tissues or cells, and m is the total number of genes. TPM (Transcripts Per Kilobase of exon model per Million mapped reads) maps read transcripts per million for every kilobase of transcripts, real numbers greater than 0, obtained by genomic sequencing. TPM for TPM of target transcript t _t The calculation process is shown as follows:

wherein ,q_t Reads generated for RNA sequencing can map to reads on target transcripts, l _t For the length of the target transcript, T represents all transcripts. After the reading on the target transcript is corrected by the length of the transcript, dividing by the sum of all transcript mapping readings corrected by the length of the transcript to calculate the TPM of the target transcript _t . One gene usually corresponds to a plurality of transcripts, and in order to remove redundancy of transcripts and to facilitate calculation, statistics are performed using the longest transcript corresponding to the gene in the present invention to represent the gene. TPM (trusted platform module) _n Namely TPM which is the longest transcript corresponding to the nth gene _t 。

We collected the gene expression level information (https:// storage. Google apis. Com/gtex_analysis_v8/rnaseq_data/GTEx_analysis_2017-06-05_v8_RNASeQCV 1.1.9_gene_tpm. Gct. Gz) in human tissues from the GTEx database, represented the median of the expression level in different tissues, and calculated the w corresponding to each codon according to the above description process _ij . The CAI value of the gene is w corresponding to each codon on CDS _ij Is a geometric mean of (c).

In the classical CAI calculation process, X _ij The number of occurrences of each codon on CDS in tens of hundreds of genes highly expressed in a species does not reflect the reality of intracellular RNA level codon usage. The popularity of second generation sequencing techniques and the development of histology have enabled accurate quantification of transcripts in cells. According to the invention, the gene expression level (TPM) is introduced to weight each transcript when counting the number of codons, so that the quantization of the codon usage environment in cells can be better realized.

For the amino acids on the S protein, w is selected _ij Codons of=1 are encoded. If the experiment requires avoidance of a particular cleavage site, then suboptimal codons are used downstream of the cleavage site for encoding.

(II) optimization of MFE levels

The stability of the folding structure of mRNA is beneficial to prolonging the half-life period of the mRNA in cells and increasing the continuous expression period of the protein. We developed an MFE genetic optimization algorithm to optimize the iteration of CDS composition of the S protein sequence to improve mRNA stability. In the iterative process, the LinearFold software is used to predict the secondary structure of the mRNA and MFE values.

The optimization algorithm of the MFE level is developed based on a genetic algorithm, and aims to directionally optimize the MFE index in the process of variation passage after generating a population, and the specific implementation steps are as follows:

1. randomly generating N corresponding CDS sequences as ancestral populations according to the provided amino acid sequences;

2. respectively generating M subsequences for each sequence in the population, and randomly introducing M synonymous mutations into each subsequence;

3. calculating MFE values corresponding to all sequences in the group, reserving N pieces with the lowest MFE values, and completing the primary generation succession of the group;

4. the 2-3 steps are cycled T times, or the lowest MFE value of the group sequence in the last 50 rounds of succession is not changed (the evolution process approaches convergence), and the final group sequence is output.

In a specific optimization procedure for the new coronavirus sequence, we performed an optimization for MFE for the full length of amino acids of the S protein using the following parameters: population size N is 200, maximum iteration number T is 1600, number M of individuals is 10, number M of synonymous mutations is introduced into each sequence is 100, and optimized CDS sequence (LowestMFE: the lowest MFE individual in population sequence) is obtained.

The population size N and the number of subsequences M in the parameters described above may be suitably adjusted according to the magnitude of the computational effort in the computing environment. The greater the values of N and M, the greater the number of population sequences per cycle, and the greater the effectiveness of the selection, but the corresponding increase in the computation of the MFE values, typically 50N 10000, 1M 1000. The value of m can be selected within the interval of 1/100 to 1/5 of the length of the optimized amino acid sequence. In the embodiment of the invention, the value of m is set to be 1/10 of the length of the amino acid sequence of the S protein, namely 1/10 of the codon usage corresponding to the amino acid is changed in the process of generating each subsequence. Too small a value reduces the optimization effect of each cycle, so that the convergence process becomes slow, and too large a value oscillates back and forth around the local optimum point and cannot converge. T can be set according to the operation speed and time requirement, and if the time is abundant and convergence is not achieved, more iterations can lead to better optimization of the MFE of the group.

