CN111166881A - Recombinant respiratory syncytial virus multi-epitope chimeric vaccine and preparation method and application thereof - Google Patents

Recombinant respiratory syncytial virus multi-epitope chimeric vaccine and preparation method and application thereof Download PDF

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CN111166881A
CN111166881A CN202010010061.1A CN202010010061A CN111166881A CN 111166881 A CN111166881 A CN 111166881A CN 202010010061 A CN202010010061 A CN 202010010061A CN 111166881 A CN111166881 A CN 111166881A
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respiratory syncytial
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李启明
邵帅
韩子泊
张靖
靳玉琴
杜丽芳
杨森森
雷泽华
马智静
栗子谦
郑凡
梁宇
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China National Biotec Research Institute Co ltd
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Abstract

The invention discloses a recombinant respiratory syncytial virus multi-epitope chimeric vaccine, wherein the main target antigen of the vaccine is a recombinant respiratory syncytial virus multi-epitope chimeric protein which consists of a hepialus hepatitis virus core protein and three different antigen epitope fragments of the recombinant respiratory syncytial virus, wherein the three different antigen epitope fragments are respectively inserted into the C end, the N end and an immunodominant region of the hepialus hepatitis virus core protein; wherein, the amino acid sequence of the antigen epitope fragment inserted into the C end and the N end of the hepes hepialid virus core protein is shown as SEQ ID No.2, and the amino acid sequence of the antigen epitope fragment inserted into the immunodominant region of the hepialid hepatitis virus core protein is shown as SEQ ID No. 3. The invention takes the hepialidae hepatitis virus core protein as an epitope presentation carrier to carry out discontinuous presentation of a plurality of epitope fragments, effectively displays different antigen epitopes at the maximum degree and high density on the surface of the virus-like particle, does not influence the self-assembly of the virus-like particle, and has higher immune response effect compared with the epitope presented at a single site.

Description

Recombinant respiratory syncytial virus multi-epitope chimeric vaccine and preparation method and application thereof
Technical Field
The invention relates to the field of biomedicine, in particular to a recombinant respiratory syncytial virus multi-epitope chimeric vaccine and a preparation method and application thereof.
Background
Respiratory Syncytial Virus (RSV) is a major pathogen causing lower Respiratory tract infections in infants and young children worldwide, causing bronchiolitis and interstitial pneumonia, causing approximately 20 million deaths per year in children under 5 years of age worldwide[1]Meanwhile, the elderly and the people with immunodeficiency are also the main population infected by RSV. At present, except for humanized monoclonal antibody Palivizumab (Palivizumab), no specific therapeutic drug and no available preventive and therapeutic RSV vaccine exist[2]. The RSV genome is 15.2kb in total length and encodes 11 proteins including 3 nucleocapsid proteins (N, P, L), 3 transmembrane proteins (F, G, SH), 3 matrix proteins (M, M2-1, M2-2) and 2 non-structural proteins (NS1, NS 2). Wherein, the G protein and the F protein respectively mediate the adhesion and fusion of the virus and the cell, and are the main target antigens for the current vaccine development. Compared with the G protein, the F protein has higher homology between the A serotype and the B serotype, and the RSV vaccine developed by the F protein has protective effect on both the A serotype and the B serotype, and is a main target point of the current RSV vaccine research.
The F protein consists of two subunits of F1 and F2, is a type I glycoprotein and exists in a trimer form. The F protein exists in two states: a metastable pre-fusion state and a more stable post-fusion state; the F protein in a pre-fusion state has higher immunogenicity. It has now been found that there are multiple sites of neutralization on the F protein: i, II, IIIIV, V, VI, VIII and
Figure BDA0002356810340000011
wherein, the antigenic site II is the combination site of monoclonal antibodies of palivizumab and Motavizumab (RSV) for preventing and treating RSV, is positioned at amino acids 254 to 277 of F1 subunit of F protein, is a conformational epitope of an 'α helix-ring- α helix' secondary structure, and the conformational state of the conformational epitope before and after F protein fusion is kept unchanged[3]Antigenic site VIII is the specific site of the F protein before fusion discovered in recent years, is positioned at 163-181 amino acids close to the 'helix-loop-helix' and partially covers antigenic sites II and II
Figure BDA0002356810340000012
The neutralizing monoclonal antibody hRSV90 for recognizing the site has better neutralizing activity to A, B subtype.
Formaldehyde inactivated RSV vaccine (Formalin-inactivat) in the last 60 s of the centuryed RSV and FI-RSV) can not prevent infant RSV infection, but also cause serious respiratory disease Enhancement (ERD), so that two infants die, and specific targets and mechanisms of the ERD phenomenon are not clear yet[4]. Therefore, how to circumvent the ERD phenomenon and improve the safety and efficacy of vaccines in RSV vaccine development becomes a primary concern. Although vaccines with F protein as the main component show good immunogenicity in animal experiments, research shows that F protein still has certain safety risk, and ERD phenomenon appears after purified F protein immunizes cotton rats. In recent years, in the case of failure of clinical trial research in the elderly population in the vaccine using the fused F protein as a main target antigen, great resistance is brought to the research and development of RSV vaccine.
Compared with an F protein subunit vaccine, the effective antigen in the epitope chimeric VLP vaccine only selects the palivizumab antibody on the RSV F protein to combine with epitope II and epitope VIII, and theoretically, the development strategy is safer for the development of the RSV vaccine. The strategy scheme is to select different neutralizing antigen epitopes on RSV F protein, and to embed the different neutralizing antigen epitopes into virus-like particles with self-organizing characteristics to form recombinant multi-epitope chimeric virus-like particles.
Human Hepatitis B Virus (HBV) belongs to the genus of orthohepadnaviridae, and its core antigen (HBcAg) has the characteristics of self-assembly property, high-density display and strong immunogenicity, and has become a high-efficiency safe and widely-applied exogenous epitope presentation carrier[5]. However, pre-existing HBV antibodies in the human population may affect the immune efficacy of HBcAg-based chimeric vaccines in the human population. Because the core antigen of the hepadnaviridae has similar spatial structure and immunogenicity, other hepatitis virus core antigens of the hepadnaviridae can also replace human hepatitis B virus to be used as an epitope display carrier for epitope presentation.
Jeanne H in 2015 for RSV vaccine development[6]Et al performed RSV F egg using Woodchuck hepadnavirus core antigen (WHcAg) as an epitope-displaying vectorThe chimeric display of the single epitope of the white antigen epitope II obtains a certain immune effect, and confirms the feasibility of the epitope display idea of the chimeric Virus Like Particle (VLP) for the research and development of RSV vaccines. However, in order to achieve a certain immune effect, the dose of antigen used for immunizing mice is significantly higher than the conventional dose (100. mu.g/dose), and the vaccine effectiveness of the chimeric epitope display technology is yet to be enhanced.
