CN106986942B - Recombinant fusion protein containing core protein of bat hepatitis virus and preparation method and application thereof - Google Patents
Recombinant fusion protein containing core protein of bat hepatitis virus and preparation method and application thereof Download PDFInfo
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- CN106986942B CN106986942B CN201710111914.9A CN201710111914A CN106986942B CN 106986942 B CN106986942 B CN 106986942B CN 201710111914 A CN201710111914 A CN 201710111914A CN 106986942 B CN106986942 B CN 106986942B
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Abstract
The invention provides a recombinant fusion protein containing core protein of a bat hepatitis virus, which consists of the core protein of the bat hepatitis virus and a foreign protein fragment inserted between amino acids of the core protein of the bat hepatitis virus, wherein the amino acid sequence of the core protein of the bat hepatitis virus is shown as SEQ ID NO. 1. The core protein of the bat hepatitis virus is used as a foreign epitope presentation vector for the first time, the recombinant fusion protein is expressed in Hansenula polymorpha to form the chimeric virus-like particle through optimized design, and the chimeric virus-like particle is used as a vaccine component, can stimulate to generate stronger immune response on the premise of ensuring the safety of the vaccine, is used for preventing RSV infection, and has important scientific and application values.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a recombinant fusion protein containing a core protein of a bat hepatitis virus, a preparation method and application thereof.
Background
Respiratory Syncytial Virus (RSV) is the major pathogen worldwide causing lower Respiratory tract infections in infants and young children. More than 90% of infants experience at least one RSV infection within two years after birth, and infants, the elderly and people with immunodeficiency are easy to have serious infection and death after 6 months. For diseases caused by RSV infection, the only currently effective drug on the market is Palivizumab (Palivizumab). RSV belongs to the family Paramyxoviridae, the genus Pneumovirus, and is a non-segmented, single-stranded, negative-strand RNA enveloped virus. The viral genome is 15.2kb in total length, comprises 10 coding genes (NS1, NS2, N, P, M, SH, G, F, M2 and L in sequence), and totally encodes 11 proteins including 3 nucleocapsid proteins (N, P, L), 3 transmembrane proteins (F, G, SH), 3 matrix proteins (M, M2-1 and M2-2) and 2 non-structural proteins (NS1 and NS 2). G, F two glycoproteins, also called adhesion protein and fusion protein, mediate adhesion and fusion of virus and cell, respectively, and have important effect on infection of host cell by virus, and these two proteins are also main target antigens for vaccine development at present. RSV can be divided into two subtypes A and B according to G protein antigen specificity, the amino acid sequence of the G protein between the two subtypes has only 53 percent of homology, while the F protein has higher amino acid homology up to 90 percent, and the F protein can be used as a vaccine antigen to prevent RSV infection of the two subtypes A and B simultaneously.
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: metastable pre-fusion and more stable post-fusion states. It has now been found that there are 6 neutralizing antibody epitopes for the F protein: I. II, IV, V and VI and the prefusion antigenic sites
Against antigenic sites
I. II and IV have corresponding reports on the development of neutralizing monoclonal antibodies, wherein the antigenic site II is the binding site of RSV preventing and treating monoclonal antibodies Palivizumab (Palivizumab) and Motavizumab (Motavizumab), is positioned at amino acid 255-278 of subunit F1, is a conformational epitope of an 'alpha helix-ring-alpha helix' secondary structure, and keeps the conformational state unchanged before and after fusion of the F protein, so that Palivizumab has neutralizing activity on RSV before and after fusion.
