CN112209995B

CN112209995B - Preparation method of SARS-CoV-2 surface protein receptor binding region

Info

Publication number: CN112209995B
Application number: CN202011117036.XA
Authority: CN
Inventors: 张静静; 安文琪; 王斌; 邢体坤; 宋路萍; 毕利利
Original assignee: Hualan Genetic Engineering Co ltd
Current assignee: Hualan Genetic Engineering Co ltd
Priority date: 2020-10-14
Filing date: 2020-10-19
Publication date: 2022-01-11
Anticipated expiration: 2040-10-19
Also published as: CN112209995A

Abstract

The invention discloses a preparation method of a SARS-CoV-2 surface protein receptor binding region. The invention provides application of a polypeptide shown as SEQ ID No.1 or related biological materials thereof in any one of the following steps: improving the secretion expression yield and the secretion expression efficiency of the SARS-CoV-2 surface protein receptor binding region in host cells; preparing a SARS-CoV-2 surface protein receptor binding region secretion protein product; the related biological material is a coding gene of the polypeptide shown in SEQ ID No.1, or an expression cassette or a recombinant vector containing the coding gene, or a recombinant bacterium or a transgenic cell line. The natural signal peptide of the S protein of the new coronavirus and the artificial signal peptide shown in SEQ ID No.1 are used for guiding the secretion expression of the RBD eukaryotic cells of the new coronavirus, and the result shows that the secretion expression level of the artificial signal peptide is obviously superior to that of the natural signal peptide, so that the method is more suitable for large-scale industrial production and reduces the production cost.

Description

Preparation method of SARS-CoV-2 surface protein receptor binding region

Technical Field

The invention relates to the field of biotechnology, in particular to a preparation method of a SARS-CoV-2 surface protein receptor binding region.

Background

The novel coronavirus (SARS-CoV-2) belongs to the beta genus of coronavirus. The novel coronavirus is an enveloped positive-strand RNA virus, the genome size is 29903nt, and four structural proteins of a surface protein (S), an envelope protein (E), a membrane protein (M) and a nucleocapsid protein (N) are mainly contained. Wherein, the main functions and research meanings of the surface protein (S) are as follows: 1) the functions of combining virus and host cell membrane receptor (ACE-2) and fusing membranes are assumed; 2) determining the host range and specificity of the virus; 3) the transmission between different hosts can be realized through the gene recombination or mutation of a receptor binding Region (RBD), and the higher lethality rate is caused; 4) important sites of action for host neutralizing antibodies; 5) a key target for vaccine design. At present, the Receptor Binding Domain (RBD) of the surface protein (S) is considered as the most important antigen target domain for inducing the body to generate neutralizing antibodies, so that the neutralizing antibodies generated by the body stimulation can be more focused on the receptor binding of the virus, and the immunogenicity and the immune efficiency of the vaccine can be improved.

The SARS-CoV-2 surface protein receptor binding area is used as key immunogen, and the eukaryotic cell secretion expression has the obvious advantages: 1) the background of secretory expression of the supernatant protein is low, and the purification process is simple; 2) soluble expression to avoid formation of inclusion bodies; 3) the signal peptidase precisely cuts and does not have redundant Met residue at the N terminal, and a protein sequence which is expected is generated. Secretory expression usually requires fusion of a signal peptide sequence at the N-terminus of the protein of interest, directing targeted secretion of the foreign protein to a specific membrane system. The signal peptide is located at the N-terminus of the secreted protein and typically consists of 15-30 amino acids, including three regions: 1) the positively charged N-terminus is referred to as the basic amino terminus; 2) the middle hydrophobic sequence is mainly neutral amino acid, can form an alpha helical structure and is also a main functional region of the signal peptide; 3) the negatively charged C-terminus, containing small amino acids, is the signal sequence cleavage site, also known as the processing region.

Disclosure of Invention

The present invention aims at providing the preparation process of SARS-CoV-2 surface protein receptor combining area.

The invention discovers an artificially synthesized signal peptide (signal peptide H, SEQ ID No.1), which guides the secretion of an S surface protein receptor binding Region (RBD) of SARS-CoV-2 to culture supernatant, and the high-purity antigen can be obtained by nickel column purification, and the secretory expression yield of the antigen is obviously higher than that of a natural signal peptide (signal peptide S) of an S protein.

In a first aspect, the invention claims the use of a polypeptide as shown in SEQ ID No.1 or a biological material related thereto in any of the following.