In the patent application No. 201710111683.1, an epitope ii single epitope is inserted into the Major Immunodominant Region (MIR) region of the core antigen of the hepes hoof hepatitis virus (RBHV), and the VLP constructed has a certain degree of immunological activity against RSV. However, the technical scheme of the patent is weak in immune response, so that the inventor inserts antigen epitope II into a plurality of epitope insertion sites of the hepialus fimbristus hepatitis virus, such as an MIR region, and inserts antigen epitope VIII into an N terminal and a C terminal on the basis of the original research, and improves the display density and variety of the antigen epitope on the surface of VLP (VLP) and the protein immunological activity to a great extent without influencing the particle assembly. Therefore, a novel preparation scheme of the recombinant respiratory syncytial virus multi-epitope chimeric vaccine is creatively invented, so that the immune effect is effectively improved.
Disclosure of Invention
One aspect of the invention provides a recombinant respiratory syncytial virus multi-epitope chimeric vaccine and a preparation method and application thereof, aiming at the problem of weak immune response of the RSV epitope chimeric VLP vaccine in the prior art.
The technical scheme provided by the invention is as follows:
a recombinant respiratory syncytial virus multi-epitope chimeric vaccine comprises a recombinant respiratory syncytial virus multi-epitope chimeric protein, and the recombinant respiratory syncytial virus multi-epitope chimeric protein consists of a hepialus hepatitis virus core protein and three different antigen epitope fragments of the recombinant respiratory syncytial virus, which are respectively inserted into the C end, the N end and an immunodominant region of the hepialus hepatitis virus core protein;
wherein the amino acid sequence of the antigen epitope fragment inserted into the C end and the N end of the Heps hepatica virus core protein is shown as SEQ ID No. 2;
the amino acid sequence of the antigen epitope fragment inserted in the immune dominant region of the hepes hoof hepatitis virus core protein is shown in SEQ ID No. 3.
The said virus belongs to hepadnaviridae, its host is bat and hoof bat, etc., and is closely related to human hepatitis B virus, woodchuck hepatitis virus and anemarrhena hepatitis virus on phylogenetic tree, and the RBHV core protein has similar structure to human hepatitis B virus core antigen, and can be self-assembled into virus-like particles (T4 or T3) by 240 or 180 RBHV core proteins. The inventor creatively invents the recombinant multi-epitope chimeric vaccine which is based on RBHV as a carrier and comprises three discontinuous respiratory syncytial virus antigen epitopes on the basis of fully taking the existing results as reference, can stimulate to generate stronger immune response, is used for preventing RSV infection, and has important scientific and application values.
In the present invention, the amino acid Sequence of the hepes hepialid virus core protein is different from that of the wild-type hepialid virus core protein, and in order to improve the protein expression amount and protein stability, the present inventors have preferably creatively truncated the N-terminal 28 amino acids of the wild-type virus core antigen (Reference Sequence: NCBI Reference Sequence: YP-009045994.1) to form a vector Sequence shown in SEQ ID No. 1. Namely, the amino acid sequence of the hepialidae hepatitis virus core protein is shown in SEQ ID No. 1.
In the present invention, the inventors have found that when a plurality of specific epitope fragments, which are the same or different, are simultaneously inserted into the N-terminus, MIR region and C-terminus of the Hepialus hepaticum virus core protein and are linked between the 78 th and 79 th amino acids by an appropriate amino acid linking arm, different combinations thereof can affect the epitope-displaying effect to different degrees. Wherein the partial combination can enable the epitope fragment to be maximally displayed on the surface of the virus-like particle, and improve the immunological activity of the cVLP.
The virus-like particle is a protein particle formed by self-assembling virus structural proteins, lacks virus nucleic acid, has no infectivity, and can enable antigen epitopes to be repeatedly displayed on the surface of VLP in high density after foreign epitopes are inserted into proper positions of the structural proteins. Therefore, the vaccine prepared by the chimeric VLP technology has the characteristics of safety and high efficiency.
Preferably, in one embodiment of the present invention, the recombinant respiratory syncytial virus multi-epitope chimeric protein assembles into a virus-like particle.
More preferably, in another embodiment of the present invention, said recombinant respiratory syncytial virus multi-epitope chimeric protein forms a virus-like particle in an expression system.
More preferably, in one embodiment of the present invention, the recombinant respiratory syncytial virus multi-epitope chimeric protein is expressed in Hansenula polymorpha to form virus-like particles.
More preferably, in another embodiment of the present invention, said viral vector comprises a hepialis hepatitis virus core protein and an amino acid fragment having the epitope sequence of SEQ ID No.2 inserted into the N-terminal and C-terminal of said hepialis hepatitis virus core protein, and an amino acid fragment having the epitope sequence of SEQ ID No.3 inserted into the MIR region of said hepialis hepatitis virus core protein.
Preferably, in one embodiment of the present invention, the epitope fragment having the epitope sequence shown in SEQ ID No.3 is linked to the amino acids of the immunodominant region of the hepialus hepatitis virus core protein via the connecting arm of GILE amino acid at the N-terminus and the connecting arm of L amino acid at the C-terminus, and the other two epitope fragments having the epitope sequence shown in SEQ ID No.2 are directly linked to the amino acids at the N-terminus and C-terminus of the hepialus hepatitis virus core protein.
Preferably, in one embodiment of the present invention, the amino acid sequence of the recombinant respiratory syncytial virus multi-epitope chimeric protein is shown in SEQ ID No. 4.
More preferably, the recombinant respiratory syncytial virus multi-epitope chimeric protein shown in SEQ ID No.4 is assembled into virus-like particles.
in the recombinant respiratory syncytial virus polyepitope chimeric vaccine of the present invention, the choice of adjuvant also plays a crucial role in the final immune response effect of the vaccine, theoretically, any suitable pharmaceutical vaccine adjuvant may achieve the objects of the present invention, but AS preferred embodiments of the present invention, the adjuvants include aluminum adjuvants, calcium phosphate adjuvants, Cholera Toxin (CT), cholera toxin B subunit (CTB), Pertussis Toxin (PT), pertussis toxin B subunit (PTB), pertussis hemagglutinin (FHA), pertussis adhesin (PRN), saporin QS-21, α -tocopherol, squalene, liposomes (lipomes), lipid A of Mono acid (MPL-A), MF59, viroid liposome (virosome), Polyglycolide (PLA) microspheres, polylactic acid-glycolic acid (PLGA) microspheres, CpG-Chol, Mono octa quaternary ammonium phosphate (DDA), immune bromide (CSF), Montanide complexes, Montanic acid-57, polyethylene-PLA) microspheres, polyethylene-glycolic acid (PLGA) microspheres, Mono CpG-Chol-CpG-Chol, Mono-32, Mono L-11, Mono L-11, Mono L-S, Mono-2, Mono L-S-2, Mono-S-L-S-A, L-S-L (ISA-S-L-S-L-S.
More preferably, in one embodiment of the invention, the adjuvant is MF59 adjuvant.
