Application of a nano-luciferase fusion antigen in the detection of African swine fever virus antibodies

Abstract

The present invention discloses an application of a nano-luciferase fusion antigen in the detection of African swine fever virus antibodies. The fusion antigen is composed of LgBiT-p30 and SmBiT-p30 proteins, which are fused and expressed by two subunits of nano-luciferase, LgBiT and SmBiT, respectively, with the African swine fever virus p30 protein. The fusion antigen can bind to a specific antibody against the African swine fever virus p30 protein to form a functional luciferase, which reacts with a substrate to produce a bright luminescent signal. The fusion antigen has high specificity and sensitivity in the detection of African swine fever virus antibodies, making the antibody detection process more efficient and rapid. The present invention realizes simple, rapid, sensitive and specific detection of African swine fever virus antibodies, and provides an innovative and reliable solution for the serological diagnosis of African swine fever.

Description

Technical Field

[0001] The present invention relates to a method for serological detection of African swine fever virus antibodies, specifically, to a method based on complementary fusion antigen of nano-luciferase subunits and its application in the detection of African swine fever virus antibodies, belonging to the field of novel immunoassay technology. Background Art

[0002] African swine fever (ASF) is an acute, febrile, highly contagious animal disease of pigs caused by African swine fever virus (ASFV). Clinically, it is characterized by high fever, reticuloendothelial hemorrhage and high mortality. The mortality rate of susceptible pigs is as high as 100%. The World Organization for Animal Health (WOAH) lists it as a notifiable animal disease, and my country lists it as a Class I animal disease. The ASFV genome is a single-molecule linear double-stranded DNA with covalently closed ends, about 170 to 190 kb in length, encoding more than 200 proteins, among which the p30 protein is one of the structural proteins encoded by CP204L and is also expressed in the early stage of infection. Studies have shown that the p30 protein has strong immunogenicity and is an important diagnostic antigen for African swine fever virus. Due to the complex infection mechanism of ASFV, there is no safe and effective African swine fever vaccine and treatment measures worldwide so far, so rapid and accurate diagnosis is the key to controlling the spread and occurrence of the disease.

[0003] For example, in the Chinese invention patent application with publication number CN116082524A, the detection method uses an African swine fever virus antigen coated on a solid phase medium, and the African swine fever virus antigen is an African swine fever virus p30-p54 fusion protein. The preparation process of the African swine fever virus p30-p54 fusion protein is cumbersome, and the optimization of conditions is required, which makes the detection steps complicated.

[0004] Up to now, due to the lack of a safe and effective vaccine, the detection of antibodies specific to the African swine fever virus means infection with the virus. In order to effectively control African swine fever, the development of highly sensitive and specific antibody detection technology and kits has important practical significance for the prevention and control of the disease. Summary of the invention

[0005] In response to the above technical problems, the purpose of the present invention is to provide a nano-luciferase subunit complementary fusion antigen combination (LgBiT / p30 and SmBiT / p30) and its application in the detection of African swine fever virus antibodies, which has the characteristics of high sensitivity, specificity, simplicity and speed.

[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 p30 protein of African swine fever virus.

[0008] Preferably, the nanoluciferase fusion antigen is composed of LgBiT-p30 fusion protein expressed by fusion of nanoluciferase subunit LgBiT and African swine fever virus p30 protein and SmBiT-p30 fusion protein expressed by fusion of nanoluciferase subunit SmBiT and African swine fever virus p30 protein.

[0009] Preferably, the nanoluciferase subunits LgBiT and SmBiT are respectively fused to the N-terminus of the African swine fever virus p30 protein.

[0010] Preferably, the amino acid sequence of the LgBiT-p30 fusion protein is shown as SEQ ID NO.1; the amino acid sequence of the SmBiT-p30 fusion protein is shown as SEQ ID NO.2.

[0011] Preferably, the gene sequence encoding the LgBiT-p30 fusion protein is shown as SEQ ID NO.3; the gene sequence encoding the SmBiT-p30 fusion protein is shown as SEQ ID NO.4.

[0012] In a second aspect, the present invention provides a recombinant plasmid, wherein the recombinant plasmid comprises the gene shown in SEQ ID NO.3 and / or the gene shown in SEQ ID NO.4.