(III) optimization of translation efficiency and mRNA stability

The HighestCAI sequence is obtained through the optimization method of the CAI level, the HighestCAI sequence is used as an ancestral sequence, the MFE is directionally optimized according to the MFE genetic optimization algorithm, and the sequence with the lowest MFE value is respectively taken as an optimization sequence considering translation efficiency and mRNA stability in a certain generation or a plurality of generations.

(IV) verification of protein expression at the cellular level

The HighestCAI sequence, the LowestMFE sequence and one or more optimized sequences which are compatible with translation efficiency and mRNA stability are respectively expressed in cells through an expression vector, the expression quantity is detected, and one or more sequences with high expression level are selected for mRNA vaccine design.

In order to explore the influence of the above-mentioned different levels of optimization strategies on protein expression, in the examples of the present invention, optimal designs of CAI (HighestCAI) and MFE (LowestMFE) were performed on the CDS sequences of the viral S genes of BA.2.12.1 and BA.4/5, respectively. To verify the effect of CAI optimization on protein expression, we generated a semi-optimal sequence with random codon usage, with CAI value of 0.7, between HighestCAI and original virus CDS, as one of the controls for optimization experiments. Meanwhile, consideration of the sequence CAI is added in a selection link of a genetic optimization algorithm aiming at the MFE, so that an optimized sequence (MFE_CAI) which has both translation efficiency and mRNA stability is obtained. I.e. using the HighestCAI sequence as ancestral sequence, a directed optimization of MFE is performed. The sequence with the lowest MFE value was taken as the candidate (mfe_cai1, mfe_cai2) in the 500 th and 1000 th generation, respectively. Finally, we performed cellular level protein expression validation of the DNA sequences designed by the different protocols described above together with the CDS sequences of the original virus, as well as the control sequences.

The screened optimized DNA sequence is used for preparing mRNA vaccine, and the steps are as follows:

1) Constructing a plasmid: the optimized DNA sequence is constructed into a plasmid vector, usually, a 5'-UTR and a 3' -UTR for improving the translation efficiency are added to both ends of the optimized DNA sequence, a promoter (promoter) sequence is added to the 5 '-end of the 5' -UTR, and a terminator (terminator) sequence is added to the 3 '-end of the 3' -UTR if necessary;

2) Template generation: performing enzyme digestion on the plasmid vector constructed in the step 1) for linearization, and obtaining linearization plasmid DNA through DNA gel electrophoresis and gel cutting recovery;

3) In vitro transcription: carrying out in vitro transcription by using the linearized plasmid DNA as a template by using an in vitro transcription kit to obtain target mRNA;

4) modification of mRNA: capping and tailing the mRNA transcribed in vitro by capping enzyme and tailing enzyme respectively to enhance the stability of the mRNA, so that the mRNA has higher translation efficiency when being used as a vaccine; at the same time, mRNA may also be subjected to nucleotide modifications, such as pseudouracil modifications and the like

5) Preparation of a delivery system: the modified mRNA is coated with a delivery system, e.g., prepared as polymer or lipid based nanoparticles (LNP), to provide an mRNA vaccine.

If necessary, RNase inhibitors may be added in the above steps 3) and 4) to prevent degradation of RNA; after the completion of the RNA synthesis in step 3), the template DNA was removed by DNase I.

The invention optimizes the codon of virus S protein gene based on the amino acid sequence of SARS-CoV-2 mutant omicron S protein, which improves the expression level of S protein in eukaryotic cell. The effect of codon usage levels and stability on gene expression has been demonstrated in several studies, and our approach also has a general effect on other gene-optimized expression. For mRNA vaccine, the efficient expression of effective antigen can reduce the inoculation dosage and the inoculation times of the vaccine, reduce the uncomfortable reaction of patients after inoculation and reduce the production cost. Therefore, the invention can provide a more effective solving way for the design of novel vaccines and provide practical guiding significance for vaccine production.

