CN111166881B - 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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CN111166881B
CN111166881B CN202010010061.1A CN202010010061A CN111166881B CN 111166881 B CN111166881 B CN 111166881B CN 202010010061 A CN202010010061 A CN 202010010061A CN 111166881 B CN111166881 B CN 111166881B
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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. Currently, there are no specific therapeutic drugs and no available prophylactic and therapeutic RSV vaccines other than the humanized monoclonal antibody Palivizumab (Palivizumab) [2]. The RSV genome is 15.2kb in 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 nonstructural proteins (NS 1, 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. The F protein has higher homology between the A serotype and the B serotype compared with the G protein, and the RSV vaccine developed by the F protein has protective effect on the A serotype and the B serotype, and is researched by the RSV vaccine at presentThe main target point.
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 GDA0004044734350000011
there are reports on the development of corresponding neutralizing monoclonal antibodies. Wherein, the antigenic site II is the combination site of RSV preventing and treating monoclonal antibody-palivizumab antibody and Motavizumab antibody (Motavizumab), is positioned at amino acids 254 to 277 of F1 subunit of the F protein, is a conformational epitope of an 'alpha helix-ring-alpha 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 recently discovered specific site for the pre-fusion F protein, located in the region of amino acids 163-181 of the "helix-loop-helix" and covering in part antigenic site II and/or `>
Figure GDA0004044734350000012
The neutralizing monoclonal antibody hRSV90 recognizing the site has better neutralizing activity to A and B subtypes.
In the last 60 years, the formaldehyde-inactivated RSV (Formalin-inactivated RSV) vaccine can not prevent RSV infection of infants, but also causes serious respiratory disease Enhancement (ERD) to cause death of two infants, and specific target points and mechanisms of the ERD phenomenon are not clear up until now [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 cases where a vaccine using the fused F protein as a main target antigen has failed clinical trial studies in the elderly population, the vaccine also hasBrings great resistance to the development of RSV vaccine.
Compared with an F protein subunit vaccine, the effective antigen in the epitope chimeric VLP vaccine only selects a palivizumab antibody on RSV F protein to combine with an epitope-epitope II and an epitope VIII, and theoretically, the development strategy is safer for developing 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 the recombinant multi-epitope embedded virus-like particles.
Human Hepatitis B Virus (HBV) belongs to the genus of orthohepadnaviridae, and a core antigen (HBcAg) of the HBV has the characteristics of self-assembly property, high-density display, strong immunogenicity and the like, and becomes an efficient, 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] The human uses Woodchuck hepatitis Virus core antigen (WHcAg) as an epitope display carrier to perform chimeric display of single epitope of RSV F protein antigen epitope II, obtains a certain immune effect, and proves the feasibility of the epitope display thought of chimeric Virus Like Particles (VLP) for RSV vaccine research and development. 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 number of 201710111683.1, an epitope II single epitope is inserted into a Main Immunodominant Region (MIR) region of a core antigen of a hoof hepatitis virus (RBHV), and the constructed cVLP has a certain degree of immunological activity on RSV. However, the technical scheme of the patent is weak in immune response, so that on the basis of the original research, the inventor inserts a plurality of epitope insertion sites of the hepes hoof hepatitis virus, such as an antigen epitope II inserted in an MIR region, and an antigen site VIII inserted at an N terminal and a C terminal, improves the display density and the display type of the antigen epitope on the surface of VLP (VLP) without influencing the particle assembly, and greatly improves the immunological activity of protein. 