[0013] In a third aspect, the present invention provides a method for preparing the nano-luciferase fusion antigen described in the first aspect, the method comprising: constructing recombinant plasmids expressing LgBiT-p30 fusion protein and SmBiT-p30 fusion protein, respectively; expressing and purifying to obtain LgBiT-p30 fusion protein and SmBiT-p30 fusion protein.

[0014] Preferably, the recombinant plasmid is constructed by:

[0015] The coding sequence of LgBiT-p30 fusion protein was double-digested through the two restriction sites of SacⅠ and NheⅠ and then ligated into the pBiT1.1-N[TK / LgBit] vector to construct the recombinant plasmid pBiT1.1N-LgBit-p30;

[0016] The coding sequence of SmBiT-p30 fusion protein was double-digested with XhoⅠ and EcoRⅠ restriction sites and then ligated into pBiT2.1-N[TK / SmBit] vector to construct the recombinant plasmid pBiT2.1N-SmBit-p30.

[0017] Preferably, the expression purification method is: HEK-293T cells are inoculated in 10 cm 2 When the cell density in the cell culture dish was about 80%, Lipofectamine TM HEK-293T cells were transfected with the recombinant plasmid pBiT1.1N-LgBit-p30 and the recombinant plasmid pBiT2.1N-SmBit-p30 using 3000 Transfection Reagent. 48 hours after transfection, the cultured cell pellets were collected, lysed by ultrasonication, and the LgBiT-p30 fusion protein and the SmBiT-p30 fusion protein were obtained after purification.

[0018] In a fourth aspect, the present invention provides an application of the nano-luciferase fusion antigen described in the first aspect in the detection of African swine fever virus antibodies for non-diagnostic purposes.

[0019] In a fifth aspect, the present invention provides a kit for detecting African swine fever virus antibodies, the kit comprising: a chemiluminescent reaction plate, the LgBiT-p30 fusion protein and the SmBiT-p30 fusion protein described in the first aspect, ProteinA / G Magnetic Beads, African swine fever virus antibody positive serum standard, African swine fever virus antibody negative serum standard, luciferase substrate, and washing solution.

[0020] In a sixth aspect, the present invention provides a method for using the kit described in the fifth aspect, the method comprising: taking the serum to be tested and incubating it with Protein A / G Magnetic Beads; after washing, adding the LgBiT-p30 fusion protein and the SmBiT-p30 fusion protein; after washing, adding 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.

[0021] Preferably, the determination is made based on the result of the nano-luciferase activity detection: if a bright luminescent signal is generated by the reaction, the detection is positive; A value greater than 4.167 is defined as positive, indicating infection with African swine fever virus, and a value less than or equal to 4.167 is defined as negative, indicating no infection with African swine fever virus.

[0022] The beneficial effects of the present invention are as follows: (1) The present invention provides a nano-luciferase subunit-labeled African swine fever virus p30 fusion protein, wherein the fusion protein comprises LgBiT-p30 labeled with the nano-luciferase subunit LgBiT and SmBiT-p30 labeled with the nano-luciferase subunit SmBiT. Based on the high sensitivity of complementary detection of nano-luciferase subunits, only a trace amount of protein is required for rapid detection. In the method of the present invention, magnetic beads are used to capture antibodies in serum samples in advance, and p30 protein is one of the earliest proteins expressed after infection with African swine fever virus. The p30 antibody content in serum is relatively high, and the detection sensitivity and specificity are greatly improved by magnetic bead capture. The fusion protein in the present invention is prepared by a eukaryotic expression system, and the expressed protein contains post-translational modifications such as phosphorylation, which is closer to the natural protein of the virus, providing a more suitable fusion antigen for the detection of African swine fever virus antibodies. There is no cross-reaction with positive sera of other common pathogens (such as porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), pseudorabies virus (PRV), foot-and-mouth disease virus type O (FMDV-O), and porcine epidemic diarrhea virus (PEDV)).