The polypeptide shown in SEQ ID No.1 or the related biological material thereof can be applied to any one of the following parts:

p1, improving the secretory expression yield of the SARS-CoV-2 surface protein receptor binding region in host cells;

p2, improving the secretion and expression efficiency of the SARS-CoV-2 surface protein receptor binding region in host cells;

p3, preparing SARS-CoV-2 surface protein receptor binding zone secretion protein preparation.

The related biological material is a coding gene of the polypeptide shown in SEQ ID No.1, or an expression cassette or a recombinant vector or a recombinant bacterium or a transgenic cell line containing the coding gene.

Wherein the host cell is a eukaryotic host cell.

Further, the eukaryotic host cell may be HEK293 cell, CHO cell, yeast cell, insect cell, and the like.

In a particular embodiment of the invention, the host cell is in particular an Expi293F cell.

Wherein, the coding gene can be any one of the following genes:

(a1) DNA molecule shown in SEQ ID No. 2;

(a2) a DNA molecule which hybridizes with the DNA molecule defined in (a1) under stringent conditions and encodes the polypeptide shown in SEQ ID No. 1;

(a3) and (b) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of homology with the DNA sequence defined in (a1) or (a2) and encodes the polypeptide shown in SEQ ID No. 1.

In a specific embodiment of the invention, the recombinant vector is a recombinant plasmid obtained by inserting a DNA fragment shown in SEQ ID No.5 (positions 1-57 of SEQ ID No.5 are coding genes of a signal peptide H, namely SEQ ID No.2) into a multiple cloning site (such as Hind III and Pac I) of a pCGS3 vector.

In a second aspect, the invention claims a fusion protein.

The fusion protein claimed by the invention is obtained by fusing the polypeptide shown in SEQ ID No.1 to the N end of the SARS-CoV-2 surface protein receptor binding region.

Furthermore, the amino acid sequence of the SARS-CoV-2 surface protein receptor binding region is shown as 20 th to 242 th positions of SEQ ID No. 4.

Furthermore, the amino acid sequence of the fusion protein is shown as 1-242 th position or 1-247 th position of SEQ ID No.4 or shown as SEQ ID No. 4.

The 1 st to 19 th sites of SEQ ID No.4 are signal peptide H (namely SEQ ID No.1), the 20 th to 242 th sites are the SARS-CoV-2 surface protein receptor binding region, the 243 nd and 247 th sites are Linker joints, and the 248 nd and 253 th sites are histidine tags.

In a third aspect, the invention claims a nucleic acid molecule encoding the fusion protein of the second aspect.

Furthermore, the nucleic acid molecule comprises a coding gene of the polypeptide shown in SEQ ID No.1 and a coding gene of a SARS-CoV-2 surface protein receptor binding region in sequence from 5 'end to 3' end.

Furthermore, the encoding gene of the polypeptide shown in SEQ ID No.1 can be any one of the following genes:

(a1) DNA molecule shown in SEQ ID No. 2;

Furthermore, the encoding gene of the SARS-CoV-2 surface protein receptor binding region can be any one of the following genes:

(b1) a DNA molecule shown as SEQ ID No. 3;

(b2) DNA molecules which hybridize under stringent conditions to the DNA molecules defined in (b1) and are of the same protein;

(b3) and (b) a DNA molecule which has 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology with the DNA sequence defined in (b1) or (b2) and encodes the same protein.

More specifically, the nucleic acid molecule is a DNA molecule shown in the 1 st to 726 th position or the 1 st to 741 th position of SEQ ID No.5 or a DNA molecule shown in SEQ ID No. 5. The 1 st to 57 th sites of SEQ ID No.5 are the coding gene (namely SEQ ID No.2) of the polypeptide shown in SEQ ID No.1, the 58 th to 726 th sites are the coding gene (namely SEQ ID No.3) of the SARS-CoV-2 surface protein receptor binding region, the 727-741 st and the 742-st and the 759 th sites are the coding gene of a Linker joint and the histidine tag.

In the above-mentioned nucleic acid molecules or encoding genes, identity means identity of nucleotide sequences. The identity of the nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, the identity of a pair of nucleotide sequences can be searched, calculation can be performed, and then the value (%) of identity can be obtained.

