CN114989266B - African swine fever virus pA104R protein immunosuppression related amino acid site and application thereof - Google Patents

Abstract

The invention discloses an African swine fever virus pA104R protein immunosuppression related amino acid site and application thereof, and belongs to the technical field of biology. The invention discovers that the amino acid sites related to the immunosuppression of the pA104R protein of the African swine fever virus are Arg 69, his 72, lys 92, arg 94 and Lys 97, and the immunosuppression capability of the pA104R protein after the mutation of the sites is obviously weakened. The loci can be used as gene editing loci for weakening African swine fever virus and as anti-African swine fever virus targets for screening antiviral drugs. The pA104R protein with the site mutation can be used for preparing African swine fever vaccines, and the mutant pA104R can play the immunoprotection function of the protein by introducing the mutant pA104R into the vaccine, so that the protein has the immunosuppression characteristic and a better protection effect is realized.

Description

African swine fever virus pA104R protein immunosuppression 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 immunosuppression 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 world animal health Organization (OIE) is listed as a legal report of diseases, which is also listed as a major precaution class of animal diseases in China.

ASF was first shown in the Kennel of Africa in 1921 and then rapidly spread across multiple countries and regions worldwide. Because China is the largest pork producing country and consuming country in the world, the ASF is particularly seriously affected by the introduction of ASF into China, and huge economic loss is caused to pig industry.

Although vaccination is an ideal method for controlling most animal diseases (Emad BeshirAta et al, 2020). However, due to the complexity of viral genomes and viral compositions, the mechanism of immunity and infection is not clear and is a major obstacle to vaccine development. Although the attenuated live vaccine with the gene deletion can induce a certain protective effect, adverse clinical reactions exist at the same time, including arthritis and pneumonia which are accompanied by some immunized animals and the risk of strong strain return (P.J.S. NChez-Cord delta n et al, 2017). Relatively safe inactivated viral particles are not resistant to viral attack (s.blome et al, 2014). To date, there are no commercial vaccines and effective antiviral drugs available for preventing and controlling ASFV infections. Therefore, by studying the function of the key genes of viruses, it is a key step in developing vaccines to analyze their role in the immune process of the organism.

The natural immune response of the body is the first line of defense of the body against viral entry and is critical to prevent viral infection and to clear the virus, with type I interferon (IFN-I) being an important aspect of the natural immune response. IFN-I can act on most cells and induce antiviral state, increase MHC expression, induce production of chemokines and cytokines, and coordinate and promote immune response. IFN-I binds to specific receptors on cell membranes, initiates a cascade of signal amplification processes, initiates a JAK-STAT signaling pathway, IFN binds to the receptors to activate JAK1 and TYK2, phosphorylates STAT1 and STAT2 to form heterodimers, and recruits IRF9 to form ISGF3 into the nucleus to bind to the interferon stimulating response element ISRE, promote expression of the interferon stimulating gene ISGs, promote natural immune response and exert antiviral function.

Viruses also form an effective strategy and mechanism for escaping host innate immunity during long-term evolution. The ASFV genome has been shown to encode a variety of proteins to regulate host cell protein expression, interfere with the host's innate immune system, thereby suppressing and evading the host's immune response, creating advantages for self-proliferation, diffusion (Dixon et al, 2004; luisa et al, 2016). Wherein, such as multi-gene family proteins MGF360, MGF505/530, DP96R and I329L can inhibit I-type interferon signal paths and escape host anti-infection immunity. Thus revealing the potential mechanism by which ASFV interacts with the host is critical to the development of effective ASFV vaccines and pharmaceuticals.

pA104R is a structural protein with histone-like characteristics encoded by African swine fever virus, and is involved in viral DNA replication, transcription, and genome packaging, a protein necessary for ASFV replication. pA104R is strongly immunogenic and is capable of inducing higher antibody levels in virally infected animals and is considered a very valuable vaccine candidate. Animal immunization with pA104R as an antigen has been reported, but no good protective effect was obtained.

