CN118146321B

CN118146321B - RSV recombinant protein vaccine and preparation method thereof

Info

Publication number: CN118146321B
Application number: CN202410357023.1A
Authority: CN
Inventors: 赵娜; 李建平
Original assignee: Puda Biotechnology Taizhou Co ltd
Current assignee: Puda Biotechnology Taizhou Co ltd
Priority date: 2024-03-27
Filing date: 2024-03-27
Publication date: 2024-08-27
Anticipated expiration: 2044-03-27
Also published as: CN118146321A

Abstract

The invention relates to the technical field of biology, in particular to a respiratory syncytial virus recombinant protein vaccine and a preparation method thereof. The RSV recombinant protein of the invention is an RSV Pre-F recombinant protein with enhanced stability through amino acid mutation modification, wherein the mutation modification mode is selected from any one of the following modes: (1) Performing amino acid point mutation on the basis of the full-length sequence of the wild pre-F protein with the sequence shown as SEQ ID NO. 1; (2) Firstly deleting a transmembrane region/an intracellular region in the full-length sequence of a wild type pre-F protein with a sequence shown as SEQ ID NO.1, connecting a fibritin/Throm/6his/Stretaq sequence at the C end of the transmembrane region/intracellular region to obtain a mutant, and then carrying out amino acid point mutation on the basis of the mutant. The invention enhances the stability of the Pre-F protein by amino acid mutation and solves the problem of poor stability of wild protein antigens.

Description

RSV recombinant protein vaccine and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a respiratory syncytial virus recombinant protein vaccine and a preparation method thereof.

Background

Respiratory syncytial virus (Respiratory Syncytial Virus, RSV) is a common virus that is transmitted primarily by air and is the primary causative agent of lower respiratory tract infections in humans. RSV infection can lead to mild to severe respiratory diseases including bronchitis, pneumonia, asthma attacks, and the like. RSV infection can lead to serious complications and even death, especially in premature infants, low birth weight infants, and children and the elderly with a weaker immune system.

RSV has posed a serious threat to human health. According to statistics, the number of times of acute lower respiratory tract infection related to global RSV in 2019 is up to 3300 ten thousand, and the number of times of hospitalization of the acute lower respiratory tract infection and death cases caused by RSV are respectively 360 ten thousand and 26300; in infants between 0 and 6 months of age, the number of acute lower respiratory infections caused by RSV, hospitalization times and hospitalization mortality cases were approximately 660 ten thousand, 140 ten thousand and 13300, respectively. In addition, the RSV infection rate of adults increases with age, the aged is one of susceptible people, the aged has poorer prognosis and heavier economic burden, the aged with the highest inpatient mortality rate is higher than 65 years old, and particularly, the aged population is increased continuously along with the aging trend of population, so that the aged population is stressed. For decades, not only is hospitalization or death caused by RSV infection a life threatening, but also the treatment costs for the medical system are great, and it is becoming particularly important and urgent to develop a vaccine capable of preventing RSV virus.

RSV is an enveloped, non-segmented, single-stranded negative-strand RNA virus of the genus pneumoviridae, and pneumovirus. The genome consists of a single-stranded negative sense RNA molecule encoding 11 proteins, including 9 structural proteins (3 glycoproteins and 6 internal proteins) and 2 non-structural proteins. Structural proteins include 3 transmembrane surface glycoproteins: attachment protein G, fusion protein F and small hydrophobic SH proteins. There are two RSV subtypes: a and B differ mainly in the G glycoprotein, while the sequence of the F glycoprotein is more conserved between the two subtypes.

RSV fusion proteins (F proteins) belong to class I transmembrane proteins, consisting of 574 amino acid residues. The F0 protein, which is initially produced as a precursor of the F protein in the host cell, is glycosylated on the Golgi apparatus, and then is hydrolyzed by intracellular furin to release a polypeptide pep27 consisting of 27 amino acids, two subunits F1 and F2 are produced at the N-and C-terminal cleavage sites of the peptide fragment, respectively, the F2 subunit consisting of the signal peptide SP and the heptapeptide repeat HRC, the F1 subunit consisting of the fusion peptide FP, the heptapeptide repeat HRA and HRB, the Domain I and Domain II transmembrane region TM and the cytoplasmic Domain CP, and F1 and F2 are linked by disulfide bonds to form a heterodimer. The three heterodimers assemble into a mature F protein trimer. RSVF protein trimers are unstable and exist in both pre-fusion (prefusion) and post-fusion (postfusion) conformations. The F protein on the surface of the virus envelope is in a metastable pre-F conformation initially, and after the virus is adsorbed on the surface of a cell membrane, the pre-F protein is triggered by factors such as a cell receptor, temperature, ion concentration and the like to change the conformation to generate a highly stable post-F trimer, and the process releases energy to mediate the fusion of the virus envelope and the cell membrane. Since the conformation of pre-F is highly unstable, the protein isolated and purified in vitro is usually post-F. However, the metastable pre-F conformation is essential for the viral-mediated membrane fusion process and is also an important antigen for inducing an immune response in humans.

