A fusion antigen for detecting African swine fever virus antibodies, preparation method and detection kit

Abstract

The present invention discloses a fusion antigen, a preparation method and a detection kit for detecting antibodies to African swine fever virus. The fusion antigen is composed of SmBiT / p30 and LgBiT / C2, which are fused and expressed by two subunits of nano-luciferase, LgBiT and SmBiT, respectively, with the p30 protein of African swine fever virus and the C2 domain of Protein G. The fusion antigen can bind to a specific antibody against the p30 protein of African swine fever virus to form a functional luciferase, which emits light under the catalytic action of a substrate. The present invention utilizes the principle of forming a functional luciferase by complementary structural subunits of nano-luciferase, and uses it for the detection of antibodies to African swine fever virus with high specificity and sensitivity, and can complete the detection of antibodies in a relatively short time, providing a technical means for the prevention, control and purification of African swine fever.

Description

Technical Field

[0001] The present invention relates to a serological method for detecting antibodies to African swine fever virus, specifically, to a fusion antigen SmBi T / p30 and LgBiT / C2 which respectively fuse the p30 antigen of African swine fever virus and the C2 domain of Protein G using nano-luciferase subunits, and its application in detecting antibodies to African swine fever virus, belonging to the field of novel immune detection technology. Background Art

[0002] African swine fever (ASF) is an acute, febrile, highly contagious disease of pigs caused by the African swine fever virus (ASFV), the only member of the Asfarviridae family and the genus Asfivirus. The virus is infectious to both domestic pigs and wild boars, and the mortality rate caused by strong strains is as high as 100%. ASF is an animal disease that must be reported to the World Organization for Animal Health (WOAH), and my country has classified it as a Class I animal disease. ASFV mainly infects the host mononuclear-macrophage system and is the only known DNA virus transmitted by insects. African swine fever virus particles are icosahedral enveloped viruses with a diameter of 260 to 300 nm. The genome size is between 170 and 190 kb, encoding more than 200 proteins, of which the p30 protein is one of the structural proteins encoded by the CP204L gene, located on the inner membrane of the virus, and is also the most expressed protein in the early stage of viral infection. Studies have shown that p30 protein has strong immunogenicity and is an important target protein for the diagnosis of African swine fever.

[0003] So far, there is no safe and effective African swine fever vaccine or treatment measure in the world. The prevention and control of the disease mainly relies on early and rapid diagnosis and strict purification measures. Therefore, the development of rapid and accurate detection technology is crucial for the prevention and control of the disease.

[0004] The clinical manifestations caused by ASFV infection in hosts vary greatly depending on the virulence of the epidemic strain, the host animal species and health status, and can range from highly lethal acute manifestations to subclinical manifestations. However, the emergence of subacute and chronic cases caused by naturally attenuated strains has further increased the difficulty of ASF prevention and control. Therefore, it is urgent to carry out the detection and monitoring of ASFV antibodies to meet the needs of disease prevention, control or purification. Summary of the invention

[0005] The object of the present invention is to provide a fusion antigen (SmBiT / p30 and LgBi T / C2) for detecting African swine fever virus antibodies, a preparation method and a detection kit for sensitive, specific and rapid detection of African swine fever virus antibodies.

[0006] Specifically include the following:

[0007] In a first aspect, the present invention provides a nanoluciferase fusion antigen, wherein the nanoluciferase fusion antigen is obtained by fusion expression of a subunit of nanoluciferase and a C2 domain of Protein G.

[0008] Preferably, the subunit of the nanoluciferase is selected from LgBiT.

[0009] Preferably, the nanoluciferase subunit LgBiT is fused to the N-terminus of the C2 domain of Protein G and is named LgBiT / C2.

[0010] Preferably, the amino acid sequence of LgBiT / C2 is as shown in SEQ ID NO.1.

[0011] Preferably, the coding gene sequence of LgBiT / C2 is shown as SEQ ID NO.2.

[0012] In a second aspect, the present invention provides a recombinant plasmid comprising the gene shown in SEQ ID NO.2.

[0013] In the third aspect, the present invention provides a nano-luciferase fusion antigen composition, which is composed of the nano-luciferase fusion antigen described in the first aspect and the SmBiT / p30 fusion protein; the SmBiT / p30 fusion protein is obtained by fusion expression of the nano-luciferase subunit SmBiT and the African swine fever virus p30 protein.

[0014] Preferably, the SmBiT is fused to the N-terminus of the African swine fever virus p30 protein.

[0015] Preferably, the amino acid sequence of the SmBiT / p30 fusion protein is as shown in SEQ ID NO.3.

[0016] In a fourth aspect, the present invention provides a kit for detecting African swine fever virus antibodies, the kit comprising: a chemiluminescent reaction plate, the nano-luciferase fusion antigen composition described in the third aspect, an African swine fever virus antibody-positive serum standard, an African swine fever virus antibody-negative serum standard, a luciferase substrate, and a washing solution.