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 which consists of a hoof-bat 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 area of the hoof-bat 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 present invention relates to a hoof bat hepatitis virus belonging to hepadnaviridae, its host is bat and hoof bat, etc., and is closely related to human hepatitis B virus, woodchuck hepatitis virus and pachinko hepatitis virus on phylogenetic tree, and the RBHV core protein is similar to human hepatitis B virus core antigen structure, and can be self-assembled into virus-like granules (T =4 or T = 3) 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 creatively truncated the N-terminal 28 amino acids of the wild-type virus core antigen (reference sequence: NCBI reference sequence: YP-009045994.1), thereby forming the vector sequence shown in SEQ ID No. 1. Namely, the amino acid sequence of the hepes hoof 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 multi-epitope chimeric vaccine, the selection of an adjuvant plays a crucial role in the final immune response effect of the vaccine. In theory, any suitable pharmaceutical vaccine adjuvant can achieve the objectives of the present invention. Preferably, however, in embodiments of the present invention, the adjuvant comprises 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, alpha-tocopherol, squalene, liposomes (lipomes), monophosphoryl lipid A (MPL-A), MF59, viroid proteoliposomes (Virosomes), polyglycolide (PLA) microspheres, polylactic-glycolic acid (PLGA) microspheres, lipid-cholesterol (DC-Chol), dimethyl dioctadecyl quaternary ammonium bromide (DDA), and mixtures thereof at least one of an immunostimulating complex (ISCOM), montanide ISA50, montanide eISA51, montanide ISA206, montanide ISA720, montanide ISA series adjuvant, AS01, AS02, AS03, AS04, AS series adjuvant, muramyl Dipeptide (MDP), bacterial lipopolysaccharide (OM-174), E.coli heat-Labile Toxin (LT), IL-1, IL-2, IL-6, IL-12, IL-15, IL-18, IFN-gamma, GM-CSF, cpG oligonucleotides, trehalose Dimycolate (TDM), and a substance containing a picornaviral adjuvant of Poly-hypoxanthine nucleotide (Poly I) and/or Poly-cytosine nucleotide (Poly C).
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.
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 the recombinant plasmid of the recombinant multi-epitope chimeric protein obtained in the step B) by EcoRI and HindIII, carrying out linearization treatment, carrying out electric transformation into NVSI-H.P-105 (delta URA3 delta LEU 2) 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 bacteria, 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 administered by any suitable means, such as intradermally (i.d.), intraperitoneally (i.p.), intramuscularly (i.m.), intranasally, orally, subcutaneously (s.c.), and the like, 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 inactivated vaccine.[J].American Journal of Epidemiology,1969,89(4):422-34.
[5]Salfeld J,Pfaff E,Noah M,et al.Antigenic determinants and functionaldomains 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-displayingvirus-like particles protect rodents from RSV challenge.[J].Journal of ClinicalInvestigation,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 100or aNewly 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 and deliveryof 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 an 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 the amino acid 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 enzyme and PCR reagent are all from TaKaRa company;
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 obtained 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 (delta URA3 delta LEU 2) Hansenula was obtained from national institute of Biotechnology, inc.;
RSV A2 strain (ATCC VR 1540) is from the American Type Culture Collection (ATCC);
hep2 cells (ATCC CCL-23) from the American Type Culture Collection (ATCC);
DMEM medium 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 and 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 Sequence of a hepes hoof hepatitis virus core protein (NCBI Reference Sequence: YP _ 009045994.1) as a Reference Sequence, cutting off 28 amino acids at the N end of the Reference Sequence to form a carrier Sequence shown as SEQ ID NO.1, inserting an epitope (shown as SEQ ID NO. 3) of a respiratory syncytial virus fusion protein (Genebank: ACO 83301.1) into an MIR region of the SEQ ID NO.1 Sequence, wherein the epitope is the amino acid epitope at positions 254-277 of the respiratory syncytial virus fusion protein, namely the binding position of a palivizumab antibody, and connecting arms are connected in series through a 'GILE' amino acid and an 'L' amino acid, and simultaneously inserting epitopes (shown as SEQ ID NO. 2) different from the amino acid epitope at positions 163-181 of the chimeric protein of the hepes hoof hepatitis virus core protein at the N end and the C end of the hepes hoof hepatitis virus core protein respectively to form the 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, a gene sequence is formed, and the sequence is subjected to whole-gene synthesis.
3. Construction of expression plasmids
A gene sequence is replaced by an S gene in an expression vector PUC25-SU yeast expression plasmid by utilizing a gene recombination technology to form a yeast expression plasmid, the plasmid takes 25S rDNA on a hansenula polymorpha genome as a homologous recombination integration arm, URA3 gene as a marker gene, and a MOX promoter is used for efficiently starting the expression of a target protein, and a plasmid construction diagram is shown in figure 1.