The PreF protein is used as one of main antigens of RSV and has good immunogenicity, but the natural PreF protein is easy to be destroyed under the conditions of high temperature, high pressure, heavy metal ions, oxidizing agent, extreme pH and the like, the structural stability of the protein is poor, and the good antigenicity of the PreF protein is difficult to be ensured, so that the structure and the function of the PreF protein need to be further studied. Studies show that the immunogenicity and stability of the PreF protein can be improved by carrying out amino acid mutation modification on the protein. Wherein the RSVF protein assumes its three-dimensional structure in a pre-fusion conformation, which presents a unique antigenic site ("antigenic site O") at its membrane distal tip, using the three-dimensional structure of pre-fusion F as a guide, utilizing engineering and construction of a stabilized form of pre-fusion F ("PreF" antigen), the immune response is many times greater in RSV neutralization than that achieved with prior RSV F protein-based immunogens, and which provides immune protection against RSV challenge in animal experiments.

However, how to design and obtain pre-F recombinant proteins with high stability and good immune effect still has a plurality of challenges, and no relevant RSV recombinant protein vaccine products are marketed in China. Thus, there is a need for immunogens derived from the RSVF protein which have improved properties compared to the corresponding natural RSVF protein, such as enhanced immunogenicity or improved stability of PreF, as well as compositions (e.g. vaccines) comprising such immunogens, all of great clinical significance.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides RSVpre-F recombinant protein vaccine and a preparation method thereof. The invention carries out reasonable optimization design on the amino acid sequence of the RSV Pre-F protein, thereby obtaining the RSV Pre-F recombinant protein vaccine with enhanced stability. The vaccine further enhances the immunogenicity and the stability of the Pre-F protein, can effectively prevent diseases caused by RSV infection, enhances the protection effect of the vaccine, and has very important significance for enlarging the research and development of domestic products and accelerating the development of clinical tests of vaccines and medicaments in China so as to furthest protect children and old people from being affected by the diseases related to the RSV infection and make up the defect of intervention measures of the RSV infection of high-risk groups.

The technical scheme for solving the technical problems is as follows:

in a first aspect of the present invention, there is provided an RSV recombinant protein, wherein the RSV recombinant protein is an RSV Pre-F recombinant protein with enhanced stability through amino acid mutation modification, and the mutation modification is selected from any one of the following modes:

(1) Performing amino acid point mutation on the basis of the full-length sequence of the wild pre-F protein with the sequence shown as SEQ ID NO.1, and mutating S at the 41 st position into C and mutating S at the 409 th position into C;

(2) Firstly deleting a transmembrane region/intracellular region in the full-length sequence of a wild pre-F protein with the sequence shown as SEQ ID NO.1, connecting a fibritin/Throm/6his/Stretaq sequence at the C end to obtain a mutant with the sequence shown as SEQ ID NO.2, carrying out amino acid point mutation on the basis of the sequence shown as SEQ ID NO.2, and mutating S at the 41 st position into C and mutating S at the 409 th position into C.

Further, the amino acid sequence of the RSV recombinant protein is shown in SEQ ID NO. 3.

Further, the RSV Pre-F recombinant protein is a trimer.

Further, the RSVPre-F recombinant protein comprises three F monomers, at least two of which comprise introduced cysteine residues in close proximity to each other and forming disulfide bonds that stabilize the RSVpre-F protein.

In a second aspect of the present invention, there is provided a method for preparing a recombinant protein of RSV according to the first aspect, comprising the steps of: expressing a nucleic acid molecule encoding the RSV protein according to the first aspect in an organism or an organism cell to obtain the RSV recombinant protein.

Further, the preparation method comprises the following steps: introducing a nucleic acid molecule encoding the RSV recombinant protein according to the first aspect into CHO cells to obtain recombinant cells; culturing the recombinant cells to obtain the RSV recombinant protein.

In a third aspect of the present invention, there is provided a biomaterial which is at least one of the following (1) to (4):

(1) A nucleic acid molecule encoding the RSV recombinant protein of the first aspect;

(2) A recombinant expression vector comprising the nucleic acid molecule of (1);

(3) A recombinant microorganism comprising (1) said nucleic acid molecule or (2) said recombinant expression vector;

(4) A recombinant cell line comprising the nucleic acid molecule of (1) or a recombinant cell line comprising the recombinant expression vector of (2).

In a fourth aspect of the present invention, there is provided the use of a recombinant RSV protein as described in the first aspect or a protein prepared according to the method as described in the second aspect or a biomaterial as described in the third aspect in any one of the following (1) - (4):

(1) As an immunogen;

(2) Preparing an anti-RSV product;

(3) Preparing a product for preventing and/or treating RSV infection;

(4) Products for preventing and/or treating diseases caused by RSV are prepared.

In a fifth aspect of the invention, there is provided a vaccine comprising as active ingredient a recombinant RSV protein according to the first aspect or a protein prepared according to the method according to the second aspect or a biomaterial according to the third aspect.

Further, the preparation form of the vaccine is water aqua or freeze-dried agent.

Further, the vaccine contains an adjuvant.

Further, the adjuvant is any one of CpG, QS21, aluminum phosphate, mixture of CpG and aluminum phosphate, or mixture of QS21 and aluminum phosphate.

In a sixth aspect of the invention there is provided the use of a vaccine as described in the fifth aspect in any one of (1) to (3) below:

(1) Preparing an anti-RSV product;

(2) Preparing a product for preventing and/or treating RSV infection;

(3) Products for preventing and/or treating diseases caused by RSV are prepared.