The invention also provides a preparation method of the recombinant respiratory syncytial virus multi-epitope chimeric vaccine, which is characterized in that the recombinant respiratory syncytial virus multi-epitope chimeric protein is prepared firstly, and the recombinant respiratory syncytial virus multi-epitope chimeric protein is matched with the adjuvant after index detection to obtain the vaccine.
Preferably, the preparation is preferably made in a hansenula polymorpha expression system.
More preferably, in one embodiment of the present invention, said preparing said recombinant respiratory syncytial virus multi-epitope chimeric protein comprises the steps of:
step 1) optimizing and synthesizing the gene sequence of the recombinant respiratory syncytial virus multi-epitope chimeric protein according to the preference of hansenula polymorpha codons and the abundance of tRNA (transfer ribonucleic acid) to obtain the gene sequence of the recombinant multi-epitope chimeric protein shown as SEQ ID No. 5;
step 2) replacing the S gene sequence in the vector PUC25-SU with the recombinant multi-epitope chimeric protein gene sequence obtained in the step 1) to obtain PUC25-RBRU I recombinant plasmid;
and 3) transforming the recombinant plasmid obtained in the step 2) into hansenula polymorpha for induced expression, and extracting and purifying to obtain the recombinant multi-epitope chimeric protein.
Specifically, the preparation method may further comprise the steps of:
step A) optimization and Synthesis of genes
Optimizing and synthesizing a gene sequence of a hepialis hepatitis virus core protein, which simultaneously contains an amino acid connecting arm and a respiratory syncytial virus multi-epitope chimeric protein fragment gene sequence, according to the preference of hansenula polymorpha codons and the abundance of tRNA;
step B) construction of recombinant plasmid
Replacing the S gene sequence in the vector PUC25-SU with the gene sequence obtained in the step A) to obtain a PUC25-RBRU II recombinant plasmid;
step C) expression and purification of recombinant multiple epitope chimeric proteins
carrying out enzyme digestion on EcoRI and Hind III on the recombinant plasmid of the recombinant multi-epitope chimeric protein obtained in the step B), carrying out linearization treatment, carrying out electric transformation on the recombinant plasmid into NVSI-H.P-105(△ URA3 △ LEU2) Hansenula to obtain a recombinant, screening by an ELISA method to obtain a positive strain with high expression quantity of the recombinant multi-epitope chimeric protein, fermenting, inducing and expressing, harvesting thalli, carrying out high-pressure crushing, centrifuging, taking a supernatant, and carrying out gel filtration chromatography purification to obtain the recombinant multi-epitope chimeric protein.
Wherein, the gene involved in the step A) is designed, optimized and synthesized according to the amino acid sequence of the chimeric protein.
The vaccines of the present invention may be used in any suitable mannerE.g., intradermal (i.d.), intraperitoneal (i.p.), intramuscular (i.m.), intranasal, oral, subcutaneous (s.c.), etc., and in any suitable delivery device[8]Is used in the preparation of the medicament. Preferably, the vaccine of the invention is administered intradermally, subcutaneously or intramuscularly.
In another aspect of the invention, the invention provides the application of the recombinant respiratory syncytial virus multi-epitope chimeric vaccine in preparing a medicament for preventing and/or treating diseases caused by respiratory syncytial virus infection.
Preferably, the disease caused by respiratory syncytial virus infection is pneumonia, bronchitis or asthma.
In another aspect of the invention, the recombinant respiratory syncytial virus multi-epitope chimeric vaccine is provided for preventing and/or treating diseases caused by respiratory syncytial virus infection.
Preferably, the disease caused by respiratory syncytial virus infection is pneumonia, bronchitis or asthma.
In another aspect of the invention, an antibody is provided, wherein the antibody is obtained by immunizing an individual with the recombinant respiratory syncytial virus multi-epitope chimeric vaccine.
In another aspect of the invention, a strain comprising a polypeptide encoding the above recombinant respiratory syncytial virus multi-epitope chimeric protein is provided. The strain is preferably a yeast strain.
The invention has the beneficial effects that:
the invention provides a recombinant respiratory syncytial virus multi-epitope chimeric vaccine, wherein the recombinant multi-epitope chimeric protein in the vaccine uses a Heps hepatitis virus core protein as a foreign epitope presentation carrier, and firstly carries out discontinuous presentation of a plurality of respiratory syncytial virus epitope fragments, so that the antigen epitope is effectively displayed on the surface of VLP to the maximum extent, the foreign epitope is repeatedly distributed on the surface of VLP in high density, the autonomous assembly of VLP is not influenced, and the vaccine has higher immune effect compared with the presentation of single epitope. The recombinant respiratory syncytial virus multi-epitope chimeric vaccine has the potential of being used as a RSV prophylactic vaccine.
Reference documents:
[1]Groothuis J R,Gutierrez K M,Lauer B A.Respiratory syncytial virusinfection in children with bronchopulmonary dysplasia.[J].Pediatrics,1988,82(2):199-203.
[2]Null D,Bimle C,Weisman L,et al.Palivizumab,a humanized respiratorysyncytial virus monoclonal antibody,reduces hospitalization from respiratorysyncytial virus infection in high-risk infants.The IMpact-RSV Study Group.[J].Pediatrics,1998,102(3Pt 1):531-537.
[3] kingdom medical molecular virology [ M ] science press, 2001.
[4]Kim H W,Canchola J G,Brandt C D,et al.Respiratory syncytial virusdisease in infants despite prior administration of antigenic inactivatedvaccine.[J].American Journal of Epidemiology,1969,89(4):422-34.
[5]Salfeld J,Pfaff E,Noah M,et al.Antigenic determinants andfunctional domains in core antigen and e antigen from hepatitis B virus.[J].Journal of Virology,1989,63(2):798-808.
[6]Schickli J H,Whitacre D C,Tang R S,et al.Palivizumab epitope-displaying virus-like particles protect rodents from RSV challenge.[J].Journal of Clinical Investigation,2015,125(4):1637-47.
[7]Prince G A,Curtis S J,Yim K C,et al.Vaccine-Enhanced RespiratorySyncytial Virus Disease in Cotton Rats Following Immunization with Lot 100ora Newly Prepared Reference Vaccine[J].Journal of General Virology,2001,82(12):2881-8.
[8]O"Hagan D T,Valiante N M.Recent advances in the discovery anddelivery of vaccine adjuvants[J].Nature Reviews Drug Discovery,2003,2(9):727-735.
