CN114716570A - Fusion protein and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a fusion protein and a nucleotide sequence of the fusion protein after codon optimization, wherein the sequence is shown as SEQ ID NO. 1. The invention also discloses a vaccine prepared by using the fusion protein. The fusion protein disclosed by the invention has good immunogenicity, can generate higher antibody titer than the conventional rabies virus G protein, and provides important reference for the research and development of rabies virus subunit vaccines. In addition, the 6 epitope polypeptides of the rabies virus G protein screened by the invention have better antigenicity, provide important reference for research of the rabies virus G protein and provide important antigenic clues for the subsequent development of monoclonal antibodies of the rabies virus G protein.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a fusion protein, and a preparation method and application thereof.
Background
Rabies Virus (RV) belongs to the Rhabdoviridae (Rhabdoviridae) genus Lyssavirus (Lyssavirus). The shape is elastic, the nucleocapsid is spirally symmetrical, the surface is provided with a coating, and the single-stranded RNA is contained in the nucleocapsid, so the nucleocapsid is a pathogen causing rabies. Rabies virus has two major antigens: one is glycoprotein antigen on the outer membrane of the virus, which can combine with acetylcholine receptor to make the virus have neurotoxicity and produce neutralizing antibody and hemagglutination inhibiting antibody in vivo, and the neutralizing antibody has protective effect; the other is an inner nucleoprotein antigen, which can generate complement-binding antibody and precipitin in vivo without protection. Rabies is an infectious disease of zoonosis caused by Rabies Virus (Rabies Virus).
The rabies virus is a ribonucleic acid type rhabdovirus, and one end of the rabies virus is round and convex; one end of the bullet is flat concave, the diameter of the bullet is 65-80nm, and the length of the bullet is about 130-240 nm. The virus is easily inactivated by sunlight, ultraviolet rays, formaldehyde, mercury rising quaternary ammonium compounds (such as benzalkonium bromide), lipid solvent, 50-70% alcohol and the like, and the suspension is inactivated by 30-60 minutes at 56 ℃ or 2 minutes at 100 ℃. The virus can keep activity for years at-70 ℃ or 0-4 ℃ after freeze-drying. Infected tissue may be stored in 50% glycerol for testing. Rabies virus contains 5 proteins, i.e., glycoprotein (G), nucleoprotein (N), dimeric enzyme (L), phosphoprotein (NS), matrix (M), and the like. The latter two are small molecule proteins. G-proteins can lead to the formation of neutralizing antibodies in vivo, which can be directed against viral attacks. The N protein causes antibodies but does not have neutralizing power, and can be used for detecting inclusion bodies in plasma.
The rabies G protein is a trimer, approximately 67kD, and is the main antigen inducing the production of virus neutralizing antibodies, and can induce the body to produce immunity to the lethal infection of rabies virus. The G protein also contains virulence determinants. The G gene is the first rabies virus gene cloned and sequenced. It is deduced from the nucleotide sequence that it encodes a polypeptide of 524 amino acids, including a signal sequence of 19 amino acids. Arginine at position 333 has an important role in viral virulence, which is associated with neuroinvasiveness and transsynaptic transmission, enabling the virus to spread more rapidly in the nervous system.
Disclosure of Invention
In order to make up for the deficiencies of the prior art, the present invention aims to provide a fusion protein and a preparation method thereof.
Accordingly, in one aspect, the present invention provides a fusion protein having an amino acid sequence of: KLTNKHSPEAAYAAYCTGVVTEAETYTNFVGYVTTTFKRKAAYAAYMAGDPRYEESLHNPYPDYHWLRTVKAAYAAYSKGSKTCGFVDERGLYKSLKGACKLAAYAAYIHDFRSDEIEHLVVEELVKKREECLAAYAAYVSFRRLSHLRKLVPGFGKAYTIFNKAAYAAYADPSTVFKDGDEAEDFVEVHLPDVHAAYAAYQARESKDNKIGKTENPHHHHHH are provided.
Preferably, the codon-optimized nucleotide sequence of the fusion protein of the present invention is shown in SEQ ID NO. 1.
