CN114989266A

CN114989266A - African swine fever virus pA104R protein immunosuppression related amino acid site and application thereof

Info

Publication number: CN114989266A
Application number: CN202210721017.0A
Authority: CN
Inventors: 王湘如; 陈启超; 李亮; 国师榜; 刘占悝; 张逸博; 牟双; 余怡丰; 陈焕春
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2022-06-16
Filing date: 2022-06-16
Publication date: 2022-09-02
Anticipated expiration: 2042-06-16
Also published as: CN114989266B

Abstract

The invention discloses an African swine fever virus pA104R protein immunosuppression related amino acid site and application thereof, belonging to the technical field of biology. The invention finds that amino acid sites related to the African swine fever virus pA104R protein immunosuppression are Arg 69, His 72, Lys 92, Arg 94 and Lys 97, and the pA104R protein immunosuppression capability after mutation of the sites is obviously weakened. The sites can be used as gene editing sites to weaken African swine fever virus and as anti-African swine fever virus targets to screen antiviral drugs. The site-mutated pA104R protein can be used for preparing an African swine fever vaccine, and the introduction of the mutated pA104R in the vaccine can play the immune protection function of the protein, eliminate the immunosuppressive property of the protein and realize better protection effect.

Description

African swine fever virus pA104R protein immune suppression related amino acid site and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an African swine fever virus pA104R protein immunosuppressive related amino acid site and application thereof.

Background

African Swine Fever (ASF) is an infectious, septic disease characterized by high fever, toxemia, hemorrhagic diathesis and high mortality. Is a virulent infectious disease caused by African Swine Fever Virus (ASFV) (s.blome et al, 2020). The disease is listed as a legal report disease by the world animal health Organization (OIE), and China also lists the disease as a type of animal epidemic disease for key prevention.

In 1921 ASF first appeared in kenya in africa and then spread rapidly in multiple countries and regions around the world. Since China is the biggest pork producing country and consumer country in the world, the import of ASF has a serious influence on China, and huge economic loss is caused to the pig raising industry.

Although vaccination is an ideal method for controlling disease in most animals (Emad BeshirAta et al, 2020). However, due to the complexity of the viral genome and viral composition, unclear mechanisms of immunity and infection are major obstacles that hinder vaccine development. The attenuated live vaccine with gene deletion can induce a certain protection effect, but has adverse clinical reactions including arthritis and pneumonia which are concomitant with some immune animals, and the risk of strain return (P.J.S. lnchez-Cord Lolo n et al, 2017). Whereas relatively safe inactivated virus particles are not able to withstand virus attacks (s.blome et al, 2014). To date, there is no commercial vaccine and effective antiviral drug available for prevention and control of ASFV infection. Therefore, by studying the functions of the key genes of the virus, analyzing the roles of the key genes in the process of body immunity is a key step for developing vaccines.

The body's innate immune response is the first line of defense against viral invasion and is critical to prevent viral infection and to clear the virus, with type I interferon (IFN-I) being an important aspect of the innate immune response. IFN-I can act on most cells and induce an antiviral state, increase MHC expression, induce the production of chemokines and cytokines, and coordinate with each other to promote an immune response. IFN-I is combined with a specific receptor on a cell membrane to initiate a cascade signal amplification process and start a JAK-STAT signal channel, and the combination of IFN and the receptor activates JAK1 and TYK2, so that STAT1 and STAT2 are phosphorylated to form heterodimer, IRF9 is recruited to form ISGF3 to enter a nucleus to be combined with an interferon stimulation response element ISRE, the expression of interferon stimulation genes ISGs is promoted, natural immune response is promoted, and an antiviral function is exerted.