Drawings
FIG. 1 is a schematic diagram showing the construction of a pUC25-RBRU I recombinant plasmid in the example of the present invention;
FIG. 2 is a SDS-PAGE analysis result of the recombinant RSV multi-epitope chimeric protein obtained in the example of the invention, wherein M is Marker, and 1 and 2 are target bands of the recombinant RSV multi-epitope chimeric protein;
FIG. 3 is a graph showing the results of dynamic light scattering of the recombinant respiratory syncytial virus multi-epitope chimeric protein cVLP obtained in the example of the present invention;
FIG. 4 is a transmission electron micrograph of cVLP after phosphotungstic acid negative staining of the recombinant respiratory syncytial virus multi-epitope chimeric protein obtained in the example of the present invention;
FIG. 5 is a graph showing the results of a binding assay between the recombinant RSV multi-epitope chimeric protein obtained in the example of the present invention and a palivizumab antibody;
FIG. 6 is a graph showing the results of a competition test between the recombinant RSV multi-epitope chimeric protein and a palivizumab antibody obtained in the example of the present invention;
FIG. 7 is a graph showing the results of the titer of the antibodies specific to the mouse immune serum protein of the recombinant respiratory syncytial virus multi-epitope chimeric protein obtained in the example of the present invention;
FIG. 8 is a graph showing the results of the titer of antibodies specific to F protein in the mouse immune serum of the recombinant RSV multi-epitope chimeric protein obtained in the example of the present invention;
FIG. 9 is a graph showing the results of neutralizing antibody titer of mouse immune sera against the recombinant respiratory syncytial virus multi-epitope chimeric protein obtained in the example of the present invention;
FIG. 10 is a graph showing the neutralizing antibody titer of the mouse immune serum of the recombinant respiratory syncytial virus multi-epitope chimeric protein and the formaldehyde-inactivated RSV vaccine obtained in the example of the present invention;
FIG. 11 is a graph showing the results of the antibody titer specific to the subtype of the mouse immune serum of the recombinant respiratory syncytial virus multi-epitope chimeric protein and the formaldehyde-inactivated RSV vaccine.
DESCRIPTION OF THE SEQUENCES
SEQ ID No.1 is the amino acid sequence of the hepialus hepatitis virus core protein;
SEQ ID No.2 is the amino acid sequence of the epitope fragment of the recombinant respiratory syncytial virus inserted into the C end and the N end of the Heps trogopus hepatitis virus core protein;
SEQ ID No.3 is the amino acid sequence of the epitope fragment of the recombinant respiratory syncytial virus inserted into the immunodominant region of the Heps fimbristi hepatitis virus core protein;
SEQ ID No.4 is an amino acid sequence of the recombinant respiratory syncytial virus multi-epitope chimeric protein;
SEQ ID No.5 is the nucleotide sequence of the recombinant respiratory syncytial virus multi-epitope chimeric protein.
Detailed Description
The invention discloses a recombinant respiratory syncytial virus multi-epitope chimeric vaccine, a preparation method and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such alterations and modifications which are obvious to those skilled in the art are deemed to be incorporated herein by reference, and that the techniques of the invention may be practiced and applied by those skilled in the art without departing from the spirit, scope and range of equivalents of the invention.
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art.
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments. The experimental procedures, in which specific conditions are not specified, in the preferred examples are generally carried out according to conventional conditions, for example, those described in the molecular cloning protocols (third edition, J. SammBruk et al, Huangpetang et al, science publishers, 2002), or according to conditions and procedures recommended by the manufacturers.
Experimental materials:
EcoRI, HindIII restriction enzymes and PCR reagents were obtained from TaKaRa;
the RSV F protein is from Beijing Yiqian Shenzhou biotechnology limited and is an insect cell recombinant expression product and a freeze-drying agent;
super Block blocking solution from Thermo corporation;
palivizumab is from Medimmune;
goat anti-human IgG-HRP is from China fir Jinqiao biotechnology, Inc. in Beijing;
the MF59 adjuvant is from national institute of Biotechnology, Inc.;
the developing solution A, the solution B and the stopping solution C are from Beijing Wantai biological pharmaceutical industry Co., Ltd;
BALB/c female mice were from Experimental animals technologies, Inc. of Wei Tony, Beijing;
the PUC25-SU yeast expression plasmid is from national institute of Biotechnology, Inc.;
NVSI-H.P-105(△ URA3 △ LEU2) Hansenula species was from national institute of Biotechnology, Japan;
RSV a2 strain (ATCC VR1540) was from the American Type Culture Collection (ATCC);
hep2 cells (ATCC CCL-23) were from the American Type Culture Collection (ATCC);
DMEM medium was from GIBCO, usa;
fetal bovine serum was from GIBCO, USA;
the dual-resistant enzyme, penicillin-streptomycin, is from GIBCO, USA;
all related gene sequencing and primer synthesis in the following examples were completed by the company Limited in the genome research center of Beijing Noso;
all the related gene synthesis and gene manipulation in the following examples were performed by Shanghai Czeri bioengineering, Inc.
The following culture medium formulas are all in percentage by mass and volume:
MD liquid medium: 1.34% of amino acid-free yeast nitrogen source and 2% of glucose;
MM liquid medium: 1.34% of amino acid-free yeast nitrogen source and 0.8% of anhydrous methanol;
SM-leu liquid Medium: 1.34% of amino acid-free yeast nitrogen source, 2% of glucose and 0.01% of leucine;
MM-leu liquid Medium: 1.34% of amino acid-free yeast nitrogen source, 0.8% of anhydrous methanol and 0.01% of leucine;
YPD liquid medium: 1% yeast extract, 2% peptone, 2% glucose;
the solid culture medium is prepared by adding 1.5% agar into the above liquid culture medium, sterilizing at high temperature under high pressure, and storing at low temperature.
The statistical analysis method was performed using SPSS version 18.0.
Example 1: construction and identification of recombinant multi-epitope chimeric protein yeast expression plasmid
1. Design of recombinant multi-epitope chimeric proteins
Taking an amino acid Sequence (NCBI Reference Sequence: YP _009045994.1) of a hepes hepialid virus core protein as a Reference Sequence, cutting off 28 amino acids at the N end of the Reference Sequence to form a vector Sequence shown as SEQ ID NO.1, inserting an epitope (shown as SEQ ID NO. 3) of a respiratory syncytial virus fusion protein (Genebank: ACO83301.1) into an MIR region of the SEQ ID NO.1 Sequence, wherein the epitope, namely amino acid epitopes 254-277 of the respiratory syncytial virus fusion protein, namely the binding position of a palivit antibody is formed by connecting a GILE and an L amino acid connecting arm in series, and simultaneously inserting epitopes (shown as SEQ ID NO. 2) different from the amino acid epitopes at the N end and the C end of the hepialid hepatitis virus core protein simultaneously, namely amino acid epitopes 163-181 of the respiratory syncytial virus chimeric protein to form an amino acid Sequence shown as SEQ ID NO. 4.
2. Gene optimization and synthesis
According to the amino acid sequence of the recombinant respiratory syncytial virus multi-epitope chimeric protein shown in SEQ ID No.4, the gene sequence is optimized according to the preference of hansenula polymorpha codons and the abundance of tRNA, so as to form a coding gene sequence shown in SEQ ID No.5, and the sequence is subjected to whole gene synthesis.