Drawings

FIG. 1 is a western blot experiment result of protein expression after optimization and original sequence of S protein CDS sequences of novel coronavirus mutants BA.2.12.1 and BA.4/5 in the example, wherein the strain BA.2.12.1 has 4 optimized sequences except the original virus sequence S1, which are respectively numbered as S2-S5, and sequentially represent a scheme CAI_MFE1 of optimal folding free energy condition (LowestMFE), control sequence (CAI=0.7), optimal codon adaptability (HighestCAI) and translation efficiency and stability; the BA.4/5 strain has 5 optimized sequences except the original virus sequence s1, the optimized sequences are respectively numbered as s2-s6, the optimized sequences sequentially represent the optimal folding free energy condition (LowestMFE) of mRNA, the control sequence (CAI=0.7), the optimal codon adaptability (HighestCAI), and two schemes of CAI_MFE1 and CAI_MFE2 which have both translation efficiency and stability; MOCK is a blank control and s1 is the original gene sequence of the optimized provirus. Bands of Full length S (FL S) and Cleaved S protein (clear S) and internal reference protein (action) are labeled on the right side of the gel.

Detailed Description

The specific construction process of the CDS optimization of the present invention is described in detail by examples below with reference to the accompanying drawings, and the use method and experimental results of the CDS optimization of the present invention are further described by examples.

In the invention, CDS sequences of virus S genes of SARS-CoV-2 omicron mutant strains BA.2.12.1 and BA.4/5 which are currently epidemic worldwide are optimally designed, meanwhile, consideration of the weight of a sequence CAI is added in a selection link of a genetic optimization algorithm aiming at MFE, an optimized sequence (MFE_CAI) which takes translation efficiency and mRNA stability into consideration is obtained, and the CDS sequences obtained in the design and an original CDS sequence are subjected to protein expression verification at a cellular level.

1. Optimization of viral S gene CDS sequences

According to the S protein amino acid sequence of SARS-CoV-2 omicron mutant strain BA.2.12.1 (see SEQ ID NO:1 in the sequence table), we optimally designed CDS sequence from two angles of codon adaptability and mRNA folding free energy. Meanwhile, UTR (untranslated region) sequence of RPL30 gene with high translation efficiency in human is added, namely:

5’-UTR:

5’-CCTTTCTCGTTCCCCGGCCATCTTAGCGGCTGCTGTTGGTTGGGGGCCGTCCCGCTCCTAAGGCAGGAAG-3’(SEQ ID NO：2)；

3’-UTR:

5’-ACCTTTTCACCTACAAAATTTCACCTGCAAACCTTAAACCTGCAAAATTTTCCTTTAATAAAATTTGCTTGTTTTAAAAA-3’(SEQ ID NO：3)。

the original CDS sequence of the virus was downloaded from NCBI database. We explored four optimization modes for ba.2.12.1. The first is codon-fitness optimization (HighestCAI), we choose w for each amino acid on the protein _ij Codons of=1. The second is the folding free energy condition optimum (LowestMFE), obtained by the MFE optimization algorithm developed in the present invention, the specific parameters are as follows: n=200, t=1600, m=10, m=100. The third and fourth methods respectively give consideration to translation efficiency and stability, and use the HighestCAI sequence as ancestral sequence to perform the directional optimization of MFE. The lowest MFE sequences (mfe_cai1, mfe_cai2) were taken at generation 500 and generation 1000, respectively. The other control sequence was a sequence generated under random codon usage (CAI value 0.7). For the S gene protein sequence of BA.4/5, we used exactly the same parameters and steps to generate the corresponding optimized and control CDS sequences.

The original CDS sequence, lowestMFE sequence, highestCAI sequence and MFE_CAI1 sequence of the S protein of the mutant strain of BA.2.12.1 are respectively shown as SEQ ID NO in a sequence table: 4. 5, 6 and 7. The amino acid sequence, the original CDS sequence, the LowestMFE sequence, the HighestCAI sequence, the MFE_CAI1 sequence and the MFE_CAI2 sequence of the BA.4/5 mutant strain S protein are respectively shown in SEQ ID NO: 8. 9, 10, 11, 12 and 13.

We inserted the 5'UTR sequence directly behind the Hind III cleavage site of the pcDNA3.1 (+) vector plasmid, while using the matched 3' UTR sequence, and the kozak sequence AGGAAG of RPL30, the optimized sequences had circumvented the Hind III and Xba I cleavage sites (BA.2.12.1 mutant S protein gene cloning cleavage sites).