[0017] In a fifth aspect, the present invention provides an application of the nano-luciferase fusion antigen composition described in the third aspect in the detection of African swine fever virus antibodies for non-diagnostic purposes.

[0018] In a sixth aspect, the present invention provides a method for using the kit described in the fourth aspect, the method comprising: taking the serum to be tested and adding the nano-luciferase fusion antigen composition described in the third aspect; after incubation, adding a luciferase substrate; measuring the luminescence intensity, and determining whether African swine fever virus antibodies are present in the serum to be tested based on the change in the luminescence intensity.

[0019] Preferably, the determination is made based on the results of the nano-luciferase activity detection: The results were judged. If S / N ≥ 3.7113, the test was positive for African swine fever virus antibodies. If S / N < 3.7113, the test was negative for African swine fever virus antibodies.

[0020] In a sixth aspect, the present invention provides a method for preparing the fusion antigen LgBiT / C2 described in the first aspect, the method comprising the steps of: constructing a recombinant plasmid pET30a-LgBiT / C2 expressing the fusion antigen LgBiT / C2; transforming the pET30a-LgBiT / C2 recombinant plasmid into Escherichia coli (Escherichia coli) BL21 (DE3) for expression and purification, and obtaining the fusion antigen LgBiT / C2 after purification.

[0021] Preferably, the construction steps of the recombinant plasmid pET30a-LgBiT / C2 are: synthesizing the gene sequence of LgBiT / C2, and cloning it into the pET30a vector using two restriction sites, NdeⅠ and Hind III, respectively; obtaining the recombinant plasmid pET30a-LgBiT / C2.

[0022] Preferably, the purification expression method is: transforming the constructed recombinant plasmid pET30a-LgBiT / C2 into BL21 (DE 3) competent cells to obtain recombinant bacteria expressing the fusion antigen LgBiT / C2, adding IPTG to induce for 6 hours, and purifying the fusion antigen LgBiT / C2 by nickel column affinity chromatography.

[0023] The beneficial effects of the present invention are:

[0024] (1) The present invention provides two fusion proteins labeled with nano-luciferase subunits, the fusion proteins comprising SmBiT-labeled African swine fever virus p30 fusion protein SmBiT / p30 and LgBiT-labeled protein G C2 domain fusion protein LgBiT / C2, based on the high sensitivity and simplicity of nano-luciferase split complementation detection, only a trace amount of protein is needed to complete rapid detection. In the method described in the present invention, p30 protein is one of the proteins expressed in the earliest stage of African swine fever virus infection, and the antibody content against p30 protein in serum is relatively high, which improves the sensitivity and specificity of detection. The present invention is a one-step incubation and does not require a washing step, which greatly improves the rapidity and simplicity of detection. This method does not have cross-reactions with positive sera of other common pig pathogens (such as porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), pseudorabies virus (PRV), O-type foot-and-mouth disease virus (FMDV-O), and porcine epidemic diarrhea virus (PEDV)).

[0025] (2) The African swine fever virus serum antibody detection method provided by the present invention has extremely high sensitivity, strong specificity, and is quick and easy to operate. It can efficiently detect African swine fever virus antibodies in samples, and its compliance rate with the most commonly used African swine fever virus antibody ELISA detection kit in the world is 100%, and there is no missed detection or suspicious interval, which provides an important technical means for the prevention and control of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the detection principle of the fusion antigen in this application;

[0027] Figure 2 It is the preferred combination of fusion antigens in this application;

[0028] Figure 3 The preferred amount of fusion antigen used in this application is preferred;

[0029] Figure 4 The preferred amount of each substance used and the reaction time in this application;

[0030] Figure 5 The specificity of the detection method in this application;

[0031] Figure 6 The sensitivity of the detection method in this application and the commercial ELISA kit analysis. DETAILED DESCRIPTION

[0032] Embodiments of the present invention will be described in detail below in conjunction with examples. It will be appreciated by those skilled in the art that the following examples are only preferred embodiments of the present invention, so as to better understand the present invention, and thus should not be considered as limiting the scope of the present invention. For those skilled in the art, the present invention may have various changes and variations, and any modifications, equivalent replacements or improvements made within the spirit and principles of the present invention, etc., should all be included within the protection scope of the present invention.

[0033] The present invention is further described in conjunction with the accompanying drawings. The experimental methods in the following embodiments are conventional methods unless otherwise specified; the experimental materials used are purchased from conventional biochemical reagent manufacturers unless otherwise specified.