Viruses have also developed effective strategies and mechanisms to escape host innate immunity over long periods of evolution. Research has shown that ASFV genome can code several proteins, which can control the expression of host cell protein and interfere with the host natural immune system, thus inhibiting and evading the host immune response, and creating advantages for self-proliferation and diffusion (Dixon et al, 2004; Luisa et al, 2016). Among them, the multigene family proteins MGF360, MGF505/530, DP96R and I329L can inhibit the type I interferon signal pathway and escape the host anti-infection immunity. Revealing the underlying mechanisms of interaction of ASFV with the host is therefore crucial for the development of effective ASFV vaccines and drugs.

pA104R is a structural protein encoded by African swine fever virus with histone-like characteristics, involved in DNA replication, transcription and genome packaging of the virus, and is a protein necessary for ASFV replication. pA104R is highly immunogenic and can induce higher antibody levels in virus-infected animals, and is considered to be a valuable vaccine candidate gene. Animal immunization using pA104R as an antigen has been reported, but no good protective effect has been achieved.

Disclosure of Invention

The invention discovers that pA104R can escape from the host to control natural immunity, block IFN-I signal conduction, inhibit the expression of interferon stimulating genes and provide favorable conditions for the proliferation of viruses. Further, the invention discovers the key amino acid position of pA104R for playing the immunosuppressive function, so that the immunosuppressive capability of pA104R is obviously weakened after the mutation, and the mutation does not influence the expression of the protein.

The invention mainly aims at providing an amino acid site related to the immunosuppression of the African swine fever virus pA104R protein, and another aim at providing an African swine fever virus pA104R mutant protein, and still another aim at providing application of the amino acid site or the mutant protein.

The purpose of the invention is realized by the following technical scheme:

an amino acid site related to the immunosuppression of the African swine fever virus pA104R protein is Arg 69, His 72, Lys 92, Arg 94 and Lys 97 of the pA104R protein. The amino acid sequence of the pA104R protein is as follows:

MSTKKKPTITKQELYSLVAADTQLNKALIERIFTSQQKIIQNALKHNQEVIIPPGIKFTVVTVKAKPARQGHNPATGEPIQIKAKPEHKAVKIRALKPVHDMLN(SEQ ID NO.1)。

an African swine fever virus pA104R mutant protein is a pA104R protein with one or more of the above-mentioned sites mutated. Furthermore, one or more of the amino acids 69, 72, 92, 94 and 97 of the African swine fever virus pA104R mutant protein is mutated into Asp, Glu or Ala and the like.

The African swine fever virus pA104R mutant protein can also be pA104R protein lacking the one or more sites, or pA104R protein lacking a fragment containing the one or more sites.

The application of the amino acid sites as gene editing sites, wherein one or more of the amino acid sites are mutated in the ASFV through gene editing, so as to weaken the immunosuppressive property of pA104R and further weaken the ASFV.

The application of the amino acid sites as anti-ASFV targets screens compounds or small molecule drugs which can target one or more of the amino acid sites, and eliminates the ASFV immunosuppressive property and enhances the antiviral immunity of organisms by targeting the sites.

The African swine fever virus pA104R mutant protein is applied to preparation of African swine fever vaccines, and the vaccines comprise ASFV attenuated vaccines, subunit vaccines, DNA vaccines, mRNA vaccines, virus vector vaccines and the like. The mutation pA104R introduced into the vaccine can play the immune protection function of the protein, and the immune suppression property of the protein is eliminated, so that the better protection effect is realized.

An African swine fever vaccine which can express the African swine fever virus pA104R mutant protein.

An anti-ASFV drug capable of targeting one or more of the above amino acid positions.

The invention has the advantages and beneficial effects that: the invention discovers the amino acid sites related to the African swine fever virus pA104R protein immunosuppression, provides a new material for preparing an African swine fever vaccine, and provides a new direction for preparing an anti-African swine fever virus medicament.

Drawings

FIG. 1 is a result of the immunogenic and immunosuppressive properties of pA 104R. A: western immunoblot results, B: dual luciferase assay results for ISRE promoter activity, C: fluorescent quantitative PCR results, wherein Vector, A104R are cells transfected with pCAGGS-HA empty and pCAGGS-HA-A104R plasmids, respectively.

FIG. 2 is the result of the determination of the immunosuppressive functional target of pA 104R. A: western immunoblot results, B: indirect immunofluorescence results, C: DNA Pulldown results, D: dual luciferase assay results for ISRE promoter activity, E: and (5) fluorescent quantitative PCR result.

FIG. 3 is the result of immunogenicity of the pA104R amino acid site mutein. The immune serum is the serum of mice immunized by the pA104R mutant protein, and the control serum is immunized by PBS as a control.

