African swine fever virus whole-protein chip and application thereof

Abstract

The invention discloses an African swine fever virus whole-protein chip and application thereof. The invention provides a protein chip with at least one of the following functions (1)-(2), 160 proteins are connected to the protein chip, and each protein independently forms a detection point; experiments prove that the invention provides an African swine fever virus whole-protein chip which is composed of a substrate and 160 proteins coded by more than 95% of African swine fever virus genes. The whole-protein chip has obvious significance in discovery of protective antigens of the African swine fever virus and research of interaction between the virus and a host. Compared with an existing research means, the method has the advantages of being global and high in throughput screening, the research design and operation process is simplified, and the research efficiency is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an African swine fever virus full protein chip and application thereof. Background Art

[0002] African swine fever (ASF) is an infectious disease of pigs caused by infection with African swine fever virus (ASFV). African swine fever has a variety of clinical manifestations, ranging from peracute, acute, subacute to chronic and asymptomatic, depending on the type of infected virus strain, the type of pig and the mode of infection. The acute form is caused by highly virulent strains, manifested by high fever, loss of appetite, skin erythema and other characteristic pathological changes related to vasculitis, with a mortality rate of up to 100%. There is currently a lack of safe and effective vaccines and drugs. ASFV particles are about 250nm in diameter, have a capsule, are regular icosahedral, have a five-layer structure, and are still infectious after removing the capsule. The viral genome is 170-190kb, encoding more than 160 proteins, including more than 60 structural proteins. The structure and function of most proteins are unknown, and the interaction relationship with the host is unclear. The correlation between immunogens that activate immune responses and immune protection is also unclear.

[0003] One of the most fundamental reasons for the passive prevention and control of African swine fever is the weakness of basic research on African swine fever virus, and there is no way to start the research and development of vaccines and drugs. The most studied B cell antigens of African swine fever virus are B646L, CP204L, E183L and EP402R, and these four antigens cannot produce effective antiviral protection no matter how they are combined and immunized in different forms. Among the more than 160 proteins of African swine fever virus, there are more than the four proteins mentioned above that can produce antibodies. What are they specifically? What are the rules in the occurrence and development of the disease? What types of antibodies can play an effective role in resisting viral infection? For African swine fever virus, a multi-protein and complex virus, traditional single-factor or single-level virus research cannot meet the needs. It is necessary to adopt multi-factor, multi-level analysis ideas and high-throughput global screening methods to understand and solve problems.

[0004] Protein chip, also known as protein microarray, is a miniaturized, fast parallel, high-throughput protein analysis system, which usually has thousands of different proteins or peptides fixed at high density on a solid phase carrier. In the past nearly two decades, this technology has been widely used in clinical and basic research. Proteome chip belongs to functional normal phase protein chip, which is to print all the proteins of a species on the chip, incubate the analysis sample with the chip, rinse and wash away the components that cannot bind to the chip protein, and then incubate with fluorescently labeled antibodies, and finally use a fluorescent scanner to read the fluorescence signal value of each point on the chip. Summary of the invention

[0005] In order to make up for the shortcomings of traditional research methods, the present invention provides an African swine fever full protein chip and a preparation method. The chip contains 160 kinds of African swine fever virus proteins, and the chip quality inspection shows that the chip is successfully prepared. The chip is used to screen the sera of infected pigs or immune pigs to discover new B cell antigens and potential protective B cell antigens. The chip is used to screen the interaction between viral proteins and pig host immune-related proteins to discover potential viral immune regulatory factors. The present invention is essentially a large-scale, global analysis and research on proteins.

[0006] The purpose of the present invention is to provide a protein chip.

[0007] In a first aspect, the protein chip provided by the present invention comprises a substrate and 160 kinds of African swine fever virus proteins fixed thereto; and each protein independently forms a detection point;

[0008] The 160 African swine fever virus proteins are DP63R, L60L, O61R, C62L, DP60R, A118R, E66L, B66L, I177L, X69R, DP71L, K78R, I73R, EP84R, CP80R, DP79L, L83L, C84L, DP96R, C122R, I9R, A104R, L11L, MGF_110-2L, I7L, H108R, I8L, D117L, E111R, B117L, MGF_110-14L, CP123L, E120R, MGF_110-12L, MGF_110-4L, MGF_110-3L, MGF_110-1L, D129L, B119L, B125R, H124R, MGF_110-8L, MGF_100-1R, C129R, A137R, MGF_110-7L, E146L, K145R, EP152R, C147L, EP153R, E165R, A151R, F165R, B169L, I10L, H171R, E183L, O174L, B175L, A179L, p14, KP177R, E184L, S183L, E199L, K196R, I196L, K205R, CP204L, D205R, MGF_110-5L-6L, I215L, H233R, A238L, A224L, I226R, A240L, MGF_360-18R, C257L, DP238L, H240R, E248R, D250R, I243L, B263R, S273R, MGF_300-1L, QP383R, MGF_110-13L, I267L, MGF_505-3R, EP296R, MGF_360-15R, CP312R, E301R, R298L, MGF_360-16R, F317L, B318L, MGF_360-8L, C315R, D339L, MGF_300-4L, D345L, F334L, p34, H339R, MGF_360-10L, MGF_360-12L, EP402R, MGF_360-13L, H359L, MGF_360-9L, EP364R, MGF_360-14L, MGF_360-21R, MGF_360-11L, MGF_360-3L, MGF_360-1L, MGF_360-2L, MGF_360-6L, p37, MGF_360-4L, B354L, NP419L, B385R, B407L, E423R, EP424R, B438L, K421R, M448R, B475L, QP509L, MGF_505-9R, B602L,MGF_505-5R, C475L, CP530R, MGF_505-6R, MGF_505-7R, MGF_505-2R, MGF_505-10R, B646L, Q706L, C717R , F778R, NP868R, A859L, B962L, C962R, F1055L, D1133L, P1192R, G1211R, EP1242L, M1249L, G1340L and p150. ,

[0009] Preferably, the number of types of African swine fever virus proteins is 160, and the protein coverage accounts for 95% of the entire African swine fever virus proteome.

[0010] The above-mentioned African swine fever virus is African swine fever virus (African swine fever virus) SY18, and the above-mentioned protein comes from this virus.

[0011] In the above protein chip, the substrate is a PATH protein chip substrate.

