CN117860880A

CN117860880A - RdRp-dependent African swine fever taRNA vaccine and construction method and application thereof

Info

Publication number: CN117860880A
Application number: CN202410034558.5A
Authority: CN
Inventors: 魏建超; 马志永; 张妍; 夏琦琦; 张海龙; 邱亚峰; 李宗杰; 刘珂; 李蓓蓓; 邵东华
Original assignee: Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
Current assignee: Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
Priority date: 2024-01-10
Filing date: 2024-01-10
Publication date: 2024-04-12

Abstract

The invention provides an RdRp-dependent African swine fever taRNA vaccine and a construction method and application thereof, and belongs to the technical field of biological medicines. The construction method of the taRNA vaccine provided by the invention comprises the following steps: constructing a replicative mRNA plasmid pJEV-T7-NS5 expressing JEV RdRp; constructing nucleotide sequences P2A-P30-T2A, P A-P54-T2A and P2A-B602L-T2A for expressing proteins P30, P54 and B602L of African swine fever virus; replacing the NS5 sequence in the pJEV-T7-NS5 with the sequence to obtain a corresponding plasmid; preparing corresponding linear mRNA by in vitro transcription of the plasmid; the linear mRNA was added with a vaccine adjuvant to prepare Cheng Feizhou swine fever taRNA vaccine. The vaccine can effectively stimulate mice to generate corresponding humoral immunity and cellular immunity response.

Description

RdRp-dependent African swine fever taRNA vaccine and construction method and application thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to an African swine fever taRNA vaccine dependent on RdRp, and a construction method and application thereof.

Background

African swine fever (African Swine Fever, ASF) is a highly infectious hemorrhagic swine infectious disease caused by African Swine Fever Virus (ASFV) characterized by high fever, hemorrhage, ataxia and depression, and has a high mortality rate in pigs at home approaching 100%. ASFV is a double stranded DNA virus with a regular icosahedron structure, the only member of the Asfivirus and Asfarviridae families, and also the only known DNA arboviruses. The explosion of ASF to the pig industry causes a significant economic loss, which severely threatens the development of the pig industry worldwide. However, no effective ASF vaccine is currently available. Control of ASF is primarily dependent on early diagnosis and elimination of infected animals, as well as on the enhancement of biosafety for disease prevention by farmers. African swine fever virus has a double-stranded DNA genome of 170-190 kb and encodes about 150-200 structural and nonstructural proteins involved in viral replication, virus-host interactions and regulation of host innate immune responses, but more than half of ASFV protein functions remain unknown, of which more than 50 are structural proteins including p72, p54, p30, B602L, CD2v, and the like.

The P30 protein is also called P32 protein, the protein produced by encoding the CP204L gene of ASFV has strong antigenicity, the protein size is 30kDa, and P30 is an intimal protein expressed by ASFV in early stage, and the expression can be detected in early stage of infection and persists in the whole infection period. Because of its strong antigenicity and immunogenicity, it is capable of inducing the production of specific antibodies, and therefore, recombinant proteins produced using baculovirus protein expression systems are often used as ELISA diagnostic antigens. The p30 protein can promote viral internalization during viral invasion, and antibodies directed against p30 can inhibit viral internalization into host cells. Therefore, the p30 protein can be used as a target protein for developing diagnosis and vaccine of African swine fever virus.

The p54 protein is a protein encoded by the E183L gene of ASFV, is a type I transmembrane protein, spans the inner envelope of the virion, has a protein size of 25kDa, and plays an important role in the generation of the envelope precursor of the virus and in the stabilization of the inner viral envelope of the mature virion. In the role of the virus with the host cell, p54 provides conditions for the transport of the virus along the cytosol within the cell, while the p54 protein also promotes apoptosis induced by ASFV in infected target and immune cells. In addition, the p54 protein is used as the main structural protein of the African swine fever virus, is the main binding site of serum antibodies, has good immunogenicity, and can also be used as a marker for early detection of the African swine fever virus.

The B602L protein is a protein encoded by the B602L gene of ASFV, also called chaperone protein, with a protein size of 62kDa, which is an advanced non-structural protein of ASFV. In the ASFV genome, the B602L reading frame is located immediately downstream of the p72 reading frame, and the major capsid protein p72 of ASFV undergoes a conformational change rapidly after synthesis, which is thought to be due to spontaneous or chaperone-assisted folding, and it has been found that expression of p72 protein alone in cells causes aggregation and precipitation, but in the presence of chaperone B602L prevents p72 aggregation and increases p72 solubility. In addition, co-expression of B602L and p72 promotes folding of p72, similar to folding kinetics after a truly infected cell. The B602L protein is able to bind to unfolded capsid proteins early after p72 protein expression, but unfold upon assembly to the endoplasmic reticulum membrane immediately after p72 conformational maturation. If the expression of the B602L protein is inhibited, the expression level of the p72 protein is reduced, and dislocation allosteric of the main capsid protein E120R is caused, so that the protein is an essential protein for the assembly of icosahedral capsids of virus particles.