4. Restriction enzyme identification and gene sequence determination of expression plasmid
Carrying out EcoRI and HindIII double enzyme digestion identification on a yeast expression plasmid containing the recombinant respiratory syncytial virus multi-epitope chimeric protein, carrying out enzyme digestion for 1 hour at 37 ℃, wherein an enzyme digestion system is shown in Table 1, carrying out 1% agarose gel electrophoresis detection on a product after enzyme digestion, thus the plasmid can be seen to be digested into two gene fragments, namely a large fragment of about 4kb and a small fragment of 1.5kb, wherein the large fragment is a yeast expression frame containing a target gene of the recombinant multi-epitope chimeric protein, and carrying out gene sequence determination on the plasmid, wherein the result shows that the result is consistent with an expected result and no target gene change.
TABLE 1 enzyme digestion System
Figure GDA0004044734350000101
Example 2: screening and identification of high-expression positive yeast strains
1. Transformation of
Culturing Hansenula polymorpha (NVSI-H.P-105 (DELTA URA3 DELTA LEU 2) 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 by 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 recombinant conversion.
2. ELISA screening
Picking single clones grown on SM-leu solid MediumThe colonies were cultured in 2ml of SM-leu liquid medium for culturing cells, and cultured at 37 ℃ for 24 hours with 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 bacterial density (OD) 600 ) After the culture reaches more than 10, centrifuging at 3000rpm to harvest thalli, discarding culture supernatant, suspending the thalli in 4ml of MM-leu culture medium for thalli culture, adding 1% absolute methanol every 6 hours at the stage, inducing the expression of target protein, and inducing for 24 hours; the cells were harvested by centrifugation, 200. Mu.l of yeast cell disruption buffer (20mM PB, pH 7.2) and 200mg of glass beads were added thereto, disrupted by high-frequency low-temperature shaking, and the resultant mixture was treated with a coating solution (Na) 2 CO 3 -NaHCO 3 Solution, pH9.6) diluting the protein supernatant liquid by 500 times, coating the diluted protein supernatant liquid on an enzyme label plate, coating the diluted protein supernatant liquid on the enzyme label plate by 100 mu l/hole for 8 hours at 4 ℃; adding PBS containing 1% BSA, 100. Mu.l/well, blocking 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, LEU2 gene is transformed into the selected yeast strain, so that the strain can grow in 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 completion of the fermentation, the cells were washed 2 times with physiological saline, and finally suspended in a disruption buffer (20mM PB,50mM NaCl, pH 7.2) 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, pH 7.3), 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-cent SDS-PAGE electrophoresis, and as a result, the band of the target protein showed a molecular weight of about 30kD, which was substantially identical to the expected band, 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-ZS 90) 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 50nm. 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.
And (3) diluting the purified recombinant multi-epitope chimeric protein, RBHV carrier protein and F protein to 0.5 mu g/ml by using a coating solution, coating the enzyme label plate by 100 mu l/hole, and performing reaction at 4 ℃ for 8h. Blocking was performed using Super Block solution, blocking at 37 ℃ for 3h, using palivizumab as the primary antibody, 2-fold gradient dilution at 0.1. Mu.g/ml initial concentration, incubation 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 8h. 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 1.5h. 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 antibody Inhibition rate (Inhibition index) of the recombinant multi-epitope chimeric protein and the corresponding carrier RBHV protein was calculated, wherein the Inhibition rate = [ (palivizumab OD value-sample OD value)/palivizumab OD value ] × 100%, that is, the Inhibition rate = (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 selected and randomly divided into groups A, B, and C3, each group consisting of 8 mice. 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 injected 0.5. Mu.g of F protein (150. Mu.l) with an equal volume of MF59 adjuvant mixture as a positive control, 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 mouse immune serum.