The 90% or greater identity in the nucleic acid molecule or encoding gene may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above-mentioned nucleic acid molecule or encoding gene, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na₃PO₄And 1mM EDTA in a mixed solutionRinsing at 50 ℃ in 2 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In a fourth aspect, the invention claims an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line comprising the nucleic acid molecule of the third aspect.

Wherein the expression cassette is a DNA capable of expressing the fusion protein described in the second aspect in a host cell, and the DNA may include not only a promoter for initiating the transcription of the target gene but also a terminator for terminating the transcription of the target gene. Further, the expression cassette may also include an enhancer sequence.

In a specific embodiment of the invention, the recombinant vector is a recombinant plasmid obtained by inserting a DNA fragment shown in SEQ ID No.5 (positions 1-57 of SEQ ID No.5 are coding genes of a signal peptide H, namely SEQ ID No.2) into a multiple cloning site (such as Hind III and Pac I) of a pCGS3 vector. Accordingly, the transgenic cell line is obtained after the recombinant plasmid is introduced into Expi293F cells.

In a fifth aspect, the invention claims the use of a fusion protein as described in the second aspect above or a nucleic acid molecule as described in the third aspect above or an expression cassette, recombinant vector, recombinant bacterium or transgenic cell line as described in the fourth aspect above in any one of:

Wherein the host cell is a eukaryotic host cell.

In a sixth aspect, the invention claims a method for preparing a SARS-CoV-2 surface protein receptor binding domain secretion protein.

The method for preparing the secretory protein of the SARS-CoV-2 surface protein receptor binding area, which is claimed by the invention, can comprise the following steps:

(A1) introducing a nucleic acid molecule as hereinbefore described in the third aspect into a host cell to obtain a recombinant cell;

(A2) culturing the recombinant cell, and obtaining SARS-CoV-2 surface protein receptor binding domain secreted protein from culture supernatant.

Wherein said nucleic acid molecule is introduced into said host cell by a recombinant vector as described hereinbefore.

In step (a1), the host cell is a eukaryotic host cell.

In the step (A2), the culturing is terminated when the cell viability is reduced to 65-75%.

In step (A2), the protein is secreted from the SARS-CoV-2 surface protein receptor binding domain obtained from the culture supernatant according to a method comprising the following steps: the culture was collected and centrifuged at 3500g for 30min, and the supernatant was collected for concentration by ultrafiltration and purification with a nickel column.

In a seventh aspect, the invention claims the use of a polypeptide as shown in SEQ ID No.1 or a biological material related thereto in any of:

q1, improving the secretory expression yield of the target protein in the host cell;

q2, improving the secretion and expression efficiency of the target protein in the host cell;

q3, preparing the target protein for secretory expression.

The target protein is coronavirus structural protein.

Further, the coronavirus is SARS-CoV-2.

Furthermore, the coronavirus structural protein is the full length S protein, the full length S1 protein, the full length S2 protein, the full length N protein, the full length M protein, the full length E protein, the full length S protein, the full length N protein, the full length M protein, the full length E protein, the full length S protein, the full length N protein, the full length E protein, the full length S protein, the full length M protein, the full length E protein, the full length S protein, the full length E protein, the full length S protein, the full length E protein, the full length S protein, the full length protein.

The invention adopts S protein natural signal peptide S (the enzyme cutting site of the receptor binding region signal peptidase is ensured to be correct theoretically, and the natural space conformation is maintained) and a manually designed signal peptide H (SEQ ID No.1) modified by a mouse antibody kappa chain signal peptide to guide the secretion and expression of a new coronavirus surface protein receptor binding Region (RBD) eukaryotic cell. Experiments prove that: the secretion expression level of the artificially synthesized signal peptide H is obviously superior to that of the natural signal peptide S, and the artificially synthesized signal peptide H is more suitable for large-scale industrial production and reduces the production cost.

Drawings

FIG. 1 is a restriction enzyme identification diagram for expression plasmid construction of natural signal peptide S and synthetic signal peptide H. Wherein, 1-3 are natural signal peptide S, 4-6 are artificially designed signal peptide H.

FIG. 2 is an SDS-PAGE identification of the cell secretion supernatant of the surface protein Receptor Binding Domain (RBD) of the novel coronavirus.

Wherein, 1 is a natural signal peptide S, 2 is an artificially designed signal peptide H, and 3 is a negative group of non-transfection expression plasmids.

