CN116970632A

CN116970632A - Recombinant plasmid for expressing SARS-CoV-2S protein, its construction method and application

Info

Publication number: CN116970632A
Application number: CN202310302706.2A
Authority: CN
Inventors: 籍晓云; 董咸池; 张焕琴
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2023-03-23
Filing date: 2023-03-23
Publication date: 2023-10-31

Abstract

The application provides a recombinant plasmid for expressing SARS-CoV-2S protein, a construction method and application thereof, belonging to the technical field of bioengineering. The recombinant plasmid takes pcDNA3.4 as a skeleton plasmid, and constructs a novel coronavirus S protein expression vector taking pcDNA3.4 as the skeleton plasmid and containing a specific Fc-tag sequence at the C end, so that the efficient expression of SARS-CoV-2S protein is realized, and the SARS-CoV-2S protein obtained by expression can keep good protein activity, and can be applied to a plurality of immunological detection platforms to assist in monitoring the infection and outbreak of a novel coronavirus. The recombinant plasmid is used as carrier of RBD polyploid in SARS-CoV-2S protein receptor binding region, and has fully verified safety and reliability, and the vaccine has good medicine property, low cost and high benefit.

Description

Recombinant plasmid for expressing SARS-CoV-2S protein, its construction method and application

Technical Field

The application belongs to the field of biological medicine, and in particular relates to a recombinant plasmid, and particularly relates to a recombinant plasmid for expressing SARS-CoV-2S protein, a construction method and application thereof.

Background

The novel coronavirus is one of coronaviruses, and can infect a large number of higher animals including humans, belonging to the genus coronavirus. Based on genomic characteristics, SARS-CoV-2 virus belongs to a member of the Sarbecovirus subgenera in the β genus of the subfamily coronavirus (CoV). The SARS-CoV-2 virus genome is about 29.8Kb long, and the 5' -end 2/3 gene encodes an enzyme involved in viral replication, leaving 1/3 of the gene encoding four structural proteins, spike (S), envelope (E), membrane (M), nucleocapsid (N), and other accessory proteins.

The technology of foreign maturation is mainly mRNA vaccine, which is to introduce mRNA expressing antigen target into the body through a specific delivery system, express protein in the body and stimulate the body to generate specific immune response. The vaccine production cycle was short, but with Delta, omacron mutants appeared, the effectiveness of the vaccine was drastically reduced. This may be associated with variations in the S protein and the storage conditions of the vaccine are demanding.

Prior studies have shown that the RBD region of spike protein is considered to be an enriched region of virus neutralizing epitopes and is capable of inducing the production of neutralizing antibodies by the body. Thus, subunit vaccine studies based on RBD regions are theoretically feasible. However, the immune effect of such recombinant subunit vaccines is not comparable to that of conventional inactivated vaccines. Therefore, how to accurately and efficiently detect and prevent novel coronaviruses becomes a problem to be solved, and antigen polyploidization is an important means for improving the immunogenicity of the novel coronaviruses.

Disclosure of Invention

The first application is to provide a recombinant plasmid for expressing SARS-CoV-2S protein.

Furthermore, the recombinant plasmid takes pcDNA3.4 as a skeleton plasmid, is inserted with polyploid for encoding novel coronavirus S protein fragments, and the amino acid sequence of the receptor binding domain of SARS-CoV-2S protein is shown as SEQ ID NO. 1.

Further, the insertion sites of the polyploid of the SARS-CoV-2S protein gene fragment on pcDNA3.4 are insertion sites Nhe1 and Not1.

Further, the pcDNA3.4 vector comprises an Fc-tag positioned at the C end, and the amino acid sequence of the Fc-tag is shown as SEQ ID NO. 2.

Further, the novel coronavirus S protein fragment polyploid is composed of at least two novel coronavirus S protein receptor binding domain fragments in tandem.

Further, the nucleotide sequence of the novel coronavirus S protein receptor binding region fragment is 319-592, 328-529, 328-596 or 322-586 of the S protein nucleotide sequence. Preferably, the C-terminus is located between positions 529 and 535 or 560 and 596 of the nucleotide sequence of the S protein. Among them, the novel coronavirus S protein receptor binding domain fragment is preferably the 319 th to 592 th nucleotide sequence of the S protein.

Further, the novel coronavirus S protein fragment polyploid may be a novel coronavirus S protein receptor binding domain fragment, i.e., diploid, triploid, tetraploid, metaploid, hexaploid, heptaploid, octaploid, nine ploid, ten ploid, etc., of RBD, preferably hexaploid to octaploid of RBD.