[0023] (2) The African swine fever virus antibody detection method based on the nano-luciferase split-complementation detection system of the present invention has the advantages of extremely high sensitivity, strong specificity, and quick and easy operation. It can efficiently detect the content of African swine fever virus p30 antibodies in samples, and the consistency rate with the internationally commonly used African swine fever virus antibody detection kit is more than 98.19%, and there is no missed detection or suspicious interval, which is easy to detect very early infection. It has broad market prospects, especially in the normalization of African swine fever prevention and control and virus antibody monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the detection principle of the fusion antigen in this application;

[0025] Figure 2 It is the preferred fusion antigen in the present invention;

[0026] Figure 3 The preferred amount of the fusion antigen used in the present invention is preferred;

[0027] Figure 4 The preferred amount of each substance used and the reaction time in the present invention;

[0028] Figure 5 ROC analysis determined the critical value of detection by this method;

[0029] Figure 6 The specificity of this method and its minimum detection limit compared with commercial ELISA kits. DETAILED DESCRIPTION

[0030] 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.

[0031] 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.

[0032] The method for detecting African swine fever virus antibodies described in the following embodiments is: take the serum to be tested and incubate it with Protein A / G Magnetic Beads, after washing, add the fusion proteins LgBiT-p30 and SmBiT-p30, after washing, add the luciferase substrate; measure the luminescence intensity, and determine whether there are African swine fever virus antibodies in the serum to be tested based on the change in luminescence intensity. If the fusion protein LgBiT-p30 labeled with the nanoluciferase subunit LgBiT and the fusion protein SmBiT-p30 labeled with the nanoluciferase subunit SmBiT are combined with two Fab fragments of the same p30 antibody, the structural complementarity of the nanoluciferase subunits LgBiT and SmBiT can be promoted to form a functional luciferase, which reacts with the substrate to produce a bright luminescent signal (such as Figure 1 As shown), the test was positive. A value greater than 4.167 is defined as positive, indicating infection with African swine fever virus, and a value less than or equal to 4.167 is defined as negative, indicating no infection with African swine fever virus.

[0033] Example 1 Preparation of African swine fever virus p30 fusion antigens LgBiT-p30 and SmBiT-p30 labeled with nanoluciferase subunits

[0034] 1. Construction of recombinant expression vector

[0035] Based on the gene sequence of CP204L of ASFV (CN / SC2019) and the embedded optimized Linker sequence, as described in SEQ ID NO.3-6. Entrusted Shanghai Shenggong Biotechnology Co., Ltd. to synthesize.

[0036] The gene sequences of SEQ ID NO.3 and 5 were constructed into the pBiT1.1-N[TK / LgBiT] vector through Sac I and Nhe I to form the LgBiT-p30 recombinant vector; the gene sequences of SEQ ID NO.4 and 6 were constructed into the pBiT2.1-N[TK / SmBiT] vector through Xho I and EcoR I to form the SmBiT-p30 recombinant vector.

[0037] The N-terminal large subunit (LgBiT) or C-terminal small subunit (SmBiT) of nanoluciferase was fused with ASFV p30 protein, respectively, connected to the N- or C-terminus through a GS linker (Gly-Ser-Ser-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Ser), and a His tag was attached to facilitate the purification of the p30 fusion protein.

[0038] 2. Transfect cells to prepare p30 fusion protein labeled with nanoluciferase subunit

[0039] Inoculate HEK-293T cells to 10 cm 2 When the cell density of the cell culture dish reaches 75%-90%, use Lipofectamine TM 10 μg of eukaryotic expression plasmid pBiT1.1-N-p30-LgBiT or pBiT2.1-N-p30-SmBiT was transfected with 3000 transfection reagent (Invitrogen). After 6 hours, the culture plate was gently shaken several times, the culture medium containing the transfection reagent was aspirated, 10 ml of fresh complete culture medium was added, and the culture was continued for 48 hours. The cells were collected by centrifugation at 1500·g, washed 3 times with PBS, and then 5.0 mL of NP-40 lysis buffer (Thermo Fisher Scientific) was added and lysed on ice for 30 minutes. After sonication and centrifugation, the His-tagged p30 fusion protein was purified by using the Mag-Beads His-Tag protein purification kit (BBI) according to the manufacturer's instructions. Centrifugation was performed at 12000·g for 10 minutes at 4°C. The purity and integrity of the target protein were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the concentration was determined using Pierce TM Protein concentration was determined by BCA protein assay kit (Thermo Fisher Scientific). The fusion protein was further validated by Western blot using specific antibodies. Meanwhile, the reactivity of p30 fusion protein was detected by Western blot using appropriate dilutions of ASFV positive serum as primary antibody and corresponding HRP-labeled goat anti-swine antibody (Abcam).