[0012] In the above protein chip, the protein chip is prepared by spotting the 160 proteins on a PATH protein chip substrate.

[0013] The above-mentioned whole protein chip includes several matrices, each of which includes the 160 African swine fever virus proteins, a positive control, a negative control and a blank control.

[0014] Preferably, in the matrix, three replicate points are set for each African swine fever virus protein and control.

[0015] Further preferably, the number of the matrix is ​​14 per chip, and the controls include positive control, negative control, and blank control, so that 14 detection windows can be formed and 14 samples can be detected simultaneously.

[0016] Preferably, the sample spots on the chip are spotted on the chip substrate using a biochip spotter. In an embodiment of the present invention, the positive control is IgG, IgM and IgA from pigs;

[0017] The negative control is a GST tag;

[0018] The blank control is the chip sample dilution solution.

[0019] Preferably, the preparation of the African swine fever virus protein is specifically as follows: a cloned plasmid connected to the protein gene sequence is transferred into brewer's yeast for overexpression, and a purified recombinant protein is obtained by affinity purification through a fusion tag, wherein the concentration of the purified recombinant protein is greater than or equal to 5 μg / mL.

[0020] The protein chip was prepared as follows: proteins were spotted on the surface of PATH substrate (purchased from Grace Bio-Labs, USA), each chip had 14 matrices, each matrix included 160 African swine fever virus proteins, negative controls, positive controls and blank controls, and African swine fever proteins and each control protein had 3 repeated spots in each array. The protein chip was constructed by overnight fixation at 4°C (proteins interacted with the substrate through adsorption).

[0021] In a second aspect, the present invention provides a kit comprising the protein chip described in the first aspect.

[0022] The above kit also includes antibodies of host serum labeled with fluorescence (such as Cy5 fluorescent anti-pig secondary antibody), chip sample diluent, chip washing solution, etc.

[0023] In a third aspect, the present invention provides use of the protein chip described in the first aspect or the kit described in the second aspect in at least one of the following:

[0024] 1) Screening or assisting in the screening of African swine fever virus protein antigens;

[0025] 2) Screening or assisting in the screening of protective antigens or antigen combinations of African swine fever virus;

[0026] 3) Construction of African swine fever virus protein antigen library;

[0027] 4) Screening or assisting in the screening of proteins or protein combinations that specifically bind to antibodies produced after the African swine fever virus or vaccine acts on the host;

[0028] 5) Construct a database of proteins that specifically bind to antibodies produced after the African swine fever virus or vaccine acts on the host;

[0029] 6) Screening or assisting in the screening of immune regulatory factors of African swine fever virus;

[0030] 7) Screening or assisting in the screening of African swine fever proteins that interact with immune-related proteins in infected hosts;

[0031] 8) Construct a database of immune regulatory factors of African swine fever virus;

[0032] 9) Detect or bind to African swine fever virus antibodies.

[0033] The antigens mentioned above are B cell antigens.

[0034] The African swine fever virus antibodies mentioned above are antibodies produced after the African swine fever virus or vaccine acts on the host, specifically, antibodies produced after the African swine fever virus infects the host or antibodies produced after the African swine fever virus vaccine is injected into the host.

[0035] The above-mentioned African swine fever virus protein antigen library can be a database, or it can also be a physical library obtained by purifying the protein antigen through prokaryotic expression.

[0036] In a fourth aspect, the present invention provides the use of the 160 African swine fever virus proteins in the protein chip described in the first aspect in preparing a product having at least one of the following functions:

[0037] 1) Screening or assisting in the screening of African swine fever virus protein antigens;

[0038] 2) Screening or assisting in the screening of protective antigens or antigen combinations of African swine fever virus;

[0039] 3) Construction of African swine fever virus protein antigen library;

[0040] 4) Screening or assisting in the screening of proteins or protein combinations that specifically bind to antibodies produced after the African swine fever virus or vaccine acts on the host;

[0041] 5) Construct a database of proteins that specifically bind to antibodies produced after the African swine fever virus or vaccine acts on the host;

[0042] 6) Screening or assisting in the screening of immune regulatory factors of African swine fever virus;

[0043] 7) Screening or assisting in the screening of African swine fever proteins that interact with immune-related proteins in infected hosts;

[0044] 8) Construct a database of immune regulatory factors of African swine fever virus;

[0045] 9) Detect or bind to African swine fever virus antibodies.

[0046] The antigens mentioned above are B cell antigens.

[0047] The African swine fever virus antibodies mentioned above are antibodies produced after the African swine fever virus or vaccine acts on the host.

[0048] The above product can be a protein chip.

[0049] The above-mentioned African swine fever virus protein antigen library can be a database, or it can be a physical library obtained by purifying the protein antigen through prokaryotic expression.

[0050] In a fifth aspect, the present invention provides a method for screening or assisting in screening African swine fever virus protein antigens or antigen combinations, comprising the following steps:

[0051] 1) reacting the protein chip described in the first aspect with serum of a host infected with African swine fever virus or serum of a host immunized with an African swine fever virus vaccine to obtain a reaction product;

[0052] 2) adding the fluorescently labeled antibody of the host serum to the reaction product of step 1) to carry out a reaction to obtain a fluorescent reaction product;

[0053] 3) Detecting the fluorescent signal of the fluorescent reaction product to determine whether a certain protein in the protein chip described in the first aspect is or is a candidate for being an African swine fever virus protein antigen or antigen combination.

[0054] The determination method described above is as follows: if the fluorescence signal value of the fluorescence reaction product of a certain protein in the protein chip described in the first aspect is greater than a threshold value, then the protein is or is a candidate for being an African swine fever virus antibody-positive protein.

[0055] Healthy host serum was used as control;

[0056] The threshold is the average value of the antibody reaction signal of each protein in the healthy host serum sample + 3 times the standard deviation.

[0057] The method described above also includes step 4): using the African swine fever virus protein antigens determined in step 3) as detection antigens, and performing enzyme-linked immunosorbent assay to detect whether an antigen-antibody binding reaction occurs between the antigens and the African swine fever virus antibodies. If a reaction occurs, the protein is an African swine fever virus protein antigen; if no reaction occurs, the protein is not an African swine fever virus protein antigen.

[0058] In the above step 4), when the determined African swine fever virus protein antigen is used as a detection antigen, it needs to be purified after prokaryotic expression for enzyme-linked immunosorbent assay. The purpose of the above prokaryotic expression and purification is to increase the expression level so that Elisa detection can be achieved.