Further, the novel coronavirus S protein receptor binding domain fragments are preferably connected in series by GG or GS sequences, more specifically, the novel coronavirus S protein receptor binding domain fragments are connected in series by GGS, GGGS, GSGS, SGGS, GGGGS, GSGGS, GGSGS, GGSGGS, GGGSGS, GGGGGS or GGGSGGS sequences.

Further, the C-terminal of the novel coronavirus S protein receptor binding domain fragment is linked to the Fc fragment of human immunoglobulin to form polyploids, and the novel coronavirus S protein receptor binding domain fragments or the polyploids of the novel coronavirus S protein fragment are linked by a protease or a protein cross-linking agent.

Further, the proteases include, but are not limited to, transpeptidase and an associated protein ligase (OaAEP 1). Further, the protein cross-linking agents include, but are not limited to, ethylene glycol, disuccinimidyl suberate, glutarate suberate succinate, dimethyl diimidate, and maleimide hexane.

Further, the RBD polyploid can be obtained by connecting novel coronavirus S protein fragments through the enzyme or protein cross-linking agent, or RBD protein in a higher ploidy form can be obtained by connecting RBD polyploid through the enzyme or protein cross-linking agent.

Specifically, if the RBD triploid fragment is ligated into a plurality of RBD triploid fragments by using a protein ligase (OaAEP 1), GL is inserted into the N-terminal and NGL is inserted into the C-terminal on the basis of the original RBD triploid sequence, thereby constructing an expression plasmid of GL-RBD-RBD-RBD-NGL. The expression vector is transfected into mammalian cells, cultured for 5 to 7 days, and culture supernatant is collected, and GL-RBD-RBD-NGL protein is purified by adopting an affinity purification method. Purified protein was added to the protein-coupled ligase at an appropriate ratio, and after incubation for an appropriate period of time, the reaction was stopped. And (3) taking a proper amount of protein, analyzing an enzyme linked result by SDS-PAGE, and finally separating the linked high-power fragments by using a molecular sieve to obtain RBD protein in a higher-power aggregation form formed by a plurality of RBD triploids.

The second application provides a preparation method of the recombinant plasmid.

Further, the preparation method comprises the following steps:

s1, inserting the S protein fragment of the encoding novel coronavirus into a pcDNA3.4 plasmid to obtain an amplified plasmid;

s2, carrying out PCR amplification by using the amplified plasmid as a template and using a first primer and a second primer to obtain a PCR product;

s3, carrying out double enzyme digestion on the pcDNA3.4 plasmid to obtain pcDNA3.4-C-8xHis-tag plasmid;

s4, connecting the pcDNA3.4-C-8xHis-tag plasmid with the PCR product to obtain a recombinant plasmid.

The third application is to provide a vaccine.

Further, the vaccine comprises the recombinant plasmid.

Further, the vaccine is an mRNA vaccine, a protein vaccine, or an adenovirus vaccine.

Further, polyploids of the novel coronavirus S protein fragment in the vaccine activate a ploidy of B cell receptors, generating novel coronavirus antibodies.

The application has the following beneficial effects:

1. the application takes pcDNA3.4 as a skeleton plasmid, is inserted with polyploid for encoding novel coronavirus S protein fragments, and particularly designs Fc-tag positioned at the C end of the plasmid, thereby obtaining higher expression quantity of SARS-CoV-2S protein, and the expressed SARS-CoV-2S protein can keep good protein activity, and can be applied to a plurality of immunological detection platforms to assist in monitoring infection and outbreak of novel coronavirus.

2. The application adopts recombinant plasmid as carrier of RBD polyploid of SARS-CoV-2S protein receptor binding region, and the plasmid as mature injection, and its safety and reliability have been fully verified, and the vaccine has good drug-forming property, and its preparation is cheap and economical.

3. The RBD domain of SARS-COV-2 spike protein, which contains the majority of neutralizing epitopes, is the primary immune target of the vaccine. However, RBD monomers are small in size, poorly immunogenic for antigen presentation, and unstable in vivo. Thus, we constructed RBD polyploids by concatenating RBD domains and fusing with Fc domains. The polyploid (i.e., 4 RBD-Fc) cannot be obtained by the conventional polymerase chain reaction because of the instability of the primer molecule due to the excessively long sequence, easy breakage, and the problem of reduced amplification efficiency due to the primer diploid. In order to break through the technical problem, the application realizes successful construction and expression of the sequence through multiple experiments and fumbling and finally through adopting a double enzyme digestion method.

Drawings

FIG. 1 is a schematic representation of a novel coronavirus S protein fragment polyploid.

FIG. 2 is a pcDNA3.4M vector map.