In the last 60 th century, severe ERD phenomenon appeared in the clinical trial of formaldehyde-inactivated RSV vaccine (FI-RSV), and the re-infection of RSV by immunized infants did not prevent the infection, but the pulmonary inflammatory reaction was aggravated, and finally 2 infants died. Therefore, how to avoid the ERD phenomenon and improve the safety and effectiveness of vaccines in the development of RSV vaccines becomes a primary consideration. Currently, a large number of RSV vaccine candidates, including live attenuated vaccines, live recombinant virus vaccines, recombinant subunit (G/F protein) vaccines, and nucleic acid (G/F) vaccines, are in preclinical and clinical research. Research and development institutions represented by Novavavax, Medmimum and GSK all use RSV F protein as a target antigen of vaccines and enter a clinical research stage, and clinical research data show that the RSV F protein has a remarkable effect on controlling and preventing RSV infection in adults.
Although the F protein has good immunogenicity, the ERD phenomenon appears in a virus attack test after the purified F protein is used for immunizing cotton rats, and the F protein still has certain safety risk. Meanwhile, not only the vaccine prepared from the F protein may have the ERD phenomenon, but also other immunogenic proteins have similar risks. Therefore, it is very important to find a safe and effective vaccine against respiratory syncytial virus.
Disclosure of Invention
The invention aims to solve the technical problems of weak immune response and potential safety hazard of vaccines in the prior art, and provides a recombinant fusion protein containing core protein of bat hepatitis virus, a preparation method and application thereof.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a recombinant fusion protein containing core protein of bat hepatitis virus, the recombinant fusion protein is composed of core protein of bat hepatitis virus and exogenous protein segment inserted between amino acids of the core protein of bat hepatitis virus, and the amino acid sequence of the core protein of bat hepatitis virus is shown in SEQ ID NO. 1.
The Bat hepatitis virus (BTHV) belongs to the hepadnaviridae, the host is the batus griseus which is closely related to human hepatitis B virus, woodchuck hepatitis virus and hamster hepatitis virus on phylogenetic trees, and BTHV core protein has a structure similar to that of human hepatitis B virus core antigen (HBc), and 240 or 180 BTHV core proteins can be autonomously assembled into virus-like particles (T is 4 or T is 3). The inventor creatively invents a recombinant fusion protein which is based on BTHV as a carrier and contains exogenous epitopes on the basis of fully taking reference to the existing fruits, expresses and forms chimeric virus-like particles (ChimericVLP) in Hansenula polymorpha through optimized design, and the cVLP is used as a vaccine component, can stimulate to generate stronger immune response on the premise of ensuring the safety of the vaccine, is used for preventing RSV infection, and has important scientific and application values.
In the present invention, the amino acid sequence of the core protein of the bat hepatitis virus is different from that of the core protein of the wild-type bat hepatitis virus, and in order to improve the protein expression amount and the protein stability, the inventor creatively truncates 28 amino acids at the N-terminal of a reference sequence (GenBank: AIW47294.1) of the wild-type protein, thereby forming a vector sequence shown in SEQ ID NO. 1.
The foreign protein fragment may be any target protein fragment, including, but not limited to, immunogenic protein fragments, such as B-cell or T-cell receptors, epitopes with neutralizing activity, protein tags (GFP or EGFP, etc.), and other peptide fragments with immunological activity.
Preferably, the foreign protein fragment is inserted between the 78 th amino acid and the 79 th amino acid of the core protein of the bat hepatitis virus, and the foreign protein fragment is connected with the core protein of the bat hepatitis virus through an amino acid connecting arm.
In the present invention, the inventors found that when a foreign protein fragment is inserted between the 78 th and 79 th amino acids of the core protein of the hepes virus and linked by a suitable amino acid linker, the foreign protein fragment can be maximally displayed on the surface of the virus-like particle, and the immunological activity of the fragment can be enhanced.
Virus-like particles (VLPs) are protein particles that are self-assembled from viral structural proteins, lack viral nucleic acids, are non-infectious, and can be repeatedly displayed on the surface of VLPs in high density after foreign epitopes are inserted into appropriate positions of the structural proteins. Therefore, the vaccine prepared by the chimeric virus-like particle technology has the characteristics of safety and high efficiency.