[0059] The above-mentioned African swine fever virus antibodies are the serum of a host infected with African swine fever virus or the serum of a host immunized with an African swine fever virus vaccine.

[0060] In a sixth aspect, the present invention provides a method for high-throughput detection of specific antibodies to the entire proteome of African swine fever virus, comprising the following steps:

[0061] 1) reacting all African swine fever virus proteins on the protein chip of the first aspect with serum of a host infected with African swine fever virus or serum of a host immunized with an African swine fever virus vaccine to obtain a reaction product on the chip;

[0062] 2) adding the fluorescently labeled antibody of the host serum to the reaction product of step 1) to carry out a reaction to obtain a fluorescently labeled reaction product;

[0063] 3) Detecting the fluorescent signal of the fluorescently labeled reaction product to determine whether the host serum contains specific binding antibodies to a certain African swine fever protein on the protein chip.

[0064] The determination method described above is as follows: if the fluorescence signal value of the fluorescently labeled reaction product corresponding to a certain African swine fever protein in the protein chip is greater than a threshold value, then the host serum contains or is likely to contain specific binding antibodies against the African swine fever protein.

[0065] Healthy host serum was used as control;

[0066] The threshold is the average value of the antibody reaction signal of each protein in the healthy host serum sample + 3 times the standard deviation.

[0067] In a seventh aspect, the present invention provides a method for screening or assisting in screening an immunomodulatory factor of African swine fever virus, comprising the following steps:

[0068] 1) reacting the protein chip described in the first aspect with a biotin-labeled host immune-related protein to obtain a protein-protein binding product;

[0069] 2) adding fluorescently labeled avidin to the protein-protein binding product to react and obtain a fluorescently labeled protein-protein binding product;

[0070] 3) Detecting the fluorescence signal of the fluorescently labeled protein and the protein binding product to determine whether a certain protein in the protein chip of the first aspect is a potential immunomodulatory factor.

[0071] The determination method described above is as follows: if the chip result signal-to-noise ratio of a protein in the protein chip described in the first aspect is greater than 3, then the African swine fever protein is or is a candidate for being an immunomodulatory factor;

[0072] The signal-to-noise ratio of the chip results is SNR(+) / SNR(-). The result of the chip reaction with the pure biotin group (biotin labeling reagent) is defined as SNR(-), and the result of the chip reaction with the biotinylated protein group (biotinylated host immune-related protein) is defined as SNR(+). The biotin labeling reagent is used as a control and recorded as the pure biotin group.

[0073] The host immunity-related proteins are PERK, IRF3, p65, LC8, IL-1β and / or IκBα.

[0074] In an eighth aspect, the present invention provides the following application:

[0075] The present invention provides the use of the R298L, P1192R and S273R proteins in the protein chip of the first aspect in preparing a product for binding or detecting the immune-related protein IRF3;

[0076] Or the present invention provides the use of the R298L, P1192R and S273R proteins in the protein chip of the first aspect as interacting proteins of the immune-related protein IRF3;

[0077] Or, the present invention provides the use of L60L, MGF360-12L and I73R proteins in the protein chip of the first aspect in preparing a product for binding or detecting the immune-related protein PERK;

[0078] Or the present invention provides the use of L60L, MGF360-12L and I73R proteins in the protein chip of the first aspect as interacting proteins of the immune-related protein PERK;

[0079] Or the present invention provides the use of R298L, P1192R, S273R, L60L, MGF360-12L and I73R proteins in the protein chip described in the first aspect as immunomodulatory factors.

[0080] The present invention has the following advantages: The African swine fever full protein chip provided by the present invention is a high-throughput analysis and detection platform. With its unique advantages, such as miniaturization, high throughput, rapid detection, low sample requirement and high sensitivity, it can achieve a rapid, efficient and systematic understanding of the biological characteristics of African swine fever virus, especially the B cell immunogenicity of African swine fever virus protein, thereby laying the foundation for its diagnosis, prevention and control, vaccine development, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 Silver staining detection of some of the prepared African swine fever virus proteins.

[0082] Figure 2 Immunoblotting detection of some of the prepared African swine fever virus proteins.

[0083] Figure 3 This is the quality control result of the prepared whole protein chip.

[0084] Figure 4 This is the result of the reaction of the whole protein chip with infected pig serum.

[0085] Figure 5 The protein screened out by Elisa validation reacts positively with infected pig serum samples.

[0086] Figure 6 This is a graph showing the results of the reaction between the whole protein chip and the serum of vaccine-immunized pigs.

[0087] Figure 7 To screen out the Elisa test results of proteins that react positively with immune pig serum samples.

[0088] Figure 8 This is the result of the interaction between the whole protein chip and the host protein. DETAILED DESCRIPTION

[0089] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0090] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0091] The technical solutions described in the present invention, unless otherwise specified, are all conventional technologies in the art; the reagents or materials described, unless otherwise specified, are all from commercial channels.

[0092] In the following examples, all solution formulations are as follows:

[0093] Table 1 is SC-URA / glucose liquid culture medium (1L)

[0094]

[0095] Table 2 is SC-URA / raffinose liquid culture medium (1L)

[0096]

[0097] Table 3 shows the lysis buffer pH 7.5 (1L)

[0098]

[0099] Lysis solution (containing protease inhibitors) Roche protease inhibitors were added to the system in Table 3, with 1 tablet (50 mg) added per 50 mL of lysis solution.