FIG. 3 is a graph showing the results of electrophoresis of the novel coronavirus S protein fragment having Fc fragment and polyploids thereof.

Fig. 4 is a size exclusion chromatogram of RBD oligomer protein: wherein mAU represents milliabsorbance units, M represents a marker, NR represents non-reducing, and R represents reducing.

Detailed Description

The following describes specific embodiments of the present application with reference to the drawings.

In the present application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, reagents, materials, and procedures used herein are reagents, materials, and conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present application, definitions and explanations of related terms are provided below.

The main reagent comprises:

plasmid pcDNA3.4M: purchased from General Biol corporation.

Receptor Binding Domain (RBD) of spike protein: synthesized by General Biol, and ligated to pcDNA3.4M universal vector by General Biol, respectively, to give the corresponding pcDNA3.4-RBD and pcDNA3.4-RBD-S plasmids.

The enzymes Xho I-HF and Nhe I-HF were both purchased from NEB company.

Sequence information:

the original RBD sequence information, i.e., the gene sequence of SARS-CoV-2 spike protein, was obtained from NCBI (National Center for Biotechnology Information, https:// www.ncbi.nlm.nih.gov /), with an Access number of NC_045512.2.

The amino acid sequence of SARS-CoV-2S protein is shown as SEQ ID NO. 1.

The amino acid sequence of the Fc-tag at the C end of the plasmid is shown as SEQ ID NO. 2.

The amplification primers during PCR were as follows:

PcDNA3.4-RBD-FP is shown in SEQ ID NO. 3.

PcDNA3.4-5' -RBD-GGGS-RP is shown as SEQ ID NO. 4.

GGGS-RBD-GGGS-FP is shown in SEQ ID NO. 5.

GGGS-RBD-GGGS-RP is shown in SEQ ID NO. 6.

GGGS-RBD-PcDNA3.4-3' -FP is shown in SEQ ID NO. 7.

GGGS-RBD-PcDNA3.4-3' -RP is shown in SEQ ID NO. 8.

PcDNA3.4-RBD-FP is shown in SEQ ID NO. 9.

RBD-finger-RP is shown as SEQ ID NO. 10.

The finger-Fc-FP is shown as SEQ ID NO. 11.

The finger-Fc-RP is shown in SEQ ID NO. 12.

PcDNA3.4-RBD-FP is shown in SEQ ID NO. 13.

RBD-finger-RP is shown as SEQ ID NO. 14.

A-RBD-D-FP is shown as SEQ ID NO. 15.

A-RBD-D-RP is shown as SEQ ID NO.16

PcDNA3.4-RBD-FP is shown in SEQ ID NO. 17.

The 3RBD-FC-RP is shown as SEQ ID NO. 18.

Example 1

The embodiment provides a construction process of a recombinant plasmid, which comprises the following steps:

1. preparing materials:

general Biol supplied RBD, RBD-S and pcDNA3.4M plasmids, and RBD-S were ligated to the pcDNA3.4M universal vector to give the corresponding pcDNA3.4-RBD and pcDNA3.4-RBD-S plasmids, wherein the plasmid map is shown in FIG. 1.

The pcDNA3.4M vector contains an amino acid sequence Fc-tag positioned at the C end, and is obtained by multiple modification in the laboratory. By comparison, the sequence can greatly increase the expression level of SARS-CoV-2 spike protein.

2. Process for constructing pcDNA3.4-2RBD-Fc

(1) Homologous recombination

The PCR tube with the above reagents added thereto was incubated at 37℃for one hour.

(2) Transformation

TOP10 competent cells were removed from the-80℃refrigerator, thawed on ice for 10 min, added with 10. Mu.L of homologous recombination product, after 25 min of ice bath, heat shock for 45s, then ice bath for 2 min, added with 800. Mu.LLB liquid medium in an ultra clean bench, and activated on a shaker at 210rpm at 37℃for 1h. After the activation, 200. Mu.L of the bacterial liquid was uniformly spread on an Amp-resistant LB solid medium in a super clean bench, and the culture was performed at 37℃for 16 hours.

(3) Shaking bacteria and plasmid extraction

Glycerol bacteria placed at-80℃were thawed on ice, 500. Mu.L was pipetted into a super clean bench and added to 100mLLB broth, and 100. Mu.L of Amp antibiotic was added, followed by overnight incubation on a thermostatic shaker at 37℃at 210 rpm. After the overnight incubation, plasmid extraction was performed using a Kangfu century company plasmid extraction kit, and plasmids were extracted according to the instructions, plasmid DNA was eluted using deionized water, and the ratio of concentration to A260:280 was measured using a UV5NANO meter and stored in a-20℃refrigerator.