Preferably, in one embodiment of the invention, the recombinant fusion protein assembles into a virus-like particle.
More preferably, in another embodiment of the invention, the recombinant fusion protein forms chimeric virus-like particles (cVLPs) in an expression system.
More preferably, in another embodiment of the present invention, the recombinant fusion protein consisting of a bat hepatitis virus core protein and a foreign protein fragment inserted between amino acids of the bat hepatitis virus core protein is expressed in hansenula polymorpha to form a chimeric virus-like particle.
More preferably, in another embodiment of the present invention, the recombinant fusion protein composed of a bat hepatitis virus core protein and a foreign protein fragment inserted between the 78 th and 79 th amino acids of the bat hepatitis virus core protein and linked to the bat hepatitis virus core protein through an amino acid linking arm is expressed in hansenula polymorpha to form a chimeric virus-like particle.
Another aspect of the present invention provides a use of the above recombinant fusion protein for preparing an antigen presenting vector.
The invention also provides an application of the recombinant fusion protein in preparing vaccines.
Preferably, the vaccine is a respiratory syncytial virus prophylactic vaccine.
Preferably, the recombinant fusion protein is expressed in Hansenula polymorpha to form a chimeric virus-like particle as part of the vaccine.
Preferably, in one embodiment of the present invention, the foreign protein fragment in the recombinant fusion protein is an immunogenic protein fragment.
More preferably, the foreign protein fragment is a respiratory syncytial virus fusion protein fragment.
Preferably, in one embodiment of the present invention, the above-mentioned foreign protein fragment inserted between the 78 th and 79 th amino acids of the core protein of the hepes hepatitis virus is an immunogenic protein fragment.
More preferably, the foreign protein fragment is a respiratory syncytial virus fusion protein fragment, and the respiratory syncytial virus fusion protein fragment is connected with the 78 th and 79 th amino acids of the hepes hepatitis virus core protein through a GILE amino acid connecting arm at the N-terminal and an L amino acid connecting arm at the C-terminal.
In the present invention, the respiratory syncytial virus fusion protein fragment can be a protein encoded by any encoding gene in the RSV genome, including but not limited to the NS1, NS2, N, P, M, SH, G, F, M2, or L gene, or combinations thereof.
Preferably, in one embodiment of the invention, the respiratory syncytial virus fusion protein fragment is the RSV F protein.
More preferably, the respiratory syncytial virus fusion protein fragment is epitope II in the RSV F protein. The amino acid sequence of the epitope II is shown in SEQ ID NO. 3.
Preferably, in one embodiment of the present invention, the amino acid sequence of the recombinant fusion protein is shown in SEQ ID NO. 2.
Preferably, the recombinant fusion protein consisting of the bat hepatitis virus core protein and the respiratory syncytial virus fusion protein epitope is assembled into virus-like particles.
More preferably, the recombinant fusion protein composed of the hepes hepatitis virus core protein and the respiratory syncytial virus fusion protein epitope is expressed in the hansenula polymorpha to form the chimeric virus-like particles.
In another aspect of the present invention, a method for preparing the above recombinant fusion protein is provided, which comprises the following steps:
step 1) optimizing and synthesizing the gene sequence of the recombinant fusion protein according to the advantages and disadvantages of Hansenula polymorpha codons and the abundance of tRNA (transfer ribonucleic acid) to obtain the optimized gene sequence of the recombinant fusion protein;
step 2) replacing the S gene sequence in the vector PUC25-SU with the recombinant fusion protein gene sequence obtained in the step 1) to obtain a PUC25-BTRU recombinant plasmid;
and 3) converting the recombinant plasmid obtained in the step 2) into hansenula polymorpha for induction expression, and extracting and purifying to obtain the recombinant fusion protein.