3. Construction of expression plasmids
The gene sequence shown in SEQ ID No.5 is used for replacing an S gene in an expression vector PUC25-SU yeast expression plasmid by utilizing a gene recombination technology to form a yeast expression plasmid containing a recombinant respiratory syncytial virus multi-epitope chimeric protein gene sequence (shown in SEQ ID No. 5), the plasmid takes 25S rDNA on a hansenula polymorpha genome as a homologous recombination integration arm, takes URA3 gene as a marker gene, and uses an MOX promoter to efficiently start target protein expression, and a plasmid construction diagram is shown in figure 1.
4. Restriction enzyme identification and gene sequence determination of expression plasmid
EcoRI and HindIII double enzyme digestion identification is carried out on a yeast expression plasmid containing the recombinant respiratory syncytial virus multi-epitope chimeric protein, enzyme digestion is carried out for 1 hour at 37 ℃, an enzyme digestion system is shown in Table 1, a product after enzyme digestion is detected by 1 percent agarose gel electrophoresis, two gene fragments can be obtained by the plasmid through enzyme digestion, the two gene fragments are respectively a large fragment of about 4kb and a small fragment of 1.5kb, the large fragment is a yeast expression frame containing a target gene of the recombinant multi-epitope chimeric protein, and the plasmid is subjected to gene sequence determination, so that the result shows that the result is consistent with the expected result and no target gene change exists.
TABLE 1 enzyme digestion System
Figure BDA0002356810340000111
Example 2: screening and identification of high-expression positive yeast strains
1. Transformation of
culturing Hansenula polymorpha (NVSI-H.P-105 (△ URA3 △ LEU2) in YPD liquid medium to obtain cell density (OD)600) When the expression level reaches 1.0, yeast competence is prepared, large fragment genes in the recombinant multi-epitope chimeric protein yeast expression plasmid subjected to double enzyme digestion of EcoRI and HindIII are converted into NVSI-H.P-105 yeast in an electric conversion mode, and finally the converted bacterial liquid is coated on an SM-leu solid culture medium and cultured for 3 days at 37 ℃ to obtain a converted recombinant.
2. ELISA screening
The monoclonal colonies grown on SM-leu solid medium were picked up and cultured in 2ml of SM-leu liquid medium, and cultured for 24 hours at 37 ℃ under shaking at 220 rpm. Transferring 200 μ l of the bacterial solution into 4ml of SM-leu liquid culture medium, and continuing culturing until the density (OD) of the bacterial cells is reached600) After reaching more than 10 rpm, the bacteria are harvested by centrifugation at 3000rpm, the culture supernatant is discarded, and the culture medium is driedThe thalli is resuspended in 4ml of MM-leu culture medium for thalli culture, 1% of anhydrous methanol is added every 6 hours at the stage, the expression of the target protein is induced, and the induction lasts 24 hours; the cells were harvested by centrifugation, 200. mu.l of yeast cell disruption buffer (20mM PB, pH7.2) and 200mg of glass beads were added, disrupted by high-frequency low-temperature shaking, and the cells were disrupted with a coating solution (Na)2CO3-NaHCO3Solution, pH9.6) and coating the protein supernatant on an enzyme label plate by diluting 500 times, coating the solution on the enzyme label plate by 100 mu l/hole for 8 hours at 4 ℃; PBS containing 1% BSA was added at 100. mu.l/well and blocked at 37 ℃ for 3 hours; the palivizumab antibody was diluted to 1. mu.g/ml, 100. mu.l/well, 37 ℃ for 1 hour; diluting goat anti-human IgG-HRP by 10000 times, 100 mul/hole, 37 ℃, 1 hour; adding 50 mul of color developing solution A and 50 mul of color developing solution B, developing for 10 minutes at room temperature, and adding 50 mul of stop solution C; and (3) reading OD values at the wavelengths of 450nm and 630nm, and selecting the strain with the highest OD value as the recombinant multi-epitope chimeric protein high-expression yeast strain. Since the selected yeast strain is supplemented with URA3 gene only and can grow in the medium supplemented with leucine only, the LEU2 gene was transformed into the selected yeast strain, so that the strain can grow in the MD basal medium.
3. Identification of target gene of strain
Extracting the genome of the recombinant multi-epitope chimeric protein high-expression yeast strain, and carrying out sequence determination on the target gene without changing the target gene.
Example 3: preparation and characterization of cVLP
1. Yeast fermentation and cell disruption
The recombinant multi-epitope chimeric protein high-expression yeast strain obtained by screening in the example 2 is inoculated in 10ml of MD liquid culture medium for shaking culture for 24 hours, then is transferred to 100ml of MD liquid culture medium for expanding culture for 24 hours, fermentation seeds are prepared, and are inoculated in a 5L fermentation tank for yeast fermentation culture, and the induction expression of target protein is carried out by using methanol. After the completion of the fermentation, the cells were washed 2 times with physiological saline, and finally suspended in a disruption buffer (20mM PB,50mM NaCl, pH7.2) and disrupted under high pressure, followed by centrifugation to obtain a protein supernatant.
2. Purification of target protein and detection of SDS-PAGE
Purifying by gel filtration chromatography with AKTA chromatography purifier (GE AKTA explorer) with Sephacryl S500-HR as medium and 900ml column volume. After equilibration of the column with 3 column volumes of equilibration buffer (50mMPB, 0.2M NaCl, pH7.3), 100ml of crude protein concentrate was pumped into the column at a pump rate of 5ml/min, after loading was complete, equilibration buffer continued to flow through the column. When the buffer solution reaches 2/3 column volume (about 550 ml), a second absorption peak appears, and the protein with the absorption peak is collected, namely the target protein.
The purified target protein was analyzed by 12% SDS-PAGE, and the band of the target protein showed a molecular weight of about 30kD, which was substantially identical to the expected size, as shown in FIG. 2.
3. Dynamic light scattering and transmission electron microscopy of vlp
The purified protein was analyzed by dynamic light scattering (Malvern, NANO-ZS90), and the results are shown in FIG. 3, which shows that the recombinant respiratory syncytial virus multi-epitope chimeric protein cVLP has good size uniformity and average hydrated particle size of about 50 nm. The target protein is dripped on a 300-mesh carbon-plated copper net film, the adsorption is carried out for 5 minutes, phosphotungstic acid is negatively dyed for 1 minute, and the particle morphology of the sample is observed by a transmission electron microscope (HITACHI, JEM-1400), so that the result is shown in figure 4, and the cVLP has the size of 30-40nm, uniform size and good morphology.
Example 4: binding and competition assays for recombinant polyepitope chimeric proteins with palivizumab antibodies
1. Palivizumab antibody binding assay
The binding ability of the recombinant multi-epitope chimeric protein to palivizumab was determined using 0.5. mu.g/ml of purified recombinant multi-epitope chimeric protein, RBHV carrier protein and F protein as the envelope antigen.