(4) Cleavage identification of pcDNA3.4-2RBD-Fc

The enzymes XhoI-HF and NheI-HF are added into the PCR tube in sequence as shown in the following table, and the mixture is uniformly mixed by a centrifuge after the sample addition is finished:

the PCR tube with the above reagents added thereto was incubated at a constant temperature of 37℃for one hour.

The DNA gel electrophoresis experiments were performed in agarose gel at a concentration of 1%, followed by digestion.

The digested sample was added to a1% concentration nucleic acid gel and electrophoresed at 120V for 45 minutes to obtain purified pcDNA3.4-2RBD-Fc.

3. Process for constructing pcDNA3.4-3RBD-Fc

(1) Homologous recombination

The PCR tube with the above reagents was incubated at 37℃for one hour

(2) Transformation

(3) Shaking bacteria and plasmid extraction

(4) Cleavage identification of pcDNA3.4-3RBD-Fc

the PCR tube added with the reagent is incubated for one hour at the constant temperature of 37 ℃, general DNA1% agarose gel electrophoresis is selected, and the digested sample is added into nucleic acid gel with the concentration of 1%, and the pressure of 120V is kept for 45 minutes.

4. Construction of pcDNA3.4-4RBD-Fc and restriction enzyme assay

(1) Homologous recombination

The PCR tube with the above reagents was incubated at a constant temperature of 37℃for one hour

(2) Transformation

(3) Shaking bacteria and plasmid extraction

(4) Enzyme digestion identification

The enzymes XhoI-HF and NheI-HF were added to the PCR tubes in the order shown in the following table, and after the sample addition was completed, the mixture was homogenized by a centrifuge as shown in the following table:

Example 2

This example provides a method for preparing competent cells.

1. Preparation:

(1)150ml 0.1mol/L CaCl ₂ sterilizing in autoclave

(2) 250ml LB liquid culture based on autoclave sterilization

(3) 20ml 85% (volume fraction) 0.1M CaCl ₂ Glycerol solution (85% 0.1M CaCl) ₂ 15% glycerol) was sterilized in autoclave

(4) Several 50ml centrifuge tubes and 1.5ml centrifuge tube are sterilized in autoclave

(A)CaCl ₂ ·H ₂ O 147.01

0.1M CaCl ₂ 500ml 7.3505g constant volume to 500ml

(B) 85% (volume fraction) 0.1M CaCl ₂ Glycerol solution (85% 0.1M CaCl) ₂ 15% glycerol

2. Operating procedure

(1) From the deep frozen strain, the strain was streaked and cultured in 5ml of LB (without antibiotics) at 37℃for 12 to 16 hours (300 rpm) with shaking (overnight culture).

(2) 5ml of the bacterial liquid (inoculated) was inoculated into 300ml of LB (37 ℃ C., preheated) and cultured in an expanded manner.

(3) Beginning about 2 hours, OD was measured ₆₀₀ Values (this culture was used only to measure the OD600 value, which indicates the cell density, and is used here to determine whether the culture is in the logarithmic growth phase). At OD600 of 0.4-0.5 (about 2.5 h), the cultures were placed in an ice bath for 20 min to stop cell division.

(4) The cultures were aliquoted into 6 50ml centrifuge tubes (sterilized and pre-chilled), centrifuged at 4000rpm at 4℃for 10 minutes.

(5) The supernatant was discarded and 30ml of 0.1M CaCl was added ₂ (sterilized and pre-cooled) the pellet was suspended and centrifuged at 4000rpm at 4℃for 10 minutes.

(6) Discarding the supernatant, adding 15ml 85%0.1M CaCl ₂ Glycerol solution (sterilized and pre-chilled) suspended pellet and dispensed into 1.5ml centrifuge tubes, about 100 μl per tube. Placing at-80deg.C for storage.

(7) The deep frozen strain was streaked and cultured in 25ml LB (without antibiotics) at 37℃for 6-8 hours (300 rpm) overnight.

(8) 4-1ml of bacterial liquid (inoculated) is taken into 2x 250ml of LB (18 ℃ C., precooled) and is cultivated in an enlarged way.

(9) About 16 hours, at 45 OD intervals ₆₀₀ Values (this culture was used only to measure the OD600 value, which indicates the cell density, and is used here to determine whether the culture is in the logarithmic growth phase). At OD600 of 0.4-0.5 (about 2.5 h), the culture was placed in an ice bath for 20 minCells were allowed to stop dividing.

(10) The cultures were aliquoted into 10 50ml centrifuge tubes (sterilized and pre-chilled), centrifuged 2500g at 4℃for 10 minutes.