Specifically, the preparation method may further comprise the steps of:
step A) optimization and Synthesis of genes
Optimizing and synthesizing a core protein gene sequence of the bat hepatitis virus, which simultaneously contains an amino acid linking arm and a respiratory syncytial virus fusion protein fragment gene sequence, according to the advantages and disadvantages 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-BTRU recombinant plasmid;
step C) expression and purification of recombinant fusion proteins
Carrying out enzyme digestion on the PUC25-BTRU recombinant plasmid obtained in the step B) by EcoRI and Hind III, then carrying out linearization treatment, carrying out electric transformation into NVSI-H.P-105 (delta URA3 delta LEU2) Hansenula to obtain a recombinant, screening by an ELISA method to obtain a recombinant fusion protein high-expression-quantity positive strain, 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 fusion protein.
Wherein, the gene related in the step A) is designed, optimized and synthesized according to the amino acid sequence of the fusion protein. Preferably, the nucleotide sequence is shown as SEQ ID NO. 4.
The invention also provides the application of the recombinant fusion protein consisting of the bat hepatitis virus core protein and the respiratory syncytial virus fusion protein epitope in the preparation of the respiratory syncytial virus preventive vaccine.
In another aspect of the present invention, a vaccine is provided, which comprises an effective dose of the recombinant fusion protein composed of the bat hepatitis virus core protein and the respiratory syncytial virus fusion protein epitope, and an adjuvant.
Among these, the aluminum hydroxide preferably contains a low-solubility aluminum component, and more preferably aluminum hydroxide. Of course, adjuvants such as MF59, aluminum phosphate, calcium phosphate, cytokines (e.g., IL-2, IL-12, GM-CSF), saponins (e.g., QS21), MDP derivatives, CpG oligonucleotides, LPS, MPL, polyphosphazenes, emulsions (e.g., Freund's, SAF), liposomes, lipopeptides, viral particles (virosomes), Iscoms, cochleates, PLG particles, poloxamer particles, virus-like particles, heat-labile enterotoxin (LT), Cholera Toxin (CT), mutant toxins (e.g., LTK63 and LTR72), microparticles and/or polymeric liposomes may also be used.
The vaccines of the present invention may be administered by any suitable means, for example intradermally (i.d.), intraperitoneally (i.p.), intramuscularly (i.m.), intranasally, orally, subcutaneously (s.c.), etc. and in any suitable delivery device (O' Hagan et al, Nature Reviews, Drug Discovery2(9), (2003), 727-. Preferably, the vaccine of the invention is administered intradermally, subcutaneously or intramuscularly.
The vaccine can be prepared into any suitable dosage form, including but not limited to freeze-dried agent, liquid agent and spray.
Another aspect of the present invention provides an antibody obtained by immunizing an individual with the above recombinant fusion protein consisting of a core protein of a bat hepatitis virus and an epitope of a respiratory syncytial virus fusion protein.
Another aspect of the present invention provides a strain comprising the above recombinant fusion protein.
The invention has the beneficial effects that:
1) the invention uses the core protein of the bat hepatitis virus as the exogenous epitope presenting carrier for the first time, can effectively display the exogenous epitope on the surface of the VLP, ensures that the exogenous epitope is repeatedly distributed on the surface of the VLP in high density, and does not influence the autonomous assembly of the VLP.
2) The recombinant fusion protein is expressed by using a hansenula polymorpha expression system, has high protein expression quantity and certain protein post-translational processing modification, can be automatically assembled into cVLP in a yeast body, and is easy to purify an expression product.
3) The recombinant fusion protein formed by the bat hepatitis virus core protein and the respiratory syncytial virus fusion protein epitope can be combined with the palivizumab monoclonal antibody, can generate a neutralizing antibody aiming at the specificity of RSV pathogen after immunizing a mouse, and provides feasibility for developing RSV vaccine.
4) The Chimeric virus-like particles (Chimeric VLP, cVLP) are formed by expression in Hansenula polymorpha through optimized design based on BTHV as a vector and recombinant fusion protein containing exogenous epitopes, and the cVLP is used as a vaccine component, can stimulate to generate stronger immune response on the premise of ensuring the safety of the vaccine, is used for preventing RSV infection, and has important scientific and application values.