As a next generation mRNA technique, self-replicating RNA, also called self-amplifying RNA (saRNA), can self-replicate using its own RNA sequence as a template, which carries, in addition to the expression of the target protein, a sequence capable of expressing an RNA polymerase (RNA-dependent RNA polymerase, rdRP). After the RNA polymerase is generated, more copies of the SARNA can be generated using the SARNA as a template. At present, there are two designs for self-amplification of RNA, one is to put the sequence encoding RNA polymerase and the sequence expressing the target protein into the same mRNA, which is common SARNA; the other is to divide the sequence encoding RNA polymerase and the mRNA sequence encoding the target protein into two parts, and introduce them into cells, and this method is called trans-amplification (trans-amplification).

Whether used as a vaccine or therapeutic, mRNA acts by expressing proteins to elicit an immune system response or therapeutic effect. Thus, the expression level of the protein is closely related to the efficacy of the treatment. One disadvantage of conventional mRNAs is that they are not stable and can be degraded in the cell for several days, resulting in an insufficient persistence of the protein expression level. If used as a therapy for the long-term treatment of a disease, the patient may be required to inject mRNA multiple times and in large amounts, which may increase the toxic side effects of mRNA therapy. The main advantage of self-amplifying RNA is that it can achieve the same protein expression level as conventional mRNA at very low doses, and studies have shown that as a vaccine, saRNA can produce the same immune response at doses hundreds or even thousands of times smaller than conventional mRNA. Since the saRNA may prolong the time that the antigen protein is present in the body, this may enhance the immune response, allowing a saRNA-based vaccine to be vaccinated with only one needle, achieving the effect of two needles of conventional mRNA vaccination. In terms of production, lower effective doses may reduce the cost of production of saRNA. As a therapy, this feature may reduce the dosage and number of injections used for mRNA therapy, thereby reducing the toxic side effects that mRNA and delivery vehicles may have on while prolonging the efficacy.

Another feature of saRNA is that it may itself have the potential to elicit an immune response. There are sensors in human cells that recognize foreign viral invasion, which are called pattern recognition receptors (pattern recognition receptors). One of the signals they recognize is double stranded RNA that appears in the cytoplasm, as this may be representative of viral RNA replication in cells. While saRNA forms double-stranded RNAs during replication, they closely resemble replicating viral RNAs, and thus may trigger the innate immune response of the cell. This may further enhance the effect of the vaccine.

However, self-amplified RNA also faces unique challenges in terms of development. Indeed, saRNA does suffer from some drawbacks associated with the inclusion of viral sequences. Since saRNA links the sequence encoding RNA polymerase to the sequence expressing the target protein, the molecular weight of the entire mRNA molecule is much greater than conventional mRNA, which limits the length of the gene of interest and complicates the creation of vectors for bacterial reproduction and vaccine packaging. Thus, a specific design or formulation adjustment of the delivery vehicle is required in terms of delivery. Too large a molecular weight may result in a decrease in delivery efficiency. In addition, the replicase portion (nsP) of alphaviruses can affect host cells and trigger immune overactivation. Another feature of saRNA is the production of intracellular double-stranded RNA (dsRNA) during replication, which activates innate immunity and can interfere with protein translation.

In response to the disadvantages of this saRNA, a trans-replicating RNA (trans-replicating RNA), which is also a form of self-replicating mRNA, has been newly developed in recent years. tarnas differ from sarnas in that tarnas separate the viral sequence encoding the RNA polymerase from the sequence expressing the antigen of interest, and the antigen RNA of interest is replicated in trans by replicase. The viral replicase may be nrRNA or saRNA, and the mRNA encoding the gene of interest is referred to as trans-replicative RNA (TR-RNA). The concept of a trans-replication system was originally proposed by Pirjo Spuul et al, which design involves the advantage of self-replicating mRNA while reducing certain drawbacks. Notably, by encoding replicases alone in the mRNA platform, limitations on the length of the antigen of interest are circumvented and the use of modified nucleotides is not limited. In addition, this method is safer because it reduces the possibility of producing recombinant viral particles. In addition, tarnas are more immunogenic, so lower doses (as low as 50 ng) than saRNA are sufficient to elicit a similar immune response. Recently, a variation of taRNA was reported. It includes two additional TR-RNAs and replicase-encoding RNAs. With the continued advancement of taRNA technology, it has been proposed that incorporation of adenine-rich regions in the 5' UTR could significantly increase mRNA replication efficiency. Such modified tarnas would result in shorter RNAs, ten-fold vaccine doses, without affecting expression levels in vitro. Evaluation of the mouse taRNA system showed that 50ng of influenza virus Hemagglutinin (HA) RNA was sufficient to elicit a neutralizing antibody response and provide protection against influenza virus challenge. And the taRNA expression level was 10 to 100 times higher than that of the standard saRNA, probably due to higher translation efficiency. However, one significant disadvantage of tarnas is the need for at least two different RNAs, one for replicase and one for the antigen of interest. Overall, the taRNA technology is still in an early stage, but has a broad practical application prospect. Pre-clinical studies are currently underway for taRNA vaccines against influenza virus. In addition, bivalent vaccines against chikungunya fever and ross river viruses have been developed using this approach.