Preferably, the fusion protein of the invention comprises 6 rabies virus G protein epitope polypeptides, and the sequences of the 6 rabies virus G protein epitope polypeptides are respectively:
epitope polypeptide 4: CTGVVTEAETYTNFVGYVTTTFKRK, respectively;
epitope polypeptide 5: MAGDPRYEESLHNPYPDYHWLRTVK, respectively;
epitope polypeptide 12: SKGSKTCGFVDERGLYKSLKGACKL;
epitope polypeptide 16: IHDFRSDEIEHLVVEELVKKREECL, respectively;
epitope polypeptide 19: VSFRRLSHLRKLVPGFGKAYTIFNK, respectively;
epitope polypeptide 27: ADPSTVFKDGDEAEDFVEVHLPDVH are provided.
Preferably, the N-terminal of the fusion protein of the present invention comprises a polypeptide capable of inducing IFN-gamma production, wherein the polypeptide has the sequence of KLTNKHSPE.
Preferably, the C end of the fusion protein of the invention contains a B cell epitope, and the sequence of the B cell epitope is QARESKDNKIGKTENP.
In still another aspect, the present invention also provides a vaccine comprising the fusion protein and a pharmaceutically acceptable adjuvant.
Preferably, the content of the fusion protein in the vaccine of the present invention is 25. mu.g/ml.
Preferably, the pharmaceutically acceptable adjuvant of the present invention is ISA 201VG adjuvant.
On the other hand, the invention also provides application of the fusion protein in preparation of rabies virus vaccines.
The fusion protein disclosed by the invention has good immunogenicity, can generate higher antibody titer than that of the conventional rabies virus G protein, and provides important reference for the research and development of rabies virus subunit vaccines. In addition, the 6 epitope polypeptides of the rabies virus G protein screened by the invention have better antigenicity, provide important reference for research of the rabies virus G protein and provide important antigenic clues for the subsequent development of monoclonal antibodies of the rabies virus G protein.
Drawings
FIG. 1 shows the prediction of the transmembrane region of the G protein with the signal peptide removed. "Inside" indicates an intracellular region, and the larger the "Inside value", the higher the probability that the amino acid is located in the intracellular region; outside indicates the extracellular domain, and a higher Outside number indicates a higher probability that the amino acid is located in the extracellular domain. The Transmembrane region is represented by Transmembrane, and the larger the number of Transmembrane, the higher the probability that the amino acid is in the Transmembrane region.
FIG. 2 is an SDS-PAGE pattern of the fusion protein. Wherein 1 is the purified fusion protein.
FIG. 3 shows the antibody titer results after the vaccine prepared from the fusion protein is used for immunizing mice.
FIG. 4 shows the result of detecting IFN-. gamma.content after the vaccine prepared from the fusion protein is used to immunize mice.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1: rabies virus G protein antigen epitope polypeptide design and screening
The G protein of GenBank: MW055078.1 was selected, and by analyzing and studying the sequence of the protein, 19 amino acids (signal peptide sequence) at the N-terminus and 55 amino acids (transmembrane region and intracellular region, see FIG. 2) at the C-terminus were removed, and 30 epitope polypeptides were designed and synthesized (Table 1). Respectively immunizing a mouse with 30 antigen epitope polypeptides, collecting mouse serum to carry out ELISA antibody titer detection, and screening the antigen epitope polypeptide with the highest antibody titer. The antibody titer of 6 epitope polypeptides was highest (table 1), and thus the 6 epitope polypeptides (4 th, 5 th, 12 th, 16 th, 19 th and 27 th) were selected as the focus of the subsequent studies.
Antibody titers were detected using a conventional ELISA method, briefly described as: preparing a G protein (described in CN 113252893A example 1) coated enzyme label plate by using a conventional eukaryotic expression foreign protein method, wherein the G protein is coated on the enzyme label plate at 100 ng/hole and 0.1 ml/hole and is coated overnight at 4 ℃; after 3 washes, blocking with PBST containing 2% BSA, incubation at 37 ℃ for 2 h; mouse serum was serially diluted 10-fold (10-10) with PBS7) Then adding the mixture into a coated enzyme label plate, incubating for 1h at 37 ℃ in a concentration of 0.1 ml/hole; washing for 3 times, adding HRP-labeled goat anti-mouse IgG secondary antibody diluted by 1:5000 times, 0.1 ml/hole, and incubating at 37 deg.C for 30 min; washing for 3 times, adding 0.1ml TMB single component color developing solution (purchased from Beijing Soilebao Biotech Co., Ltd.), and incubating at 37 deg.C for 10 min; adding stop solution (2M sulfuric acid), and measuring the OD450nm value; a positive result was determined when the OD450nm value was greater than 0.1.
