CN116159133B - Pig A-type foot-and-mouth disease mRNA vaccine and preparation method thereof - Google Patents
Pig A-type foot-and-mouth disease mRNA vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of foot-and-mouth disease vaccines, and particularly relates to a pig A type foot-and-mouth disease mRNA vaccine and a preparation method thereof. The invention takes an inactivated antigen of porcine foot-and-mouth disease virus A (Re-A/WH/09 strain) as a template, and the amplified structural protein VP1 gene is connected with a pSFV1 vector to construct a recombinant plasmid pSFV1-VP1; the mRNA obtained by the recombinant plasmid through linearization, in vitro transcription and other methods is mixed with the prepared liposome nano-particles to prepare the foot-and-mouth disease mRNA vaccine; the neutralizing antibodies, IL-4 and IFN-gamma of the mRNA vaccine after the guinea pigs are immunized are obviously higher than those of a negative control group; can be used for 50 times of GPID 50 The Re-A/WH/09 strain attack provided immune protection. The mRNA vaccine prepared by the invention can induce stronger humoral immunity and cellular immune response, has good immunogenicity, and provides a new way for developing an emergency preventive vaccination for foot-and-mouth disease.
Description
Technical Field
The invention belongs to the field of foot-and-mouth disease vaccines, and particularly relates to a pig A type foot-and-mouth disease mRNA vaccine and a preparation method thereof.
Background
Foot-and-mouth disease (FMD) is an acute, virulent and high-contact infectious disease of artiodactyl caused by Foot-and-mouth disease virus virus (FMDV). After the host is infected, blisters can be caused on the parts of the mouth, hooves, papillae and the like, and gradually spread to form fester, thereby affecting the production performance. FMDV has 7 serotypes A, O, C, asia I and SAT I-III, and there is no cross protection between serotypes. Currently, the A-type FMD has a plurality of outbreaks in China, and causes huge loss to the breeding industry. Vaccination remains the most effective means of preventing and controlling the disease. However, such vaccines suffer from the disadvantages of high manufacturing cost, short immunization duration, potential detoxification, and the like. Therefore, the development of safer, efficient, inexpensive novel FMD vaccines is a focus of increasing researchers.
FMDV belongs to a single-strand positive-strand, envelope-free virus, with the ORF in the genome encoding a polyprotein. The polyprotein is cleaved by proteases to form 3 viral structural proteins (VP 0, VP3 and VP 1) and 8 nonstructural proteins. VP1, the major capsid protein, contains a large number of epitopes, especially Arg-Gly-Asp (RGD) motifs, which are capable of stimulating both an effective humoral and cellular immune response in the body, and is believed to be the key site for FMDV infection to invade cells and bind to cellular receptors, and is capable of stimulating the body to produce effective neutralizing antibodies.
The invention develops a novel FMDV mRNA vaccine which can be rapidly prepared to cope with sudden epidemic situation by taking VP1 gene of FMDV as a candidate gene of RNA vaccine, and provides reference for developing the novel FMD vaccine.
Disclosure of Invention
Aiming at the technical problems, the invention aims to provide a pig A type foot-and-mouth disease mRNA vaccine which can induce stronger humoral immunity and cellular immune response and has good immunogenicity and a preparation method thereof, and the invention specifically comprises the following steps:
in a first aspect, the present invention provides a porcine type a foot and mouth disease mRNA vaccine comprising an mRNA molecule and a liposome nanoparticle; the mRNA molecule codes for foot-and-mouth disease virus structural protein VP1.
Preferably, the mRNA molecule obtaining method is: connecting a gene encoding foot-and-mouth disease virus structural protein VP1 with a pSFV1 vector to construct a recombinant plasmid pSFV1-VP1; mRNA molecules obtained by linearization and in vitro transcription treatment of the recombinant plasmid.
Preferably, the sequence of the mRNA molecule is shown in SEQ ID NO. 1.
Preferably, the liposome nanoparticle consists of ionized lipids, DSPC, cholesterol, PEG lipids in a molar ratio of 50:10:39:1.7.