[0100] Table 4 is cleaning solution I pH 7.5 (1L)

[0101]

[0102] Table 5 is cleaning solution II pH 7.5 (1L)

[0103]

[0104] Table 6 is the elution buffer pH 7.5 (1L)

[0105]

[0106] Table 7 is the chip sample dilution

[0107]

[0108] Table 8 is the chip washing solution

[0109]

[0110] Table 9 is the chip sealing solution

[0111]

[0112]

[0113] Table 10 is PBST

[0114]

[0115] Table 11 is carbonate buffer pH 9.6

[0116]

[0117] Prokaryotic expression and purification methods:

[0118] 160 coding nucleic acid sequences of African swine fever virus proteins were cloned into the NotI and BamHI restriction sites of the prokaryotic expression vector pet32a plasmid (Fenghui Biotechnology, FH1635), and transformed into the prokaryotic expression bacteria BL21. The constructed recombinant protein expression bacteria were streaked on a plate containing Amp resistance, single clones were picked, cultured in a test tube, and then the bacterial solution was transferred to a large volume of 1-5L LB liquid culture medium at a 1% inoculation volume for amplification, and the corresponding antibiotics were added and cultured at 37°C with an oscillation of 180rpm until the bacterial solution OD 600 The concentration of the inducing agent IPTG was between 0.4 and 0.6, and the induction agent IPTG was added to a final concentration of 1mmol / L. The induction culture was continued at 16℃ for 18h. The bacteria were collected by centrifugation at 8000rpm and 4℃ for 4min. The collected bacteria were resuspended in PBS and crushed with an ultrasonic crusher in an ice bath for 40min. After centrifugation at 10000g and 4℃ for 30min, the precipitate was discarded. The supernatant was purified by His tag affinity chromatography column. 160 prokaryotic expression libraries of African swine fever virus proteins with a purity of 80-90% and a concentration of 0.1-5mg / mL were obtained.

[0119] Example 1. Preparation of whole protein chip

[0120] (I) Obtaining a Saccharomyces cerevisiae library expressing African swine fever virus protein

[0121] 160 African swine fever virus protein sequences were referenced to ASFV-SY18 (GenBank: MH766894.1, 20-OCT-2018).

[0122] 160 African swine fever virus proteins include DP63R, L60L, O61R, C62L, DP60R, A118R, E66L, B66L, I177L, X69R, DP71L, K78R, I73R, EP84R, CP80R, DP79L, L83L, C84L, DP96R, C122R, I9R, A104R, L11L, MGF_110-2L, I7L, H108R, I8L, D117L, E111R, B117L, MGF_110-14L, CP123L, E120R, MGF_110-12L, MGF_110-4L, MGF_110-3L, MGF_110-1L, D129L, B119L, B125R, H124R, MGF_110-8L, MGF_100-1R, C129R, A137R, MGF_110-7L, E146L, K145R, EP152R, C147L, EP153R, E165R, A151R, F165R, B169L, I10L, H171R, E183L, O174L, B175L, A179L, p14, KP177R, E184L, S183L, E199L, K196R, I196L, K205R, CP204L, D205R, MGF_110-5L-6L, I215L, H233R, A238L, A224L, I226R, A240L, MGF_360-18R, C257L, DP238L, H240R, E248R, D250R, I243L, B263R, S273R, MGF_300-1L, QP383R, MGF_110-13L, I267L, MGF_505-3R, EP296R, MGF_360-15R, CP312R, E301R, R298L, MGF_360-16R, F317L, B318L, MGF_360-8L, C315R, D339L, MGF_300-4L, D345L, F334L, p34, H339R, MGF_360-10L, MGF_360-12L, EP402R, MGF_360-13L, H359L, MGF_360-9L, EP364R, MGF_360-14L, MGF_360-21R, MGF_360-11L, MGF_360-3L, MGF_360-1L, MGF_360-2L, MGF_360-6L, p37, MGF_360-4L, B354L, NP419L, B385R, B407L, E423R, EP424R, B438L, K421R, M448R, B475L, QP509L, MGF_505-9R, B602L,MGF_505-5R, C475L, CP530R, MGF_505-6R, MGF_505-7R, MGF_505-2R, MGF_505-10R, B646L, Q706L, C717R , F778R, NP868R, A859L, B962L, C962R, F1055L, D1133L, P1192R, G1211R, EP1242L, M1249L, G1340L, p150. ,

[0123] The above four proteins, p14 (CP2475L shown at positions 118364-118825 of MH766894.1), p34 (CP2475L shown at positions 117392-118363 of MH766894.1), p37 (CP2475L shown at positions 118826-119938 of MH766894.1) and p150 (CP2475L shown at positions 119939-124687 of MH766894.1) are obtained by splitting the CP2475L protein according to the cleavage site of the S273R protease (the splitting method was reported in EMBO J. 1993 Jul; 12 (7): 2977-87).

[0124] The coding genes of the above 160 proteins were optimized and synthesized by facultatively optimizing the sequences of Escherichia coli and yeast, and the spytag coding sequence was added to the 3' end of each protein coding gene to obtain the protein coding gene with added spytag.

[0125] The above-mentioned protein coding genes with added spytags were respectively connected to the thrombin site and Hind III restriction site of the target vector pEGH-A (the vector was disclosed in "Jian Zhu, Heng Zhu, et al: J. Virol. May 2009 vol. 83 no. 105219-5231") to obtain 160 plasmids, in which the protein coding genes with added spytags and the GST tag on the vector were fused to express 160 recombinant African swine fever virus proteins, all of which were recombinant proteins with a GST tag and Linker at the N-terminus and a spytag at the C-terminus. The naming examples are as follows: GST-Linker-DP63R-spytag.

[0126] The amino acid sequences of the above-mentioned recombinant African swine fever virus proteins are composed of a GST tag (sequence 1, MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAII RYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLDGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK), a Linker (sequence 2, GSACEL), the corresponding African swine fever virus proteins and a spytag (sequence 3, AHIVMVDAYKPTK).

[0127] The above 160 plasmids expressing recombinant African swine fever virus proteins were then transferred into the competent cells of Saccharomyces cerevisiae Y258 (this yeast strain was disclosed in "Heng Zhu, Michael Snyder, et al: Science. 2001 Sep 14; 293 (5537)") to obtain 160 recombinant Saccharomyces cerevisiae. The 160 recombinant Saccharomyces cerevisiae constituted a Saccharomyces cerevisiae library expressing African swine fever virus proteins.

[0128] (II) Construction of GST-tagged African swine fever virus protein expression library in Saccharomyces cerevisiae

[0129] 1) High-throughput expression of recombinant African swine fever virus protein

[0130] The 160 recombinant Saccharomyces cerevisiae prepared in (i) above were taken out from the bacterial preservation tube and activated in the SC-URA / glucose plate. The bacteria were picked and inoculated in 96-well deep-well plates, 500 μL SC-URA / glucose liquid medium in each well, 200 rpm, 30°C for 18 hours. In the 12-channel plate, 6 mL SC-URA / raffinose medium was first added to each channel, and then 6 μL of the cultured bacterial solution was inoculated. The recombinant Saccharomyces cerevisiae of each protein was inoculated into 2 channels. Cultured at 30°C and 90rpm for about 24 hours, OD600 reached about 0.6, and galactose with a final concentration of 2% (mass volume percentage g / mL) was added, and induced at 30°C and 90rpm for 6 hours. The yeast after induced expression was collected at 3900 rpm for 5 min, the supernatant was discarded, 200 μL of sterile ddH2O was added, and the mixture was resuspended. After centrifugation at 3900 rpm for 5 min, the supernatant was discarded, the bacteria were collected, sealed with sealing film, and frozen in a -80°C refrigerator.