2. Gene acquisition

1. Gene synthesis: the optimized S protein genes of omicron mutant strains BA.2.12.1 and BA.4/5 are sent to Nanjing Jinsri biotechnology Co., ltd for gene synthesis, and plasmid freeze-dried powder is returned to about 4 mug;

2. conversion: at 40. Mu.L ddH ₂ Thawing the dry powder plasmid by O solution, adding 1 mu L of DH5 alpha escherichia coli engineering bacteria, carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 1min, carrying out ice bath for 5min, adding into non-resistant sterile LB, culturing for 1h in a 37 ℃ constant temperature shaking incubator, transferring into 50mL of ampicillin resistant LB, and culturing overnight;

3. harvesting the cells: transferring the bacterial liquid cultured overnight into a 50mL centrifuge tube, centrifuging for 10min at 3000g, discarding the supernatant, and collecting the precipitated cells;

4. plasmid extraction: plasmid neutralization was performed using the plasmid neutralization kit from axygen company, briefly as follows: adding matched RNAse into buffer S1, uniformly mixing, adding 5mL into the precipitated cells, and re-suspending the cells; 5mL buffer S2 was added and mixed slightly upside down to lyse the cells; after standing for 3min, adding buffer S3 5mL, and mixing uniformly upside down to neutralize S2, so as to prevent excessive lysis from causing genome DNA pollution; adding buffer B, reversing, mixing, and centrifuging for 10min at 3000 g. After fixing the adsorption column, pouring the supernatant after centrifugation into the adsorption column, pumping out the liquid, adding buffer W1 7mL, after pumping out the liquid, adding buffer W2 8mL which is provided with absolute ethyl alcohol according to the requirements of the specification, and pumping out the liquid again; removing the adsorption film, washing again with 300 μL buffer W2, centrifuging, and adding 300 μL ddH preheated at 65deg.C ₂ O, after soaking, 12000g is centrifuged for 30s, the adsorption film is removed, and the liquid in the tube is plasmid extracting solution. Plasmid concentration and 280/260 260/230 ratio were measured with a Nano drop ultramicrophotometer and labeled.

3. S protein expression

1. Cell preparation: HEK 293T cells

(1) Cell resuscitation: one HEK 293T cell frozen and stored is taken out from a liquid nitrogen tank, quickly thawed and melted in a constant temperature water bath at 37 ℃, and 10mL of complete culture medium (DMEM culture medium containing 10% FBS) is taken to 15 in a biosafety cabinetThe cells were removed by centrifugation at 500g for 5min in a mL centrifuge tube. The supernatant after centrifugation was discarded, and the cells were resuspended in 10mL of complete medium and inoculated to 75cm ² In a cell culture flask, 5% CO at 37 DEG C ₂ Culturing in an incubator for 2 days until the cell density is about 95%;

(2) Cell passage: the cell culture medium with the cell density of 95% is discarded, 5mL of cells are washed by PBS and then discarded, 1mL of 0.25% trypsin-EDTA is added to digest the cells for 2min, 10mL of complete culture medium is used for neutralization, the uniformly mixed cells are blown and sucked, the cells are transferred into a 15mL centrifuge tube, 500g of centrifuge tube is used for 5min, the supernatant is discarded, the cells are resuspended in the complete culture medium, and one sixth of passage is carried out, and the cells can grow up every day. After resuscitating, the cells can be subjected to experimental operation after one or two passages;

(3) And (3) paving: 293T cells with cell density of about 95% were washed with PBS, digested with 0.25% trypsin-EDTA and resuspended, and then seeded in six well plates at 37℃with 5% CO ₂ The incubator was cultured overnight.

2. Cell transfection

(1) Transfection reagent: PEI (sigma company) dilutes PEI to 1mg/mL with normal saline and then passes through a 0.22 mu m filter membrane for standby;

(2) Transfection: taking serum-free DMEM culture medium as mother liquor, preparing six holes by 500 mu L of each hole, adding 2 mu g of plasmid into each hole, adding 4 mu L of transfection reagent PEI, uniformly mixing and standing for 15min; discarding the complete culture medium in the six-well plate, replacing with serum-free DMEM culture medium, adding the prepared and standing transfection liquid into each well, marking, mixing, and mixing with 5% CO at 37deg.C ₂ Culturing in an incubator; transfection was performed for 6 hours and replaced with complete medium.