Preferably, the liposome nanoparticle to mRNA molecule N/P molar ratio is 8:1.
in a second aspect, the invention provides a method for preparing an mRNA vaccine for swine A-type foot-and-mouth disease, which comprises the following steps:
(1) The VP1 gene and the pSFV1 vector are respectively subjected to double enzyme digestion by restriction endonucleases BamH I and Sma I and then are connected to obtain a recombinant plasmid pSFV1-VP1;
(2) Linearizing the recombinant plasmid pSFV1-VP1 by Spe I cleavage to obtain a linearization vector;
(3) Replacing UTP in the linearization vector with N1-methyl pseudolaridine-5' -triphosphates (m 1 ψTP) for in vitro transcription;
(4) Capping modification is carried out on the transcribed mRNA to obtain an mRNA molecule;
(5) The liposome nanoparticle is mixed with mRNA molecules to prepare the mRNA vaccine.
Preferably, the VP1 gene in step (1) is obtained by the following method: extracting RNA of an inactivated antigen of foot-and-mouth disease virus type A (Re-A/WH/09 strain), and performing reverse transcription to synthesize cDNA; the cDNA is used as a template, and the VP1 gene is obtained by amplification by utilizing a specific primer of the VP1 gene.
Preferably, the mRNA molecule obtained in step (4) has the sequence shown in SEQ ID NO. 1.
Preferably, the liposome nanoparticle and mRNA molecule in step (5) are in a molar ratio of N/P of 8:1, mixing.
Preferably, the liposome nanoparticle in the step (5) is composed of ionized lipid, DSPC, cholesterol, PEG lipid in a molar ratio of 50:10:39:1.7.
The beneficial effects of the invention are as follows: firstly, taking an inactivated antigen of porcine foot-and-mouth disease virus A (Re-A/WH/09 strain) as a template, and connecting an amplified structural protein VP1 gene with a pSFV1 vector to construct a recombinant plasmid pSFV1-VP1; the mRNA obtained by the recombinant plasmid through linearization, in vitro transcription and other methods is mixed with the prepared liposome nano-particles to prepare the foot-and-mouth disease mRNA vaccine. The neutralizing antibodies, IL-4 and IFN-gamma of the mRNA vaccine after the guinea pigs are immunized are obviously higher than those of a negative control group; can be used for 50 times of GPID 50 The Re-A/WH/09 strain attack provides 3/4 protection, which shows that the mRNA vaccine prepared by the invention can induce stronger humoral immunity and cellular immunity response, has good immunogenicity, and provides a new way for developing an emergency preventive vaccination for foot-and-mouth disease.
Drawings
FIG. 1 is a flow chart of an animal experiment;
FIG. 2 PCR amplification of FMDV VP1 gene; wherein 1-2 are PCR amplification products of VP1 gene; 3 is M: DL2000 DNA markers;
FIG. 3 double restriction identification of recombinant plasmid pSFV1-VP1; wherein M is DNA molecular mass standard DL5,000; 1-2 is recombinant plasmid enzyme cutting product;
FIG. 4 Western blot results after transfection of BHK-21 cells;
FIG. 5 is a view of an LNP transmission electron microscope;
FIG. 6 Babc/C mouse injection site anatomy;
FIG. 7 LNPs/pCMV-N-EGFP transfected BHK-21 cells;
FIG. 8 serum neutralizing antibody titers of guinea pigs after immunization;
FIG. 9 results of IL-4 and IFN-gamma ELISA assays of guinea pig spleen cells after immunization;
figure 10 protection rate against challenge in guinea pigs after immunization.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The porcine foot-and-mouth disease virus A (Re-A/WH/09 strain) inactivating antigen described in the examples below is provided by the Ministry of production of China Weite biotechnology Co., ltd; BHK-21 cells were stored by the Ministry of middle farm Witt research and development; t (T) 4 The ligases, restriction endonucleases BamH I, sma I and Spe I were all purchased from NEB company; primeScript TM II 1st Strand cDNA Synthesis Kit，Premix Taq TM (LA Taq TM Version 2.0 plus dye), DL5000 DNA markers were all purchased from baori doctor materials technology (beijing) limited; pSFV1 vector is purchased from Invitrogen corporation (U.S.A.); guinea pig gamma interferon (IFN-gamma) and interleukin 4 (IL-4) ELISA detection kits were purchased from Beijing belosol Biotech Co.
The experimental animals described in the examples below were female guinea pigs of 2 months of age and weighing 300 to 500g, purchased from the animal center of the animal research institute of Lanzhou, china, national academy of agricultural sciences.