Drawings
FIG. 1 is a schematic diagram of the construction of a pUC25-BTRU recombinant plasmid;
FIG. 2 is a diagram showing the results of restriction enzyme identification of the recombinant plasmid pUC25-BTRU, wherein Lane is the restriction enzyme result of the plasmid pUC 25-BTRU;
FIG. 3 is a diagram showing the PCR identification result of the screened yeast strains with high protein expression positive, wherein Lane1 is the PCR electrophoresis result of the strains;
FIG. 4 is a diagram showing the identification results of the components of the recombinant fusion protein designed in the present invention, wherein,
left panel, total thallus analysis after induction expression;
lane1 is uninduced thallus;
lane2 is the induced thallus;
in the middle figure, SDS-PAGE analysis of the purified recombinant fusion protein is carried out;
lane3 is the electrophoresis result of the purified target protein;
the right picture is Western-blot analysis of the recombinant fusion protein;
lane4 is the immunoblotting result of the target protein;
FIG. 5 is a graph showing the binding results of the recombinant fusion protein to the palivizumab antibody;
FIG. 6 is a graph showing the results of dynamic light scattering of recombinant fusion protein cVLP;
FIG. 7 is a transmission electron micrograph of cVLP of the recombinant fusion protein after phosphotungstic acid negative staining;
FIG. 8 is a graph showing the results of pathogen-specific neutralizing antibody titer levels generated after immunization of mice with the recombinant fusion protein;
DESCRIPTION OF THE SEQUENCES
SEQ ID NO.1 is the amino acid sequence of the core protein of the bat hepatitis virus;
SEQ ID NO.2 is the amino acid sequence of the recombinant fusion protein of the present invention;
SEQ ID NO.3 is the amino acid sequence of the epitope in the recombinant fusion protein of the invention;
SEQ ID NO.4 is the nucleotide sequence of the recombinant fusion protein of the present invention.
Detailed Description
The invention discloses a recombinant fusion protein containing a core protein of a bat hepatitis virus, a preparation method and application thereof, and a person skilled in the art can realize the recombinant fusion protein by appropriately improving 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 are all from TaKaRa;
the RSVsF protein is from Beijing Yiqian Shenzhou biotechnology, and is an insect cell recombinant expression product and a freeze-drying agent;
palivizumab is from Medimmune;
goat anti-human IgG-HRP is from China fir Jinqiao biotechnology, Inc. in Beijing;
the aluminum hydroxide adjuvant is from SERVA Electrophororesis GmbH company;
balb. c female mice were from experimental animal technology ltd, vietonema, beijing;
the PUC25-SU yeast expression plasmid is from the company of limited responsibility of the Beijing institute of biological products;
NVSI-H.P-105(Δ URA3 Δ LEU2) Hansenula species was from the company Limited responsibility of the Biochemical research institute, Beijing;
the RSV Long strain (ATCC VR26) is from the American Type Culture Collection (ATCC);
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 culture medium: 1.34% of amino acid-free yeast nitrogen source and 2% of glucose;
MM medium: 1.34% of amino acid-free yeast nitrogen source and 0.8% of anhydrous methanol;
SM-leu Medium: 1.34% of amino acid-free yeast nitrogen source, 2% of glucose and 0.01% of leucine;
MM-leu Medium: 1.34% of amino acid-free yeast nitrogen source, 0.8% of anhydrous methanol and 0.01% of leucine;
YPD 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, and sterilizing at high temperature under high pressure.