FIG. 3 is a graph of the analysis of ash removal rate on secretion of cells of the surface protein Receptor Binding Domain (RBD) of the novel coronavirus. Wherein S is a natural signal peptide S secretion supernatant, and H is an artificially designed signal peptide H secretion supernatant.

FIG. 4 shows SDS-PAGE purification identification of new coronavirus surface protein Receptor Binding Domain (RBD) protein purification. Wherein, 1 is S signal peptide expression secretion supernatant, 2 is S signal peptide secretion expression purification sample; 3 is H signal peptide expression secretion supernatant, and 4 is H signal peptide secretion expression purification sample.

FIG. 5 is a gray scale analysis chart of purified sample of the new coronavirus surface protein Receptor Binding Domain (RBD). Wherein S is a natural signal peptide S purified sample, and H is an artificially designed signal peptide H purified sample.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The main reagents and their manufacturer information in the following examples are as follows:

SARS-CoV-2(2019-nCoV) Spike RBD Gene: beijing Yi Qiao Shenzhou science and technology, Inc.;

Expi293F^TMcells: thermo corporation;

pCGS3 expression vector: merck corporation;

Expi293^TMexpression Medium: thermo corporation;

ExpiFectamine^TM293Transfection Kit: thermo corporation;

Opti-MEM^TMi Reduced Serum Medium: thermo corporation;

protein marker: auspicious;

ready-to-use seamless cloning kit: biometrics (Shanghai) Inc.;

Ni-NTA protein purification kit: biometrics (Shanghai) Inc.;

centrifugal Filters Ultratcel-10K: millipore Corp;

centrifugal Filters 0.5ml 10K Ulracel: millipore Corp;

PBS ph7.4(1 ×): gibco corporation;

gel imaging system: ProteinSimple Co

Cell counting instrument: roche Inc.;

superclean bench: suzhou Antai air technologies, Inc.;

electric heating constant temperature water bath: fisher Scientific Inc.;

CO₂constant temperature shaking table: KRYSTAL corporation;

HYG-A full constant temperature shake flask cabinet: taicang City laboratory facilities;

model DYY-6C electrophoresis apparatus: six instrument factories in Beijing;

DYCP-31DN type horizontal electrophoresis tank: six instrument factories in Beijing;

a micropipette: eppendorf Ltd.

Example 1 expression plasmid construction

In the embodiment, the natural signal peptide S of SARS-CoV-2(2019-nCoV) S protein and the coding gene of artificially synthesized signal peptide H (SEQ ID No.1) modified by the kappa chain signal peptide of a mouse antibody are respectively fused to the 5' end of the coding gene of SARS-CoV-2S protein RBD protein to construct the eukaryotic recombinant expression vector.

The fragment of interest was amplified using SARS-CoV-2(2019-nCoV) Spike RBD Gene (positions 58-726 of SEQ ID No.5, positions 46-714 of SEQ ID No. 6) as the first template.

Primer 1 and primer 5 for the first round of amplification; the amplified fragment is used as a second round template, and a second round of amplification is carried out by using a primer 2 and a primer 5 to obtain an H-RBD target fragment.

Primer 3 and primer 5 for the first round of amplification; the amplified fragment is used as a second round template, and a second round of amplification is carried out by using a primer 4 and a primer 5 to obtain an S-RBD target fragment.

Adopting a ready-to-use seamless cloning kit for recombinant connection: and carrying out double enzyme digestion linearization on pCGS3 by HindIII and PacI, amplifying target fragments S-RBD and H-RBD, and cloning the amplified target fragments to a pCGS3 expression vector through recombination.

Primer 1: 5'-TGGTGCTGATGTTCTGGATTCCTGCTGCTAGATCTAGGGTCCAACCAACAGAGAG-3', respectively;

primer 2: 5' -CACCGTCCTTGACACGAAGCTTGCCACCATGGCCTTGCCTGTTTGGCTGTTGGTGCTGATGTTCTGGATT-3’；

Primer 3: 5'-TGCTGCTGCCCCTGGTGAGCAGCCAGTGCAGGGTCCAACCAACAGAGAG-3', respectively;

primer 4: 5' -CACCGTCCTTGACACGAAGCTTGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGC-3’；

Primer 5: 5' -CAGTTAGCCTCCCCCTTAATTAATTAATGATGGTGGTGATGGTGAG-3’。

The restriction enzyme identification results of the two recombinant expression vectors are shown in FIG. 1. As can be seen from the figure, lanes 1-3 are pCGS3-S-RBD expression plasmid, after enzyme digestion, the vector 7120bp, the target gene 759 bp; lanes 4-6 are pCGS3-H-RBD vector 7120bp, target gene 771bp, size of enzyme cutting band is expected.