Disclosure of Invention

The invention discovers that pA104R can escape from a host to produce natural immunity, block IFN-I signal transduction, inhibit the expression of interferon stimulated genes and provide favorable conditions for the proliferation of viruses. Furthermore, the invention discovers a key amino acid site of pA104R which plays an immunosuppressive function, the immunosuppressive ability of pA104R is obviously weakened after the mutation, and the mutation does not influence the expression of the protein.

The primary object of the present invention is to provide an amino acid site related to the immunosuppression of the African swine fever virus pA104R protein, another object of the present invention is to provide an African swine fever virus pA104R mutant protein, and still another object of the present invention is to provide an application of the amino acid site or mutant protein.

The aim of the invention is achieved by the following technical scheme:

amino acid sites related to the immunosuppression of the African swine fever virus pA104R protein are 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 mutein is a pA104R protein mutated in one or more of the above sites. Furthermore, one or more of 69, 72, 92, 94 and 97 amino acids of the African swine fever virus pA104R mutant protein are mutated into Asp, glu or Ala and the like.

The African swine fever virus pA104R mutant protein may also be a pA104R protein deleted at one or more of the above sites, or a pA104R protein deleted in a fragment containing one or more of the above sites.

The use of the above amino acid sites as gene editing sites, wherein one or more of the amino acid sites is mutated in ASFV by gene editing to attenuate the immunosuppressive properties of pA104R and thereby attenuate the ASFV.

The application of the amino acid sites as anti-ASFV targets, screening compounds or small molecule drugs capable of targeting one or more of the amino acid sites, and eliminating ASFV immunosuppression characteristics by targeting the sites to enhance the antiviral immunity of the organism.

The application of the African swine fever virus pA104R mutant protein in preparing African swine fever vaccine comprises an ASFV attenuated vaccine, a subunit vaccine, a DNA vaccine, an mRNA vaccine, a viral vector vaccine and the like. The mutant pA104R introduced into the vaccine can play the immunoprotection function of the protein, eliminate the immunosuppressive property of the protein and realize better protection effect.

An african swine fever vaccine capable of expressing the african swine fever virus pA104R mutein described above.

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

The invention has the advantages and beneficial effects that: the invention discovers that the African swine fever virus pA104R protein immunosuppression related amino acid locus provides a new material for preparing African swine fever vaccine and provides a new direction for preparing African swine fever virus resistant medicines.

Drawings

FIG. 1 shows the results of the immunogenicity and immunosuppressive properties of pA 104R. A: western blot results, B: double luciferase assay detection ISRE promoter activity results, C: fluorescent quantitative PCR results, wherein Vector, A104R are cells transfected with pCAGGS-HA empty and pCAGGS-HA-A104R plasmids, respectively.

FIG. 2 shows the results of the determination of the pA104R immunosuppressive function target. A: western blot results, B: indirect immunofluorescence results, C: DNA Pulldown results, D: double luciferase assay detection ISRE promoter activity results, E: fluorescent quantitative PCR results.

FIG. 3 shows the result of immunogenicity of the pA104R amino acid site mutein. Wherein the immune serum is the serum of the mice immunized by the immune pA104R mutant protein, and the control serum is immunized by PBS (phosphate buffered saline) as a control.

Detailed Description

The following examples are provided to further illustrate the present invention and should not be construed as limiting the invention, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the invention are intended to be equivalent substitutes. The technical means used in the detailed description are conventional, if not specified, procedures well known to those skilled in the art.

TABLE 1 primers used in the examples below

Example 1pA104R has immunogenic and immunosuppressive properties

The ASFV inactivated nucleic acid is used as a template to amplify the A104R gene (the amplification primer is HA-A104R-F, hA-A 104R-F), the gene is inserted between restriction enzyme EcoRI and XhoI cleavage sites of a pCAGGS-HA vector to obtain a pCAGGS-HA-A104R plasmid, the pCAGGS-HA-A104R plasmid is amplified by using escherichia coli after sequencing and comparison, and the plasmid is extracted by using an Omega endotoxin removal plasmid extraction kit.