[0040] Example 2 Sample Detection

[0041] 1. Dual selection of fusion protein sensors

[0042] Nanoluciferase LgBiT and SmBiT subunits were attached to the N- or C-terminus of p30 protein, respectively, and specific antibodies were detected by luciferase split complementation assay. This resulted in four different combinations, including fusion proteins N-LgBit-p30 and N-SmBit-p30 (LN-p30 and SN-p30), fusion proteins N-LgBit-p30 and p30-SmBit-C (LN-p30 and SC-p30), fusion proteins p30-LgBit-C and N-SmBit-p30 (LC-p30 and SN-p30), and fusion proteins p30-LgBit-C and p30-SmBit-C (LC-p30 and SC-p30).

[0043] To evaluate the efficiency of LgBiT and SmBiT subunit fusion in different directions (N- or C-terminal of p30 protein), we incubated 20 μl ASFV-positive serum with 30 μl protein A / G magnetic beads (MCE, USA) for 30 min, while using ASFV-negative serum as a control. Subsequently, IgG-Protein A / G magnetic beads were washed with PBS, and 20 or 40 ng of LgBit- and SmBit-p30 fusion sensors were added simultaneously in a total volume of 80 μl and incubated at 37°C for 30 min.

[0044] According to Nano- NanoLuc substrate was prepared according to the instructions of Live Cell Assay System (Promega) and 20 μl of the prepared substrate was added. The luminescent signal was read by Navigator Microplate Luminometer (Promega). Figure 2 As shown, compared with other fusion antigen combinations, the signal-to-noise ratio (S / N) of the N-LgBit-p30 and N-SmBit-p30 fusion antigen combination (LN-p30 and SN-p30) is the highest, and the detection sensitivity is better. Therefore, this application selects the N-LgBit-p30 and N-SmBit-p30 fusion antigen combination that provides the highest signal-to-noise ratio (S / N) for African swine fever virus antibody detection. Figure 3 As shown, the higher the amount of N-LgBit / p30 and N-SmBit / p30 fusion protein combination (LN-p30 and SN-p30) used, the higher the signal-to-noise ratio (S / N).

[0045] Among them, the amino acid sequence of the fusion protein N-LgBit-p30 is shown in SEQ ID NO.1, which consists of 374 amino acids, of which amino acids 1 to 159 are the amino acid sequence of the nanoluciferase subunit LgBiT; amino acids 160 to 173 are the GS connecting peptide; amino acids 174 to 362 are the amino acid sequence of the ASFV-p30 antigen; amino acids 363 to 366 are the GS connecting peptide; and amino acids 367 to 374 are the amino acid sequence of the 8X·His tag.

[0046] The amino acid sequence of the fusion protein N-SmBit-p30 is shown in SEQ ID NO.2, 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.

[0047] 2. Optimization of the dosage of each substance and reaction time in this method

[0048] In order to avoid protein aggregation leading to detectable signals, the serum should be diluted to an appropriate level for antibody detection. By testing different amounts of ASFV-positive and -negative sera (20, 10, 5, 2.5 and 1 μl), the optimal serum dosage for LgBit-p30 and SmBit-p30 fusion proteins was determined to be 5 μl ( Figure 4 shown).

[0049] Similarly, different amounts of protein A / G magnetic beads (5, 10, 20, 40, and 50 μl) were tested, and the optimal amount of magnetic beads was determined to be 10 μl based on the maximum signal-to-noise ratio ( Figure 4 shown).

[0050] The incubation time for IgG capture was determined (5, 10, 15, 20, and 25 min), and the IgG capture time was determined to be 15 min ( Figure 4 shown).