Example 1: construction and identification of recombinant fusion protein yeast expression plasmid
1. Design of recombinant fusion proteins
Taking a bat hepatitis virus core protein amino sequence (GenBank: AIW47294.1) 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 a respiratory syncytial virus fusion protein (Genebank: ACO83301.1) amino acid epitope (shown as SEQ ID NO. 3) at position 254 and 277 between the amino acid at position 78 and position 79 of the SEQ ID NO.1 sequence, wherein the epitope is the binding position of the palivizumab antibody, and is formed by connecting a connecting arm of a 'GILE' amino acid and an 'L' amino acid in series to form an amino acid sequence shown as SEQ ID NO. 2.
2. Gene optimization and synthesis
According to the amino acid sequence of the recombinant fusion protein shown in SEQ ID NO.2, the optimization of the gene sequence is carried out according to the advantages and disadvantages of Hansenula polymorpha codons and the abundance of tRNA, the coding gene sequence shown in SEQ ID NO.4 is formed, and the sequence is subjected to gene synthesis.
3. Construction of expression plasmids
According to a plasmid construction schematic diagram shown in figure 1, a target gene is replaced by an S gene in an expression vector PUC25-SU yeast expression plasmid by using a gene recombination technology to form a yeast expression plasmid PUC25-BTRU containing a recombinant fusion protein gene sequence (shown as SEQ ID NO. 4), wherein the plasmid uses 25S rDNA on a Hansenula polymorpha genome as a homologous recombination integration arm, uses a URA3 gene as a marker gene, and uses an MOX promoter to efficiently start the expression of the target protein.
4. Enzyme digestion identification and gene sequence determination of PUC25-BTRU expression plasmid
The expression plasmid of PUC25-BTRU yeast is subjected to EcoR I and HindIII double enzyme digestion identification, 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 subjected to 1% agarose gel electrophoresis detection, as shown in figure 2, the plasmid can be seen to be digested to obtain two gene fragments, namely a large fragment of about 4kb and a small fragment of 2kb, the large fragment is a yeast expression frame containing a target gene of the recombinant fusion protein, and the plasmid is subjected to gene sequence determination, so that the result shows that the expected result is consistent with the expected result and no change of the target gene is caused.
TABLE 1 enzyme digestion System
Example 2: screening and identification of high-expression positive yeast strains
1. Transformation of
Culturing NVSI-H.P-105 (delta URA3 delta LEU2) Hansenula polymorpha in a YPD liquid culture medium, preparing yeast competence when the thallus density (OD600) reaches 1.0, transforming large fragment genes in PUC25-BTRU plasmid subjected to double enzyme digestion by EcoRI and Hind III into NVSI-H.P-105 yeast in an electric transformation mode, finally coating transformed bacteria liquid on an SM-LEU solid culture medium, and culturing for 3-5 days at 37 ℃ to obtain a transformed recombinant.
2. ELISA screening
The monoclonal colonies grown on the SM-leu solid medium were picked up and cultured in 2ml of SM-leu liquid medium, and the cells were cultured at 37 ℃ for 24 hours with shaking at 250 rpm. Transferring 200 mul of bacterial liquid into 4ml of SM-leu liquid culture medium for continuous culture, after the density (OD600) of the thallus reaches more than 10, centrifuging at 3000rpm to obtain the thallus, and suspending in 4ml of MM-leu culture medium for thallus culture, adding 1% of anhydrous methanol every 6 hours at the stage, inducing the expression of the target protein, and inducing for 24 hours; centrifuging to obtain thallus, adding 200 μ l yeast thallus disruption buffer (20mM PB, pH 7.2) and 200mg glass beads, high-frequency low-temperature shaking for disruption, and coating with coating solution (Na)2CO3-NaHCO3Solution, pH value 9.6) diluting the protein supernatant by 500 times and coating the diluted protein supernatant on an enzyme label plate, wherein the concentration of the diluted protein supernatant is 100 mu l/hole, and the protein supernatant is coated for 8 hours at 4 ℃; PBS containing 1% BSA was added at 100. mu.l/well and blocked at 37 ℃ for 3 hours; diluting palivizumab to 1 μ g/ml, 100 μ l/well, 37 ℃, 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 wavelength of 450nm and the wavelength of 630nm, and selecting the strain with the highest OD value as the recombinant fusion 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 is transformed into the selected yeast strain, so that the strain can grow in the MD basal mediumMedium growth.