NS5 of encephalitis virus (JEV) plays a key role in prior immune evasion. NS5 is the largest flaviviral protein containing N-terminal methyltransferase (MTase) and C-terminal polymerase RNA-dependent RNA polymerase (RdRp), and has been demonstrated to interact with RdRp using crystal structure, biochemistry, and reverse genetics, with a complete MTase and RdRp interaction interface consisting of six closely spaced hydrophobic amino acids with a conserved GTR sequence hypothetically mediated interface formation. GTR mediates MTase-RdRp interactions mainly through hydrogen bonds, and the MTase and RdRp regulate and control viral replication characteristics through the interactions. Furthermore, there are 10 very non-conserved amino acid sequences (GTR linker) following the GTR sequence, which are able to regulate NS5 conformation and function, and whose flexibility is critical for viral replication. Thus, the MTase-RdRp interface and the GTR junction region in the NS5 protein are necessary for JEV RdRp extension, initiation and JEV replication.

Since the concept of self-replicating mRNA was proposed, replication was performed using the replicase of alphaviruses, including nsP1, nsP2, nsP3 and nsP4. Among them, the most conserved protein nsP4 in alphaviruses, which acts as an RNA-dependent RNA polymerase (RdRp), is responsible for amplifying the copy number of the original in vitro transcribed mRNA. Because the nsP fragment is about 7kb in length and the complete alphavirus saRNA system is as long as 10kb, mRNA is too long, the vaccine preparation difficulty is increased, and the size of antigen is limited, therefore, a replicase with small molecular weight is found, and the preparation of mRNA vaccine for expressing p30, p54 and B602L proteins of ASFV can greatly improve the efficiency and performance of vaccine preparation.

Disclosure of Invention

The invention aims to provide an African swine fever taRNA vaccine dependent on RdRp, a construction method and application thereof, and the vaccine can effectively stimulate mice to generate corresponding humoral immunity and cellular immunity response, provides a new thought for research and development and innovation of the African swine fever vaccine, and has a certain application value in clinic.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a construction method of an African swine fever taRNA vaccine depending on RdRp, which comprises the following steps:

(1) Constructing a replicative mRNA plasmid pJEV-T7-NS5 expressing JEV RdRp;

(2) Constructing nucleotide sequences P2A-P30-T2A, P A-P54-T2A and P2A-B602L-T2A for expressing proteins P30, P54 and B602L of African swine fever virus;

(3) Replacing the NS5 sequence in the pJEV-T7-NS5 with the P2A-P30-T2A, P2A-P54-T2A or P2A-B602L-T2A sequence to obtain plasmids pJEV-T7-P30, pJEV-T7-P54 and pJEV-T7-B602L;

(4) In vitro transcription of pJEV-T7-NS5, pJEV-T7-p30, pJEV-T7-p54 and pJEV-T7-B602L Linear mRNAs of NS5, p30, p54 and B602L were prepared;

(5) The linear mRNAs of NS5, p30, p54 and B602L were added with vaccine adjuvants to prepare Cheng Feizhou classical swine fever taRNA vaccine.

Preferably, the nucleotide sequence of the pJEV-T7-NS5 plasmid in the step (1) is shown as SEQ ID NO. 1.

Preferably, the nucleotide sequences P2A-P30-T2A, P A-P54-T2A and P2A-B602L-T2A in the step (2) are respectively shown in SEQ ID NO. 3-5.

Preferably, the vaccine adjuvant comprises an ionizable lipid, distearoyl phosphatidylcholine, cholesterol, and PEG-lipid; the mass mole ratio of the ionizable lipid, distearoyl phosphatidylcholine, cholesterol and PEG-lipid is (48-52): (8-12): (37-40): (1-2).