[0051] The incubation time of the samples was determined (5, 10, 15, 20 and 25 min), and the sample incubation time was determined to be 20 min based on the maximum signal-to-noise ratio ( Figure 4 shown).

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

[0053] The threshold of this method was determined by testing known ASFV antibody positive and negative sera. Using the above optimal conditions and procedures, 216 ASFV antibody negative and 108 ASFV antibody positive samples were tested, and the Receiver Operating Characteristic (ROC) curve was used to determine that the detection threshold of this method was 4.167. When S / N ≥ 4.167, it was determined to be positive, and S / N < 4.167 was determined to be negative. The results are as follows Figure 5 shown.

[0054] 4. Cross-reactivity and sensitivity

[0055] By testing the positive sera of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), porcine circovirus type 2 (PCV2) and foot-and-mouth disease virus type O (FMDV-O), it was shown that this method has no cross-reaction with the positive sera of the above-mentioned swine viruses.

[0056] The dilution ratio of African swine fever virus positive serum was tested and compared with the commercial ELISA kit. The minimum detection limit of this method reached 1:51200, and the minimum detection limit of the commercial ELISA kit reached 1:3200. The sensitivity of this method was 16 times higher than that of the commercial kit. Figure 6 shown.

[0057] 5. Consistency detection

[0058] This method was used to test 166 pig clinical sera and compared with a commercial ELISA kit (INGEZIMPPACOMPACK3, INGENSA, Spain). The results showed that this method detected 22 positive samples and 144 negative samples for African swine fever virus antibodies; the commercial kit detected 23 positive samples and 143 negative samples for African swine fever virus antibodies. The concordance rate of the two methods reached 98.19%, indicating that this method has a high concordance rate with the commercial kit.

[0059] Table 1. Comparison of this method and commercial kits in detecting clinical samples

[0060]

[0061] 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, characterized in that: The nanoluciferase fusion antigen is composed of the LgBiT-p30 fusion protein expressed by the fusion of the nanoluciferase subunit LgBiT and the African swine fever virus p30 protein, and the SmBiT-p30 fusion protein expressed by the fusion of the nanoluciferase subunit SmBiT and the African swine fever virus p30 protein; the amino acid sequence of the LgBiT-p30 fusion protein is shown in SEQ ID NO.1; the amino acid sequence of the SmBiT-p30 fusion protein is shown in SEQ ID NO.

2.

2. The nano-luciferase fusion antigen according to claim 1, characterized in that: The coding gene sequence of the LgBiT-p30 fusion protein is shown in SEQ ID NO.3; the coding gene sequence of the SmBiT-p30 fusion protein is shown in SEQ ID NO.

4.

3. A recombinant plasmid set, characterized in that: The recombinant plasmid group comprises the gene shown in SEQ ID NO.3 and the gene shown in SEQ ID NO.

4.

4. A method for preparing a nano-luciferase fusion antigen as claimed in any one of claims 1-2, characterized in that: The method comprises: constructing recombinant plasmids for expressing LgBiT-p30 fusion protein and SmBiT-p30 fusion protein respectively; and obtaining LgBiT-p30 fusion protein and SmBiT-p30 fusion protein through expression and purification.

5. The preparation method according to claim 4, characterized in that: The construction method of the recombinant plasmid is: The coding sequence of LgBiT-p30 fusion protein was expressed by Sac Ⅰ and Nhe Ⅰ The two restriction sites were double-digested and connected to the pBiT1.1-N[TK / LgBit] vector to construct the recombinant plasmid pBiT1.1N-LgBit-p30; The coding sequence of SmBiT-p30 fusion protein was used XOt Ⅰ and EcoR Ⅰ The two restriction sites were double-digested and connected to the pBiT2.1-N[TK / SmBit] vector to construct the recombinant plasmid pBiT2.1N-SmBit-p30.

6. Use of the nano-luciferase fusion antigen as described in any one of claims 1-2 in the detection of African swine fever virus antibodies for non-diagnostic purposes.

7. A kit for detecting antibodies to African swine fever virus, characterized in that: The kit comprises: a chemiluminescent reaction plate, the LgBiT-p30 fusion protein and the SmBiT-p30 fusion protein described in claim 1, Protein A / G Magnetic Beads, 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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