3. Identification of target gene of strain
Extracting the genome of the recombinant fusion protein high-expression yeast strain, performing PCR amplification of a target gene by using primers shown in table 2, performing 1% agarose gel electrophoresis detection on a PCR product as shown in tables 3 and 4, wherein the result is shown in figure 3, a DNA fragment with an expected size can be amplified, and the gene sequence of the PCR product is determined without changing the target gene.
TABLE 2 primer information Table for strain identification
TABLE 3 PCR reaction System
TABLE 4 PCR reaction conditions
Example 3: preparation and characterization of cVLP
1. Yeast fermentation and cell disruption
The recombinant fusion 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 fermentation was completed, the cells were washed 2 times with physiological saline, and finally suspended in a disruption buffer (20mM PB,50mM NaCl, pH 7.2) for high-pressure disruption, followed by centrifugation to obtain a protein supernatant. The total mycoprotein before and after induction is subjected to 10% SDS-PAGE electrophoretic analysis, the result is shown in the left picture of figure 4, and the arrow indicates the target protein band.
2. Target protein purification and Western-blot detection
Purifying by gel filtration chromatography with AKTA chromatographic purifier (GE AKTA explorer), wherein Sephacryl S500-HR is selected as medium, the column volume is 900ml, and the specific process is as follows:
A. column balancing: equilibrating the column with 3 column volumes of equilibration buffer (50mM PB +0.2M Nacl, pH7.3) to 280nm absorbance with no significant change, less than 0.5mAU, zeroes the absorbance of the detector.
B. Loading: pumping 100ml of crude pure protein solution into the chromatographic column at the pump speed of 5ml/min, and after the sample loading is finished, continuously flowing the equilibrium buffer solution through the chromatographic column.
C. Collecting a sample: when the absorption value at 280nm gradually increases from the buffer to the volume of 1/3 columns, the samples are automatically collected according to 5 ml/tube, and the collected samples are subjected to SDS-PAGE electrophoretic analysis.
D. Column recovery and recycling: treating the chromatographic column with 0.2M sodium hydroxide solution, then balancing the chromatographic column with the balance buffer solution, and continuing the chromatographic purification of the next protein.
The purified target protein was analyzed by 10% SDS-PAGE, and the result is shown in the middle panel of FIG. 4, in which the arrow indicates the target protein band, which has a molecular weight of about 25kD and is substantially the same as the expected size. And meanwhile, carrying out Western-blot detection on the purified target protein, transferring the target protein to a PVDF membrane according to a conventional method, and detecting by using a palivizumab antibody, wherein the result is shown in the right graph of figure 4, and the arrow indicates a target protein developing zone.
3. Binding assays with palivizumab antibodies
The purified cplp was diluted from 1 μ g/ml with a 2-fold gradient and coated on an enzyme plate while using RSVsF protein as a positive control and adding palivier antibody at a concentration of 1 μ g/ml to verify the degree of binding of cplp protein to palivier antibody, and as a result, cplp could be efficiently bound to palivier antibody as shown in fig. 5.
4. Dynamic light scattering and transmission electron microscopy of vlp
The purified protein was analyzed by dynamic light scattering (Malvern, NANO-8S90) and the results are shown in FIG. 6, which shows that VLP has good size uniformity and a hydrated particle size of about 26 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 VLP is seen to be 20-30nm in size, uniform in size and good in morphology as shown in figure 7.