The purified recombinant multi-epitope chimeric protein, RBHV carrier protein and F protein are diluted to 0.5 mu g/ml by using coating solution, 100 mu l/hole is coated on an ELISA plate, and the temperature is 4 ℃ for 8 h. Blocking was performed using Super Block solution, blocking at 37 ℃ for 3h, using palivizumab as the primary antibody, diluted in a 2-fold gradient at an initial concentration of 0.1. mu.g/ml, incubated at 37 ℃ for 1.5h, 1: goat anti-human IgG-HRP antibody diluted in 10000 ratios was used as a secondary antibody, incubated at 37 ℃ for 1 hour, developed, and absorbance (OD) values were read at a wavelength of 450nm and 630 nm. The result is shown in fig. 5, the recombinant multi-epitope chimeric protein shows better palivizumab antibody binding capacity, and is slightly stronger than the F protein under the same dosage, which indicates that the recombinant multi-epitope chimeric protein has better epitope display effect.
2. Competitive assay with RSV F protein palivizumab antibody
The ability of the recombinant multi-epitope chimeric protein to compete with the F protein for binding to the palivizumab antibody was determined using 0.5. mu.g/ml of the F protein as the coating antigen.
RSV F protein was diluted to 0.5. mu.g/ml using coating solution, 100. mu.l/well coated microplate, 4 ℃ for 8 h. The microplate was blocked with Super Block solution for 3h at 37 ℃. The recombinant multi-epitope chimeric protein and the corresponding carrier RBHV protein are diluted to 80 mu g/ml by Super Block solution, then 2-fold gradient dilution is carried out, and the diluted solution is mixed with equal volume of dilution solution containing 0.1 mu g/ml palivizumab antibody, 100 mu l/hole is carried out, each dilution degree is carried out for 3 multiple holes, the temperature is 37 ℃, and the time is 1.5 h. Mixing the following components in parts by weight: goat anti-human IgG-HRP antibody diluted in 10000 proportion was used as a secondary antibody, developed at 37 ℃ for 1 hour, and OD values were read at wavelengths of 450nm and 630 nm. The palivizumab Inhibition ratio (Inhibition index) of the recombinant polyepitope chimeric protein and the corresponding carrier RBHV protein was calculated, wherein the Inhibition ratio is [ (palivizumab OD value-sample OD value)/palivizumab OD value ] × 100%, i.e., the Inhibition ratio is (1-sample OD value/palivizumab OD value) × 100%. As shown in FIG. 6, the recombinant multi-epitope chimeric protein showed a competitive activity with the palivizumab antibody of the F protein, and showed a certain dose relationship, and as the protein concentration decreased, the competitive inhibition rate of the F protein decreased.
Example 5: preparation and immunization of recombinant multi-epitope chimeric vaccine
1. Vaccine preparation
The concentration of the stock solution protein is determined after the recombinant multi-epitope chimeric protein is filtered and sterilized by 0.22 mu m. Diluting the recombinant multi-epitope chimeric protein to 200 mu g/ml, weighing a certain volume of the recombinant multi-epitope chimeric protein according to the immunization dosage, mixing the recombinant multi-epitope chimeric protein with MF59 adjuvant with the same volume to prepare the vaccine, and slowly and reversely mixing the vaccine for 20min at room temperature in a dark place.
2. Vaccine immunization
24 SPF-grade BALB/c female mice 6-8 weeks old were randomly divided into A, B, C3 groups of 8 mice each. The following design schemes were used for immunization: group A is injected with 0.3ml of recombinant multi-epitope chimeric vaccine per vaccine; group B was injected with 0.5. mu. g F protein (150. mu.l) as a positive control with an equal volume of MF59 adjuvant mixture, 0.3 ml/mouse; group C was injected with saline (150. mu.l) and an equal volume of MF59 adjuvant mixture as an adjuvant control, 0.3 ml/mouse. The immunization was performed by the intraperitoneal route, and each group was immunized 3 times at 2 weeks intervals. Two weeks after the end of the last immunization, blood was collected by cutting the cone and serum was separated.
Example 6: immunological evaluation of recombinant multi-epitope chimeric vaccines
1. Protein specific antibody detection
The recombinant multi-epitope chimeric protein is used as a coating antigen, and an indirect ELISA method is used for detecting a protein specific antibody in the immune serum of the mouse.
The recombinant multi-epitope chimeric protein and the F protein are diluted to 0.1 mu g/ml by using a coating solution, and 100 mu l/hole is coated in an enzyme label plate for 8 hours at 4 ℃. Blocking by using Super Block solution, blocking for 3h at 37 ℃, diluting the serum to be detected by using Super Block in a 3-fold gradient manner, incubating for 1.5h at 37 ℃, and carrying out 1: goat anti-mouse IgG-HRP antibody diluted in 10000 proportion is used as a secondary antibody, and the secondary antibody is incubated for 1h at 37 ℃ for color development. OD values were read at wavelengths of 450nm and 630nm, with serum vlp-specific antibody titers at the maximal dilution factor of serum above Cut-off values (negative control OD Mean + 2-fold Standard Deviation (SD)), and results were expressed as antibody Geometric Mean Titer (GMT) and 95% confidence interval (confidenceinval, CI), and tested for differences between groups using a two-sided independent sample t-test. The results are shown in fig. 7, the geometric mean titer of the protein-specific antibody of the immune serum of the recombinant multi-epitope chimeric vaccine is 5.66Log10, and the geometric mean titer of the protein-specific antibody of the immune serum of the F protein MF59 is 5.34Log10, and the results show that the recombinant multi-epitope chimeric vaccine produces higher levels of protein-specific antibody after immunizing mice.
2. RSV F protein specific antibody detection
The RSV F protein specific antibody in the immune serum of the mouse is detected by an indirect ELISA method by taking 0.1 mu g/ml F protein as a coating antigen.
The F protein was diluted to 0.1. mu.g/ml using the coating solution, 100. mu.l/well coated microplate, 4 ℃ for 8 h. Blocking by using a SuperBlock solution, blocking for 3h at 37 ℃, diluting the serum to be detected by using a SuperBlock 3-fold gradient, incubating for 1.5h at 37 ℃, and 1: goat anti-mouse IgG-HRP antibody diluted in 10000 proportion is used as a secondary antibody, and the secondary antibody is incubated for 1h at 37 ℃ for color development. OD values were read at wavelengths of 450nm and 630nm, and the maximum dilution factor of serum higher than Cut-off (negative control OD mean +2SD) was the serum F protein-specific antibody titer, and the results were expressed as GMT and 95% CI, and a difference test between groups was performed using a two-sided independent sample t-test. The results are shown in fig. 8, the geometric mean titer of the F protein-specific antibody of the sera immunized by the recombinant multi-epitope chimeric vaccine is 2.94Log10, and the results show that the recombinant multi-epitope chimeric vaccine can generate a certain level of F protein-specific antibody after immunizing Balb/c mice.