TABLE 1G protein epitope Polypeptides
Example 2: design and preparation of rabies virus G protein fusion protein
Combining 6 epitope polypeptides screened in example 1, connecting two polypeptides by "AAYAAY", adding an amino acid sequence (KLTNKHSPE, which can induce IFN-gamma generation) at the N-terminal and an amino acid sequence (QARESKDNKIGKTENP, which is one of B cell epitopes) at the C-terminal, thereby forming a complete fusion protein, wherein the sequences of the fusion protein are as follows:
KLTNKHSPEAAYAAYCTGVVTEAETYTNFVGYVTTTFKRKAAYAAYMAGDPRYEESLHNPYPDYHWLRTVKAAYAAYSKGSKTCGFVDERGLYKSLKGACKLAAYAAYIHDFRSDEIEHLVVEELVKKREECLAAYAAYVSFRRLSHLRKLVPGFGKAYTIFNKAAYAAYADPSTVFKDGDEAEDFVEVHLPDVHAAYAAYQARESKDNKIGKTENPHHHHHH。
delivering the fusion protein sequence to Nanjing Jinslei Biotechnology GmbH for codon optimization and whole gene synthesis of Escherichia coli; the synthesized gene is connected between the restriction sites of BamHI and EcoRI of pET22b vector, and the sequence of the synthesized gene is shown in SEQ ID NO. 1. The recombinant plasmid is transformed into E.coli BL21(DE3) host, and is preserved as strain after resistance screening and recombinant plasmid identification, and the strain is used as engineering bacteria for expressing recombinant protein.
Inoculating the engineering bacteria into LB liquid culture medium containing 20 microgram/ml kanamycin at the proportion of 1%, carrying out shaking culture at the temperature of 37 ℃ at the speed of 200r/min for 2-4 hours, adding IPTG (isopropyl-beta-thiogalactoside) with the final concentration of 1.0mM for continuous induction for 4 hours when bacterial liquid OD600nm is 0.6-0.8. The induced recombinant bacteria are centrifuged for 10 minutes at 5000g, the supernatant is discarded, and the precipitate is dissolved for 10 minutes by PBS and then is crushed by an ultrasonic cell crusher. After disruption, the supernatant was discarded by centrifugation (12000r/min) at 4 ℃ for 30 minutes, the inclusion body pellet was lysed with a lysis buffer (50mM Tris, 8M Urea,0.5M NaCl) for 10 minutes, and the supernatant was purified by centrifugation at 12000r/min for 30 minutes and then passed through a nickel column. Washing the column with 10 times of column volume of lysis solution; adding the supernatant into a nickel column, and slowly loading the protein solution; washing with 10 times column volume of lysate to remove foreign protein; the purified fusion protein was collected by elution with 5 column volumes of eluent (50mM Tris, 8M Urea,0.5M NaCl, 300mM imidazole) and stored at below-70 ℃. The fusion protein is subjected to electrophoresis detection by using SDS-PAGE protein gel, a clear band is formed around 26KD, and the purity is more than 90 percent (figure 2); protein concentration was measured using BCA and fusion protein concentration was 1.58 mg/ml.
Example 3: preparation and application of rabies virus fusion protein vaccine
Preparing a vaccine: the fusion protein prepared in example 2 was diluted to 50. mu.g/ml and emulsified with ISA 201VG adjuvant (purchased from French Seidex Co.) in a mass ratio of 1:1 to prepare a vaccine containing the fusion protein at a concentration of about 25. mu.g/ml. The test is carried out according to the appendix of the existing Chinese veterinary pharmacopoeia, and no bacteria, mycoplasma and exogenous virus pollution exist; the viscosity is 25.8cP and less than 200 cP; 10ml of vaccine is sucked and added into a centrifuge tube, and the centrifuge tube is centrifuged for 15 minutes at 3000r/min, so that the tube bottom is separated out, and the stability is good. And storing the prepared and qualified vaccine at 2-8 ℃ for later use. With reference to this vaccine preparation method, the eukaryotic expressed G protein used in the ELISA assay in the examples was used to prepare the vaccine for use in alignment.