HEK293T cells were plated into corresponding cell culture dishes, and when the cells grew to 80%, pCAGGS-HA-A104R plasmid and pCAGGS-HA empty control were transfected into the cells, plasmid transfection was performed using jetPRIME Versatile DNA/siRNA transfection reagent of Polyplus Transfection and performed according to the instructions. After 24h transfection, the medium was discarded, a 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 the supernatant was collected as a cell protein sample by centrifugation at 15000g for 10min at 4 ℃, and subjected to SDS-PAGE gel electrophoresis and western immunoblotting: 10-15% of separation gel and 5% of concentrated gel are prepared, tris glycine electrophoresis buffer solution is added to operate at 80V, the voltage is adjusted to 120V when bromophenol blue indicates that the separation gel is entered, and the target strip is stopped when the target strip is migrated to a proper position. After the electrophoresis, the gel and PVDF membrane were loaded into an electrophoresis tank according to the instructions of a Bio-RAD electrophoresis apparatus. The transfer condition is a constant current 330mA transfer for 1h. The protein-transferred PVDF membrane was blocked in TBST (containing 5% BSA) at room temperature for 2 hours, followed by incubation with ASFV-positive serum as antibody, and color development was performed using a chemiluminescent imaging system. As shown in FIG. 1-A, pA104R was able to react strongly with positive serum compared to the control group, demonstrating that pA104R was more immunogenic and was able to stimulate an immune response in the body during viral infection.

ISRE promoter activity was detected by double luciferase assay: the pCAGGS-HA-A104R plasmid and the pCAGGS-HA empty plasmid were transfected into HEK293T cells co-transfected with the pIRRE-Luc luciferase plasmid and the pRL-TK internal reference plasmid, and after 24 hours of transfection, IFN- α (1000U/mL) was added to stimulate for 8 hours, and ISRE promoter activity was detected by referring to Promega company double luciferase assay kit instructions. As shown in the experimental results in FIG. 1-B, compared with the control group pA104R in the double luciferase experiment, the promoter activity of the ISRE can be obviously inhibited, so that the pA104R has the characteristic of inhibiting IFN-I signal transduction.

To confirm further inhibition of native host immunity by pA104R, IFN- α (1000U/mL) was added to HEK293T cells transfected with pCAGGS-HA-A104R plasmid and pCAGGS-HA empty, and the cells were washed 3 times with pre-chilled sterile PBS, the waste liquid was discarded, 1mL TRIpure Reagent reagent (Beijing Edley Biotechnology Co., ltd.) was added to the cells, and after sufficient lysis, the cells were transferred to an RNase-free 1.5mL centrifuge tube for RNA extraction according to the product instructions. A cDNA template was prepared using HiScript II Q RT SuperMix for qPCR reverse transcription kit (Nanjinouzan Biotechnology Co., ltd.) after the concentration and purity of the obtained total RNA were measured and qPCR was performed to detect gene expression: according to the coding sequences of target genes ISG54, ISG56 and OAS1 searched in NCBI database, using Beacon identifier 8 software to make primer design (table 1) suitable for SYBR method fluorescence quantitative PCR, uniformly setting primer annealing temperature to 60 ℃, amplification product length to 80-200bp, primer length to 18-24nt, and using GAPDH as reference gene. 3 repeats are carried out on each sample, after the reaction is finished, the dissolution curve analysis is carried out by using software matched with a fluorescent quantitative PCR instrument, and the analysis is carried out byThe relative gene expression differences were analyzed by the method. The experimental results are shown in FIG. 1-C, where ISGs expression in control cells can be significantly increased under the action of IFNα, but ISGs expression in pA104R transfected cells was significantly inhibited. This is consistent with the results of the dual luciferase assay, and thus it was determined that pA104R has immunosuppressive function.