The invention has the following technical effects:

(1) The invention carries out reasonable optimization design on the amino acid sequence of the RSV protein by genetic engineering means, and the RSVPre-F recombinant protein modified by amino acid mutation has increased stability compared with the corresponding wild type RSV F protein, wherein the stability is measured by the combination of the mutant and antibodies PA1 and PA 2. The vaccine mainly carries out mutation modification on the amino acid of the PreF protein, so that the protein can still keep the structural stability and the antigen cluster function under different environments including high temperature, acidity and high osmotic pressure, and can still keep the better antigenicity even after undergoing chemical reaction.

(2) The invention enhances the stability of the structure of the Pre-F protein by designing the amino acid mutation of the RSV specific antigen, and solves the problem of poor stability of the wild protein antigen. Experiments prove that: the modified Pre-F protein prepared by the invention has obviously better stability at different temperatures, pH values and osmotic pressure than the Pre-F protein before modification, and the modified Pre-F protein still shows higher antigen binding activity after high temperature, acidity and high osmotic pressure treatment.

(3) According to the invention, through carrying out amino acid mutation design on RSVPre-F protein, the structural stability of the protein is ensured, and meanwhile, the RSV antibody with neutralization activity in a living body can be effectively induced, so that the living body is endowed with effective immune protection. Experiments prove that: compared with RSVpreF protein before modification, after the amino acid modified PreF recombinant protein vaccine prepared by the invention is used for immunizing mice, serum with high protection titer can be obtained, and the serum of the mice can generate higher neutralizing antibody titer against RSVA2 virus.

Drawings

FIG. 1 shows the temperature stability test (PA 1) of Pre-F protein before and after modification.

FIG. 2 shows the temperature stability test (PA 2) of Pre-F protein before and after modification.

FIG. 3 shows the pH stability test (PA 1) of Pre-F protein before and after modification.

FIG. 4 shows the pH stability test (PA 2) of Pre-F protein before and after modification.

FIG. 5 shows the osmotic stability test (PA 1) of Pre-F protein before and after modification.

FIG. 6 shows the osmotic stability test (PA 2) of Pre-F protein before and after modification.

FIG. 7 shows the results of protein antibody titer assays in murine immune serum of preparation A and preparation B.

Detailed Description

In order to more clearly demonstrate the technical scheme, objects and advantages of the present invention, the technical scheme of the present invention is described in detail below with reference to the specific embodiments and the accompanying drawings. Unless otherwise indicated, the instrumentation, reagents, materials, etc., used in the present invention are all available through conventional commercial means.

Example 1: preparation of RSV recombinant proteins

Construction of (one) proteins

The term "wild type" as used in the present invention means that it exists in nature without any modification or processing of the product by man. Those skilled in the art will appreciate that the wild-type RSV F protein can be a variety of sequences that may differ slightly, but have substantially identical biological activity. The wild-type full-length F protein mentioned in the present invention refers to the sequence provided by GenBank, and the specific sequence is shown in SEQ ID No.1 (Fusion glycoprotein F0 os= Human respiratory syncytial virus A (strain A2) ox= 11259 GN =fpe=1 SV =1).

(1) Mutant modification of amino acids

The RSV Pre-F protein with enhanced stability is obtained through amino acid mutation modification, and the amino acid mutation mode comprises the following two modes:

(1) Performing amino acid point mutation on the basis of the full-length sequence of the wild pre-F protein; s at position 41 is mutated to C, S at position 409 is mutated to C;

(2) Deleting a transmembrane region/an intracellular region in the full-length sequence of the wild pre-F protein, connecting a fibritin/Throm/6his/Stretaq sequence at the C end of the transmembrane region/intracellular region to obtain a mutant sequence as shown in SEQ ID NO.2, and carrying out amino acid point mutation on the basis of the sequence of SEQ ID NO. 2. Specific modes of mutation include the following: the S mutation at the 41 st position of the amino acid sequence of the F protein is C before the wild fusion, the S mutation at the 409 st position is C, and the full-length mutant of the F protein is obtained, and the amino acid sequence of the full-length mutant is shown as SEQ ID NO. 3.

In this example, RSVPre-F recombinant protein was obtained in a second mode.

The sequences of SEQ ID NO.1-SEQ ID NO.3 are shown below, respectively:

SEQ ID NO.1：

>sp|P03420|FUS_HRSVA Fusion glycoprotein F0 OS＝Human respiratory syncytial virus A(strainA2)OX＝11259 GN＝F PE＝1 SV＝1

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLS LIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

SEQ ID NO.2：

>RSVF

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK

SEQ ID NO.3：

>RSV F S41C S409C

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVCKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITCLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK.

(2) Synthesis of RSV Pre-F target gene

Determining a corresponding coding sequence according to the designed amino acid sequence SEQ ID NO.3, adding a restriction endonuclease XbaI sequence at the 5 'end of the segment gene, adding a restriction endonuclease EcoRI sequence at the 3' end, and chemically synthesizing the designed nucleotide sequence according to the codon preference of the host cell.