Mouse immunization: female Balb/c mice of 6 weeks old are randomly grouped into 5 mice each group, and are grouped and immunized according to Table 2, and mouse serum is collected before the first immunization, 14 days after the first immunization (before the second immunization) and 28 days after the first immunization (14 days after the second immunization), and the mouse serum titer and IFN-gamma are detected.
Table 2 mice immunization groups
| Group of | Immunization dose and regimen | Antigen content | Number of immunizations |
| Group 1 (fusion protein) | 0.2 ml/leg intramuscular injection | 5 ug/piece | Immunization for one time |
| Group 2 (fusion protein) | 0.2 ml/leg intramuscular injection | 5 ug/piece | Booster immunizations 2 weeks apart |
| Group 3(G protein) | 0.2 ml/leg intramuscular injection | 5 ug/piece | Immunization for one time |
| Group 4(G protein) | 0.2 ml/leg intramuscular injection | 5 ug/piece | Booster immunizations 2 weeks apart |
| Control group | / | / | / |
The conventional ELISA method is used for detecting the serum antibody titer of the mice, and the brief description is as follows: preparing a G protein (described in CN 113252893A example 1) coated enzyme label plate by using a conventional eukaryotic expression foreign protein method, wherein the G protein is coated on the enzyme label plate at 100 ng/hole and 0.1 ml/hole and is coated overnight at 4 ℃; after 3 washes, blocking with PBST containing 2% BSA, incubation at 37 ℃ for 2 h; serial 2-fold dilutions (100, 200, 400, 800, 1600.) of mouse serum were made with PBS, added to the coated elisa plate at 0.1 ml/well, and incubated at 37 ℃ for 1 h; washing for 3 times, adding HRP-labeled goat anti-mouse IgG secondary antibody diluted by 1:5000 times, 0.1 ml/hole, and incubating at 37 deg.C for 30 min; washing for 3 times, adding 0.1ml TMB single component color developing solution (purchased from Beijing Soilebao Biotech Co., Ltd.), and incubating at 37 deg.C for 10 min; adding stop solution (2M sulfuric acid), and measuring the OD450nm value; positive was judged when the P/N value (sample OD450nm value/blank well OD450nm value) was greater than 2.1 and the sample ODOD450nm value > 0.1.
The antibody detection results show (fig. 3): compared with two groups of single immunization groups, the antibody titer of the group 1 (fusion protein) is higher than that of the group 3(G protein), and the two groups have significant difference; compared with the two groups of secondary immunization groups, the antibody titer of the group 2 (fusion protein) is higher than that of the group 4(G protein), and the two groups have significant difference; the antibody titer in the single immunization group was higher than that in the secondary immunization group. In conclusion, the fusion polypeptide designed and prepared by the invention has better immune effect after being used for preparing the vaccine.