Detailed Description

The following examples are intended to further illustrate the present invention and should not be construed as limiting the present invention, 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 shall be included within the scope of the present invention. Unless otherwise specified, the technical means used in the detailed description are conventional means well known to those skilled in the art.

TABLE 1 primers used in the examples described below

Example 1pA104R having immunogenic and immunosuppressive properties

Amplifying an A104R gene (an amplification primer is HA-A104R-F, HA-A104R-F) by taking ASFV inactivated nucleic acid as a template, inserting the ASFV inactivated nucleic acid between restriction enzymes EcoRI and XhoI of a pCAGGS-HA vector to obtain a pCAGGS-HA-A104R plasmid, amplifying the pCAGGS-HA-A104R plasmid by using escherichia coli after sequencing comparison without errors, and extracting the plasmid by using an Omega endotoxin-removing plasmid extraction kit.

HEK293T cells were plated on corresponding cell culture dishes, and when 80% of the cells grew, pCAGGS-HA-A104R plasmid and pCAGGS-HA no-load control were transfected into the cells, plasmid Transfection was performed using the jetPRIME Versatile DNA/siRNA Transfection reagent from Polyplus Transfection company, and the procedure was performed according to the instruction. After 24h of transfection, the medium was discarded, cell lysate containing protease inhibitors and phosphatase inhibitors was added to the cells, after sufficient lysis on ice, the lysate was gently scraped off with a cell scraper and transferred to a 1.5mL centrifuge tube and centrifuged at 15000g at 4 ℃ for 10min to collect the supernatant as a cell protein sample, and SDS-PAGE gel electrophoresis and western blotting were performed: preparing 10-15% separation gel and 5% concentration gel, adding Tris glycine electrophoresis buffer, running at 80V, adjusting the voltage to 120V when bromophenol blue is indicated to enter the separation gel, and stopping when a target band is transferred to a proper position. After the electrophoresis was completed, the gel and PVDF membrane were loaded into the electrophoresis tank according to the instructions of the Bio-RAD electrophoresis apparatus. The transfer condition was constant current 330mA transfer for 1 h. The PVDF membrane to which the proteins were transferred was blocked in TBST (containing 5% BSA) at room temperature for 2h, followed by incubation with ASFV-positive serum as an antibody and development with a chemiluminescent imaging system. The result is shown in figure 1-A, compared with the control group pA104R, the control group pA104R can have strong reaction with positive serum, which indicates that pA104R has better immunogenicity and can stimulate the body to generate immune response in the process of virus infection.

ISRE promoter activity was detected by dual luciferase assay: the pCAGGS-HA-A104R plasmid and the pCAGGS-HA no-load plasmid are transfected into HEK293T cells co-transfected with pISRE-Luc luciferase plasmid and pRL-TK internal reference plasmid, IFN-alpha (1000U/mL) is added after 24h of transfection to stimulate for 8h, and ISRE promoter activity detection is carried out according to the specification of a dual-luciferase detection kit of Promega. The experimental results are shown in FIG. 1-B, compared with control group pA104R, ISRE promoter activity can be obviously inhibited in the dual-luciferase experiment, so pA104R has the characteristic of inhibiting IFN-I signal transduction.

To further confirm inhibition of pA104R on host innate immunity, IFN- α (1000U/mL) was added to HEK293T cells transfected with pCAGGS-HA 104R plasmid and pCAGGS-HA for 8h, the medium was discarded, the cells were washed with pre-cooled sterile PBS 3 times and the waste solution was discarded, 1mL of TRIpure Reagent (Beijing Edley Biotech Co., Ltd.) was added to the cells, the cells were lysed thoroughly and transferred to a RNase-free 1.5mL centrifuge tube for RNA extraction according to the product instructions. After the concentration and purity of the obtained total RNA were measured, a HiScript II Q RT Supermix for qPCR reverse transcription kit (Nanjing Novozan Biotech Co., Ltd.) was used to prepare a cDNA template and to perform qPCR to detect gene expression: based on the coding sequences of the target genes ISG54, ISG56 and OAS1 searched in NCBI database, Beacon Dispner 8 software was used to design primers suitable for SYBR fluorescent quantitative PCR (Table 1), and the annealing temperature of the primers wasThe unified setting is 60 ℃, the length of the amplification product is limited to 80-200bp, the length of the primer is limited to 18-24nt, and GAPDH is used as the reference gene. Each sample is repeated for 3 times, after the reaction is finished, the dissolution curve analysis is carried out by using the matched software of the fluorescence quantitative PCR instrument, and