(3) Plasmid amplification and target gene extraction

The pUC19 plasmid vector is connected with the synthesized gene after double restriction by EcoRI and XbaI, and is led into an amplified host DH5 alpha, and a LB (amp+) agar solid medium is used for screening monoclonal; the monoclonal containing the target gene is inoculated in LB (amp+) liquid culture medium, cultured and amplified at 37 ℃ and 200rpm, and plasmid pUC19-preF is extracted by using a mass preparation kit of Sigma-Aldrich GenEluteTMHP plasmid; the extracted plasmid was digested with EcoRI and XbaI restriction enzymes, and the target gene fragment was recovered by TaKaRa MiniBestAgarose Gel Extraction Kit.

(4) Construction of eukaryotic expression vectors

The mammalian cell expression plasmid pGN-M, which contains the CMV promoter and the dihydrofolate reductase (DHFR) gene, was digested with EcoRI and XbaI restriction enzymes, and the vector DNA fragment was recovered using the TaKaRa MiniBEST DNA FRAGMENT Purification Kit Ver.4.0; the vector DNA fragment and the target gene fragment are connected in a cohesive end mode and are led into a DH5 alpha amplification host, and a monoclonal containing eukaryotic expression plasmid pGN-M_ preF is obtained through screening; amplified plasmids were extracted using endotoxin-free plasmid extraction kit TaKaRa MidiBEST Endo-FREE PLASMID PurificationKit, and the amplified plasmids were designated as RSVpre-F.

Expression of (II) proteins and cloning screening

CHO K1 cells purchased from ATCC were used as host cells and were cultured in DMEM medium (Sigma-Aldrich) with 10% new born calf serum after resuscitating the cells, 1 passage every 3 days. After passage 2, cells were observed to grow well, and then CHO K1 cells were plated in three 9.6cm ² wells at 0.75X10 ⁶ cells/well with Iscove's optimized DMEM medium (Sigma-Aldrich) and 10% fetal bovine serum (IMEM+FBS) (Gibco). Cells were loaded with 4. Mu.g of pcDNA vector in each well in a humidity-saturated incubator at 5% CO ₂ and 37℃and DNA was mixed with Lipofectamine 2000 (Sigma-Aldrich) and added to two of the wells, lipofectamine 2000 alone was added to the third well as a negative control. After 48 hours, the medium was removed and centrifuged at 200 Xg for 5 minutes, and the centrifuged supernatant was stored at-20 ℃. IMDM+FBS medium and 10. Mu.g/mL Blasticidin-HCl (Invitrogen) were added to one well of transfected cells, and the other transfected cell well was washed with PBS, then lysed with 50mM Tris-HCl, pH8, 150mM NaCl,1% (v/v) Triton X-100 containing complete,EDA-free protease inhibitor cocktail (Roche Diagnostics). The lysate was stored at-20℃by centrifugation at 16000 Xg for 10 min at 4 ℃. Western blot was used to detect if recombinant proteins were present in the supernatant and lysate. After 5 days of culture in selective medium, the cells were eluted with trypsin (Invitrogen) and then serially diluted on 9cm Petri dishes to isolate monoclonal cells. In the next 7-11 days, 42 individual clones were picked and transferred to wells on a 96-well plate. Culture supernatants were assayed using a Westernblot to screen for high-expression proteins. Clones secreting the highest amount of RSVpre-F protein were subjected to the next round of screening, and finally the cells were expanded and 30 new clones were screened for storage.

Selected clones were amplified into three T175 flasks (NETS). Trypsin digestion was added, washed with PBS, resuspended in 250mL spinner flasks with 100mL ProCHO ₄ (Lonza) plus 1X ProHT, 4mM L-glutamine and 2% FBS (Lonza). The culture was performed in a humidified incubator at 37℃under 5% CO ₂ at a stirring speed of 90rpm with the lid slightly opened to ensure air diffusion. The cells were sampled daily, stained with trypan blue (Sigma-Aldrich), counted and passaged every 3-5 days, after a plateau when the viable cell concentration was above 0.3X10 ⁶ cells/mL and the viable cell count exceeded 90%. When cells adapt and grow well, BFS is gradually removed, at which point cells are considered to be well suited for serum-free suspension growth.

(III) production of RSV pre-F protein in a bioreactor

A3 liter bioreactor was placed with 1.5 liter perfusion culture and a rotary filtration (10 μm) separator. The culture parameters were set as follows: the temperature was controlled at 37℃by a heating blanket, the pH was adjusted to 6.9 by CO ₂ or 0.3M sodium hydroxide, the stirring speed was 200-300RPM, and dissolved oxygen (dO ₂) was adjusted to 40% of saturated air with a maximum flow of a mixture of N ₂ and O ₂ at 200 mL/min. The perfusion rate was 0.3 to 0.8V dilution per day, and cells were sampled from the culture fluid daily for cell counting. Trypan blue staining was used and the glucose and lactate concentrations in the supernatant were measured off-line.

A total of 12.5 l of cell-free culture broth was collected, centrifuged at 8000 Xg for 30min at 4℃and filtered through a 0.45 μm membrane and concentrated by ultrafiltration using a 10kDa membrane pack. Ultrafiltration was performed with buffer, and the sample solution was concentrated to a volume of 0.5 liter, added with 0.5 liter of PBS, and then concentrated to 0.5 liter. The above steps were repeated 5 times.