Example 1 preparation of mRNA vaccine
mRNA preparation
1.1 primer design
VP1 primers for amplifying FMDV were designed manually from the FMDV/A genomic sequence at NCBI:
an upstream primer: f: CGC (common gateway control)GGATCCGCCACCATGACCACCTCCGCAGGTGA；
A downstream primer: r: TCC (TCC)CCCGGGCTACTGTTTCACAGGCGCA。
And BamH I and Sma I cleavage sites (underlined) were inserted into the upstream and downstream primers, respectively, and all primers were synthesized by Nanjin St Biotechnology Co., ltd.
1.2 RNA extraction and cDNA Synthesis
Extracting RNA of an antigen inactivated by foot-and-mouth disease virus A (Re-A/WH/09 strain) by using a Trizol method, performing cDNA synthesis according to a TaKaRa reverse transcription kit, and storing in a refrigerator at-80 ℃ for later use.
1.3 PCR amplification and product purification
The full length of VP1 gene is amplified by using cDNA of FMDV as template and utilizing specific primer of VP1 gene. Amplification system: LA Taq Mix 12.5. Mu.L, 1. Mu.L of each of the upstream and downstream primers (10. Mu. Mol/L), 2. Mu.L of the template, and ddH were added 2 O to 25. Mu.L. The reaction conditions are as follows: 94 ℃ for 2min;94 ℃,30s,50 ℃,30s,72 ℃ and 40s for 32 cycles; finally, the extension is carried out for 10min at 72 ℃.
After electrophoresis of the amplified product on a 1% agarose gel, the target band was excised with a sterile scalpel, and the PCR product was subjected to a procedure according to the PCR purification kit (OMEGA).
PCR amplification was performed using reverse transcribed type A foot-and-mouth disease virus (Re-A/WH/09 strain) cDNA as a template, and the target band of about 639bp (shown in FIG. 2) was seen as a result of electrophoresis, and no nonspecific band appeared.
1.4 plasmid ligation, transformation and characterization
The purified PCR product was digested with restriction endonucleases BamH I and Sma I, respectively, and gel-recovered, and ligated overnight at 4℃according to the ligation system.
The whole process is operated on ice, and the connection reaction system is as follows:
then the recombinant plasmid is transformed into E.coli DH5 alpha competent cells, and the plasmid is extracted after picking single colony culture by screening of ampicillin resistance. And (3) after the recombinant plasmid is identified by PCR and double enzyme digestion, selecting a positive sample and sending the positive sample to Shanghai biological engineering company for sequencing.
After the positive PCR product and pSFV1 vector are transformed by double digestion, bacterial liquid is subjected to PCR method, double digestion identification (shown in FIG. 3) and sequencing analysis, which shows that the target gene is correctly inserted into the vector.
1.5 linearization, in vitro transcription, capping modification and purification of recombinant plasmids
Recombinant plasmids were extracted in large quantities, and after purity determination, the plasmids were linearized using SpeI cleavage.
In vitro transcription was performed according to the SP6RNA polymerase-mediated transcription kit (Invitrogen U.S.) with replacement of UTP with N1-methylpseudoridine-5' -triphosphates (m 1 ψTP) (Thermo).
Kit mMESSAGEmMACHINE for mRNA after transcription TM The SP6 kit (Invitrogen U.S.) was subjected to capping modification.
MEGAclear for modified mRNA TM Kit Purification for Large Scale Transcription Reactions kit (Invitrogen, U.S.) was purified.
1.6 in vitro cell transfection of recombinant plasmids
mRNA transcribed in vitro was then purified according to Lipofectamine TM MeAfter BHK-21 is transfected by the ssenger MAX (Invitrogen US) instruction book, cells are scraped by a cell scraping blade gently for centrifugation and sample collection, rabbit anti-FMDV hyperimmune serum (prepared in the laboratory) is used as a primary antibody, and a guinea pig anti-rabbit marked by HRP is used as a secondary antibody for Western blot detection.
After the mRNA obtained by in vitro transcription is transfected into BHK-21 cells for 24 hours, the cells are collected and Western blot verification is carried out. The results showed that after mRNA transfection of the cells, the band of interest appeared at about 35kD, consistent with the expected size, whereas the control was devoid of bands (shown in FIG. 4). The results indicate that recombinant mRNA can be expressed in BHK-21 cells.