3. Protein collection

(1) Cell collection: 48 hours after transfection, the culture medium in the six-well plate was discarded, 1.5mL of the culture medium was added to each well, and the cells were then blown down in 1.5mL of EP tube, and the cells were washed by centrifugation at 12000g for 30s, and the supernatant was discarded;

(2) Cell lysis: RIPA lysate

Lysing cells on ice, adding 120 mu L of RIPA lysate into each tube, blowing off and mixing the cells, standing on ice for 30 minutes, and shaking in the middle to fully lyse;

(3) Collecting protein: after sufficient lysis, each tube was centrifuged at 12000g for 30min at 4℃and 100. Mu.L of supernatant was removed to a new EP tube for use;

(4) Protein quantification: thermo protein quantitative kit

Preparing mother liquor: liquid a, liquid b=49:1

Taking one ELISA plate, adding 10 mu L of protein sample into each hole, adding 200 mu L of protein quantitative mother solution, mixing uniformly with light shaking, standing at 37 ℃ and incubating for 30 minutes; setting a standard substance protein concentration gradient for drawing a standard curve; detecting absorbance by using an enzyme-labeled instrument after sample incubation is completed, and detecting the wavelength 560;

(5) Sample preparation: protein sample concentrations were determined according to a standard curve, and the concentrations of each sample were kept consistent by dilution with RIPA, and the samples were boiled at 98 ℃ for 10 minutes with the addition of amoebonite.

4. Expression detection

(1) 10% SDS-PAGE gel configuration: a PAGE gel is prepared according to the requirements of the specification by adopting a yase kit, and the method is briefly as follows: placing 1.5mm rubber plates, diluting A, B liquid of lower layer rubber according to a ratio of 1:1 to prepare mixed liquid, adding a coagulant according to a ratio of 1:100, pouring 7.5mL of lower layer rubber mixed liquid into each rubber plate, and sealing with isopropanol; after standing for 20 minutes, discarding the upper isopropanol layer, gently flushing with tap water for three times, gently flushing with purified water for three times, and sucking clean filter paper to remove more residual purified water; preparing upper glue mixed liquid, preparing upper glue A, B liquid 1:1 mixed liquid, adding coagulant 1:100, filling the upper layer of the glue plate with the coagulant, inserting a glue comb with a hole of 1.5mm 15, and standing for 20 minutes to obtain the gel plate for western detection.

(2) And (3) Running buffer configuration: 10 Xrunning buffer stock (2.5L) was prepared: tris 75.5g, glycine 470g, SDS 25g, dissolved with stirring and diluted 1X when used.

(3) Western: adding the prepared samples into a glue hole according to a certain sequence, and adding marker indicator protein into the glue hole for 80V for 30 minutes and 120V for 1 hour and 20 minutes;

(4) Preparing a transfer film liquid: 10X transfer stock (2L) was prepared: diluting 60g of Tris and 28g of glycine with purified water, and stirring and uniformly mixing; diluting to 1X when in use, and adding methanol to a final concentration of 20%; pre-cooling the prepared film transferring liquid for standby at 4 degrees;

(5) Transfer film (wet transfer): after the running is finished, film transferring operation is carried out, and the film transferring operation is briefly as follows: the transfer film clamping plate comprises two sponges and two thick filter papers, when transferring the film, the glue plate is pried off, glue is attached to an NC film (nitrocellulose film), two sides are respectively provided with one filter paper and one sponge, and the whole sequence of the clamping plate is positive electrode-sponge-filter paper-NC film-glue-filter paper-sponge-negative electrode; transferring the film to 80V for 2 hours;

(6) Closing: after the transfer of the membrane, the transferred membrane is taken out, and is sealed for 1 hour on a shaking table by using 5% skimmed milk (dissolved and diluted by TBST);

(7) Incubation resistance: the blocked NC membrane was cut according to the marker size and incubated with antibodies against the S protein on a shaker for 2 hours, in this experiment, the S protein was incubated with Sino Biological Inc company SARS-CoV-2 (2019 nCoV) Spike, rabbit PAb (40591-T62) and SARS-CoV-2 (2019 nCoV) Spike S2, rabbit PAb (40590-T62) antibodies, 1:2000 diluted with blocking solution; incubating anti-beta-actin from mouse antibody of sigma company, and diluting 1:3000;

(8) Washing the film: replacing the primary antibody with TBST, and washing the membrane for 3 times by a shaking table for 15 minutes each time;

(9) Secondary antibody incubation: incubating with corresponding HRP (horseradish peroxidase) labeled secondary antibody according to primary antibody source, namely, the S protein corresponding secondary antibody is anti-rabbit secondary antibody, the actin corresponding antibody is anti-mouse secondary antibody, diluting the antibody 1:5000, and shaking for 1 hour;

(10) Washing the film: the same membrane is washed after the membrane is washed;

(11) Color development: HRP substrate was diluted in the manner described. The bio-rad chromogenic kit adopted by the method comprises the steps of diluting a chromogenic solution A, B in a mode of 1:1, taking out a film, draining more water, adding the chromogenic solution onto the film, shaking until the whole film is covered, keeping away from light for 1 minute, and scanning the film for imaging in a chemiluminescent imager.