(IV) RSV pre-F protein purification

The sample solution was loaded onto a Q-Sepharose fast flow (GE Bioscience) column and the column was washed with 20mM Tris-HCl pH 7.5. The column was then washed with 20mM Tris-HCl pH7.5, to which 200mM sodium chloride was added, to further remove the adsorbed protein impurities. The pre-F protein was eluted with a solution that increased the concentration of sodium chloride solution to 300 mM. Ammonium sulfate was added to the pool to a concentration of 800mM, loaded onto Butyl-Sepharose (GE Bioscience) column, the column was washed with phosphate buffer (PBS, 6mM Na ₂HPO₄,1.5mM KH₂PO₄, 0.15M sodium chloride pH 6.8) with 800mM ammonium sulfate, the column was washed with a PBS solution containing 400mM ammonium sulfate, and finally pre-F protein was eluted with purified water. And finally loading SEPHACRYL S-400HR (GE Bioscience), washing the column with PBS, collecting protein peaks, adding a cosolvent, freeze-drying on a vacuum freeze-dryer, and storing at-70 ℃ for later use.

The post-RSV preF protein and pre-RSV preF protein were prepared separately using the methods described above.

Example 2: temperature stability test of modified Pre-F protein obtained in example 1

1) The proteins to be tested (Pre-modified Pre-F protein prepared in reference example 1 and post-modified Pre-F protein prepared in example 1, respectively) were diluted to 20ug/mL with 1 XPBS pH7.4 buffer, placed in a 1.5mL centrifuge tube, and the total volume was 1mL.

2) Incubation at different temperatures was performed according to the following table.

3) After the incubation, each sample was placed at 4℃for temporary storage.

4) ELISA tests were performed according to the protocol:

Samples stored at 4℃were diluted to 1ug/mL with 1X PBS pH7.4, 100 uL/well and coated overnight at 4℃with an ELISA plate (NUNC 442404). The ELISA plate was spun down and incubated at 37℃for 2 hours with 1% BSA-PBS added at 150 uL/well. The plate washer was programmed to wash the plate 3 times, and the detection antibody PA1 (Medimmune origion)/PA 2 (Merck origion), 100 uL/well, was added as designed and incubated at 37℃for 2 hours. The plate washer washes the plate 3 times according to the program, according to 1: the AP-labeled goat anti-human secondary antibody was added at 2000 dilution, 100 uL/well, and incubated at 37℃for 1 hour. The plate washer was programmed to wash the plate 3 times, and pNPP substrate solution, 100 uL/well, was added, and the microplate reader was set to a wavelength of 405nm, and read.

The results are shown in FIGS. 1-2. As can be seen from fig. 1-2, the binding activities have significant differences, and the detection OD values of the PA1 and PA2 antibodies of the modified Pre-F protein at 4 ℃, 50 ℃ and 70 ℃ are significantly higher than those of the Pre-modified protein, which indicates that the modified Pre-F protein prepared by the invention can still maintain higher antigen binding activity after different temperature treatments, i.e., compared with the Pre-F protein before modification, the temperature stability of the modified Pre-F protein prepared by the invention is significantly enhanced.

Example 3: PH stability test of modified Pre-F protein obtained in example 1

1) Liquid preparation

① 25MM acetate buffer, pH3.5.

Formulation method (calculated as 100mL formulation):

Component (A)	Dosage of	Concentration of
			Sodium acetate (mw: 82.03 g/mol)	98.953mg	1.206mM
Acetic acid (mw: 60.05 g/mol)	1.429g	23.79mM
			Sodium chloride (mw: 58.44 g/mol)	0.9g	0.9％

1. 98.953Mg of sodium acetate was accurately weighed and added to a 200mL beaker.

2. 1.429G of acetic acid was weighed accurately and added to a 5mL centrifuge tube.

3. About 80mL of purified water was added to sodium acetate to dissolve it sufficiently, and acetic acid was added.

4. The pH of the solution was adjusted to 3.5.

5. Constant volume to 100mL, and preserving at room temperature for 3 months.

② 25MM acetate buffer, pH5.0.

Formulation method (calculated as 100mL formulation):

Component (A)	Dosage of	Concentration of
			Sodium acetate (mw: 82.03 g/mol)	1.381g	16.83mM
Acetic acid (mw: 60.05 g/mol)	490.3mg	8.166mM
			Sodium chloride (mw: 58.44 g/mol)	0.9g	0.9％

1. 1.381G of sodium acetate was accurately weighed into a 200mL beaker.

2. 490.3Mg of acetic acid was accurately weighed into a 5mL centrifuge tube.

4. The pH of the solution was adjusted to 5.0.

5. Constant volume to 100mL, and preserving at room temperature for 3 months.

③ 25MM Tris-HCl buffer, pH8.0.

Formulation method (calculated as 100mL formulation):

Component (A)	Dosage of
		Tris(mw:121.14g/mol)	302.85mg
1N HCl	1.42mL

1. 80ML of purified water was added to a 200mL beaker.

2. 302.85Mg of Tris was accurately measured.

3.1 ML of 1N hydrochloric acid (theoretical 1.42 mL) was added, followed by addition in small amounts, and the pH of the solution was adjusted to 8.0.