Example 2pA104R immunosuppression functional target determination

Since IFN-I signaling is primarily a heterodimer of STAT1 and STAT2 phosphorylated in the cytoplasm and recruited to IRF9 to form an ISGF3 heterotrimer and then into the nucleus for immunization. Thus, to determine the action target of pA104R, STAT1, STAT2 and IRF9 protein levels as well as phosphorylation levels were first examined. Transfection of the pCAGGS-HA-A104R plasmid and pCAGGS-HA empty control in HEK293T cells protein samples were harvested 2h after stimulation with IFNα (1000U/mL) for WB experiments, as shown in FIG. 2-A, indicating that pA104R had no effect on STAT1, STAT2 and IRF 9. An indirect immunofluorescence experiment was performed again under the same conditions: HEK293T cells were inoculated into confocal dishes, transfected with pCAGGS-HA-A104R plasmid for 24h, treated with IFNα for 2h, the supernatant was discarded, and washed 1-2 times with pre-chilled PBS. 4% paraformaldehyde was added for fixation for 20min. After washing, 0.25% Triton X-100 was added for 15min. After washing, the mixture was blocked by adding 5% BSA (diluted with PBS) for 1h. After washing, the antibody was added for incubation for 1h, after washing 3 times, DAPI was added for incubation for 5min, and then washed again, followed by observation under a laser confocal microscope. As a result, as shown in fig. 2-B, STAT1 localization in cells under ifnα stimulation was transferred from cytoplasm to nucleus, whereas the presence or absence of pA104R had no effect on this, and thus inhibition of natural immunity by pA104R was not responsible for ISGF3 protein phosphorylation and nuclear transport.

After entering the 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 immunity role. Because pA104R has DNA binding property, it is hypothesized whether pA104R binds to ISRE through its DNA binding property, thus antagonizing blocking of signaling caused by binding of ISGF3 to ISRE, pEGFP-N1-STAT1, pEGFP-N1-STAT2 and pEGFP-N1-IRF9 plasmids were co-transformed in HEK293T cells (cloning human STAT1, STAT2, IRF9 gene sequences into pEGFP-N1 plasmids to enable corresponding expression of STAT1, STAT2, IRF9 proteins) and pCAGGS-HA-A104R plasmids, and after 24h transfection, adding IFN alpha (1000U/mL) to stimulate 8h protein collection samples for DNA pulldown experiments: biotin-labeled ISRE sequence Biotin-ISRE-F, biotin-ISRE-R (Table 1) was synthesized by Nanjing Jinsrey corporation, complementary single-stranded DNA was diluted to 100. Mu.M, mixed at 1:1, denatured at 100℃for 1h, then naturally annealed to form double-stranded DNA, and the streptavidin magnetic beads added to MCE corporation were subjected to spin incubation at 4℃for 4-6h, then washed 5 times, and then detected by WB assay. As a result, as shown in FIG. 2-C, biotin-labeled ISRE was able to bind to ISGF3 normally, but was not affected by pA104R, so pA104R did not suppress innate immunity through DNA binding capacity.