The recombinant expression vector pCGS3-S-RBD has the following structural description: and (3) inserting a DNA fragment shown in SEQ ID No.6 between enzyme cutting sites HindIII and PacI of the pCGS3 vector to obtain a recombinant vector (5 'of the SEQ ID No.6 is added with a Kozak sequence for optimizing expression, and the 3' end is added with a termination code). The 1 st to 45 th sites of SEQ ID No.6 are coding genes of natural S signal peptide, the 46 th to 714 th sites are RBD coding genes, 715-729 is Linker joint, 730-747 is histidine tag.

The recombinant expression vector pCGS3-H-RBD has the following structural description: and (3) inserting a DNA fragment shown in SEQ ID No.5 between enzyme cutting sites HindIII and PacI of the pCGS3 vector to obtain a recombinant vector (5 'of the SEQ ID No.5 is added with a Kozak sequence for optimizing expression, and the 3' end is added with a termination code). The 1 st to 57 th sites of SEQ ID No.5 are coding genes of an artificial signal peptide H, the 58 th to 726 th sites are RBD coding genes, 727-741 is a Linker, 742-759 is a histidine tag.

SEQ ID No.5 encodes the protein shown in SEQ ID No. 4. The 1 st to 19 th sites of SEQ ID No.4 are artificial signal peptide H, the 20 th to 242 th sites are RBD, the 243 nd and 247 th sites are Linker, and the 248 nd and 253 th sites are histidine tags.

Example 2 Gray-Scale analysis of SDS protein electrophoretograms

The SDS protein electrophorogram was subjected to grayscale analysis using Image J software. The operation step is that Image → Type → 32-Bit is converted into a gray-scale Image; process → Background → OK remove Background color; the rectangle tool selects a Lane → Analyze → Gel → Select First Lane to determine the analysis Lane, and repeatedly selects a plurality of lanes for analysis; analyze → Gel → Plot Lane generating peak area; and selecting a peak map corresponding to the target band by using a linear tool, and calculating the area of the corresponding peak map by using a Wand tool to obtain the percentage of the expression quantity in the total protein.

Example 3 transient expression of RBD protein

First, host cell

The seed bank cells of the host cells Expi293F were taken from the liquid nitrogen tank, thawed rapidly in a 37 ℃ water bath, the thawed cell suspension was aseptically transferred to 125ml vials containing 30ml of pre-warmed complete growth medium, shake culture conditions: 37 ℃ and 8% CO₂120rpm, amplitude of 25mm and humidity of more than or equal to 80 percent. And taking cell suspension after 15-30 min to detect the cell density and the survival rate.

When the cell survival rate is recovered to more than 90 percent, the cell density reaches 3-5 multiplied by 10⁶cells/ml, in an amount of 0.3 to 0.5X 10⁶cells/ml were used for the inoculation amplification.

Second, cell transfection

The host cells were transfected with pCGS3-S-RBD and pCGS3-H-RBD constructed in example 1, respectively.

1. One day before transfection

Cells were plated at 2.5-3X 10 h before transfection⁶cells/ml were re-inoculated and cultured for 24 h.

2. Day of transfection

(1) The cell density should be 4.5-5.5 × 10⁶cells/ml, the activity rate should be more than or equal to 95 percent. Cells were diluted to 3X 10 with fresh pre-warmed complete growth medium⁶cells/ml。

(2) Preparation of transfection reagent and DNA Complex

1) DNA dilution

The plasmids (pCGS 3-S-RBD and pCGS3-H-RBD constructed in example 1) were diluted to 1. mu.g/. mu.l with sterile water, and the amount of plasmid required for transfection of 50ml of cells, that is, 50. mu.l of plasmid was taken in accordance with the amount of 1. mu.g plasmid transfected into 1ml of cells, and 3ml of Opti-MEM was added^TMI Reduced Serum medium for use.

2) Dilution of transfection reagents

Transfection reagent Expifeacmine 293 was used before use^TMReagent is mixed by gently inverting up and down, and the amount of transfection Reagent required for transfecting 50ml of cells, namely 160. mu.l Expifeacylamine 293 is taken^TMReagent was mixed in 2.8ml of Opti-MEMTM I Reduced Serum medium by gently inverting it upside down, and allowed to stand at room temperature for 5 min.