(11) The supernatant was discarded, and 10 x 20ml of ITB (sterilized and pre-cooled) was added to suspend the pellet, which was centrifuged at 2500g at 4℃for 10 minutes.

(12) The supernatant was discarded, suspended and precipitated by adding 10 x 4ml of ITB (sterilized and pre-cooled), added 3ml dmso in total, allowed to stand for 10 minutes, and dispensed into 1.5ml centrifuge tubes, about 100 μl per tube. Placing at-80deg.C for storage. Competent cells, 293F cells, were finally obtained.

Example 3

Based on example 2, three plasmids pcDNA3.4-2RBD-Fc, pcDNA3.4-3RBD-Fc and pcDNA3.4-4RBD-Fc were transferred into competent cells, 293F cells, respectively.

1. Extraction of pcDNA3.4-2RBD-Fc, pcDNA3.4-3RBD-Fc, pcDNA3.4-4RBD-Fc plasmid

Three plasmids in example 1 were extracted using the Kangfu century company plasmid extraction kit and following the instructions.

2. PEI transfection method for transfection of plasmid

(1) Preparation of the culture Medium

OPM-293CD05Medium stored at 4℃was placed in a biosafety cabinet and allowed to warm to room temperature.

(2) Host cell preparation

The cell density count of yesterday is 2x10 ⁶ The cell density of HEK-293 cells was re-measured and the cell density of HEK-293 cells was re-diluted to 2X10 with medium warmed to room temperature ⁶ 。

(3) Plasmid transfection

3 sterile 15mLEP tubes were each added with 5mL of sterile PBS, followed by a separate addition of plasmid that had been filtered with a 0.22. Mu.M filter (100. Mu.g of the desired transfection plasmid per 100mL of culture medium of HEK-293 cells with a cell density of 2X 106) and the appropriate PEI transfection reagent (300. Mu.L PEI per 100. Mu.g of plasmid transfected). After 5 minutes of standing at room temperature, the solution in the 15mLEP tube containing the plasmid was slowly added in hanging drop to the 15mL EP tube containing PEI and allowed to stand for 15 minutes. After 15 minutes, the liquid in 15mL EP tube was all slowly added in hanging drops to HEK-293 cell fluid and incubated in a light-resistant shaker at 37℃at 125rpm at 8% carbon dioxide.

The state of the transfected cells was checked every day, and after the dead cells were more than 50% of the total cells or cultured for 96 hours, the cell fluid was collected and subjected to the next purification experiment.

Example 4

On the basis of example 3, this example provides a method for purifying SARS-CoV-2S protein as described above.

1. Collecting the supernatant

The 9 cell supernatants collected in example 3 were centrifuged at 2500rpm for 10 minutes, the pellet was discarded, the supernatant was centrifuged at 8000rpm for 60 minutes, and the final supernatant was suction filtered with 0.22. Mu.M filter paper. 1M Tris-HCl (pH=7.8) buffer was added to the supernatant after suction filtration, and the pH was adjusted to neutral.

2. Preparation of ProteinAbeads

The appropriate volume of Protein A beads stored in 20% ethanol solution was pipetted into the purification column, and the Protein A beads were rinsed with 2 column volumes of deionized water and then with 2 column volumes of PBS.

3. Purifying protein supernatant by column

Protein A beads and rotor in the purification column were added to the supernatant and placed in a magnetic stirrer located in a refrigerator at 4℃for stirring for 2h. After the completion of stirring, the mixture of Protein A beads and supernatant was fed to a purification column, the flow rate was adjusted, and the slow-flowing flow-through liquid was collected in a clean vessel. After the mixed solution is completely added, the flowing-through solution is added into the column again and then flows through once again.

4. Eluting the target protein

(1) Eluting the hybrid protein

Washing off the Protein with 10mM wash solution of 10 Protein A beads volumes, collecting the washed liquid, after washing 10 Protein A beads volumes, 1 Protein A beads volume per wash, sucking 10uL when the liquid in the column is about to run out, measuring solubility with 50uLCBB solution, if there is color change, indicating that the Protein has not been washed out, continuing to add PBS wash solution for washing. Otherwise, the PBS impurity washing liquid can be considered to be washed completely, and the next purification operation can be carried out.

(2) Elute protein of interest

After washing the hetero-protein, eluting the target protein with sodium citrate eluent. 2 Protein A beads volumes eluted one tube, a total of 4 tubes. The eluate was immediately neutralized with 2m tris-Hcl (ph=8.5).