the method analyzes relative gene expression difference. The experimental results are shown in figure 1-C, and the expression of ISGs in the cells of the control group can be significantly increased under the action of IFN α, but the expression of ISGs in the pA104R cell transfection group is significantly inhibited. This is consistent with the results of the dual luciferase assay and thus it was determined that pA104R has immunosuppressive activity.

Example 2pA104R determination of immunosuppressive functional targets

The transmission of IFN-I signals is realized by that STAT1 and STAT2 are phosphorylated in cytoplasm to form heterodimer, IRF9 is recruited to form ISGF3 heterotrimer, and then the heterotrimer enters the nucleus to play an immune role. Thus to determine the target of action of pA104R, STAT1, STAT2 and IRF9 protein levels and phosphorylation levels were first examined. The plasmid pCAGGS-HA-A104R and pCAGGS-HA empty load control were transfected into HEK293T cells, and protein samples were collected after 2h of IFN alpha (1000U/mL) stimulation for WB experiments, and the results are shown in FIG. 2-A, which indicates that pA104R HAs no effect on STAT1, STAT2 and IRF 9. Indirect immunofluorescence experiments were also performed under the same conditions: HEK293T cells were plated into confocal culture dishes, transfected with pCAGGS-HA-A104R plasmid for 24h, treated with IFN α for 2h, then the supernatant was discarded, and washed 1-2 times with pre-cooled PBS. Adding 4% paraformaldehyde for fixation for 20 min. After washing, 0.25% Triton X-100 was added for 15 min. After washing, blocking was performed by adding 5% BSA (PBS dilution) for 1 h. And adding the antibody after washing, incubating for 1h, washing for 3 times, adding DAPI, incubating for 5min, washing again, and observing under a laser confocal microscope. As a result, as shown in FIG. 2-B, STAT1 was localized in cells by cytoplasmic translocation to the nucleus under IFN α -stimulated conditions, whereas the presence or absence of pA104R did not affect this, so that inhibition of innate immunity by pA104R did not act on ISGF3 protein phosphorylation and nuclear trafficking.

After entering the cell nucleus, ISGF3 trimer needs to be combined with a specific DNA sequence (an interferon stimulation response element ISRE) and then starts the expression of an interferon stimulation gene ISGs to play an antiviral immune role. Since pA104R HAs DNA binding property, and it is suspected whether pA104R binds to ISRE through the DNA binding property thereof, so as to antagonize the blocking of signal transmission caused by the binding of ISGF3 and ISRE, pEGFP-N1-STAT1, pEGFP-N1-STAT2 and pEGFP-N1-IRF9 plasmids (human STAT1, STAT2 and IRF9 gene sequences are cloned into pEGFP-N1 plasmids to enable the corresponding expression of STAT1, STAT2 and IRF9 proteins) and pCAGGS-HA-A104R plasmids are co-transferred in HEK293T cells, and IFN alpha (1000U/mL) is added after 24h of transfection to stimulate 8h to collect protein samples for DNA pulldown experiment: biotin-labeled ISRE sequence Biotin-ISRE-F, Biotin-ISRE-R (table 1) was synthesized by seiry corporation, mikroorganism, inc, diluted to 100 μ M, mixed at 1:1, denatured at 100 ℃ for 1h, then naturally annealed to form double-stranded DNA, added with streptavidin magnetic beads from MCE corporation, placed at 4 ℃ for rotary incubation for 4-6h, washed 5 times, and detected by WB experiments. As a result, as shown in FIG. 2-C, the biotin-labeled ISRE was able to bind to ISGF3 normally, but was not affected by pA104R, and thus pA104R did not inhibit innate immunity by DNA binding ability.