3) And adding the diluted transfection reagent into the plasmid, slightly reversing the transfection reagent up and down, uniformly mixing the transfection reagent and the plasmid, and reacting the mixture at room temperature for 10-20 min. The mixed transfection reagent and DNA complex was slowly added to the cell culture. 37 ℃ and 8% CO₂Culturing at 120rpm, amplitude of 25mm and humidity of 80% or more.

3. Day one after transfection

The enhancer is added 18-22 h after transfection according to the amount of 50ml of transfected cells. Namely, 300. mu.l of Expifeacylamine was taken^TM293 Transfecton Enhancer 1 and 3ml Expifeacmine^TM293Transfection Enhancer 2 was mixed well and slowly added to the cell culture.

4. Collection of culture supernatants

Cell viability was monitored daily after transfection and cultures were terminated for 4 days when viability dropped to 65% -75%. The culture was collected and centrifuged at 3500g for 30min, the supernatant was collected and filtered through a 0.22 μm filter, and SDS-PAGE identification and gray scale analysis showed that the expression level of RBD protein in the signal peptide H group was increased by 24.99% as compared with that in the signal peptide S group (FIGS. 2 and 3). It can be seen that the artificial signal peptide H of the present invention has a higher secretory expression yield of the novel coronavirus RBD protein than the natural signal peptide.

Example 4 RBD protein purification

First, ultrafiltration concentration

The supernatant (i.e., the supernatant obtained after "collecting the culture and centrifuging 3500g for 30 min" in step 4 of example 3) was subjected to ultrafiltration concentration at 4 ℃ with a 10KD ultrafiltration membrane 6000g for 20min, and the final cell supernatant was concentrated to 20-30ml, leaving 10. mu.l of the supernatant for SDS-PAGE protein electrophoresis detection.

Purifying with nickel column

(1) Concentrating the supernatant by ultrafiltration and mixing/Wash Buffer according to the volume ratio of 1: 1, uniformly mixing, standing for 20min, and fully incubating until the mixture is purified by a column;

(2) balancing the column with a Binding/Wash Buffer of twice the column volume, the Buffer flowing through the pre-packed column by gravity;

(3) adding the ultrafiltration concentrated supernatant and the Binding/Wash Buffer mixing solution into a column, and flowing through a pre-packed column by gravity. If the residual sample exists, the sample can be loaded again, the flow-through liquid is circulated once again, the flow-through liquid is collected into the centrifuge tube, and 10 mu l of sample is reserved for SDS-PAGE protein electrophoresis detection;

(4) the column was washed with a Binding/wash buffer of twice the column volume and the flow-through was collected. Repeating this step using a new collection tube until the absorbance of the flow-through solution at 280nm approaches the baseline;

(5) the histidine-tagged protein on the column was eluted with an Elution Buffer of twice the column volume. Repeating the steps until the absorbance of the flow-through liquid is close to a baseline at 280nm, collecting eluent to be purified, and reserving 10 mu l of sample for protein concentration determination and SDS-PAGE protein electrophoresis detection;

(6) the protein concentration in the eluate was determined by A280(nm) ultraviolet absorption.

Third, ultrafiltration replacement

(1) The protein solution after nickel column purification was added to Millipore (UFC5010BK, 0.5ml, 10K) and centrifuged in portions of 10000g for 3min until about 150. mu.l of solution remained.

(2) 300. mu.l of PBS (pH7.4) was gently added, and centrifugation at 10000g was carried out until 150. mu.l remained, and the procedure was repeated three times.

(3) The final volume of the tube was about 1-2ml, and 5. mu.l of the tube was used for protein concentration determination and SDS-PAGE protein electrophoresis detection.

(4) Protein concentration is determined by A280(nm) ultraviolet absorption method, purity of purified samples is more than 95% by SDS-PAGE detection (figure 4 and figure 5), and aluminum salt is adopted; or CpG: or a liposome; or oily adjuvant can be used to produce novel coronavirus immune composition for preventing novel pneumonia.