Preferably, the ratio of linear mRNA to total adjuvant is 1:35 to 45.

Preferably, the linear mRNA is mixed with a vaccine adjuvant and dialyzed to obtain the taRNA vaccine.

The invention also provides an African swine fever taRNA vaccine obtained by the construction method.

The invention also provides an application of the African swine fever taRNA vaccine in preparing a medicine for preventing African swine fever virus.

The invention prepares a novel taRNA vaccine which is dependent on JEV RdRp and expresses ASFV p30, p50 and B602L proteins, respectively constructs a replicative plasmid containing JEV RdRp and a plasmid expressing ASFV p30, p50 and B602L proteins, and is used for preparing mRNA, preparing the taRNA vaccine by combining lipid nano particles, and evaluating the immune effect of the taRNA vaccine in mice. Compared with the traditional mRNA vaccine, the taRNA vaccine has a self-amplification element, and compared with the saRNA, the taRNA separates the sequence for coding RdRP from the target protein sequence, so that the molecular weight of mRNA is reduced, a plurality of antigens can be presented at the same time, and the delivery efficiency of mRNA is obviously improved. The method has application value in research and development and innovation of African swine fever vaccine, and can promote transformation application of the African swine fever vaccine.

Drawings

FIG. 1 is a graph showing the results of agarose gel electrophoresis identification of a fusion PCR product of a 5'UTR-NS5-3' UTR fragment in example 1 (M: marker IV);

FIG. 2 is an agarose gel electrophoresis chart of example 2; wherein (a) is a P2A-P30-T2A (734 bp) and P2A-P54-T2A (683 bp) PCR product identification result diagram (M: D2000) and (B) is a P2A-B602L-T2A (1721 bp) PCR product identification result diagram (M: marker IV);

FIG. 3 is a graph showing the results of the digestion and identification of the pJEV-T7-NS5 plasmid in example 2 (M: marker IV);

FIG. 4 is a diagram showing the WB results of 293T cells transfected with cap-NS5-mRNA, cap-p30-mRNA, cap-p54-mRNA and cap-B602L-mRNA in example 4 (M: 26616 protein Marker); wherein (a) is NS5 protein expression, (B) is p30 protein expression, and (c) is p54 and B602L protein expression;

FIG. 5 is a graph showing the results of detection of specific p30 antibody levels in mouse serum after immunization with LNP-NS5-p30-p54-B602L-taRNA vaccine of example 6;

FIG. 6 is a graph showing the results of detection of specific p54 antibody levels in mouse serum after immunization with LNP-NS5-p30-p54-B602L-taRNA vaccine of example 6;

FIG. 7 is a graph showing the results of detection of specific B602L antibody levels in mouse serum after immunization with LNP-NS5-p30-p54-B602L-taRNA vaccine of example 6;

FIG. 8 is a graph showing the results of lymphocyte proliferation in the spleen of mice after immunization with the LNP-NS5-p30-p54-B602L-taRNA vaccine of example 6.

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

The apparatus used in the present invention is shown in table 1.

Table 1 test instrument table

The major reagents and biomaterials of the present invention are shown in Table 2.

TABLE 2 Main reagents

Example 1

Construction of a replicative mRNA plasmid (pJEV-T7-NS 5) expressing the JEV RdRp region

Three pairs of specific fusion primers are designed by taking the existing JEV cDNA in the laboratory as a template, the 5'UTR, the NS5 and the 3' UTR of JEV are respectively amplified, xbaI and BbeI restriction sites are respectively introduced into the upstream and downstream of the first pair of primers, salI and MunI restriction sites are respectively introduced into the upstream and downstream of the third pair of primers,

primers were synthesized by Shanghai Seisakusho Biotechnology Co., ltd, and specific primer sequence information is shown in Table 3.

TABLE 3 primer sequences

The 5'UTR and 3' UTR regions of JEV were amplified using the first and third pairs of primers in Table 3, respectively, and the second pair of primers amplified the NS5 gene of JEV. The specific amplification system is as follows:

the specific reaction procedure is as follows:

after PCR amplification, 1% agarose gel electrophoresis was performed, and the target fragments were recovered by gel Extraction using E.Z.N.A.gel Extraction Kit, respectively. Then, fusion PCR was used to fuse fragment 1 (96 bp) and fragment 2 (2953 bp), wherein both fragment 1 and fragment 2 were PCR amplified products as described above, and the specific reaction system was as follows:

the specific reaction procedure is as follows:

2. Mu.L of each of the primers 5'UTR-XbaI-F and 3' -SalI-NS5-R was added to the reaction tube, and the fragment 1+2 was further amplified, and after mixing, reacted under the following conditions:

and (3) after the reaction is finished, carrying out electrophoresis identification on the amplified product by using 1% agarose gel, cutting off a target fragment, and carrying out gel recovery on the target fragment by using an E.Z.N.A.gel Extraction Kit to obtain a fragment 1+2 after PCR fusion. Subsequently, the fragment 1+2 and the fragment 3 (335 bp) were fused in the same manner using the 5'UTR-XbaI-F and 3' UTR-MunI-R primers, and after the fusion was completed, a target fragment having a total length of 3384bp containing the NS5 gene was obtained, and the result of electrophoresis was shown in FIG. 1, designated 5'UTR-NS5-3' UTR, and the sequence was shown in SEQ ID NO. 2. Double digestion is carried out on a vector and a target fragment (3384 bp) by using restriction endonucleases XbaI and MunI respectively, the vector is JEV replicon plasmid (pJEV-T7) with a T7 promoter constructed in the early stage of the laboratory, the reaction is carried out for 2 hours in a water bath at 37 ℃, gel recovery is carried out after 1% agarose gel electrophoresis, the concentration is measured, and the mixture is stored to-20 ℃ for standby. The enzyme digestion system is as follows:

ligating the target fragment by using T4 DNA ligase, wherein the mass ratio of the vector to the 5'UTR-NS5-3' UTR fragment is 1:3 the volume of each fragment to be added was calculated and the metal bath at 16℃was connected overnight. The connection system is as follows:

the ligation products were transformed into DH 5. Alpha. Competent cells as follows:

(1) mu.L of DH 5. Alpha. Competent cells were taken and left to stand on ice for 5min.

(2) mu.L of the ligation product was added to 50. Mu.L of DH 5. Alpha. Competent cells, gently flicked, and mixed for 30min in an ice bath.

(3) Heat-shock in a water bath at a temperature of 42 ℃ for 90s, ice-bath for 2-3min, adding 1mL of LB culture medium (without adding antibiotics), and placing in a shaking table at a temperature of 37 ℃ for renaturation for 1h.

(4) Centrifugation at 5000rpm for 3min, 900. Mu.L of supernatant was discarded, and the remaining bacterial liquid was plated on ampicillin-resistant LB plates and incubated overnight in a constant temperature bacterial incubator at 37 ℃.

Colony PCR identification was performed using the 5'UTR-F and 3' UTR-R primers of Table 3, a monoclonal colony with the correct identification result was inoculated into 5mL LB medium containing ampicillin, shaking culture was performed for 3 hours, then 2mL of the bacterial liquid was added into a conical flask containing 200mL LB medium (ampicillin-containing) at 37℃and shaking culture was performed for 18 hours in a constant temperature shaker at 200rpm, plasmids were extracted using a Tiangen-taking endotoxin-large extraction kit and then were sent to the engineering (Shanghai) stock company for sequencing, and the plasmid with the correct sequencing was designated pJEV-T7-NS5, the sequence of which was shown as SEQ ID NO.1

Example 2

Construction of mRNA plasmids expressing ASFVp30, p54 and B602L proteins

The complete P30 (124801 ～ 125405, 603 bp), P54 (162259 ～ 162811, 552 bp) and B602L (100716 ～ 102306, 1590 bp) genes of ASFV SY-18 strain (GenBank: MH 766894.3) published on NCBI were synthesized by Nanjing Seisakusho Biotechnology Co., ltd. For homosapiens cells, cloned into pUC57 vector, and the synthetic plasmids further contained the upstream P2A sequence and downstream T2A sequence of the target protein, designated as P2A-P30-T2A (734 bp), P2A-P54-T2A (683 bp) and P2A-B602L-T2A (1721 bp) (SEQ ID NO. 3-5), respectively. And the target protein sequence is amplified by taking the synthesized plasmid as a template and 5'-BbeI-P2A-F and 3' -SalI-T2A-R as primers, and the upstream primer and the downstream primer are respectively introduced into BbeI and SalI cleavage sites. Primers were synthesized by Shanghai Seisakusho Biotechnology Co., ltd, and specific primer sequences are shown in Table 4:

TABLE 4 primer sequences

The specific amplification system is as follows:

the specific reaction procedure is as follows:

after the reaction, the reaction products were identified by electrophoresis using 1% agarose gel as in FIG. 2, and the target band was excised and the current gene was gel recovered and measured for concentration. Then, the PCR products P2A-P30-T2A (734 bp), P2A-P54-T2A (683 bp) and P2A-B602L-T2A (1721 bp) introduced with the cleavage sites were subjected to BbeI and SalI double cleavage, and the pJEV-T7-NS5 plasmid constructed as above was subjected to cleavage by the same method and cleavage sites, to cut out two genes altogether, and the vector and JEV 5'UTR and 3' UTR parts (JEV-T7-5 '-DELTANS 5-3',4151 bp) were retained, and were subjected to a water bath reaction at 37℃for 2 hours as shown in FIG. 3, the specific cleavage system was as follows;

after the reaction is completed, the enzyme digestion products are verified by 1% agarose gel, the result is checked by a gel imager after 120V for 30min, the corresponding target strips are cut off, and the target strips are recovered and the concentration thereof is measured according to the specification of the gel recovery kit. The desired fragments P2A-P30-T2A, P A-P54-T2A and P2A-B602L-T2A were then ligated with the digested vector pJEV-T7-NS5 using T4 ligase, with a 16℃metal bath overnight, the ligation system being as follows:

Colony PCR was identified using the 5'UTR-F and 3' UTR-R primers of Table 3, and the monoclonal colonies with correct identification results were inoculated into 5mL LB medium containing ampicillin, followed by shaking culture for 3 hours by adding 2mL of the bacterial liquid into a conical flask containing 200mL LB medium (ampicillin-containing) at 37℃and shaking culture for 18 hours in a constant temperature shaker at 200rpm, extracting plasmids using a Tiangen extraction kit, and sequencing them by the company of Probiotics (Shanghai) Co., ltd, and the plasmids with correct sequencing were designated pJEV-T7-p30, pJEV-T7-p54, and pJEV-T7-B602L.

Example 3

In vitro transcription to produce mRNAs for NS5, p30, p54 and B602L

The recombinant plasmids of pJEV-T7-NS5, pJEV-T7-p30, pJEV-T7-p54 and pJEV-T7-B602L constructed in examples 1 and 2 were subjected to Mun I cleavage site cleavage linearization, 25. Mu.g of the plasmid was taken, and the cleavage system was subjected to water bath cleavage at 37℃for 2 hours as follows:

after the enzyme digestion is completed, the concentration of the enzyme is directly recovered by using a gel recovery kit.

mRNA of the target gene is prepared by in vitro transcription by using the T7 promoter upstream of the target gene of the mRNA synthesis plasmid.

Mu.g of linearized plasmid was taken and used with mMESSAGE from Sieimer technology Co., ltdThe T7Ultra Kit (capped version) in vitro transcription Kit is used for synthesizing mRNA, and the specific steps are as follows:

after thoroughly mixing well, incubation was carried out at 37℃for 2h.

Adding 1 mu L TURBO DNase, removing template DNA, mixing, incubating at 37deg.C for 15min to obtain mRNA of target gene, which is named cap-NS5-mRNA, cap-p30-mRNA, cap-p54-mRNA and cap-B602L-mRNA, respectively.

Example 4

WB validation of transfected mRNA expressed NS5, p30, p54 and B602L proteins

To verify that the in vitro transcribed mRNA of example 3 correctly expressed the pre-set protein in the cells, the transfection of mRNA into 293T cells was verified as follows:

(1) 293T cells were passaged into six well plates and transfected when the cells were grown to a density of 70-80% in high-sugar DMEM containing 10% FBS, 1% SP.

(2) Clean EP tubes were taken, 1mL of Opti-MEM and 6. Mu.L of DMRIE-C transfection reagent were added, vortexed and incubated for 15min at room temperature.

(3) 20. Mu.L of each of cap-NS5-mRNA, cap-p30-mRNA, cap-p54-mRNA and cap-B602L-mRNA obtained in example 3 was added to the medium of the same tube step (2) in a total of 80. Mu.L, gently swirled and mixed, and incubated at room temperature for 5 minutes.

(4) Cells were washed once with Opti-MEM medium, washing was discarded, and the incubated mixture (1 mL+6. Mu.L+80. Mu.L total) from step (3) was added to the cell plate and the plate was gently shaken to distribute the liquid evenly. After 6h, the solution was changed to high sugar DMEM containing 2% FBS and 1% SP, and the mixture was continued at 37℃with 5% CO ₂ And (5) standing and culturing for 24 hours in a constant temperature incubator.

(5) Negative controls were also set. Linearization and in vitro transcription were performed according to the method of example 3 on the empty vector plasmid used in the early construction of pJEV-T7 in the laboratory, the in vitro transcription product was added to the medium described in step (2), gently stirred and mixed, incubated at room temperature for 5min, and transfected as negative control according to the method described in step (4).

After 24h of culture, the cells were collected for WesternBlot, as follows:

a. after 24h incubation, the medium was discarded, the cell plates were washed twice with PBS, after which 600. Mu.L of PBS was added and the cells were scraped with a cell scraper and collected in a 1.5ml centrifuge tube.