Preparation of mRNA vaccine
2.1 preparation of Liposome nanoparticle, coating, electron microscope observation, particle size and potentiometry
Ionizing lipid according to the mole ratio: DSPC: cholesterol: PEG lipid=50:10:39:1.7 Liposome nanoparticles (lipid nanoparticle, LNP) were prepared.
LNP prepared was used with mRNA in N/P molar ratio = 8:1 (n=the amount of total nitrogen in DLinDMA, p=the amount of total phosphorus in mRNA wherein the average molecular weight of the individual nucleotides in RNA is 330 Da).
After the LNP was diluted in an appropriate ratio, transmission electron microscopy was performed. Particle size and potential were analyzed using Malvern Zetasizer Nano ZS (uk).
By morphological observation of the LNP prepared by transmission electron microscopy, approximately spherical particles (shown in FIG. 5) with a diameter of about 135.8nm were observed, with a potential of 36.4mV.
2.2 LNP safety and in vitro cell transfection Effect evaluation
To evaluate LNP safety, prepared LNP was subcutaneously injected 5-8 weeks old, 5 Babc/C female mice weighing 23+ -1.5 g, at a dose of 0.5mL, and continuously observed for 7 days.
The safety test results showed that all LNP vaccinated Babc/C mice did not show death due to injection or significant local adverse systemic reaction (shown in fig. 6).
After coating the LNP with the plasmid pCMV-N-EGFP, the LNP was used as a plasmid in N/P (mol/mol) 10:1 after 12h inoculation of BHK-21 cells, observations were made under a fluorescence microscope.
To verify the effect of LNP transfection on cells, the results showed that the test group visible specific green fluorescence, while the control group did not (shown in fig. 7). The results indicate that the prepared LNP has better delivery effect.
2.3 immune animal test and immunoprotection Effect evaluation
The animal test procedure is shown in figure 1. The mRNA vaccine group, the LNP and the PBS control group are respectively immunized with guinea pigs, 14 mice in each group are intramuscular injected into the inner thigh, 4 mice in each group are boosted 1 time on 14 days after the initial immunization, and the heart of the guinea pigs is periodically sampled after the immunization to respectively determine the neutralizing antibody titer of serum. At 35d (days post vaccination, dpv) post immunization, 2 isolated cultured spleen cells were sacrificed per group and levels of IL-4 and IFN-gamma secreted by the spleen cells were detected by ELISA. And simultaneously, measuring the change condition of the T lymphocyte subpopulation by using a flow cytometer.
When the immunized animals are bred to 35dpv, 12 animals in each of four groups of experimental animals are divided into 3 groups at random, wherein one group is continuously bred for detecting subsequent experiments such as antibody, and the other two groups are respectively bred with 0.2mL of 50 times GPID 50 Foot and mouth disease virus (FMDV/Re-A/WH/09 strain) was injected (50% of guinea pig infection dose) into the toes of the left hind leg of immunized guinea pigs. The protective efficiency of each group of vaccines was assessed according to the criteria reported by Wang et al (2011) by continuous observation 14d (fig. 1).
Serum was collected at weeks 2, 4, 5 and 6 after immunization, and the neutralizing antibody titer was determined. The results showed that with the passage of immunization time, the neutralizing antibody level was continuously increased, and the neutralizing titer in the first 5 th week serum reached the peak of the detection period, which was 1:64, significantly higher than the negative control, began to decline at week six (shown in fig. 8). The results indicate that mRNA vaccines can stimulate the body to produce higher levels of neutralizing antibodies.
The serum of the immunization 35d is taken for cytokine detection, and the test result shows that (shown in figure 9), the mRNA vaccine can stimulate the organism to produce IFN-gamma and IL-4, and the level of the mRNA vaccine is obviously higher than that of a control group (P is less than 0.05). The test results show that the mRNA vaccine can stimulate strong cellular immunity.
The immunized guinea pigs were challenged at 35dpv,the protective effect of the mRNA vaccine on the challenged guinea pigs was evaluated. The results showed that all guinea pigs in PBS and LNP groups developed significant FMD onset symptoms. In animals immunized with mRNA vaccine with 50-fold GPID compared to control group 50 When the virus is challenged, only 1 disease symptom exists, and 3/4 protection is provided (shown in figure 10), which shows that the mRNA vaccine can provide a certain protection effect for FMDV/Re-A/WH/09 animals challenged.