Although inhibition of pA104R against innate immunity is independent of its DNA binding properties, mutations at amino acid positions (amino acids 69, 72, 92 and 94, 97) associated with DNA binding of pA104R protein are able to restore its immunosuppressive capacity. We constructed pA104R protein amino acid point mutation plasmid construction: the plasmid is subjected to point mutation by a PCR method, a corresponding point mutation primer (table 1) is adopted to amplify a pCAGGS-HA-A104R wild type plasmid as a template, the amplified product is subjected to enzyme digestion by Dpn I enzyme, and the enzyme digestion product is converted into competent cells DH5 alpha, so that mutant plasmids pCAGGS-HA-A104R-R/H69/72D (namely, the 69 th amino acid and 72 th amino acid of pA104R protein are mutated into Asp) and pCAGGS-HA-A104R-K/R92/94/97E (namely, the 92 th amino acid, 94 th amino acid and 97 th amino acid of pA104R protein are mutated into Glu) are constructed. Double luciferase and qPCR experiments were performed on wild-type pCAGGS-HA-A104R and mutant plasmids pCAGGS-HA-A104R-R/H69/72D and pCAGGS-HA-A104R-K/R92/94/97E, and ISRE promoter activity and ISGs expression were examined (methods described above). As a result, as shown in FIG. 2-D, pA104R was able to inhibit ISRE promoter activity in a double luciferase assay, whereas pA104R mutant was able to significantly revert to this inhibition. As shown in FIG. 2-E, the mutant of pA104R in the qPCR experiment result also remarkably reverts the inhibition of pA104R to ISGs expression, and in addition, the mutation of the amino acid site of pA104R protein in the WB result of the double luciferase experiment in FIG. 2-D can be found to not influence the normal expression of the protein. Thus, although the inhibition of innate immunity by pA104R is independent of DNA binding, the amino acid positions (amino acids 69, 72, 92 and 94, 97) associated with DNA binding are closely related to the function of pA104R in immunosuppression, which may be that pA104R inhibits innate immunity by apparent modification, while amino acids 69, 72, 92 and 94, 97 are exactly the key positions for apparent modification.

EXAMPLE 3 immunogenicity of pA104R amino acid site muteins

To verify whether the immunogenicity of the pA104R amino acid site mutein was disruptedBad, construct pA104R amino acid site mutation prokaryotic expression plasmid and carry on prokaryotic expression: sequentially carrying out two-round point mutation on pCAGGS-HA-A104R plasmid by using two pairs of mutation primers to obtain plasmids with mutations of amino acids 69, 72, 92, 94 and 97 of pA104R, amplifying pA104R sequence with mutation of amino acid site by using the primers His-A104R-F, his-A104R-R (table 1) as a template, inserting the primers between restriction enzyme BamHI and enzyme cleavage site of XhoI of PET-30a vector to obtain PET-30a-A104R plasmid, transforming competent cell BL21 (DE 3), inoculating positive bacterial liquid into LB liquid culture medium containing antibiotics according to a ratio of 1:100, placing the LB liquid culture medium at a constant temperature of 37 ℃ for shake culture for 2-3h, and culturing until OD ₆₀₀ The value is between 0.5 and 0.6, the IPTG with the final concentration of 0.6mM is added, and the mixture is placed in a shaking table at the constant temperature of 16 ℃ for shake culture for 16 to 20 hours. The bacterial solution was centrifuged at 4000g at 4℃for 5min and the cells were resuspended in 1/10 of the volume of the binding buffer. The cells were crushed at 4℃with a low-temperature ultra-high-pressure cell crusher, repeatedly crushed 3 to 5 times, and centrifuged at 8000g at 4℃for 10min, and the supernatant was collected and purified according to the GE nickel affinity chromatography column procedure. Fully emulsifying the purified His-A104R protein with the equal volume of protein and Freund's complete adjuvant, subcutaneously injecting 50 mug of protein into the nape of a 6-week-old female BALB/c mouse at multiple points for one-way injection, performing three-way injection with the same amount of protein emulsified by Freund's incomplete adjuvant after 10 days, and then taking blood from tail vein for indirect ELISA detection: with a coating solution (1.59 g NaCO) ₃ ，2.93g NaHCO ₃ Adding a proper amount of ddH ₂ O was dissolved and 1L) of the diluted antigen was fixed, and the ELISA plate was coated with 100 μl/well and left overnight at 4 ℃. The antigen solution was discarded, washed 3 times with PBST (PBS containing 0.1% Tween-20) and 200. Mu.L of each well was added, and gently swirled at room temperature for 5min. And (5) removing the liquid in the hole as much as possible. 5% skim milk lock in PBS was used for overnight lock at 4deg.C. The blocking solution was discarded, washed 3 times with PBST, and the immunized mouse serum and the blank mouse serum were respectively subjected to gradient dilution with PBS, and added to ELISA reaction plates at a concentration of 100. Mu.L per well, respectively, and incubated at 37℃for 1 hour. Serum was discarded, washed 3 times with PBST, goat anti-mouse HRP-IgG enzyme-labeled secondary antibody diluted 1/8000 was added, 100. Mu.L/well, and incubated at 37℃for 30min. And discarding the enzyme-labeled secondary antibody, washing with PBST for 3 times, and finally sequentially drying the liquid in the hole as much as possible. Adding TMB color development liquidColor development was performed at room temperature for 10min in the dark. Results as shown in fig. 3, the mouse antibody titer can reach 1 after the third immunization: 500000 shows that the immunogenicity of the pA104R protein after amino acid site mutation is not affected. Therefore, the pA104R with the mutation of the immunosuppression related site is a vaccine candidate gene with great potential, and avoids the negative influence caused by the immunosuppression characteristic while stimulating the exertion of the body immunity.