Although inhibition of natural immunity by pA104R is independent of its DNA binding properties, amino acid position mutations (amino acids 69, 72, 92 and 94, 97) associated with DNA binding of the pA104R protein can revert to its immunosuppressive capacity. We constructed the pA104R protein amino acid point mutation plasmid: the plasmid is subjected to point mutation by a PCR method, a corresponding point mutation primer (table 1) is adopted to amplify by taking pCAGGS-HA-A104R wild-type plasmid as a template, an amplification product is subjected to enzyme digestion by Dpn I enzyme, the enzyme digestion product is transformed into a competent cell DH5 alpha, and a mutant plasmid pCAGGS-HA-A104R-R/H69/72D (namely that the 69 th and 72 th amino acids of the pA104R protein are mutated into Asp) and pCAGGS-HA-A104R-K/R92/94/97E (namely that the 92 th, 94 th and 97 th amino acids of the pA104R protein are mutated into Glu) are constructed. The wild type pCAGGS-HA-A104R, mutant plasmids pCAGGS-HA-A104R-R/H69/72D and pCAGGS-HA-A104R-K/R92/94/97E were subjected to a dual luciferase experiment and a qPCR experiment, and ISRE promoter activity and ISGs expression were detected (the same method as above). The results are shown in FIG. 2-D, pA104R was able to inhibit ISRE promoter activity in the dual luciferase assay, whereas the mutation of pA104R was able to significantly revert to this inhibition. The mutation of pA104R also significantly reverted the inhibition of ISGs expression by pA104R as shown in fig. 2-E in the qPCR experiment results, and in addition, the mutation of amino acid position of pA104R protein was found not to affect normal expression of the protein in the WB results of the dual luciferase experiment as shown in fig. 2-D. Thus, the amino acid sites (amino acids 69, 72, 92 and 94, 97) of pA104R related to DNA binding for inhibiting natural immunity are closely related to the function of pA104R for immunosuppression, which is probably that pA104R inhibits natural immunity through apparent modification, and the amino acids 69, 72, 92 and 94, 97 are the key sites for apparent modification.

Example 3 amino acid site mutein of pA104R is immunogenic

In order to verify whether the immunogenicity of the pA104R amino acid site mutant protein is damaged, a pA104R amino acid site mutant prokaryotic expression plasmid is constructed for prokaryotic expression: two pairs of mutation primers are used for sequentially carrying out two rounds of point mutation on pCAGGS-HA-A104R plasmid to obtain plasmid with mutated amino acids at 69 th, 72 th, 92 th, 94 th and 97 th positions of pA104R, the plasmid is used as a template to amplify pA104R sequence with mutated amino acid positions by using primers His-A104R-F, His-A104R-R (table 1), the sequence is inserted between restriction enzyme BamHI and XhoI enzyme cutting positions of PET-30a vector to obtain PET-30a-A104R plasmid, competent cells BL21(DE3) are transformed, positive bacteria liquid is inoculated into LB liquid culture medium containing antibiotics according to the ratio of 1:100, the mixture is placed at 37 ℃ for shake culture for 2-3h at constant temperature, and is cultured until OD is OD ₆₀₀ Adding IPTG to a final concentration of 0.6mM and shaking-culturing at 16 deg.C for 16-20 h. The cells were centrifuged at 4000g and 4 ℃ for 5min and the cells were resuspended in 1/10 cell volume of binding buffer. And (3) crushing the thalli at 4 ℃ by using a low-temperature ultrahigh-pressure cell crusher, repeatedly crushing for 3-5 times, centrifuging for 10min at 8000g and 4 ℃, collecting supernatant, and purifying the protein according to the steps of a GE nickel affinity chromatography column. The purified His-A104R protein was well emulsified with equal volume of protein and Freund's complete adjuvant, and the primary immunization was performed by subcutaneous multi-point injection of 50. mu.g of protein into the back and neck of 6-week-old female BALB/c mice, followed by 10-day intervals and then by incomplete Freund's injectionCarrying out secondary-immunization and tertiary-immunization on the same amount of protein emulsified by the whole adjuvant, then collecting blood from tail veins, and carrying out indirect ELISA detection: with coating solution (1.59g NaCO) ₃ ，2.93g NaHCO ₃ Adding appropriate amount of ddH ₂ After dissolving O, 1L of diluted antigen is added, and an ELISA reaction plate is coated with 100. mu.L/well and placed at 4 ℃ overnight. Antigen solution was discarded, washed 3 times with PBST (0.1% Tween-20 in PBS), 200. mu.L per well, and gently shaken at room temperature for 5 min. Liquid in the holes is thrown away as far as possible. 5% skim milk in PBS was used as a blocking solution overnight at 4 ℃. The blocking solution was discarded, PBST was washed 3 times, and the immunized mouse serum and the blank mouse serum were each diluted with PBS in a gradient, added to an ELISA reaction plate in an amount of 100. mu.L per well, and incubated at 37 ℃ for 1 hour. Serum was discarded, PBST washed 3 times, goat anti-mouse HRP-IgG enzyme-labeled secondary antibody diluted 1/8000 was added, 100. mu.L/well, and incubated at 37 ℃ for 30 min. Enzyme-labeled secondary antibody is discarded, PBST is washed for 3 times, and finally liquid in the holes is sequentially dried as much as possible. Adding TMB developing solution, and developing at room temperature in dark for 10 min. The results are shown in fig. 3, and after the third immunization, the antibody titer of the mice can reach 1: 500000, indicating that the immunogenicity of the pA104R protein after amino acid position mutation is not affected. Therefore, pA104R with the mutation of the immunosuppression-related site is a vaccine candidate gene with great potential, and the negative influence caused by the immunosuppressive property of the gene is avoided while the body immune function is stimulated.