Example 4: immunological effect study of the recombinant fusion protein of the present invention
32 SPF-grade BALB/c female mice, 6-8 weeks old, were randomly divided into A, B, C and D4 groups of 8 mice each. The following design schemes were used for immunization: group A was injected with 0.5. mu.g of recombinant fusion protein (cVLP) in admixture with 250. mu.g of aluminum hydroxide adjuvant; group B injected 0.5. mu.g of RSVsF protein in admixture with 250. mu.g of aluminum hydroxide adjuvant; group C was injected with saline and 250. mu.g of aluminum hydroxide adjuvant as a control group. The immunization was performed by the intraperitoneal route, and each group was immunized 3 times at 2 weeks intervals. Mice status and body weight changes were observed daily after immunization of each group of mice. Two weeks after the completion of immunization, the blood was collected by cutting the cone and serum was separated. The titer level of the RSV-specific neutralizing antibody in serum is detected by using a virus micro-neutralization test method, and the result is shown in figure 8, after the recombinant fusion protein (cVLP) is mixed with an aluminum hydroxide adjuvant, a higher-level RSV-specific neutralizing antibody titer can be generated, which indicates that the recombinant fusion protein has the potential of being used as an RSV prophylactic vaccine.
Comparative example: comparative analysis of recombinant fusion proteins of the invention with other types of recombinant fusion proteins
According to the design scheme of example 1, a cVLP using a core antigen of woodchuck hepatitis virus (WHC) as a presentation carrier and a cVLP using a core antigen of human hepatitis B virus (HBC) as a presentation carrier are respectively designed and prepared according to the methods described in examples 1, 2 and 3 to finally obtain a WHC-cVLP and an HBC-cVLP, and two groups, 8 in each group, are added on the basis of the animal immunization method in example 4, and the group D is injected with 0.5 μ g of WHC-cVLP and 250 μ g of aluminum hydroxide adjuvant mixture; group E was injected with 0.5. mu.g of HBC-cVLP mixed with 250. mu.g of aluminum hydroxide adjuvant and immunized by the intraperitoneal route, 3 times for each group at 2-week intervals. 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 a result, as shown in FIG. 8, it was revealed that the RSV-specific neutralizing antibody titer of BTHV-cVLP was about 10.0, that of WHC-cVLP was about 6.5, that of HBC-cVLP was about 3.0, and that BTHV-cVLP produced a higher level of neutralizing antibody than those produced by WHC-cVLP and HBC-cVLP. The results indicate that BTHV-cVLP has obvious advantages in the aspect of exogenous epitope presentation, and the recombinant fusion protein has the potential of being used as a RSV prophylactic 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; limited liability company of Beijing institute of biological products
<120> a recombinant fusion protein containing core protein of bat hepatitis virus, preparation method and application thereof
<130> None
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<170> PatentIn version 3.5
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Claims (6)
1. A recombinant fusion protein containing core protein of bat hepatitis virus is characterized in that the amino acid sequence of the recombinant fusion protein is shown in SEQ ID NO. 2.
2. Use of a recombinant fusion protein according to claim 1 for the preparation of a vaccine against respiratory syncytial virus.
3. A method of producing a recombinant fusion protein according to claim 1, comprising the steps of:
step 1) optimizing and synthesizing the gene sequence of the recombinant fusion protein according to the advantages and disadvantages of Hansenula polymorpha codons and the abundance of tRNA (transfer ribonucleic acid) to obtain the optimized gene sequence of the recombinant fusion protein;
step 2) replacing the S gene sequence in the vector PUC25-SU with the recombinant fusion protein gene sequence obtained in the step 1) to obtain a PUC25-AGRU recombinant plasmid;
and 3) converting the recombinant plasmid obtained in the step 2) into hansenula polymorpha for induction expression, and extracting and purifying to obtain the recombinant fusion protein.
4. The use according to claim 2, wherein the vaccine is a respiratory syncytial virus prophylactic vaccine.
5. A vaccine comprising an effective amount of the recombinant fusion protein of claim 1, and an adjuvant.
6. A strain expressing the recombinant fusion protein of claim 1.
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