Centrifuging at 12000rpm for 3min, discarding supernatant, adding lysate containing 1% protein inhibitor, blowing with a pipetting gun, mixing, and performing ultrasonic cleavage (25% power, instantaneous cleavage) with an ultrasonic breaker.

c. After sonication, the supernatant was centrifuged at 12000rpm at 4℃for 10min, and the transferred supernatant was added to a new 1.5mL centrifuge tube by volume at 5X SDS Loading Buffer, and the mixture was boiled for 10min after mixing.

d. And (3) glue preparation: 15% SDS-PAGE separating gel was prepared according to Table 5, after the gel had solidified, 5% SDS-PAGE concentrating gel was prepared according to Table 6, and then sample combs were inserted and used after solidification.

TABLE 5 formulation of 15% SDS-PAGE separating gel

Table 6 5% SDS-PAGE concentrate

e. SDS-PAGE electrophoresis is carried out by 15% polypropylene gel, after loading, the voltage is regulated to 120V for 90min after the constant voltage of 80V is 30min, and the gel is continuously run.

f. Transferring: transferring by wet transfer method, taking out gel after electrophoresis, and transferring protein strip into NC film. Sequentially placing the sponge, the three layers of filter paper, the separating gel, the NC film, the three layers of filter paper and the sponge in sequence, placing the sponge in a transfer printing groove, adding an ice box and the prepared 1X film transferring liquid, and carrying out constant-current transfer printing for 120min at 240 mA.

g. Closing: after the membrane transfer is finished, the NC membrane is taken out, a sealing liquid (5% of skimmed milk) is added, the membrane is placed on a shaking table and sealed for 3 hours at room temperature, and after the sealing is finished, the membrane is rinsed three times by TBST.

h. Incubating primary antibodies: rabbit-derived JECNS 5 protein polyclonal antibody (1:2000 dilution) was used as primary antibody and incubated overnight on a 4℃shaker. After the incubation, the incubation was completed, washed three times with TBST for 10min each on a shaker.

i. Incubating a secondary antibody: HRP-labeled goat anti-rabbit antibody (1:10000 dilution) was used as secondary antibody and incubated on a shaker for 50min at room temperature. After the incubation, the incubation was completed, washed three times with TBST for 10min each on a shaker.

j. After completion, the results were exposed and analyzed with a gel imaging system.

As shown in FIG. 4, it was revealed from FIG. 4 that the expression of JECNS 5 protein was detected in cells co-transfected with cap-NS5-mRNA, cap-p30-mRNA, cap-p54-mRNA and cap-B602L-mRNA, and the expression of p30, p54 and B602L was detected in African swine fever positive serum at a size of about 101 kDa. The results show that in the taRNA system based on RdRp encoded by JEV NS5 protein, p30, p54 and B602L of ASFV can be expressed normally.

Example 5

Preparation of lipid nanoparticle mRNA vaccine LNP-NS5-p30-p54-B602L-taRNA

mRNA is a negatively charged biological macromolecule, is difficult to transport passively across negatively charged cell membranes, lipid Nanoparticles (LNPs) can be used to deliver mRNA and can prevent nucleic acids from being degraded, is an effective drug delivery means for mRNA, and comprises the following steps:

(1) Ionizable lipid (Dlin-MC 3-DMA), distearoyl phosphatidylcholine (DSPC), cholesterol, and PEG-lipid (PEG 2000-DMG) were mixed in a molar ratio of 50:10:38.5:1.5, and a formulation with a total mass of 200 μg was dissolved in 30 μl ethanol.

(2) The ethanol solution was rapidly injected into 90. Mu.L of 20mM sodium acetate buffer containing 5. Mu.g of each of cap-NS5-mRNA, cap-p30-mRNA, cap-p54-mRNA and cap-B602L-mRNA prepared in example 3 under vortexing conditions. Mixing for 20s in a microfluidic mixer, and standing for 10min to obtain the nano-particles.

(3) And dialyzing the prepared mixed solution of the sodium ethylacetate containing the nano particles in 10mM PBS solution for 3 hours to remove the ethanol, and performing ultrafiltration concentration to obtain the final product LNP-NS5-p30-p54-B602L-taRNA.

Example 6

To evaluate the immune effect of the JEV RdRp-based taRNA vaccine expressing ASFVp30, p54 and B602L constructed in the present invention, the LNP-NS5-p30-p54-B602L-taRNA vaccine prepared in example 5 and PBS were immunized into BALB/c mice, and their effects of inducing cellular immunity and humoral immunity in the mice were evaluated. The specific implementation steps are as follows:

immunization of BALB/c mice 20 SPF-class 4 week old female BALB/c mice were randomly divided into 2 groups of 10, one group was intramuscular injected with LNP-NS5-p30-p54-B602L-taRNA vaccine 5 μg/and the other group was intramuscular injected with equal amount of PBS as a control.