3. Neutralizing antibody titer detection of immune sera
The titer of the specific neutralizing antibody of the RSV A2 strain in immune serum is detected by a trace neutralization test method, the result is shown in figure 9, the titer of the neutralizing antibody of the immune serum of the recombinant multi-epitope chimeric vaccine is 9.875Log2, which shows that the vaccine can generate high-level RSV specific neutralizing antibody after immunizing Balb/c mice, and the recombinant multi-epitope chimeric vaccine has the potential of being used as an RSV preventive vaccine.
Example 7: comparison of recombinant multi-epitope chimeric vaccine and Formaldehyde inactivated vaccine
1. Preparation and immunization of formaldehyde inactivated vaccine and recombinant multi-epitope chimeric vaccine
The RSV A2 strain was cultured using Hep2 cells, and the virus was harvested after 3 days at 37 ℃. Method according to Lot100[7]Preparing formaldehyde inactivated RSV vaccine (FI-RSV), and after the preparation is completed, remaining at 4 ℃ for later use. Recombinant multi-epitope chimeric vaccines were prepared as described in example 5.
24 SPF-grade BALB/c female mice 6-8 weeks old were randomly divided into A, B, C3 groups of 8 mice each. The following design schemes were used for immunization: group A is injected with 0.3ml of recombinant multi-epitope chimeric vaccine per vaccine; FI-RSV is injected into the group B, and the volume is 0.1 ml/mouse; group C was injected with saline (150. mu.l) and an equal volume of MF59 adjuvant mixture as an adjuvant control, 0.3 ml/mouse. The immunization was performed by the intraperitoneal route, and each group was immunized 3 times at 2 weeks intervals. Two weeks after the end of the last immunization, blood was collected by cutting the cone and serum was separated.
2. Neutralizing antibody titer detection of immune sera
The RSV A2 strain-specific neutralizing antibody titer was detected according to the method described in example 6, and the result is shown in FIG. 10, the neutralizing antibody titer of the immune serum of the recombinant multi-epitope chimeric vaccine was 10.00Log2, and the neutralizing antibody titer of the immune serum of FI-RSV was 6.0Log2, which are significantly different (P < 0.001), indicating that the immunization of Balb/c mice with the recombinant protein vaccine produced high level of RSV-specific neutralizing antibody compared with the inactivated formaldehyde vaccine.
3. IgG1 and IgG2a antibody subtype analysis of immune sera
The F protein is used as a coating antigen, and RSV F protein specific IgG and IgG2a antibodies in immune serum of a mouse are detected by an indirect ELISA method.
The F protein was diluted to 0.1. mu.g/ml using the coating solution, 100. mu.l/well coated microplate, 4 ℃ for 8 h. Blocking by using a SuperBlock solution, blocking for 3h at 37 ℃, diluting the serum to be detected by using a SuperBlock 3-fold gradient, incubating for 1.5h at 37 ℃, and 1: goat anti-mouse IgG1-HRP and goat anti-mouse IgG2a-HRP antibodies diluted in 10000 proportion are respectively used as secondary antibodies, and the secondary antibodies are incubated for 1h at 37 ℃ and developed. OD values were read at 450nm/630nm dual wavelength, with serum F protein specific IgG1 and IgG2a antibody titers as the maximum dilution factor of serum above Cut-off value (negative control OD mean +2SD), and results are expressed as GMT and 95% confidence interval CI. The result is shown in fig. 11, the IgG1 and IgG2a subtype antibody titer levels in the immune serum of the recombinant multi-epitope chimeric vaccine are more balanced than that of the FI-RSV vaccine, which suggests that the vaccine generates more balanced Th1 and Th2 cellular immune responses after immunization, thereby avoiding immunopathology risks to a certain extent and having better safety.
Comparative example 1: the recombinant respiratory syncytial virus multi-epitope chimeric vaccine (non-continuous multi-epitope) of the invention and cVLP immunological activity comparison analysis in the patent (application No. 201710111683.1)
1. Palivizumab antibody binding assay
The single epitope chimeric protein RBHV-cVLP was prepared according to the design protocol of example 1 and the method described in examples 2 and 3, and the purified RBHV-cVLP was diluted to 0.5. mu.g/ml, coated on an ELISA plate at 100. mu.l/well, 4 ℃ for 8h using the coating solution according to the Parley bead antibody binding assay method in example 4. Blocking was performed using Super Block solution, blocking at 37 ℃ for 3h, using palivizumab as the primary antibody, diluted in a 2-fold gradient at an initial concentration of 0.1. mu.g/ml, incubated at 37 ℃ for 1.5h, 1: goat anti-human IgG-HRP antibody diluted in 10000 proportion is used as a secondary antibody, and the secondary antibody is incubated for 1h at 37 ℃ for color development. As shown in fig. 5, the recombinant multi-epitope chimeric protein showed good palivizumab antibody binding ability as the single-epitope chimeric protein RBHV-cplp, suggesting that the insertion of the N-terminal and C-terminal foreign epitopes did not affect the assembly characteristics of the particles.
2. Competitive assay with RSV F protein palivizumab antibody
The single epitope chimeric protein RBHV-cVLP finally obtained according to the example prepared according to the method described in example 1, 2 and 3 according to the design scheme of example 1, and the RSV F protein was diluted to 0.5. mu.g/ml, coated on a microplate at 100. mu.l/well, and 8h according to the palivizumab antibody competition assay method in example 4. The microplate was blocked with Super Block solution for 3h at 37 ℃. RBHV-cVLP was diluted to 80. mu.g/ml using Super Block solution, then 2-fold gradient diluted, mixed with equal volume of dilution containing 0.1. mu.g/ml palivizumab antibody, 100. mu.l/well, 3 replicates per dilution, 37 ℃, 1.5 h. Mixing the following components in parts by weight: goat anti-human IgG-HRP antibody diluted in 10000 proportion was used as a secondary antibody, and color development was performed at 37 ℃ for 1 hour. The palivizumab antibody inhibition rate of the RBHV-cVLP is calculated. As shown in FIG. 6, the recombinant multi-epitope chimeric protein showed good competitive activity with the F protein palivizumab antibody, as with the single-epitope chimeric protein RBHV-cVLP.
3. Neutralizing antibody titer detection of immune sera
According to the design scheme of example 1, the single epitope chimeric protein RBHV-cVLP is prepared according to the methods described in examples 1, 2 and 3, the single epitope chimeric protein RBHV-cVLP is finally obtained, and group D is injected with 30 ug (150 uL) of the mixture of RBHV-cVLP and an equal volume of MF59 adjuvant according to the vaccine preparation and animal immunization method in example 5, and is immunized by the intraperitoneal route, wherein each group is immunized 3 times at intervals of 2 weeks. Mice status and body weight changes were observed daily after immunization of each group of mice. Two weeks after immunization, the broken cone blood is collected and serum is separated, and the RSV specific neutralizing antibody titer level in the serum is detected by using a virus micro-neutralization test method. The result is shown in figure 9, the RSV specific neutralizing antibody titer of the recombinant respiratory syncytial virus multi-epitope chimeric protein is about 9.875Log2, while the RSV specific neutralizing antibody titer of the RBHV-cVLP is about 8.0Log2, and the neutralizing antibody level generated after the recombinant respiratory syncytial virus multi-epitope chimeric protein adsorbs the MF59 adjuvant is improved by 3 times compared with the RBHV-cVLP.