The result shows that the invention obtains mRNA molecules taking FMDV VP1 gene as a core, and after transfection of BHK-21 cells, protein expression of mRNA transfected cells is verified by Western blot experiment; an FMDV structural protein VP1 gene mRNA vaccine is prepared. Intramuscular injection of mRNA vaccine in guinea pigs, and detection of neutralizing antibodies, IL-4, IFN-gamma and challenge protection tests show that the mRNA vaccine induces humoral immunity and cellular immunity in guinea pigs, and 50 times of GPID (general purpose input/output) 50 3/4 protection is provided for the guinea pigs of the experimental group during the virus attack, the invention provides a new thought for developing novel candidate FMDV vaccine, and provides a reference for effective prevention and control of FMD.
Claims (4)
1. A swine a-type foot-and-mouth disease mRNA vaccine, characterized in that the mRNA vaccine comprises an mRNA molecule and a liposome nanoparticle; the mRNA molecule codes for foot-and-mouth disease virus structural protein VP1; the liposome nano-particles consist of ionized lipid, DSPC, cholesterol and PEG lipid with the molar ratio of 50:10:39:1.7; the N/P molar ratio of the liposome nanoparticle to the mRNA molecule is 8:1, a step of; the sequence of the mRNA molecule is shown as SEQ ID NO. 1.
2. The mRNA vaccine of claim 1, wherein the mRNA molecule is obtained by the method of: connecting a gene encoding foot-and-mouth disease virus structural protein VP1 with a pSFV1 vector to construct a recombinant plasmid pSFV1-VP1; mRNA molecules obtained by linearization and in vitro transcription treatment of the recombinant plasmid.
3. A method for preparing a swine a-type foot-and-mouth disease mRNA vaccine, the method comprising:
(1) VP1 Gene and pSFV1 vector were used with restriction nuclei, respectivelyEndonuclease enzymeBamH I and is provided withSmaI, double enzyme cutting and then connecting to obtain recombinant plasmids pSFV1-VP1;
(2) By usingSpeI, enzyme digestion is carried out to linearize the recombinant plasmid pSFV1-VP1, and a linearization vector is obtained;
(3) Replacing UTP in the linearization vector with N1-methyl pseudoudidine-5' -triphosphates for in vitro transcription;
(4) Capping modification is carried out on the transcribed mRNA to obtain an mRNA molecule; the sequence of the mRNA molecule is shown as SEQ ID NO. 1;
(5) The mole ratio of liposome nano particles to mRNA molecules is 8:1, mixing to prepare mRNA vaccine; the liposome nanoparticle consists of ionized lipid, DSPC, cholesterol and PEG lipid with the molar ratio of 50:10:39:1.7.
4. The method of claim 3, wherein the VP1 gene in the step (1) is obtained by: extracting RNA of an inactivated antigen of a foot-and-mouth disease virus Re-A/WH/09 strain, and synthesizing cDNA by reverse transcription; the cDNA is used as a template, and the VP1 gene is obtained by amplification by utilizing a specific primer of the VP1 gene.
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| CN115025212A (en) * | 2022-06-20 | 2022-09-09 | 天康制药(苏州)有限公司 | mRNA vaccine for preventing porcine reproductive and respiratory syndrome and preparation method thereof |
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| CN115025212A (en) * | 2022-06-20 | 2022-09-09 | 天康制药(苏州)有限公司 | mRNA vaccine for preventing porcine reproductive and respiratory syndrome and preparation method thereof |
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| Expression of the foot-and-mouth disease virus VP1 protein using a replication-competent recombinant canine adenovirus type 2 elicits a humoral antibody response in a porcine model;YE LIU等;VIRAL IMMUNOLOGY;第19卷(第2期);第202-209页 * |
| MT863266.1;GenBank;GenBank;全文 * |
| 基于猪口蹄疫O型病毒结构蛋白VP1基因的mRNA疫苗制备及免疫活性研究;董金杰等;农业生物技术学报;第30卷(第6期);摘要、第1220页右栏、第1221页1.3-1.9、第1226页图4、6-8 * |
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