The serum cytokine IFN-. gamma.content was determined according to the Kit instructions (Mouse IFN-. gamma.ELISA Kit from Biotech, Inc., Beijing Sorbobao). The results show (fig. 4) that group 1 (fusion protein) was higher and significantly different than group 3(G protein) in the two single immunization groups; compared with the two groups of secondary immune groups, the group 2 (fusion protein) is higher than the group 4(G protein), and has significant difference; the single immunization group was higher than the secondary immunization group. In conclusion, the fusion polypeptide designed and prepared by the invention has better immune effect after being used for preparing the vaccine, and can induce and generate IFN-gamma with higher titer.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Wei slowly
<120> fusion protein, preparation method and application thereof
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 669
<212> DNA
<213> nucleotide sequence of codon-optimized fusion protein
<400> 1
aaactgacca acaaacactc cccggaagcc gcgtatgctg cgtattgcac cggtgttgtt 60
accgaagcag aaacctatac caatttcgtg ggttacgtga ccacgacctt caagcgcaaa 120
gctgcatacg cggcttacat ggctggtgat ccgcgctatg aggaatccct gcacaacccg 180
tatcctgatt atcactggct gcgtactgtg aaagcggctt atgctgccta ctccaaaggc 240
tctaaaacct gcggttttgt tgatgaacgt ggcctgtaca aatctctgaa aggtgcttgc 300
aaactggctg cctacgctgc ttacatccat gatttccgtt ccgatgaaat tgaacacctg 360
gtggtcgaag aactggtgaa aaaacgtgaa gaatgcctgg ctgcatatgc tgcctacgtt 420
tccttccgtc gcctgagcca cctgcgcaaa ctggtgcctg gctttggtaa agcttatacc 480
atcttcaaca aagcggctta cgcagcgtac gcggacccat ctacggtttt caaagatggc 540
gacgaagcag aagacttcgt tgaagttcac ctgccagacg tgcacgctgc ttacgccgca 600
taccaggcac gcgaaagcaa agacaataaa atcggcaaaa ccgaaaaccc gcaccatcat 660
caccatcat 669
Claims (9)
1. A fusion protein, wherein the amino acid sequence of said fusion protein is:
KLTNKHSPEAAYAAYCTGVVTEAETYTNFVGYVTTTFKRKAAYAAYMAGDPRYEESLHNPYPDYHWLRTVKAAYAAYSKGSKTCGFVDERGLYKSLKGACKLAAYAAYIHDFRSDEIEHLVVEELVKKREECLAAYAAYVSFRRLSHLRKLVPGFGKAYTIFNKAAYAAYADPSTVFKDGDEAEDFVEVHLPDVHAAYAAYQARESKDNKIGKTENPHHHHHH。
2. the fusion protein of claim 1, wherein the codon-optimized nucleotide sequence of the fusion protein is set forth in SEQ ID No. 1.
3. The fusion protein of claim 1, wherein the fusion protein comprises epitope polypeptides of 6 rabies G proteins, and the sequence of the epitope polypeptides of the 6 rabies G proteins are respectively:
epitope polypeptide 4: CTGVVTEAETYTNFVGYVTTTFKRK, respectively;
epitope polypeptide 5: MAGDPRYEESLHNPYPDYHWLRTVK, respectively;
epitope polypeptide 12: SKGSKTCGFVDERGLYKSLKGACKL;
epitope polypeptide 16: IHDFRSDEIEHLVVEELVKKREECL, respectively;
epitope polypeptide 19: VSFRRLSHLRKLVPGFGKAYTIFNK, respectively;
epitope polypeptide 27: ADPSTVFKDGDEAEDFVEVHLPDVH are provided.
4. The fusion protein of claim 1, wherein the N-terminus of the fusion protein comprises a polypeptide sequence KLTNKHSPE that induces IFN- γ production.
5. The fusion protein of claim 1, wherein the C-terminus of the fusion protein comprises a B-cell epitope and the sequence of the B-cell epitope is QARESKDNKIGKTENP.
6. A vaccine comprising the fusion protein of claim 1 and a pharmaceutically acceptable adjuvant.
7. The vaccine of claim 6, wherein the amount of fusion protein in the vaccine is 25 μ g/ml.
8. The vaccine of claim 6, wherein the pharmaceutically acceptable adjuvant is ISA 201VG adjuvant.
9. Use of a fusion protein according to any one of claims 1 to 5 in the preparation of a rabies vaccine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115785281A (en) * | 2022-09-27 | 2023-03-14 | 兴盟生物医药(苏州)有限公司 | Preparation method and application of fusion protein containing rabies virus G protein |
| CN116789858A (en) * | 2023-06-26 | 2023-09-22 | 广东兴亚生物科技有限公司 | Pseudorabies virus recombinant fusion protein and preparation method and application thereof |
-
2022
- 2022-04-22 CN CN202210429064.8A patent/CN114716570A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115785281A (en) * | 2022-09-27 | 2023-03-14 | 兴盟生物医药(苏州)有限公司 | Preparation method and application of fusion protein containing rabies virus G protein |
| CN116789858A (en) * | 2023-06-26 | 2023-09-22 | 广东兴亚生物科技有限公司 | Pseudorabies virus recombinant fusion protein and preparation method and application thereof |
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