From the results of the above examples, it can be seen that the present invention employs the S protein natural signal peptide S and the artificially designed signal peptide H (SEQ ID No.1) for directing the secretory expression of the Receptor Binding Domain (RBD) of the novel coronavirus surface protein in eukaryotic cells. The secretion expression level of the artificially synthesized signal peptide H is obviously due to the natural signal peptide S, so that the artificially synthesized signal peptide H is more suitable for large-scale industrial production, and the production cost is reduced.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> Hualan genetic engineering Co., Ltd

<120> preparation method of novel coronavirus surface protein receptor binding domain

<130> GNCLN202119

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 19

<212> PRT

<213> Artificial sequence

<400> 1

Met Ala Leu Pro Val Trp Leu Leu Val Leu Met Phe Trp Ile Pro Ala

1 5 10 15

Ala Arg Ser

<210> 2

<211> 57

<212> DNA

<213> Artificial sequence

<400> 2

atggccttgc ctgtttggct gttggtgctg atgttctgga ttcctgctgc tagatct 57

<210> 3

<211> 669

<212> DNA

<213> Artificial sequence

<400> 3

agggtccaac caacagagag cattgtgagg tttccaaaca tcaccaacct gtgtccattt 60

ggagaggtgt tcaatgccac caggtttgcc tctgtctatg cctggaacag gaagaggatt 120

agcaactgtg tggctgacta ctctgtgctc tacaactctg cctccttcag caccttcaag 180

tgttatggag tgagcccaac caaactgaat gacctgtgtt tcaccaatgt ctatgctgac 240

tcctttgtga ttaggggaga tgaggtgaga cagattgccc ctggacaaac aggcaagatt 300

gctgactaca actacaaact gcctgatgac ttcacaggct gtgtgattgc ctggaacagc 360

aacaacctgg acagcaaggt gggaggcaac tacaactacc tctacagact gttcaggaag 420

agcaacctga aaccatttga gagggacatc agcacagaga tttaccaggc tggcagcaca 480

ccatgtaatg gagtggaggg cttcaactgt tactttccac tccaatccta tggcttccaa 540

ccaaccaatg gagtgggcta ccaaccatac agggtggtgg tgctgtcctt tgaactgctc 600

catgcccctg ccacagtgtg tggaccaaag aagagcacca acctggtgaa gaacaagtgt 660

gtgaacttc 669

<210> 4

<211> 253

<212> PRT

<213> Artificial sequence

<400> 4

Met Ala Leu Pro Val Trp Leu Leu Val Leu Met Phe Trp Ile Pro Ala

1 5 10 15

Ala Arg Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn

20 25 30

Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

35 40 45

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

50 55 60

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

65 70 75 80

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

85 90 95

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

100 105 110

Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

115 120 125

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

130 135 140

Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser

145 150 155 160

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

165 170 175

Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro

180 185 190

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro

195 200 205

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

210 215 220

Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val

225 230 235 240

Asn Phe Gly Gly Gly Gly Ser His His His His His His

245 250

<210> 5

<211> 759

<212> DNA

<213> Artificial sequence

<400> 5

atggccttgc ctgtttggct gttggtgctg atgttctgga ttcctgctgc tagatctagg 60

gtccaaccaa cagagagcat tgtgaggttt ccaaacatca ccaacctgtg tccatttgga 120

gaggtgttca atgccaccag gtttgcctct gtctatgcct ggaacaggaa gaggattagc 180

aactgtgtgg ctgactactc tgtgctctac aactctgcct ccttcagcac cttcaagtgt 240

tatggagtga gcccaaccaa actgaatgac ctgtgtttca ccaatgtcta tgctgactcc 300

tttgtgatta ggggagatga ggtgagacag attgcccctg gacaaacagg caagattgct 360

gactacaact acaaactgcc tgatgacttc acaggctgtg tgattgcctg gaacagcaac 420

aacctggaca gcaaggtggg aggcaactac aactacctct acagactgtt caggaagagc 480

aacctgaaac catttgagag ggacatcagc acagagattt accaggctgg cagcacacca 540

tgtaatggag tggagggctt caactgttac tttccactcc aatcctatgg cttccaacca 600

accaatggag tgggctacca accatacagg gtggtggtgc tgtcctttga actgctccat 660

gcccctgcca cagtgtgtgg accaaagaag agcaccaacc tggtgaagaa caagtgtgtg 720

aacttcgggg gtggaggctc tcaccatcac caccatcat 759

<210> 6