Sequence listing

<110> university of agriculture in Huazhong

<120> African swine fever virus pA104R protein immunosuppression related amino acid site and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 104

<212> PRT

<213> African swine fever virus

<400> 1

Met Ser Thr Lys Lys Lys Pro Thr Ile Thr Lys Gln Glu Leu Tyr Ser

1 5 10 15

Leu Val Ala Ala Asp Thr Gln Leu Asn Lys Ala Leu Ile Glu Arg Ile

20 25 30

Phe Thr Ser Gln Gln Lys Ile Ile Gln Asn Ala Leu Lys His Asn Gln

35 40 45

Glu Val Ile Ile Pro Pro Gly Ile Lys Phe Thr Val Val Thr Val Lys

50 55 60

Ala Lys Pro Ala Arg Gln Gly His Asn Pro Ala Thr Gly Glu Pro Ile

65 70 75 80

Gln Ile Lys Ala Lys Pro Glu His Lys Ala Val Lys Ile Arg Ala Leu

85 90 95

Lys Pro Val His Asp Met Leu Asn

100

Claims

1. An amino acid site related to immunosuppression of African swine fever virus pA104R protein, which is characterized in that: arg 69, His 72, Lys 92, Arg 94 and Lys 97 of pA104R protein; the amino acid sequence of the pA104R protein is shown in SEQ ID NO. 1.

2. An African swine fever virus pA104R mutant protein, which is characterized in that: the pA104R protein mutated at one or more of the amino acid positions recited in claim 1.

3. The African swine fever virus pA104R mutein of claim 2, wherein: one or more mutations in the amino acid sites are Asp, Glu or Ala.

4. The African swine fever virus pA104R mutein of claim 2, wherein: a pA104R protein that is a deletion of one or more of the amino acid positions of claim 1, or a pA104R protein that is a deletion of a fragment containing one or more of the amino acid positions.

5. Use of the amino acid site of claim 1 as a gene editing site.

6. The use of the amino acid site of claim 1 as a target against African swine fever virus.

7. Use of the pA104R mutein of any one of claims 2 to 4 for the preparation of an African swine fever vaccine.

8. Use according to claim 7, characterized in that: the vaccine comprises ASFV attenuated vaccine, subunit vaccine, DNA vaccine, mRNA vaccine and virus vector vaccine.

9. An African swine fever vaccine, which is characterized in that: capable of expressing the pA104R mutein of any one of claims 2 to 4.

10. A drug for resisting African swine fever virus is characterized in that: which is capable of targeting one or more of the amino acid positions of claim 1.