(3) Concentrating the protein of interest

The eluted target protein was concentrated to 500uL by centrifugation through ultrafiltration tube, followed by centrifugation at 12000rpm for 10 min, and then further purified by AKTA purifier-using super dex (TM) 200 increment 10/300GL as pre-cartridge, PBS as buffer in system, set parameters: the flow rate was 0.3 mL/min, the maximum pre-column pressure was 1.8MPa, 1 tube per 0.5mL of collection, and the total volume was 24mL.

(4)SDS-PAGE

Taking 5ug sample in the collecting tube of the highest peak of A280 in AKTA purifier, mixing with non-reducing protein loading buffer (NR) and reducing protein loading buffer (R), and boiling at 98deg.C for 5 min to obtain a protein electrophoresis sample. 120V electrophoresis is carried out for 60 minutes, and after coomassie brilliant blue staining solution is stained for 1 hour, tap water is used for soaking overnight for decolorization.

As can be seen from FIG. 4, a representative size exclusion chromatography of the recombinant RBD oligomer on a Superdex 200 Increate 10/300GL column shows UV absorption at 280 nm. The inset shows SDS-PAGE of eluted protein samples.

Example 5

The present embodiment provides a novel coronavirus S protein fragment polyploid consisting of at least two novel coronavirus S protein receptor binding domain fragments in tandem.

The novel coronavirus S protein exists in a trimeric form, with about 1300 amino acids in each monomer, where 300 amino acids constitute a "receptor binding domain segment" (RBD), where the S protein binds ACE 2.

In this example, the nucleotide sequence of the novel coronavirus S protein receptor binding domain fragment is at positions 319-592, 328-529, 328-596 or 322-586 of the S protein nucleotide sequence. Preferably, the C-terminus is located between positions 529 and 535 or 560 and 596 of the nucleotide sequence of the S protein.

It should be noted that, because the S protein sequence is very long, if a fragment is randomly intercepted in the S protein, numerous possibilities exist, so that after a great deal of researches on the spatial structure and action characteristics of the S protein and a great deal of experiments, the conclusion that the fragment can exert the strongest binding effect to the receptor and has the best effect when the novel coronavirus S protein receptor binding region fragment is 319-592, 328-529, 328-596 or 322-586 of the S protein is obtained. Taking diploid as an example, when RBD fragments are 319 th to 592 th, 328 th to 529 th, 328 th to 596 th or 322 th to 586 th of S protein, two RBD fragments can be just combined with two ends of a receptor in a space structure, and if the RBD fragments are too short, even if the RBD fragments form diploid, the RBD fragments can be combined with one end of the receptor, and the combination ability is weak; if the RBD fragment is too long, the RBD fragment is not easy to express, the expression effect is poor, and the receptor binding capacity and the like of the RBD fragment are negatively influenced.

The novel coronavirus S protein fragment polyploid can be diploid, triploid, tetraploid, quintuple, hexaploid, heptaploid, octaploid, nonaploid, tenfold, etc. of the novel coronavirus S protein receptor binding domain fragment, namely RBD. Hexaploid to octaploid of RBD are preferred.

In particular, the novel coronavirus S protein receptor binding domain fragments are preferably connected in series by GG or GS sequences, more particularly, the novel coronavirus S protein receptor binding domain fragments are connected in series by GGS, GGGS, GSGS, SGGS, GGGGS, GSGGS, GGSGS, GGSGGS, GGGSGS, GGGGGS or GGGSGGS sequences.

The novel coronavirus S protein fragment polyploid provided by the embodiment is composed of at least two novel coronavirus S protein receptor binding Region (RBD) fragments, has high expression quantity and good stability, can be applied to antibody detection of novel coronavirus and vaccine preparation, has strong applicability and wide application range, wherein the novel coronavirus S protein fragment polyploid is applied to antibody detection of novel coronavirus, can effectively improve specificity, sensitivity and sensibility of antibody detection, and further effectively improve accuracy of antibody detection, and can stimulate organisms to quickly produce antibodies when being applied to vaccine preparation.

Example 6

The present embodiment provides a novel coronavirus S protein fragment polyploid consisting of at least two novel coronavirus S protein receptor binding domain fragments.

In this example, the C-terminus of the novel coronavirus S protein receptor binding domain fragment is linked to the Fc fragment of human immunoglobulin.

Wherein one or more of the novel coronavirus S protein receptor binding domain fragments constitute a polyploid unit that forms a dimer by the Fc fragment in which the novel coronavirus S protein receptor binding domain fragments are linked, and treating the dimer as a novel coronavirus S protein fragment polyploid.