<211> 747

<212> DNA

<213> Artificial sequence

<400> 6

atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcagggt ccaaccaaca 60

gagagcattg tgaggtttcc aaacatcacc aacctgtgtc catttggaga ggtgttcaat 120

gccaccaggt ttgcctctgt ctatgcctgg aacaggaaga ggattagcaa ctgtgtggct 180

gactactctg tgctctacaa ctctgcctcc ttcagcacct tcaagtgtta tggagtgagc 240

ccaaccaaac tgaatgacct gtgtttcacc aatgtctatg ctgactcctt tgtgattagg 300

ggagatgagg tgagacagat tgcccctgga caaacaggca agattgctga ctacaactac 360

aaactgcctg atgacttcac aggctgtgtg attgcctgga acagcaacaa cctggacagc 420

aaggtgggag gcaactacaa ctacctctac agactgttca ggaagagcaa cctgaaacca 480

tttgagaggg acatcagcac agagatttac caggctggca gcacaccatg taatggagtg 540

gagggcttca actgttactt tccactccaa tcctatggct tccaaccaac caatggagtg 600

ggctaccaac catacagggt ggtggtgctg tcctttgaac tgctccatgc ccctgccaca 660

gtgtgtggac caaagaagag caccaacctg gtgaagaaca agtgtgtgaa cttcgggggt 720

ggaggctctc accatcacca ccatcat 747

Claims

Use of the polypeptide of SEQ ID No.1 or a biological material related thereto in any of:

p1, improving the secretory expression yield of the SARS-CoV-2 surface protein receptor binding region in host cells;

p2, improving the secretion and expression efficiency of the SARS-CoV-2 surface protein receptor binding region in host cells;

p3, preparing a SARS-CoV-2 surface protein receptor binding region secretion protein product;

the related biological material is a coding gene of the polypeptide shown in SEQ ID No.1 or an expression cassette or a recombinant vector or a recombinant bacterium or a transgenic cell line containing the coding gene;

the host cell is a eukaryotic host cell.
2. Use according to claim 1, characterized in that: the coding gene is a DNA molecule shown in SEQ ID No. 2.
3. The fusion protein is obtained by fusing the polypeptide shown in SEQ ID No.1 to the N end of the SARS-CoV-2 surface protein receptor binding region.
4. The fusion protein of claim 3, wherein: the amino acid sequence of the fusion protein is shown as 1-242 th site or 1-247 th site of SEQ ID No.4 or shown as SEQ ID No. 4.
5. A nucleic acid molecule encoding the fusion protein of claim 3 or 4.
6. The nucleic acid molecule of claim 5, wherein: the nucleic acid molecule consists of the coding gene of the polypeptide shown in SEQ ID No.1 and the coding gene of the SARS-CoV-2 surface protein receptor binding area in sequence from 5 'end to 3' end.
7. The nucleic acid molecule of claim 6, wherein: the encoding gene of the polypeptide shown in SEQ ID No.1 is a DNA molecule shown in SEQ ID No. 2.
8. The nucleic acid molecule of claim 6, wherein: the coding gene of the SARS-CoV-2 surface protein receptor binding region is a DNA molecule shown in SEQ ID No. 3.
9. The nucleic acid molecule of claim 6, wherein: the nucleic acid molecule is a DNA molecule shown in the 1 st to 726 th site or the 1 st to 741 th site of SEQ ID No.5 or a DNA molecule shown in SEQ ID No. 5.
10. An expression cassette, recombinant vector, recombinant bacterium or transgenic cell line comprising the nucleic acid molecule of any one of claims 5 to 9.
11. Use of the fusion protein of claim 3 or 4 or the nucleic acid molecule of any one of claims 5 to 9 or the expression cassette, recombinant vector, recombinant bacterium or transgenic cell line of claim 10 in any one of:

p1, improving the secretory expression yield of the SARS-CoV-2 surface protein receptor binding region in host cells;

p2, improving the secretion and expression efficiency of the SARS-CoV-2 surface protein receptor binding region in host cells;

p3, preparing a SARS-CoV-2 surface protein receptor binding region secretion protein product;

the host cell is a eukaryotic host cell.
12. A method for preparing SARS-CoV-2 surface protein receptor binding zone secretion protein includes the following steps:

(A1) introducing a nucleic acid molecule according to any one of claims 5 to 9 into a host cell to obtain a recombinant cell;

(A2) culturing the recombinant cell to obtain SARS-CoV-2 surface protein receptor binding region secreted protein from culture supernatant;

the host cell is a eukaryotic host cell.