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 8h at 4 ℃. Blocking by using a SuperBlock solution, blocking for 3h at 37 ℃, diluting the serum to be detected by using the SuperBlock solution in a 3-fold gradient manner, incubating for 1.5h at 37 ℃,1: goat anti-mouse IgG-HRP antibody diluted in 10000 proportion was used as a secondary antibody, incubated at 37 ℃ for 1h, and developed. OD values were read at wavelengths of 450nm and 630nm, with serum vlp-specific antibody titers at the highest dilution fold 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 serum immunized with the recombinant multi-epitope chimeric vaccine is 5.66Log10, the geometric mean titer of the protein-specific antibody of the serum immunized with 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 8h. Blocking is carried out by using Super Block solution, blocking is carried out for 3h at 37 ℃, the serum to be detected is diluted by using Super Block in a 3-fold gradient manner, incubation is carried out for 1.5h at 37 ℃,1: goat anti-mouse IgG-HRP antibody diluted in 10000 proportion was used as a secondary antibody, incubated at 37 ℃ for 1h, and developed. 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 +2 SD) was taken as the serum F protein-specific antibody titer, and the results were expressed as GMT and 95% CI, and the inter-group difference test was performed using the double-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 specific neutralizing antibody titer of the RSV A2 strain in immune serum is detected by a trace neutralization test method, the result is shown in figure 9, the neutralizing antibody titer 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 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. As described in example 5The method of (3) for preparing a recombinant multiple epitope chimeric vaccine.
24 SPF-grade BALB/C female mice 6-8 weeks old were selected and randomly divided into groups A, B, and C3, each group consisting of 8 mice. 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 measured 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 is significantly different (P < 0.001), indicating that Balb/c mice immunized with the recombinant protein vaccine produced high levels of RSV-specific neutralizing antibodies 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 8h. Blocking is carried out by using Super Block solution, blocking is carried out for 3h at 37 ℃, the serum to be detected is diluted by using Super Block in a 3-fold gradient manner, incubation is carried out for 1.5h at 37 ℃,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 above the Cut-off value (negative control OD mean +2 SD), and the 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 those of the FI-RSV vaccine, which suggests that the vaccine generates more balanced Th1 and Th2 cell immune responses after immunization, immune pathological risks can be avoided to a certain extent, and the vaccine has better safety.
Comparative example 1: the invention relates to a comparison analysis of immunological activities of cVLP (recombinant respiratory syncytial virus) multi-epitope chimeric vaccine (non-continuous multi-epitope) and a patent (application number 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, 2-fold gradient dilution at 0.1. Mu.g/ml initial concentration, incubation at 37 ℃ for 1.5h,1: goat anti-human IgG-HRP antibody diluted in 10000 proportion is used as a secondary antibody, incubated for 1h at 37 ℃ and developed. 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. Competition 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 ℃,1.5h. 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 was 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 of examples 1, 2 and 3, the single epitope chimeric protein RBHV-cVLP is finally obtained, and group D is injected with 30 mu g (150 mu L) of the mixture of the 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. As shown in FIG. 9, the RSV-specific neutralizing antibody titer of the recombinant respiratory syncytial virus multi-epitope chimeric protein of the invention 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 of the invention adsorbs 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.
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Claims (10)

1. A recombinant respiratory syncytial virus multi-epitope chimeric vaccine comprises a recombinant respiratory syncytial virus multi-epitope chimeric protein, and is characterized in that the amino acid sequence of the recombinant respiratory syncytial virus multi-epitope chimeric protein is shown as SEQ ID No. 4.
2. The recombinant respiratory syncytial virus multi-epitope chimeric vaccine of claim 1, wherein said recombinant respiratory syncytial virus multi-epitope chimeric protein assembles into virus-like particles.
3. The recombinant respiratory syncytial virus multi-epitope chimeric vaccine of claim 1, wherein said recombinant respiratory syncytial virus multi-epitope chimeric vaccine further comprises an adjuvant.