[0131] 2) High-throughput purification of African swine fever virus protein

[0132] The cells prepared in 1) above and stored at -80°C were taken out and placed on ice, 200 μL of zirconium beads (purchased from Shanghai Ruian Industrial Co., Ltd., China) were added, and then 300 μL of lysis solution (containing protease inhibitors) were added, and the cells were shaken on a high-throughput cell disruptor for 6 times, each time for 1 min, and placed on ice for 1 min to obtain cell lysis solution. At the same time, the NUNC 96-well filter plate was sealed with 1% agar.

[0133] The bacterial lysate was centrifuged at 3900 rpm for 5 min, the supernatant was aspirated and transferred to a NUNC 96-well filter plate with a sealed bottom, 300 μL of lysis buffer (containing protease inhibitors) was added again, the vortex was repeated once, and the two supernatants were combined.

[0134] Prepare an appropriate amount of glutathione affinity purification column material (Zhongke Senhui Microsphere Technology Co., Ltd.), wash it three times with lysis buffer (containing protease inhibitors), then add it to the above supernatant, and finally add 500 μL of lysis buffer (containing protease inhibitors) to make up to 1.2 mL, and incubate it at 4°C on a shaker for 2 h.

[0135] After the incubation, quickly remove the agar on the bottom, quickly put the filter plate into a clean 96-well plate, centrifuge at 1000rpm for 30s, collect the supernatant after incubation in the 96-well plate, seal it with a sealing film and store it at -40℃. Then wash the column material three times with 250μL of cleaning solution I and cleaning solution II respectively. Add 80μL of elution buffer to the center of the filter plate with a suspended gun and let it stand at 4℃ for 15min, centrifuge and collect the eluate in a 96-well PCR plate. The eluate obtained is the target protein solution.

[0136] A total of 160 target protein solutions were obtained, in which the solvent was an elution buffer, and the solutes were 160 target proteins at a concentration of about 5 ng / μL, respectively, and the target proteins were various recombinant African swine fever virus proteins.

[0137] 3) Silver staining quantification of African swine fever virus protein and Western blotting quality identification

[0138] Prepare two 10% SDS-PAGE gels, 1.0 mm, 15 wells, one for silver staining and one for Western Blotting.

[0139] Take 8 μL of the purified target protein solution obtained in 2) above, add 2 μL of 5× loading buffer, prepare BSA sample as a silver staining quantitative control, and boil the sample for 10 minutes. Add 9 μL of the prepared sample, BSA sample, and 1.5 μL of protein marker to each well in turn. 80V 20min, 130V 1h. Silver stain one of the gels with a silver staining kit (Biyuntian): fixation, alcohol washing, water washing, sensitization, silver staining, water washing, color development, and termination. Transfer another gel at 15V for 40min, block with 5% skim milk, incubate with 1 μg / mL anti-GST rabbit anti-tag antibody (abcam ab19256), incubate with 1 μg / mL HRP goat anti-rabbit antibody (ABclonal AS014), react with ECL luminescent liquid (Biyuntian P0018S) to emit light, and save the picture.

[0140] Figure 1 Silver staining detection of some of the prepared African swine fever virus proteins. Figure 2 This is a partial result of immunoblotting of the prepared African swine fever virus protein; it can be seen that the various African swine fever virus proteins with N-terminal GST tags of the expected size were obtained, such as GST-L83L, etc. And after testing, the amount of the prepared protein was 5μg / mL.

[0141] The remaining proteins were also verified to be correct.

[0142] (III) Preparation and quality inspection of African swine fever protein chips

[0143] 1) Preparation: Take out 5-10 μL of each target protein solution prepared in 2) of the above (II) and transfer it to a 384-well plate. At the same time, set up positive controls (porcine IgG (purchased from Novus NBP1-97054), porcine IgM (purchased from Novus NBP1-96788), porcine IgA (purchased from ADI 20017-4-1)), negative controls (GST tags), and blank controls (elution buffer).

[0144] Using a microarray chip spotting system (Super Marathon microarray spotter purchased from Arrayjet, UK), proteins were spotted on the surface of PATH substrate (purchased from Grace Bio-Labs, USA). Each chip has 14 matrices, each matrix includes 160 African swine fever virus proteins, negative controls, positive controls and blank controls. African swine fever proteins and each control protein have 3 repeated spots in each array. The spotted chips were stored at -80°C for later use.

[0145] 2) Quality inspection: Use chip washing solution containing 3% (mass volume percentage g / mL) bovine serum albumin as blocking solution, block the protein chip obtained in 1) at room temperature for 1h, 50rpm. Use rabbit anti-GST antibody (abcam ab19256) as primary antibody, add the primary antibody to chip washing solution (1:1000 dilution), add 100μL to each well, and incubate the primary antibody at room temperature for 1h, 50rpm. Wash the chip washing solution 3 times, 5min each time. Use Cy5-conjugated goat anti-rabbit antibody (abcam ab6939) as secondary antibody, add the secondary antibody to chip washing solution (1:1000 dilution), add 100μL to each well, and incubate the secondary antibody at room temperature for 1h, 50rpm. Wash the chip washing solution 3 times, 5min each time. Wash with ultrapure water for 1min, spin dry on SliderWasher, and scan and record the results using GenePix4200A scanner.

[0146] The results are as follows Figure 3 As shown, the quality control shows that the signal-to-noise ratio of the fluorescence intensity of each protein spotted is significant, and the chip construction is complete. The enlarged image on the right is one of the 14 array distances on the chip.

[0147] Example 2: Detection of infected pig serum using whole protein chip

[0148] (I) Analysis of infected pig serum using African swine fever whole protein chip

[0149] The sera of 5 African swine fever virus-infected pigs and 5 healthy control pigs were allowed to stand at room temperature for 2 hours, and then centrifuged at 10,000 g for 20 minutes. The supernatant was taken as the serum sample to be tested and immediately tested.