Specifically, the polyploid unit may be an RBD fragment (haploid) or an RBD polyploid. For example, two novel coronavirus diploids can form a novel coronavirus tetraploid by an Fc fragment; two novel coronavirus triploids are capable of forming a novel coronavirus hexaploid by the Fc fragment; two novel coronavirus tetraploids are capable of forming a novel coronavirus octaloid by an Fc fragment; two novel coronavirus fivefold can form a novel coronavirus fivefold through the Fc fragment, etc.

In the embodiment, the Fc fragment is innovatively added to the RBD fragment and the C end of the RBD polyploid, so that the RBD fragment and the RBD fragment, the RBD fragment and the RBD polyploid can be flexibly combined to form a dimer with stronger activity and higher binding capacity with ACE2, namely a novel coronavirus S protein fragment polyploid, and more choices are provided for detecting and preventing novel coronaviruses.

Example 7

In this example, novel coronavirus S protein receptor binding domain fragments or novel coronavirus S protein fragment polyploids are linked by a protease or protein cross-linking agent.

Wherein the protease includes but is not limited to transpeptidase and conjunctive protein ligase, and the protein cross-linking agent includes but is not limited to polyethylene glycol, disuccinimidyl suberate, glutarate suberate succinate, dimethyl diimidate, dimethyl imidoester and maleimide hexane.

In this example, RBD polyploids were obtained by ligating RBD fragments with the above-mentioned enzyme or protein crosslinking agent, or RBD polyploids as described in example 5 or 6 were ligated with the above-mentioned enzyme or protein crosslinking agent to obtain RBD proteins in a higher aggregate form.

The fragment size of 4RBD-Fc was 4108bp, four fragments were linked by GGGS and bound to FC at the C-terminus, while the RBD multimer without Fc was linked by GGGS linker and GGGS did not change the structure of the protein. Glycine is commonly found in flexible natural linkers, and the addition of a polar serine residue can maintain the stability of the linker. Thus, the most commonly used flexible linkers contain Gly and serine residues, one example of which is the (Gly-Gly-Gly-Gly-serine) n sequence.

In the experiments, the RBD multimers with Fc were easier to construct and also easier to sequence in the construction of PcDNA3.4-2RBD-Fc, but increasing the RBD fragments or increasing the amount of RBD fragments did not result in PcDNA3.4-3RBD-Fc and PcDNA3.4-4RBD-Fc during the construction of PcDNA3.4-3 RBD-Fc. In order to overcome the technical problem, the inventor firstly designs a primer at the N end to put SP on RBD, designs a primer at the C end to be connected with FC, and designs a primer at the middle RBD segment to be connected with a connector, so as to search that the molar ratio of the segments is connected with PCDNA3.4M carrier. The PcDNA3.4-3RBD-Fc, wherein the number of the bases of the PcDNA3.4-4RBD-Fc is 3000 base pairs and 4000 base pairs respectively, whether the correct sequence is obtained or not can not be determined through sequencing, the inventor determines whether the construction is successful or not by adopting a double enzyme digestion method, and finally experimental conditions are searched for a plurality of times, so that the successful expression of the sequence is obtained.

Commonly used tags include flag, HA, his, myc, V5, etc., all having molecular weights within 1-2 kd. Thus, the tag is an important tool for finding protein interaction domains or functional domains. It is conventional practice to split a protein structurally into an N-terminal, a C-terminal and a central region core. For example, protein A and another protein B have interactions, and in order to determine which domain of protein A is involved in the interaction process, different truncations of protein A can be constructed, and the interactions of the different truncations with protein B can be determined by Co-IP analysis. For example, the full length and N-terminal and ΔC-terminal of subsequent experiments can pull down protein B, and other truncations are not performed, which indicates that the N-terminal domain is involved in the binding process. Since the epitopes of these different truncations have no homology regions, only tag antibodies can be used for IP experiments.

The application is to express SARS-CoV-2S protein with high efficiency, through repeated design of experiments, a novel coronavirus S protein expression vector with pcDNA3.4 as skeleton plasmid and specific Fc-tag sequence at C end is constructed, thus realizing SARS-CoV-2S protein expression with high efficiency, and the expressed SARS-CoV-2S protein can keep good protein activity, and can be applied to multiple immunological detection platforms to assist in monitoring infection and outbreak of new coronavirus.

Meanwhile, RBD is dimerized through Fc domain, so that protein stability and size can be increased, and efficient lymph node targeting and FcR+ dendritic cell capturing are promoted.

While the preferred embodiments and examples of the present application have been described in detail with reference to the accompanying drawings, the present application is not limited to the above-described embodiments and examples, and various changes may be made within the knowledge of those skilled in the art without departing from the spirit of the present application.