Experimental results:

the results of the western blotting are shown in FIG. 1, which shows that the expression of the original viral CDS sequence protein (lanes 2 and 8 from left to right) is similar to that of the Mock group (lanes 1 and 7), indicating that the expression amount of the protein is extremely small. The expression of the S proteins of BA.2.12.1 and BA.4/5 can be significantly improved, both based on the optimization method of CAI and MFE (lanes 3, 5, 9 and 11). Optimization of both CAI and MFE also significantly increased protein expression levels (lanes 6, 12, 13). This result demonstrates the expression barrier that the original viral sequence may encounter in making an mRNA vaccine and reveals the importance of codon usage for antigen protein expression. At the same time, the results also confirm the potential role of the sequence optimization method proposed in the study in the optimization design of mRNA vaccine.

Claims

1. A method for codon optimization of mRNA encoding the S protein of the novel coronavirus SARS-CoV-2, comprising the step of optimizing the CDS sequence encoding the S protein by optimization at CAI level and optimization at MFE level in sequence, wherein the method comprises the steps of:

(1) Optimization of CAI levels: the optimization of the CAI level is based on the amino acid sequence of S protein, and codon adaptation index optimization is carried out on the CDS of the original coding sequence of the S protein to obtain a HighestCAI sequence; the method comprises the following steps: firstly, introducing a gene expression quantity TPM to weight each transcript when counting the number of codons, namely:

wherein ,X _ij represents the firstiAmino acid number onejThe number of occurrences of the seed codon,Codon _ln represents a codonlIn the first placenNumber of occurrences on individual genes, TPM _n Representing the mRNA expression level of the gene in the host tissue or cell,mis the total number of genes; then according toX _ij Calculating the relative usage frequency RSCU of synonymous codons:

wherein ,RSCU _ij represents the firstiAmino acid number onejThe relative frequency of use of the seed codons,n _i is the firstiThe number of codons corresponding to the amino acids; the relative fitness w corresponding to each codon is calculated according to the RSCU value _ij I.e. the ratio of the RSCU value corresponding to the current codon to the codon with the highest frequency of use corresponding to the amino acid:

w _ij a value between 0 and 1, the closer to 1 indicating a higher codon fitness; the CAI value of the gene is w corresponding to each codon on CDS _ij Selecting a HighestCAI sequence with the highest CAI value, namely optimal codon adaptability;

(2) Optimization of MFE level: the optimization of the MFE level is to optimize the stability of an mRNA folding structure of a coding sequence, obtain a lowestMFE sequence with the minimum folding free energy MFE, and specifically realize the optimization through an MFE genetic optimization algorithm, and comprises the following steps:

1) Using the HighestCAI sequence resulting from optimization of CAI levels as an ancestral sequence;

2) Generating M subsequences for the ancestral sequences, randomly introducing M synonymous mutations into each subsequence, wherein the M value is 1/100-1/5 of the length of the S protein amino acid sequence;

3) Calculating MFE values corresponding to all sequences, reserving N pieces with the lowest MFE values, and completing the primary generation succession of the group;

4) And (3) cycling the steps 2) to 3) for T times, or outputting the final group sequence without changing the lowest MFE value of the group sequence in the last 50 rounds of succession, wherein the sequence with the lowest MFE value is LowestMFE sequence, namely the optimized sequence.

2. The method of claim 1, wherein in the MFE genetic optimization algorithm, N is 200, m is 10, m is 100, and the maximum number of iterations T is 1600.

3. An mRNA vaccine of an S protein of SARS-CoV-2, wherein the S protein is an S protein of a mutant strain ba.2.12.1, the mRNA vaccine comprises an mRNA encoding the S protein, and the sequence of the mRNA is as shown in SEQ ID NO: 5. 6 or 7.

4. An mRNA vaccine of an S protein of SARS-CoV-2, wherein the S protein is an S protein of a mutant strain ba.4/5, the mRNA vaccine comprises an mRNA encoding the S protein, and the sequence of the mRNA is as shown in SEQ ID NO: 10. 11, 12 or 13.