4. Constant volume to 100mL, and preserving at room temperature for 3 months.

④ 25MM Tris-HCl buffer, pH10.0.

Formulation method (calculated as 100mL formulation):

Component (A)	Dosage of
		Tris(mw:121.14g/mol)	302.85mg
1N HCl	33uL

1. 80ML of purified water was added to a 200mL beaker.

2. 302.85Mg of Tris was accurately measured.

3. 20UL of 1N hydrochloric acid (33 uL of theory) was added, followed by a small amount of addition, to adjust the pH of the solution to 10.0.

4. Constant volume to 100mL, and preserving at room temperature for 3 months.

⑤ 25MM PBS buffer control, pH7.5.

Formulation method (calculated as 100mL formulation):

Component (A)	Dosage of
		Na₂HPO₄	142mg
KH₂PO₄	27mg
		NaCl	800mg
KCl	20mg

1. 80ML of purified water was added to a 200mL beaker.

2. Accurately weighing Na ₂HPO₄142mg、KH₂PO₄ mg, naCl 800mg and KCl 20 mg.

3. And adding a small amount of HCl, and adjusting the pH value of the solution to 7.5.

4. Constant volume to 100mL, and preserving at room temperature for 3 months.

2) Proteins to be tested (Pre-modified Pre-F protein prepared in reference example 1 and post-modified Pre-F protein prepared in example 1, respectively) were diluted to 20ug/mL with buffers of different pH values, placed in a 1.5mL centrifuge tube, and the total volume was 1mL.

3) Samples were incubated at different pH according to the following table.

4) After the incubation, the pH of each sample was neutralized to 7.5 with acid or alkali (pH test paper assay), and the samples were kept at 4℃for temporary storage. ELISA tests (procedure with temperature stability test) were performed according to the protocol.

The results are shown in FIGS. 3-4. The detection results of fig. 3 and 4 show that: the Pre-F protein before and after modification has obvious difference in binding activity with antibodies (PA 1 and PA 2) after different pH treatments, wherein FIG. 3 shows that the detection OD values of the PA1 antibodies of the Pre-F protein after modification are obviously higher than those of the Pre-F protein before modification at pH3.5, pH5.0, pH7.5, pH8.0 and pH10, and FIG. 4 shows that the detection OD values of the PA2 antibodies of the Pre-F protein after modification are obviously higher than those of the Pre-F protein before modification at pH5.0, pH7.5, pH8.0 and pH10, so that the modified Pre-F protein prepared by the invention can still maintain higher antigen binding activity after different pH treatments, namely, compared with the Pre-F protein before modification, the pH stability of the modified Pre-F protein prepared by the invention is obviously enhanced.

Example 4: osmotic stability test of modified Pre-F protein obtained in example 1

1) Liquid preparation

① 10MM Tris-HCl buffer pH7.5.

Formulation method (calculated as 25mL formulation):

Component (A)	Dosage of
		25MM Tris-HCl buffer, pH8.0	10mL
1N HCl	6.45mL

1. 10ML of 25mM Tris-HCl buffer (pH 8.0) was added to a 50mL centrifuge tube.

2. Purified water was added to a volume of 25mL.

3. 6ML of 1N hydrochloric acid (theoretical value: 6.45 mL) was added, followed by addition in a small amount, and the pH of the solution was adjusted to 7.5.

4. Constant volume to 25mL, and preserving at room temperature for 3 months.

② 80MM Tris-HCl buffer, pH7.5.

Formulation method (calculated as 100mL formulation):

Component (A)	Dosage of
		Tris(mw:121.14g/mol)	969.12mg
1N HCl	6.45mL

1. 80ML of purified water was added to a 200mL beaker.

2. 969.12Mg of Tris was accurately measured and added.

4. Constant volume to 100mL, and preserving at room temperature for 3 months.

③1.5M MgCl₂ Solution

Formulation method (calculated as 100mL formulation):

Component (A)	Dosage of
		3M MgCl ₂ solution	20mL
Purified water	20mL

1. Accurately weighing 20mL of 3M MgCl ₂ solution and adding the solution into a50 mL centrifuge tube.

2. Purified water was added to 20mL and mixed well.

3. Stored at room temperature and has a shelf life of 3 months.

④3M MgCl₂ Solution

Formulation method (calculated as 100mL formulation):

Component (A)	Dosage of
		Magnesium chloride hexahydrate (mw: 203.30 g/mol)	60.99g

1. 60.99G of magnesium chloride hexahydrate was accurately weighed into a 200mL beaker.

2. Purified water was added to about 80mL and stirred to dissolve thoroughly.

3. Constant volume to 100mL, and preserving at room temperature for 3 months.

⑤ 150MM Tris-HCl buffer, pH7.5.

Formulation method (calculated as 100mL formulation):

Component (A)	Dosage of
		Tris(mw:121.14g/mol)	1.815g
1N HCl	6.45mL

1. 80ML of purified water was added to a 200mL beaker.

2. 1.815Mg of Tris was accurately measured and added.

4. Constant volume to 100mL, and preserving at room temperature for 3 months.

⑥ 4M NaCl solution

Formulation method (calculated as formulation 125mL capacity):

Component (A)	Dosage of
		Sodium chloride (mw: 58.44 g/mol)	29.22g

1. 29.22G of sodium chloride was weighed accurately and added to a 200mL beaker.