4. The recombinant respiratory syncytial virus multi-epitope chimeric vaccine of claim 3, the adjuvant is selected from 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, alpha-tocopherol, squalene, liposomes (lipomes), monophosphoryl lipid A (MPL-A), MF59, viroid particle lipoproteins (Virosomes), polyglycolide (PLA) microspheres, polylactic-glycolic acid (PLGA) microspheres, lipid-cholesterol (DC-Chol), dimethyldioctadecyl quaternary ammonium bromide (DDA), and mixtures thereof at least one of immunostimulating complex (ISCOM), montanide ISA50, montanide ISA51, montanide ISA206, montanide ISA720, montanide ISA series adjuvant, AS01, AS02, AS03, AS04, AS series adjuvant, muramyl Dipeptide (MDP), bacterial lipopolysaccharide (OM-174), escherichia coli heat-Labile Toxin (LT), IL-1, IL-2, IL-6, IL-12, IL-15, IL-18, IFN-gamma, GM-CSF, cpG oligonucleotide, trehalose Dimycolate (TDM), and a substance containing a picornaviral adjuvant of Poly-hypoxanthine nucleotide (Poly I) and/or Poly-cytosine nucleotide (Poly C).
5. The recombinant respiratory syncytial virus multi-epitope chimeric vaccine according to claim 4, wherein the adjuvant is MF59 adjuvant.
6. A preparation method of the recombinant respiratory syncytial virus multi-epitope chimeric vaccine according to any one of claims 1 to 5, characterized in that the recombinant respiratory syncytial virus multi-epitope chimeric protein is prepared firstly, and the vaccine is obtained by matching the recombinant respiratory syncytial virus multi-epitope chimeric protein with an adjuvant after index detection.
7. The method as claimed in claim 6, wherein the recombinant RSV multi-epitope chimeric protein is prepared in Hansenula polymorpha expression system.
8. Use of the recombinant respiratory syncytial virus multi-epitope chimeric vaccine according to any one of claims 1-5 in the preparation of a medicament for the prevention and/or treatment of a disease caused by respiratory syncytial virus infection.
9. The use according to claim 8, wherein the disease caused by respiratory syncytial virus infection is pneumonia, bronchitis or asthma.
10. A strain comprising a polypeptide encoding the recombinant respiratory syncytial virus polyepitopic chimeric protein of any one of claims 1-5.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104293741A (en) * 2014-10-10 2015-01-21 武汉大学 Respiratory syncytial virus virus-like particle vaccine as well as preparation method and application thereof
CN106905434A (en) * 2017-02-28 2017-06-30 国药中生生物技术研究院有限公司 A kind of recombination fusion protein comprising hoof bat hepatitis B core protein and its preparation method and application
CN106986942A (en) * 2017-02-28 2017-07-28 国药中生生物技术研究院有限公司 A kind of recombination fusion protein comprising bat hepatitis B core protein and its preparation method and application
CN108456686A (en) * 2017-02-17 2018-08-28 厦门大学 It is a kind of to be used to show peptide carrier of desired polypeptides and application thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011020079A1 (en) * 2009-08-13 2011-02-17 Calmune Corporation Antibodies against human respiratory syncytial virus (rsv) and methods of use
TWI683826B (en) * 2016-11-22 2020-02-01 國立臺灣大學 Recombinant rsv antigens

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104293741A (en) * 2014-10-10 2015-01-21 武汉大学 Respiratory syncytial virus virus-like particle vaccine as well as preparation method and application thereof
CN108456686A (en) * 2017-02-17 2018-08-28 厦门大学 It is a kind of to be used to show peptide carrier of desired polypeptides and application thereof
CN106905434A (en) * 2017-02-28 2017-06-30 国药中生生物技术研究院有限公司 A kind of recombination fusion protein comprising hoof bat hepatitis B core protein and its preparation method and application
CN106986942A (en) * 2017-02-28 2017-07-28 国药中生生物技术研究院有限公司 A kind of recombination fusion protein comprising bat hepatitis B core protein and its preparation method and application

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Structural basis of respiratory syncytial virus neutralization by motavizumab;Jason S McLellan 等;《Nature structural & molecular biology》;20100124;第17卷(第2期);Nature structural & molecular biology *

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