Sequence listing

SEQ ID NO.1：

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNS

ASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYK

LPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGS

TPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKK

STNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQT

LEILDITPCS

SEQ ID NO.2：

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE

YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK

LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO.3：

GATCGAACCCTTGCTAGCATGTTCGTGTTTCTGGTGTTACTGC SEQ ID NO.4：

TGCACCCGTGAACCTCCACCTGAGCAAGGGGTGATGTCCA SEQ ID NO.5：AGGTTCACGGGTGCAGCCTACCGAA

SEQ ID NO.6：CCACCTGAGCAAGGGGTGATGTCCASEQ ID NO.7：

ACCCCTTGCTCAGGTGGAGGTTCACGGGTGCAGCCTACCGAASEQ ID NO.8：

TCAGTGATGGTGGTGATGGTGTGAGCAAGGGGTGATGTCCASEQ ID NO.9：

GATCGAACCCTTGCTAGCATGTTCGTGTTTCTGGTGTTACTGCSEQ ID NO.10：

GGCTCGGAGCCGCCGCCGCCTGAGCAAGGGGTGATGTCCASEQ ID NO.11:

GGCGGCGGCGGCTCCGAGCCCCCGAAATCTTGTGASEQ ID NO.12:

TAGACTCGAGCGGCCGCTCATTTACCCGGAGACAGGGAGAGSEQ ID NO.13：

GATCGAACCCTTGCTAGCATGTTCGTGTTTCTGGTGTTACTGCSEQ ID NO.14：

GGCTCGGAGCCGCCGCCGCCTGAGCAAGGGGTGATGTCCASEQ ID NO.15:

AGGTTCACGGGTGCAGCCTACCGAASEQ ID NO.16:

CCACCTGAGCAAGGGGTGATGTCCASEQ ID NO.17：

GATCGAACCCTTGCTAGCATGTTCGTGTTTCTGGTGTTACTGCSEQ ID NO.18：

GGCTCGGAGCCGCCGCCGCCTGAGCAAGGGGTGATGTCCA

Claims

1. A recombinant plasmid for expressing SARS-CoV-2S protein is characterized in that pcDNA3.4 is used as skeleton plasmid, polyploid for encoding novel coronavirus S protein fragment is inserted into the recombinant plasmid, and the amino acid sequence of SARS-CoV-2S protein is shown as SEQ ID NO. 1.

2. The recombinant plasmid according to claim 1, wherein the polyploid encoding SARS-CoV-2S protein gene fragment has insertion sites on pcDNA3.4 that are insertion sites Nhe1 and Not1.

3. The recombinant plasmid according to claim 1, wherein the pcDNA3.4 vector comprises an amino acid sequence Fc-tag at the C-terminus of the vector, and the amino acid sequence of the Fc-tag is shown in SEQ ID NO. 2.

4. The recombinant plasmid of claim 1, wherein the polyploid of said novel coronavirus S protein fragment is comprised of at least two novel coronavirus S protein receptor binding domain fragments in tandem; the N end of the nucleotide sequence of the novel coronavirus S protein receptor binding region fragment is positioned between 319 th and 328 th positions of the S protein nucleotide sequence, and the C end is positioned between 529 th and 596 th positions of the S protein nucleotide sequence; the novel coronavirus S protein receptor binding domain fragments are connected in series through GGS, GGGS, GSGS, SGGS, GGGGS, GSGGS, GGSGS, GGSGGS, GGGSGS, GGGGGS or GGGSGGS sequences.

5. The recombinant plasmid of claim 4, wherein the C-terminal end of the novel coronavirus S protein receptor binding region fragment is linked to the Fc fragment of a human immunoglobulin to form polyploids, wherein the novel coronavirus S protein receptor binding region fragment is linked to each other or the polyploids of the novel coronavirus S protein fragment are linked to each other by a protease or a protein cross-linking agent.

6. The recombinant plasmid of claim 5, wherein the protein cross-linking agent comprises a glycol, disuccinimidyl suberate, glutarate suberate succinate, dimethyldiimidate, dimethylimidoester, and maleimidohexane.

7. A method for preparing a recombinant plasmid according to any one of claims 1 to 6, comprising the steps of:

s1, inserting the SARS-CoV-2S protein fragment into pcDNA3.4 plasmid to obtain amplified plasmid;

8. A vaccine comprising the recombinant plasmid of any one of claims 1-6.

9. The vaccine of claim 8, wherein the vaccine is an mRNA vaccine, a protein vaccine, or an adenovirus vaccine.

10. The vaccine of claim 8, wherein polyploid of novel coronavirus S protein fragments in the vaccine activates a ploidy B cell receptor, producing novel coronavirus antibodies.