[0034] The method for detecting African swine fever virus antibodies described in the following examples is: take the serum to be tested, add the SmBiT-labeled African swine fever virus p30 fusion protein SmBiT / p30 and the LgBiT-labeled Protein G C2 domain fusion protein LgBiT / C2, incubate, add the luciferase substrate; measure the luminescence intensity, and determine whether the serum to be tested contains African swine fever virus antibodies based on the change in luminescence intensity. If SmBiT / p30 binds to the Fab region of the p30 antibody and the LgBiT / C2 protein binds to the Fc fragment of the p30 antibody, it will promote the reconstruction of the nanoluciferase SmBiT and LgBiT to form a functional luciferase, which emits light under the catalytic action of the substrate (such as Figure 1 As shown), the test was positive. The results were judged. If S / N ≥ 3.7113, the test was positive for African swine fever virus antibodies. If S / N < 3.7113, the test was negative for African swine fever virus antibodies.

[0035] Example 1 Preparation of Nano-Luciferase Subunit Labeled Fusion Antigens SmBiT / p30 and LgBiT / C2

[0036] 1. Construction of recombinant expression vector

[0037] According to the nanoluciferase subunit SmBiT and ASFV CN / SC / 2019 virus CP204L (p30) gene sequence, they were connected through the optimized GS connecting peptide, as described in SEQ ID NO. 4. The gene sequence of SEQ ID NO. 4 was commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd., and was connected to the pET30a (+) vector through the NdeⅠ and Hind III double restriction sites to form the pET30a-SmBiT / p30 recombinant vector.

[0038] According to the gene sequence of the nanoluciferase subunit LgBiT and the C2 domain of Protein G, they were connected through an optimized GS connecting peptide, as described in SEQ ID NO. 2. The gene sequence of SEQID NO. 2 was commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd., and was connected to the pET30a (+) vector through the NdeⅠ and Hind III double restriction sites to form the pET30a-LgBiT / C2 recombinant vector.

[0039] The subunits of nanoluciferase SmBiT and LgBiT were fused with the p30 antigen of ASFV and the C2 domain of Protein G, respectively. The SmBiT subunit was connected to the N- or C-terminus of the p30 antigen through a GS connecting peptide (Gly-Ser-Ser-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Ser); the LgBiT subunit was connected to the N- or C-terminus of the C2 domain of Protein G through a GS connecting peptide (Gly-Ser-Ser-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Ser); and His tags were added to facilitate the purification of the fusion proteins SmBiT / p30 and LgBiT / C2.

[0040] 2. The recombinant vector was transformed into BL21 (DE3) competent cells to prepare nanoluciferase subunit-labeled fusion proteins SmBi T / p30 and LgBiT / C2.

[0041] The recombinant vectors pET30a-SmBiT / p30 and pET30a-LgBiT / C2 were transformed into BL21 (DE3) competent cells, respectively, to obtain recombinant bacteria expressing SmBiT / p30 and LgBiT / C2. IPTG with a final concentration of 1 mM was added for induction for 6 h, and the two fusion antigens SmBiT / p30 and LgBiT / C2 were purified by nickel column affinity chromatography.

[0042] Example 2 Sample Detection

[0043] 1. Screening of fusion antigen sensors

[0044] The nanoluciferase subunit SmBiT was fused to the N- or C-terminus of the p30 protein of African swine fever virus, and the nanoluciferase subunit LgBiT was fused to the N- or C-terminus of the C2 domain of Protein G, and specific antibodies were detected by split complementation of the nanoluciferase subunits. A total of four different combinations were produced, including SmBiT / p30 and LgBiT / C2, p30 / SmBiT and LgBiT / C2, C2 / LgBiT and SmBiT / p30, and C2 / LgBiT and p30 / SmBiT. To determine the efficiency of nanoluciferase reconstruction in different combinations, 10μl ASFV positive serum was added to 50ng of SmBiT / p30 and 100ng of LgBiT / C2 fusion antigens in a total volume of 80μl, and ASFV negative serum was used as a control, and incubated at 37°C for 30min. Add 20 μl of the prepared nanoluciferase substrate and read the luminescent signal.

[0045] The results are as follows Figure 2 As shown, compared with other fusion antigen combinations, when the LgBiT / C2 and SmBit / p30 fusion antigen combination (LN-C2 and SN-p30) is selected, the signal-to-noise ratio (S / N) is the highest and the detection sensitivity is better. Therefore, the present application selects the LgBiT / C2 and SmBit / p30 fusion antigen combination that provides the highest signal-to-noise ratio (S / N) for African swine fever virus antibody detection.

[0046] Among them, the amino acid sequence of the fusion protein LgBiT / C2 is shown in SEQ ID NO.1, which consists of 240 amino acids, the 1st to 159th amino acids are the nanoluciferase subunit LgBiT, the 160th to 173rd amino acids are the GS connecting peptide, the 174th to 228th amino acids are the C2 domain of Protein G, the 229th to 232nd amino acids are the GS connecting peptide, and the 233th to 240th amino acids are 8X·HIS tags.