2. 100ML of purified water was added and dissolved with sufficient agitation.

3. Constant volume to 125mL, and storing at room temperature for 3 months.

2) Proteins to be tested (Pre-modified Pre-F protein prepared in reference example 1 and post-modified Pre-F protein prepared in example 1, respectively) were diluted to 20ug/mL with buffers of different osmotic pressure values, placed in a 1.5mL centrifuge tube, and the total volume was 1mL.

3) Samples were incubated for different osmolarity values according to the following table.

4) The 10mM and 80mM groups were diluted with Tris buffer and the 1500mM and 3000mM groups were diluted with MgCl ₂ buffer, respectively.

5) After the incubation, the 10mM and 80mM groups were adjusted to 150mM with 4M NaCl buffer, and 1500mM and 3000mM groups were diluted 10-fold and 20-fold with purified water, respectively, and placed at 4℃for temporary storage.

ELISA tests were performed according to the protocol. (operation and temperature stability test).

The results are shown in FIGS. 5-6. The detection results of fig. 5 and 6 show that: the Pre-F protein before and after modification has obvious difference in binding activity with antibodies (PA 1 and PA 2) after incubation treatment with different osmotic pressure values, and the detection OD values of the modified Pre-F protein at 10mM, 80mM, 150mM, 1500mM and 3000mM of the PA1 and PA2 antibodies are obviously higher than those of the Pre-protein before modification, which indicates that the modified Pre-F protein prepared by the invention can still keep higher antigen binding activity after incubation treatment with different osmotic pressure values, namely, compared with the Pre-F protein before modification, the stability of the modified Pre-F protein prepared by the invention under different osmotic pressure is obviously enhanced.

Therefore, compared with the Pre-F protein before modification, the modified Pre-F protein prepared by the method still maintains the structural stability and the antigen cluster function under different environments including high temperature, acidity and high osmotic pressure, and can still preserve the better antigenicity even after undergoing chemical reaction.

Example 5: preparation of immune preparation A (modified Pre-F protein)

The modified Pre-F protein obtained in example 1 was used to sample 5. Mu.g, cpG (Genscript) mg was added to the sample to a final amount of 0.1mg, phosphate buffer pH5.8 was added, and the resulting mixture was sterilized and filtered through a 0.22 μm membrane; adding sterile aluminum phosphate gel (Benetag), stirring at 4deg.C for 1 hr, aseptically packaging at 0.5 mL/bottle, and preserving at 4deg.C.

Example 6: preparation of immune preparation B (Pre-F protein before modification)

Using the Pre-modification Pre-F protein obtained in example 1, the sample content was 5. Mu.g, cpG (Genscript) was added, the final amount was 0.1mg, phosphate buffer pH5.8 buffer was added, and the mixture was sterilized and filtered with a 0.22 μm membrane; adding sterile aluminum phosphate gel (Benetag), stirring at 4deg.C for 1 hr, packaging in 0.5 mL/bottle under aseptic condition, and preserving at 4deg.C for immunization.

Example 7: RSV protein vaccine immunized mice before and after modification and blood collection

Taking 4-6 week female BALB/c mice, randomly grouping 10 mice in each group, totally 2 groups, and taking each group of separate immune preparations, wherein each group of separate immune preparations is prepared by using the preparation A and the preparation B prepared in the examples 5 and 6, performing subcutaneous immunization once every other two weeks, performing immunization twice, performing blood sampling after 35 days of immunization, standing blood at room temperature for 4 hours, centrifuging at 10000RPM (revolutions per minute), sucking off-core supernatant serum, and preserving at-70 ℃ for detection.

Example 8: ELISA detection of pre-F protein antibody titer in modified RSV recombinant protein vaccine mouse immune serum before modification

1Mg/mL (1 XPBS solution) of purified pre-F protein stock solution was prepared and stored in a refrigerator at 4 ℃. The protein stock was diluted to 4. Mu.g/mL of coating buffer, 100. Mu.L of coating solution was added to each well to coat ELISA plates, and incubated overnight at room temperature. Washing with plate washing buffer 4 times, adding 150 μl of blocking buffer, incubating at 37deg.C for 2 hours, washing with 300 μl of plate washing buffer per well 3 times, and storing at 4deg.C for one week.

The corresponding test serum obtained by injecting the vaccine into the mice in example 7 was diluted to working sample serum, diluted by an appropriate factor, added to the first row of wells of ELISA plate, 100. Mu.l per well, serial dilutions were performed 2-fold down from the first row, and incubated at 37℃for 2 hours. Wash 300 μl per well with plate wash buffer 3 times according to 1: AP-labeled anti-murine secondary antibody was added at 2000 dilution, 100 uL/well, and incubated at 37℃for 1 hour. The plate washer was programmed to wash the plate 3 times, and pNPP substrate solution, 100 uL/well, was added, and the microplate reader was set to a wavelength of 405nm, and read.