Sequence listing

<110> university of agriculture in China

<120> an 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 (7)

1. An african swine fever virus pA104R mutein characterized in that: is any one of the following proteins:

protein with the pA104R protein with the 69 th Arg and 72 th His mutated into Asp, glu or Ala;

protein of pA104R, lys at 92 th, arg at 94 th and Lys at 97 th are mutated into Asp, glu or Ala;

protein of pA104R protein with mutation of 69 th Arg, 72 th His, 92 th Lys, 94 th Arg and 97 th Lys to Asp, glu or Ala;

the amino acid sequence of the pA104R protein is shown as SEQ ID NO. 1.

2. An application of an amino acid site related to the immunosuppression of an African swine fever virus pA104R protein as a gene editing site, which is characterized in that: the amino acid site is any one of the following combinations:

arg at 69 th and His at 72 th of pA104R protein are mutated into Asp, glu or Ala;

lys at 92 th position, arg at 94 th position and Lys at 97 th position of pA104R protein are mutated into Asp, glu or Ala;

the Arg at 69 th, his at 72 th, lys at 92 th, arg at 94 th and Lys at 97 th of pA104R protein are mutated into Asp, glu or Ala;

the amino acid sequence of the pA104R protein is shown as SEQ ID NO. 1;

the application is for the purpose of non-disease diagnosis and treatment.

3. An application of an amino acid site related to the immunosuppression of an African swine fever virus pA104R protein as an anti-African swine fever virus target, which is characterized in that: the amino acid site is any one of the following combinations:

arg at 69 th and His at 72 th of pA104R protein are mutated into Asp, glu or Ala;

lys at 92 th position, arg at 94 th position and Lys at 97 th position of pA104R protein are mutated into Asp, glu or Ala;

the Arg at 69 th, his at 72 th, lys at 92 th, arg at 94 th and Lys at 97 th of pA104R protein are mutated into Asp, glu or Ala;

the amino acid sequence of the pA104R protein is shown as SEQ ID NO. 1;

the application is for the purpose of non-disease diagnosis and treatment.

4. Use of the pA104R mutein of claim 1 for the preparation of an african swine fever vaccine.

5. The use according to claim 4, characterized in that: the vaccine comprises ASFV attenuated vaccine, subunit vaccine, DNA vaccine, mRNA vaccine and virus vector vaccine.

6. An african swine fever vaccine, characterized in that: which is capable of expressing the pA104R mutein of claim 1.

7. An anti-african swine fever virus drug, characterized in that: the gene can target an amino acid site related to the immunosuppression of the African swine fever virus pA104R protein, wherein the amino acid site is any one of the following combinations:

arg at 69 th and His at 72 th of pA104R protein are mutated into Asp, glu or Ala;

lys at 92 th position, arg at 94 th position and Lys at 97 th position of pA104R protein are mutated into Asp, glu or Ala;

the Arg at 69 th, his at 72 th, lys at 92 th, arg at 94 th and Lys at 97 th of pA104R protein are mutated into Asp, glu or Ala;

the amino acid sequence of the pA104R protein is shown as SEQ ID NO. 1.