The results indicate that the recombinant respiratory syncytial virus multi-epitope chimeric vaccine has obvious advantages in immune effect, and the recombinant respiratory syncytial virus multi-epitope chimeric vaccine has the potential of being used as a RSV preventive vaccine.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> national institute of Biotechnology, Biotechnology Limited
<120> recombinant respiratory syncytial virus multi-epitope chimeric vaccine and preparation method and application thereof
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Claims (14)

1. A recombinant respiratory syncytial virus multi-epitope chimeric vaccine comprises recombinant respiratory syncytial virus multi-epitope chimeric protein, and is characterized in that the recombinant respiratory syncytial virus multi-epitope chimeric protein consists of a hepialus hepatitis virus core protein and three different antigen epitope fragments of the recombinant respiratory syncytial virus which are respectively inserted into the C end, the N end and an immunodominant region of the hepialus hepatitis virus core protein;
wherein the amino acid sequence of the antigen epitope fragment inserted into the C end and the N end of the Heps hepatica virus core protein is shown as SEQ ID No. 2;
the amino acid sequence of the antigen epitope fragment inserted in the immune dominant region of the hepes hoof hepatitis virus core protein is shown in SEQ ID No. 3.
2. The recombinant respiratory syncytial virus multi-epitope chimeric vaccine according to claim 1, wherein the amino acid sequence of the hepialus hepatitis virus core protein is shown in SEQ ID No. 1.
3. The recombinant respiratory syncytial virus multi-epitope chimeric vaccine according to claim 1 or 2, wherein said recombinant respiratory syncytial virus multi-epitope chimeric protein assembles into virus-like particles.
4. The recombinant respiratory syncytial virus multi-epitope chimeric vaccine according to claim 3, wherein the recombinant respiratory syncytial virus multi-epitope chimeric protein is expressed in Hansenula polymorpha to form virus-like particles.
5. The recombinant respiratory syncytial virus multi-epitope chimeric vaccine according to claim 1 or 2, wherein the amino acid sequence of the recombinant respiratory syncytial virus epitope fragment represented by SEQ ID No.3 inserted into the hepes hepatis virus core protein immunodominant region is linked to the amino acid of the hepes hepatis virus core protein immunodominant region through the GILE amino acid linking arm at the N-terminus and the L amino acid linking arm at the C-terminus, and the recombinant respiratory syncytial virus epitope fragment represented by SEQ ID No.2 inserted into the N-terminus and the C-terminus of the hepes hepatis virus core protein is directly linked to the N-terminus or the C-terminus of the hepatis virus core protein.
6. The recombinant respiratory syncytial virus multi-epitope chimeric vaccine according to claim 1 or 2, characterized in that the amino acid sequence of the recombinant respiratory syncytial virus multi-epitope chimeric protein is shown as SEQ ID No. 4.
7. The recombinant respiratory syncytial virus multi-epitope chimeric vaccine of claim 6, wherein said recombinant respiratory syncytial virus multi-epitope chimeric protein assembles into virus-like particles.
8. the chimeric vaccine according to claim 1 or 2, wherein the chimeric vaccine further comprises an adjuvant, preferably selected from the group consisting of aluminum adjuvant, calcium phosphate adjuvant, Cholera Toxin (CT), cholera toxin B subunit (CTB), Pertussis Toxin (PT), pertussis toxin B subunit (PTB), pertussis Filamentous Hemagglutinin (FHA), pertussis adhesin (PRN), saporin QS-21, α -tocopherol, squalene, lipoid, liposome (lipomes), monophosphoryl lipid a (MPL-a), MF59, viroid liposome (virosomees), Polyglycolide (PLA) microspheres, polylactic-glycolic acid (PLGA) microspheres, lipid-cholesterol (DC-Chol), dimethyldioctadecyl quaternary ammonium bromide (DDA), immunostimulating complex (isc), Montanide ISA50, Montanide ISA51, tan sa206, Montanide ISA720, Montanide series AS 3825, AS series 02, AS-C series of AS, serine-l, heparin series ISA-C, heparin-C series, heparin-co-IL-C series (IL-l), heparin-C), heparin (IL-C), heparin series (IL-C), heparin-C series (IL-C), heparin (IL-C), and optionally containing at least one or more preferably at least one of a heat resistant adjuvant, silicone (IL-C adjuvant, silicone (IL-C series).
9. A method for preparing the recombinant respiratory syncytial virus multi-epitope chimeric vaccine according to any one of claims 1 to 8, wherein the recombinant respiratory syncytial virus multi-epitope chimeric protein is prepared firstly, and is subjected to index detection and then is compatible with an adjuvant to obtain the vaccine;
the preparation of the recombinant respiratory syncytial virus multi-epitope chimeric protein is preferably carried out in a hansenula polymorpha expression system.
10. The method for preparing according to claim 9, wherein the preparing the recombinant respiratory syncytial virus multi-epitope chimeric protein comprises the following steps:
step 1) optimizing and synthesizing the gene sequence of the recombinant respiratory syncytial virus multi-epitope chimeric protein according to the preference of hansenula polymorpha codons and the abundance of tRNA (transfer ribonucleic acid) to obtain the gene sequence of the recombinant multi-epitope chimeric protein shown as SEQ ID No. 5;
step 2) replacing the S gene sequence in the vector PUC25-SU with the recombinant multi-epitope chimeric protein gene sequence obtained in the step 1) to obtain PUC25-RBRU I recombinant plasmid;
and 3) transforming the recombinant plasmid obtained in the step 2) into hansenula polymorpha for induced expression, and extracting and purifying to obtain the recombinant multi-epitope chimeric protein.
11. Use of the recombinant respiratory syncytial virus multi-epitope chimeric vaccine according to any one of claims 1 to 8 in the preparation of a medicament for the prevention and/or treatment of a disease caused by respiratory syncytial virus infection, preferably pneumonia, bronchitis or asthma.
12. Use of the recombinant respiratory syncytial virus multi-epitope chimeric vaccine according to any one of claims 1 to 8 for the prevention and/or treatment of a disease caused by respiratory syncytial virus infection, preferably pneumonia, bronchitis or asthma.
13. An antibody obtained by immunizing an individual with the recombinant respiratory syncytial virus multi-epitope chimeric vaccine of any one of claims 1-8.
14. A strain comprising a polypeptide encoding the recombinant respiratory syncytial virus polyepitopic chimeric protein of any one of claims 1-7.
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