[0150] The whole protein chip prepared in Example 1 can be used for serum analysis.

[0151] Specific steps:

[0152] The whole protein chip prepared in Example 1 was taken out from -80°C and rewarmed to room temperature for 10 minutes. The chip was placed in a washing box, and chip blocking liquid was added, and the chip was blocked at 50 rpm for 1 hour at room temperature. The excess liquid on the chip was quickly dried and placed in a wet box.

[0153] Use fences to separate the 14 protein matrices on the chip into independent detection wells. The serum sample to be tested is diluted with the chip sample diluent configured above at a volume ratio of 1:100, and 100 μL of the diluted solution containing the serum to be tested is added to the detection well of the above-mentioned sealed whole protein chip. React at room temperature for 1 hour. The sample to be tested is configured within 15 minutes before use. Take the chip out of the wet box, place it in the washing box, add the chip washing solution, wash at 50 rpm at room temperature for 5 minutes, and repeat 3 times. Quickly shake off the excess liquid on the chip and place it in the wet box. Add Cy5 fluorescent anti-pig secondary antibody (purchased from JacksonimmunoResearch 114-175-003) with a final concentration of 1 μg / mL to each well on the chip and react for 1 hour in the dark. Take the chip out of the wet box, place it in the washing box, add the chip washing solution, wash at 50 rpm at room temperature for 5 minutes, and repeat 3 times. Wash once with ultrapure water for 5 minutes. Centrifuge and dry, and read the data under the 635nm channel with a Genepix scanner.

[0154] The antibody response threshold for each protein was set as the average value of the antibody response signal for each protein in the serum samples of healthy control pigs + 3 times the standard deviation.

[0155] In the infected pig serum samples, proteins with signal values ​​greater than the antibody reaction threshold of the corresponding protein are antibody-positive proteins, and proteins with signal values ​​less than or equal to the antibody reaction threshold of the corresponding protein are antibody-negative proteins.

[0156] Some of the results are as follows Figure 4 As shown, proteins such as A137R, B602L, CP312R, E165R, C129R, A104R, H240R, P14, P150, and E183L specifically bind to infected pig sera (with white dots), but have no specific binding to healthy pig sera.

[0157] The above results show that these proteins can bind to African swine fever virus-specific antibodies in the serum of pigs infected with African swine fever virus, are B cell antigens of African swine fever virus, and are used to construct an African swine fever virus protein antigen library.

[0158] (II) Elisa validation of proteins that react positively with infected pig serum samples

[0159] In order to verify the results of the present invention, the proteins A137R, B602L, CP312R, E165R, C129R, A104R, H240R, P14, P150, and E183L that were screened out as positive for reacting with infected pig serum samples were detected by Elisa.

[0160] First, the African swine fever virus protein to be verified was expressed and purified using the Escherichia coli prokaryotic expression system. The African swine fever protein was diluted to 1-10 μg / mL with carbonate buffer and added to a 96-well polystyrene plate as a detection antigen. It was incubated at 4°C overnight to coat the antigen in the 96-well plate. The next day, the coating buffer was discarded, washed 3 times with PBST to remove unbound antigens, and blocked with 1% BSA in PBS buffer at 37°C for 2h. The blocking solution was discarded, washed 4 times with PBST, and infected pig serum was diluted 1:1000 with PBST and added to a 96-well plate, 3 replicates, and healthy control pig serum was used as a negative control, incubated at 37°C for 2h. The pig serum was discarded, washed 6 times with PBST, and unbound antibodies were removed. The HRP-labeled anti-swine IgG secondary antibody (Zhuangmeng Bio) was diluted 1:5000 with PBST, and then added to a 96-well plate and incubated at 37°C for 2h. The antibody incubation solution was discarded, washed 6 times with PBST, and unbound antibodies were removed. TMB colorimetric substrate (Biyuntian) was added, color development was carried out for 10 min, 2 M sulfuric acid was added to terminate the reaction, and the absorbance at 450 nm was measured using an enzyme reader.

[0161] The absorbance value indirectly reflects the level of antibodies in pig serum that specifically bind to the test antigen. If the absorbance value of a test antigen in the infected pig serum group and the healthy control group is statistically different, it is considered that the infected pig serum has antibodies that can bind to the antigen.

[0162] The results are as follows Figure 5 As shown, the results are consistent with the results of the method of the present invention. The serum of pigs infected with African swine fever virus does contain specific binding antibodies to these proteins, which proves that the analysis method of the present invention is reliable; it also proves that these antigens can be used to detect African swine fever virus or bind to African swine fever virus antibodies.

[0163] Example 3: Detection of vaccine-immunized pig serum using whole protein chip

[0164] (I) Analysis of serum from pigs vaccinated with African swine fever vaccine using a whole protein chip

[0165] After sera from 12 pigs immunized with African swine fever subunit vaccine (immunized pigs) and sera from 20 healthy controls were placed at room temperature for 2 hours, centrifuged at 10,000 g for 20 minutes, and the supernatant was taken as the serum sample to be tested and immediately tested.

[0166] The whole protein chip prepared in Example 1 can be used for serum analysis.

[0167] The specific operation steps are shown in Example 2 (I).

[0168] The antibody response threshold for each protein was set as the average value of the antibody response signal for each protein in the serum samples of healthy control pigs + 3 times the standard deviation.

[0169] In the immune pig serum samples, proteins with signal values ​​greater than the antibody reaction threshold of the corresponding protein are antibody-positive proteins, and proteins with signal values ​​less than or equal to the antibody reaction threshold of the corresponding protein are antibody-negative proteins.

[0170] Some of the results are as follows Figure 6 As shown, proteins such as B602L, E183L, F317L, CP204L, EP153R, B646L, A104R, and P34 specifically bind to immune pig serum (with white dots), but have no specific binding to healthy pig serum.

[0171] The above shows that these proteins can bind to African swine fever virus antibodies in the sera of immune pigs.

[0172] (II) Elisa verification chip detected positive proteins in pig serum samples

[0173] In order to verify the results of the present invention, the proteins screened out as above and reacting positively with the immune pig serum samples were tested by ELISA.