The detection results are shown in fig. 7: the protein antibody titer in serum of mice immunized with different preparations is obviously different, wherein the antibody titer of mice immunized with RSVpreF recombinant protein vaccine (preparation A) after amino acid modification is obviously higher than that of RSVpreF protein vaccine (preparation B) before modification; the serum of the immunized mice is diluted by different times, the antibody titer of the RSVpreF recombinant protein vaccine (preparation A) after amino acid modification is still obviously higher than that of the RSVpreF protein vaccine (preparation B) before modification under each concentration, and the antibody titer of RSVpreF protein before and after modification is reduced along with the gradual increase of the serum dilution times. Therefore, compared with RSVpreF protein before modification, the RSV PreF recombinant protein vaccine modified by amino acid can obtain serum with higher protection titer, which indicates that the RSV PreF recombinant protein vaccine modified by amino acid prepared by the invention has better immune effect.

Example 9: RSVA2 virus neutralization assay

(1) Preparation immunized mouse and blood sampling

(2) RSVA2 virus neutralization titer assay

RSV virus type A was cultured using Hep-2 cells in DMEM medium containing 10% bovine serum. According to 20000/hole of Hep2 cell number, 100 uL/hole of complete culture medium, inoculating to 96-well plate, culturing in 5% CO ₂ at 37 ℃ for 24h to cell density of 60-80%; changing the culture medium, sucking the culture medium out of the 96-well plate, adding the maintenance medium, and 100 uL/well; the serum was heat-inactivated in a water bath at 56℃for 30 minutes. Sub-packaging at least 4 tubes in total according to 15 uL/tube, and storing serum at-80 ℃; the antiserum is diluted in a 96-well V-shaped plate, and 70uL of antiserum is diluted according to proper dilution times, and each dilution is repeated for 2 wells; the virus was diluted to 100 TCID50 with DMEM and 70 uL/well in the 96-well V-plate. Incubating for 1h at 37 ℃; 100 uL/well aspirates were transferred to the Hep2 cell plates described above; culturing at 37deg.C in 5% CO ₂ for about 3-5 days; CPE was observed daily and cells were stained with 5% glutaraldehyde containing 0.25% crystal violet.

TABLE 1 detection results of RSV A2 neutralization assay

The result of the RSVA2 virus neutralization titer test is shown in Table 1, and Table 1 shows that there is a clear difference in serum neutralization titer values of Pre-F immunized mice before and after modification, wherein the average value of the neutralization titer of Pre-F protein after modification is 6241, which is about 1.38 times the serum neutralization titer value of Pre-F immunized mice before modification. Therefore, the amino acid modified RSV Pre-F recombinant protein vaccine prepared by the invention can be injected into mice to obtain high protection titer serum, and the mice serum can generate higher neutralizing antibody titer against the main epidemic strain A of RSV.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention, and it should be covered by the scope of the claims of the present invention.

Claims

1. The RSV recombinant protein is characterized in that the RSV recombinant protein is an RSV Pre-F recombinant protein with enhanced stability through amino acid mutation modification, and the mutation modification mode is as follows:

Firstly deleting a transmembrane region/intracellular region in the full-length sequence of a wild pre-F protein with the sequence shown as SEQ ID NO.1, connecting a fibritin/Throm/6his/Stretaq sequence at the C end to obtain a mutant with the sequence shown as SEQ ID NO.2, carrying out amino acid point mutation on the basis of the sequence shown as SEQ ID NO.2, and mutating S at the 41 st position into C and mutating S at the 409 th position into C.

2. The RSV recombinant protein according to claim 1, wherein said RSV recombinant protein has an amino acid sequence as set forth in SEQ ID No. 3.

3. The RSV recombinant protein according to claim 1, wherein said RSV Pre-F recombinant protein is a trimer.

4. The method of producing a recombinant protein of RSV of any one of claims 1-3, comprising the steps of: expressing a nucleic acid molecule encoding the RSV protein according to any of claims 1-3 in an organism or in a cell of an organism, resulting in said RSV recombinant protein.

5. The method of preparation according to claim 4, characterized in that it comprises the steps of: introducing a nucleic acid molecule encoding the RSV recombinant protein according to any one of claims 1-4 into CHO cells to obtain recombinant cells; culturing the recombinant cells to obtain the RSV recombinant protein.

6. A biomaterial characterized by being at least one of the following (1) to (4):

(1) A nucleic acid molecule encoding the RSV recombinant protein according to any one of claims 1-4;

7. Use of the recombinant RSV protein according to any one of claims 1-3 or the protein prepared according to the method of claim 4 or 5 or the biomaterial according to claim 6 in any one of the following (1) - (3):

(1) Preparing an anti-RSV product;

(2) Preparing a product for preventing and/or treating RSV infection;

8. A vaccine comprising as active ingredient the recombinant RSV protein according to any one of claims 1-3 or the protein produced according to the method of claim 4 or 5 or the biomaterial according to claim 6.

9. The vaccine of claim 8, wherein the vaccine is formulated as an aqueous or lyophilized formulation.

10. The vaccine of claim 8, wherein the vaccine comprises an adjuvant.

11. The vaccine of claim 10, wherein the adjuvant is any one of CpG, QS21, aluminum phosphate, a mixture of CpG and aluminum phosphate, or a mixture of QS21 and aluminum phosphate.

12. Use of the vaccine of claim 8 in any one of the following (1) - (3):

(1) Preparing an anti-RSV product;

(2) Preparing a product for preventing and/or treating RSV infection;