[0047] The amino acid sequence of the fusion protein SmBit / p30 is shown in SEQ ID NO.3, which consists of 227 amino acids. Amino acids 1 to 12 encode the nanoluciferase subunit SmBiT; amino acids 13 to 26 are GS connecting peptides; amino acids 27 to 215 encode ASFV-p30 antigens; amino acids 216 to 219 are GS connecting peptides; and amino acids 220 to 227 encode 8X·His tags.

[0048] 2. Optimization of the dosage of each substance and reaction time in this method

[0049] The optimal dosage of fusion antigen SmBiT / p30 and LgBiT / C2 was optimized, and different dosages of fusion antigen Sm BiT / p30 (50, 25, 12.5, 6.25, 3.125 and 1.5625 ng) and LgBiT / C2 (100, 50, 25, 12.5 and 6.25 ng) were set. By detecting the positive and negative serum of African swine fever virus antibodies, the optimal dosages of SmBiT / p30 and LgBiT / C2 were determined to be 6.25 ng and 50 ng, respectively, according to the maximum S / N value. Figure 3 shown.

[0050] The serum dosage was optimized. Using the optimal dosage of fusion antigens SmBiT / p30 and LgBiT / C2, the optimal serum dosage for the SmBiT / p30 and LgBiT / C2 fusion sensor was determined to be 1 μl based on the maximum S / N value by detecting different dosages (20, 10, 5, 2.5, 1 and 0.5 μl) of African swine fever virus antibody positive and negative serum. Figure 4 shown.

[0051] The optimal reaction time between SmBiT / p30 and LgBiT / C2 fusion antigens and serum was determined (5, 10, 20, 30, 45 and 60 min). By testing the positive and negative serum of African swine fever virus antibodies, the optimal reaction time was determined to be 10 min based on the maximum S / N value. Figure 4 shown.

[0052] 3. Determination of the detection threshold of this method

[0053] The critical value of this method was determined by testing 192 negative sera with known background of African swine fever virus antibodies. Based on the luminescence intensity of the tested samples and calculation of the S / N value, this method uses the average value of the S / N of the 192 samples (1.1057) plus 3 times the standard deviation (0.8685) as the critical value (3.7113). If the sample S / N ≥ 3.7113, it is determined to be positive for African swine fever virus antibodies; if S / N < 3.7113, it is determined to be negative for African swine fever virus antibodies. Figure 5 shown.

[0054] 4. Cross-reactivity and sensitivity

[0055] The specificity of the method was determined by testing positive sera of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), foot-and-mouth disease virus type O (FMDV-O), porcine Actinobacillus pleuropneumoniae (APP) and Haemophilus parasuis (HPS). The results showed that the method had no cross-reaction with the positive sera of the above-mentioned porcine pathogens.

[0056] By testing the dilution ratio of African swine fever virus positive serum and comparing it with the commercial ELISA kit, the results showed that the minimum detection limit of this method was 1:2048, and the minimum detection limit of the commercial kit was 1:256; the analytical sensitivity of this method was 8 times higher than that of the commercial kit. Figure 6 shown.

[0057] 5. Consistency detection

[0058] This method and commercial ELISA kits were used to simultaneously test 90 pig clinical serum samples. Both this method and commercial ELISA kits detected 26 positive samples and 64 negative samples for African swine fever virus antibodies. The results of this method and commercial kits were consistent for 90 samples, with a compliance rate of 100%.

[0059] The present invention illustrates the detailed features and detailed methods of the present invention through the above embodiments, but the present invention is not limited to the above detailed features and detailed methods, that is, it does not mean that the present invention must rely on the above detailed features and detailed methods to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the components selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A nano-luciferase fusion antigen composition, characterized in that: The nano-luciferase fusion antigen composition consists of a nano-luciferase fusion antigen LgBiT / C2 and a SmBiT / p30 fusion protein; the SmBiT / p30 fusion protein is obtained by fusing the nano-luciferase subunit SmBiT to the N-terminus of the African swine fever virus p30 protein; the LgBiT / C2 is obtained by fusing the nano-luciferase subunit LgBiT to the N-terminus of the C2 domain of Protein G.

2. The nano-luciferase fusion antigen composition according to claim 1, characterized in that: The amino acid sequence of the LgBiT / C2 is shown in SEQ ID NO.1; the amino acid sequence of the SmBiT / p30 fusion protein is shown in SEQ ID NO.

3.

3. A kit for detecting African swine fever virus antibodies, characterized in that: The kit comprises: a chemiluminescent reaction plate, the nano-luciferase fusion antigen composition according to any one of claims 1-2, an African swine fever virus antibody positive serum standard, an African swine fever virus antibody negative serum standard, a luciferase substrate, and a washing solution.

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