[0174] First, the African swine fever virus protein to be detected was expressed and purified using the E. coli prokaryotic expression system. The African swine fever protein was diluted to 5 μg / mL with carbonate buffer and added to a 96-well polystyrene plate as a detection antigen. The plate was incubated at 4°C overnight to coat the antigen in the 96-well plate. The next day, the coating buffer was discarded, the plate was washed 3 times with PBST to remove unbound antigens, and the plate was blocked with 1% BSA in PBS buffer at 37°C for 2 hours. The blocking solution was discarded, the plate was washed 4 times with PBST, and the immune pig serum was diluted with PBST at a ratio of 1:2 10 -1:2 19 10 dilution gradients were added to a 96-well plate, healthy control pig serum was used as a negative control, and incubated at 37° C. for 2 h. The rest of the operation method was the same as that of Example 2 (II).

[0175] The antibody titer was the serum dilution factor at which the absorbance value was greater than 2.1 times the average absorbance of healthy control serum.

[0176] If the antibody titer of a test antigen is not 0, it is considered that the serum contains antibodies that can bind to the antigen.

[0177] The results are as follows Figure 7 As shown, the results are consistent with the results of the method of the present invention. B602L, E183L, F317L, CP204L, EP153R, B646L, A104R, P34, etc. can specifically bind to immune serum, proving that the analysis method of the present invention is reliable.

[0178] Therefore, this example shows that the protein chip can be used to detect the serum of pigs immunized with the African swine fever virus candidate vaccine and analyze their antibody production.

[0179] Example 4: Analysis of protein interactions between African swine fever virus and host using a full protein chip

[0180] Protein interaction between viruses and hosts is an important molecular event in the process of virus infection of host cells, which can provide key information and clues for understanding related mechanisms such as immune escape, viral replication, viral release, and viral virulence. In this example, the Escherichia coli prokaryotic expression system was used to express and purify 6 immune-related host proteins (PERK (GenBank: XM_003124925.4, 13-MAY-2017), IRF3 (GenBank: NM_213770.1, 20-DEC-2022), p65 (GenBank: NM_001114281.1, 27-DEC-2021), LC8 (GenBank: NM_001195114 .2, 02-JUL-2020), IL-1β (GenBank: NM_214055.1, 20-DEC-2022), IκBα (GenBank: NM_001005150.1, 20-DEC-2022)), and the whole protein chip reaction of Example 1 (iii) were used to screen African swine fever proteins that interact with the above-mentioned 6 immune-related host proteins. These African swine fever proteins may be involved in immune escape-related reactions and are potential immune regulatory factors.

[0181] The whole protein chip prepared in Example 1 can be used to analyze virus-host protein interactions.

[0182] Specific steps:

[0183] The six host proteins were mixed with biotin labeling reagent (EZ-link Sulfo-NHS-LC-Biotin) at a molar ratio of 1:5, and 1M glycine solution was added after incubation at 4°C for 2 hours to terminate the reaction. Excess biotin labeling reagent and glycine were removed by stirring and dialysis at 4°C overnight. Through this step, biotin labeling was added to the host protein to obtain the biotinylated protein to be tested, which was recorded as the biotinylated protein group. The biotin labeling reagent was used as a control, which was recorded as the pure biotin group.

[0184] The whole protein chip prepared in Example 1 was taken out from -80°C and warmed to room temperature for 10 minutes. The chip was placed in a washing box, and chip blocking liquid was added, and the chip was blocked at 50 rpm for 1 hour. The excess liquid on the chip was quickly dried and placed in a wet box.

[0185] The biotinylated protein or biotin labeling reagent to be tested is diluted to 20μg / ml with the chip sample diluent, and 200μL is added to the closed enclosure space. React at 4℃ overnight. Take the chip out of the wet box, place it in the washing box, add the chip washing solution, wash at 50rpm for 5min at room temperature, and repeat 3 times. Quickly shake off the excess liquid on the chip and place it in the wet box. Add 1:1000 diluted SA-Cy3 to each well on the chip and react for 1h in the dark. Take the chip out of the wet box, place it in the washing box, add the chip washing solution, wash at 50rpm for 5min, and repeat 3 times. Wash once with ultrapure water for 5min. Centrifuge and dry, and read the data under the 532nm channel with a Genepix scanner.

[0186] The result of the chip reacting with the pure biotin group (biotin labeling reagent) is defined as SNR(-), and the result of the chip reacting with the biotinylated protein group (biotinylated host immune-related protein) is defined as SNR(+). SNR(+) / SNR(-) is used to represent the interaction strength of a certain protein.

[0187] The positive reaction threshold was set such that the signal value ratio of each protein spot in the biotinylated protein group and the pure biotin group was >3, that is, SNR(+) / SNR(-)>3.

[0188] Among different biotinylated proteins, if the SNR(+) / SNR(-) of a certain African swine fever protein in the whole protein chip is greater than 3, then the African swine fever protein can interact with the biotinylated protein, and the African swine fever protein is or is a candidate for an immune regulatory factor; if the SNR(+) / SNR(-) of a certain African swine fever protein in the whole protein chip is less than or equal to 3, then the African swine fever protein does not interact with the biotinylated protein, and the African swine fever protein is not or is not a candidate for an immune regulatory factor.

[0189] Some of the results are as follows Figure 8 As shown, R298L, P1192R, and S273R proteins interact with the host protein IRF3, and L60L, MGF360-12L, and I73R proteins interact with the host protein PERK.

[0190] The above indicates that these proteins can bind to immune-related host proteins and are potential immune regulatory factors for the virus.

[0191] It shows that protein chips can be used to screen proteins that interact with the host.