Serum samples collected from immunized mice on days 0, 7, 14, 21, 28, 35, and 42 were collected through the orbit for specific antibody detection, and 3 mice per group were sacrificed at 49d for lymphocyte proliferation experiments by aseptically isolating spleen lymphocytes.

The levels of p30, p54 and B602L protein-specific antibodies in serum after immunization of LNP-NS5-p30-p54-B602L-taRNA vaccine were determined using an ELISA assay. Diluting the serum of the mice to be detected into PBST at a ratio of 1:100, adding 100 mu L of the serum of each hole into the micropores coated with p30, p54 and B602L proteins, and incubating at 37 ℃ for 1h; the wells were discarded and washed three times with PBST; using PBST1: diluting HRP-labeled goat anti-mouse secondary antibody with 5000 mu L per well, and incubating at 37 ℃ for 1h; the wells were discarded and washed three times with PBST; adding 100 mu LTMB color development liquid into each hole in a dark place, and incubating for 15min at 37 ℃; 100 mu L of stop solution is added to each well; absorbance at OD450 mm was measured using an microplate reader.

As shown in FIGS. 5 to 7, the levels of ASFVp30, p54 and B602L protein-specific antibodies in the samples were determined, and the levels of antibodies were increased in p30, p54 and B602L in the test group immunized with the LNP-NS5-p30-p54-B602L-taRNA vaccine compared with the negative control group. Thus, specific antibodies can be generated by activating humoral immunity through immunization with taRNA vaccines that rely on JEV RdRp to express ASFVp30, p54, and B602L.

Further, isolated spleen lymphocytes from mice were diluted with RPMI-1640 medium containing 10% FBS to adjust the cell density to 1X 10 ⁶ mu.L of lymphocytes per well (3 replicates per sample cell) were plated uniformly per mL into 96-well plates. Then 50. Mu.L of specific stimulation antigens p30, p54 and B602L protein (final concentration 10. Mu.g/mL) were added to each well, respectively, and 50. Mu.L of RPMI-1640 was added as a control group. The 96-well plate was placed in 5% CO at 37 ℃C ₂ After culturing in a cell incubator for 72 hours, 20. Mu.L of CCK-8 solution was added to each well, incubation was continued for 3 hours in a 37℃5% CO2 cell incubator, OD values of each well were detected using an ELISA reader at OD450 mm, and the stimulation index of each group was calculated. The specific calculation formula is as follows: stimulation Index (SI) = (average OD value of specific antigen stimulated wells-background OD value)/(average OD value of no antigen stimulated wells-background OD value).

As shown in fig. 8, after the specific antigens p30, p54 and B602L stimulated lymphocytes from both groups of mice, lymphocytes proliferated, and the Stimulation Index (SI) of p30, p54 and B602L was significantly higher in the immunized group than in the PBS group. The results show that the taRNA vaccine expressing ASFV p30, p54 and B602L through immune dependence JEV RdRp can effectively improve the cellular immune response level of mice.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. A method for constructing an african swine fever taRNA vaccine dependent on RdRp, comprising the steps of:

(1) Constructing a replicative mRNA plasmid pJEV-T7-NS5 expressing JEV RdRp;

2. The method according to claim 1, wherein the nucleotide sequence of the pJEV-T7-NS5 plasmid in step (1) is shown in SEQ ID NO. 1.

3. The method of claim 1, wherein the nucleotide sequences P2A-P30-T2A, P A-P54-T2A and P2A-B602L-T2A in step (2) are shown in SEQ ID NO. 3-5, respectively.

4. A method of construction according to claim 3, wherein the vaccine adjuvant comprises an ionizable lipid, distearoyl phosphatidylcholine, cholesterol and PEG-lipid; the mass mole ratio of the ionizable lipid, distearoyl phosphatidylcholine, cholesterol and PEG-lipid is (48-52): (8-12): (37-40): (1-2).

5. The method of claim 4, wherein the ratio of linear mRNA to total adjuvant is 1:35 to 45.

6. The method of claim 5, wherein the linear mRNA is mixed with a vaccine adjuvant and dialyzed to obtain the taRNA vaccine.

7. An african swine fever taRNA vaccine obtained by the construction method of any one of claims 1 to 6.

8. Use of an african swine fever taRNA vaccine of claim 7 in the preparation of a medicament for preventing african swine fever virus.