Claims

1. A protein chip, comprising a substrate and 160 African swine fever virus proteins connected thereto; and each protein forms a separate detection point; The 160 African swine fever virus proteins are DP63R, L60L, O61R, C62L, DP60R, A118R, E66L, B66L, I177L, X69R, DP71L, K78R, I73R, EP84R, CP80R, DP79L, L83L, C84L, DP96R, C122R, I9R, A104R, L11L, MGF_110-2L, I7L, H108R, I8L, D117L, E111R, B117L, MGF_110-14L, CP123L, E120R, MGF_110 -12L, MGF_110-4L, MGF_110-3L, MGF_110-1L, D129L, B119L, B125R, H124R, MGF_110-8L, MGF_100-1R, C129R, A137R, MGF_110-7L, E 146L, K145R, EP152R, C147L, EP153R, E165R, A151R, F165R, B169L, I10L, H171R, E183L, O174L, B175L, A179L, p14, KP177R, E184L, S 183L, E199L, K196R, I196L, K205R, CP204L, D205R, MGF_110-5L-6L, I215L, H233R, A238L, A224L, I226R, A240L, MGF_360-18R, C257 L, DP238L, H240R, E248R, D250R, I243L, B263R, S273R, MGF_300-1L, QP383R, MGF_110-13L, I267L, MGF_505-3R, EP296R, MGF_360-1 5R, CP312R, E301R, R298L, MGF_360-16R, F317L, B318L, MGF_360-8L, C315R, D339L, MGF_300-4L, D345L, F334L, p34, H339R, MGF_36 0-10L, MGF_360-12L, EP402R, MGF_360-13L, H359L, MGF_360-9L, EP364R, MGF_360-14L, MGF_360-21R, MGF_360-11L, MGF_360-3L, MGF_360-1L, MGF_360-2L, MGF_360-6L, p37, MGF_360-4L, B354L, NP419L, B385R, B407L, E42 3R, EP424R, B438L, K421R, M448R, B475L, QP509L, MGF_505-9R, B602L, MGF_505-5R, C475L, CP 530R, MGF_505-6R, MGF_505-7R, MGF_505-2R, MGF_505-10R, B646L, Q706L, C717R, F778R, NP8 68R, A859L, B962L, C962R, F1055L, D1133L, P1192R, G1211R, EP1242L, M1249L, G1340L and p150.

2. A kit comprising the protein chip according to claim 1.

3. Use of the protein chip according to claim 1 or the kit according to claim 2 in at least one of the following or in the preparation of a product having at least one of the following functions: 1) Screening or assisting in the screening of African swine fever virus protein antigens; 2) Screening or assisting in the screening of protective antigens or antigen combinations of African swine fever virus; 3) Construction of African swine fever virus protein antigen library; 4) Screening or assisting in the screening of proteins or protein combinations that specifically bind to antibodies produced after the African swine fever virus or vaccine acts on the host; 5) Construct a database of proteins that specifically bind to antibodies produced after the African swine fever virus or vaccine acts on the host; 6) Screening or assisting in the screening of immune regulatory factors of African swine fever virus; 7) Screening or assisting in the screening of African swine fever proteins that interact with immune-related proteins in infected hosts; 8) Construct a database of immune regulatory factors of African swine fever virus; 9) Detect or bind to African swine fever virus antibodies.

4. Use of the 160 African swine fever virus proteins in claim 1 in preparing a protein chip having at least one of the following functions: 1) Screening or assisting in the screening of African swine fever virus protein antigens; 2) Screening or assisting in the screening of protective antigens or antigen combinations of African swine fever virus; 3) Construction of African swine fever virus protein antigen library; 4) Screening or assisting in the screening of proteins or protein combinations that specifically bind to antibodies produced after the African swine fever virus or vaccine acts on the host; 5) Construct a database of proteins that specifically bind to antibodies produced after the African swine fever virus or vaccine acts on the host; 6) Screening or assisting in the screening of immune regulatory factors of African swine fever virus; 7) Screening or assisting in the screening of African swine fever proteins that interact with immune-related proteins in infected hosts; 8) Construct a database of immune regulatory factors of African swine fever virus; 9) Detect or bind to African swine fever virus antibodies.

5. A method for screening or assisting in screening African swine fever virus protein antigens or antigen combinations, comprising the following steps: 1) reacting the protein chip according to claim 1 with serum of a host infected with African swine fever virus or serum of a host immunized with an African swine fever virus vaccine to obtain a reaction product; 2) adding the fluorescently labeled antibody of the host serum to the reaction product of step 1) to carry out a reaction to obtain a fluorescent reaction product; 3) Detecting the fluorescent signal of the fluorescent reaction product to determine whether a certain protein in the protein chip of claim 1 is or is a candidate for being an African swine fever virus protein antigen or antigen combination.

6. The method according to claim 5, characterized in that: The method also includes step 4): using the African swine fever virus protein antigens determined in step 3) as detection antigens, respectively, and detecting whether an antigen-antibody binding reaction occurs between the antigens and African swine fever virus antibodies by enzyme-linked immunosorbent assay; if a reaction occurs, the protein is an African swine fever virus protein antigen; if no reaction occurs, the protein is not an African swine fever virus protein antigen.

7. A method for high-throughput detection of specific antibodies to the whole protein of African swine fever virus, comprising the following steps: 1) reacting all African swine fever virus proteins of the protein chip according to claim 1 with serum of a host infected with African swine fever virus or serum of a host immunized with an African swine fever virus vaccine to obtain a reaction product on the chip; 2) adding the fluorescently labeled antibody of the host serum to the reaction product of step 1) to carry out a reaction to obtain a fluorescently labeled reaction product; 3) Detecting the fluorescent signal of the fluorescently labeled reaction product to determine whether the host serum contains specific binding antibodies to a certain African swine fever protein in the protein chip of claim 1.

8. A method for screening or assisting in screening an immunomodulatory factor of African swine fever virus, comprising the following steps: 1) reacting the protein chip according to claim 1 with a biotin-labeled host immune-related protein to obtain a protein-protein binding product; 2) adding fluorescently labeled avidin to the protein-protein binding product of step 1) to react and obtain a fluorescently labeled protein-protein binding product; 3) Detecting the fluorescence signal of the fluorescently labeled protein and the protein binding product to determine whether a certain protein in the protein chip of claim 1 is an immunomodulatory factor.

9. Use of the R298L, P1192R and S273R proteins in the protein chip of claim 1 in the preparation of a product for binding or detecting the immune-related protein IRF3; Or the use of the R298L, P1192R and S273R proteins in the protein chip of claim 1 as interacting proteins of the immune-related protein IRF3; Or, use of the L60L, MGF360-12L and I73R proteins in the protein chip of claim 1 in the preparation of a product for binding or detecting the immune-related protein PERK; Or the use of L60L, MGF360-12L and I73R proteins in the protein chip of claim 1 as interacting proteins of the immune-related protein PERK; Or the use of the R298L, P1192R, S273R, L60L, MGF360-12L and I73R proteins in the protein chip of claim 1 as immunomodulatory factors.

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