Dual-antigen sandwich antibody detection kit for African swine fever virus and its preparation method

Abstract

The present invention provides a double antigen sandwich antibody detection kit for African swine fever virus, which comprises a test strip, comprising a bottom plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorbent pad in sequence on the bottom plate, the gold label pad adsorbing a recombinant African swine fever virus P30 protein labeled with colloidal gold; the nitrocellulose membrane comprises a detection line and a quality control line, the detection line is immobilized with a recombinant African swine fever virus P30 protein, the quality control line is immobilized with a polyclonal antibody to the African swine fever virus P30 protein, and the recombinant African swine fever virus P30 protein is shown in SEQ.ID No. 3. The kit can realize early detection of infection, and the detection sensitivity is higher than that of the kit in the prior art, which is convenient for timely, accurate and convenient clinical monitoring and early warning.

Description

Technical Field

[0001] The present invention belongs to the field of immunoassays, and in particular relates to an African swine fever virus double antigen sandwich antibody detection kit and a preparation method thereof. Background Art

[0002] African swine fever (ASF) is an acute, febrile, highly contagious disease of pigs caused by the African swine fever virus (ASFV). It is characterized by a short course, high morbidity, and a high mortality rate (up to 100%). Clinical symptoms and pathological changes are similar to those of acute swine fever, making it easy to misdiagnose. Symptoms include high fever, congestion and cyanosis of the skin, abortion, edema, and organ hemorrhage.

[0003] The disease is classified as a Category I animal disease in my country and is a notifiable animal disease by the World Organization for Animal Health (OIE). my country reported its first case of African swine fever in 2018, and it quickly spread to all provinces and regions. China accounts for 50% of the world's pig population, and the spread of ASFV in China poses a serious threat to the global pig farming industry. To date, the primary diagnosis of African swine fever has been based on viral gene detection, using RT-PCR and partial genome sequence analysis. This is time-consuming and cannot provide a timely and accurate diagnosis of infection. Therefore, there is an urgent need for colloidal gold test strips that can quickly, accurately, and effectively detect antibodies. This would allow for real-time testing on pig farms and prevent unnecessary transmission due to factors such as sample transportation.

[0004] At the same time, the existing technology urgently needs a test kit with high sensitivity and high specificity that can detect early infection of African swine fever, and can provide timely, accurate and convenient clinical monitoring and early warning, as well as monitor the early immune effects of vaccines. Summary of the Invention

[0005] The object of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide an African swine fever virus double antigen sandwich antibody detection kit, wherein the kit comprises an African swine fever virus double antigen sandwich antibody detection test strip, wherein the African swine fever virus double antigen sandwich antibody detection test strip comprises a bottom plate, wherein the bottom plate is sequentially provided with a sample pad, a gold label pad, a nitrocellulose membrane and a water-absorbing pad, and the gold label pad is adsorbed with a recombinant African swine fever virus P30 protein labeled with colloidal gold; the nitrocellulose membrane comprises a detection line and a quality control line, the detection line is immobilized with the recombinant African swine fever virus P30 protein, and the quality control line is immobilized with an African swine fever virus P30 protein polyclonal antibody, wherein the recombinant African swine fever virus P30 protein is shown in SEQ ID No. 3.

[0006] The African swine fever virus double antigen sandwich antibody detection kit of the present invention uses recombinant African swine fever virus P30 protein as an antigen and detects African swine fever virus antibodies in samples by a double antigen sandwich method. It has high sensitivity and high specificity and can monitor and warn in the early stages of infection.

[0007] The preparation of the colloidal gold-labeled antigen protein in the African swine fever virus double antigen sandwich antibody detection test strip and the immobilized antigen protein on the detection line in the African swine fever virus double antigen sandwich antibody detection kit of the present invention can be achieved by different molecular biological expression methods, or by the same molecular biological expression method. The molecular biological expression method includes but is not limited to a prokaryotic expression system, a mammalian cell expression system, an insect cell expression system, a yeast expression system, or direct synthesis; more preferably, the prokaryotic expression system is an E. coli expression system, the mammalian cell expression system is a CHO cell expression system, and the insect cell expression system is a baculovirus expression system.

[0008] As an embodiment of the present invention, in the kit of the present invention, the colloidal gold-labeled recombinant African swine fever virus P30 protein and the recombinant African swine fever virus P30 protein immobilized on the detection line are respectively expressed in different expression systems or obtained by preparation methods; the colloidal gold-labeled recombinant African swine fever virus P30 protein and the recombinant African swine fever virus P30 protein immobilized on the detection line are respectively recombinant African swine fever virus P30 proteins expressed by the following expression systems or directly synthesized: prokaryotic expression system, mammalian cell expression system, insect cell expression system, yeast expression system.

[0009] The recombinant African swine fever virus P30 protein of the present invention expressed by a pair of different expression systems can ensure better detection sensitivity when used as a double antigen for sandwich method detection of antibodies.

[0010] As a more preferred embodiment of the present invention, the prokaryotic expression system is an E. coli expression system, the mammalian cell expression system is a CHO cell expression system, and the insect cell expression system is a baculovirus expression system.

[0011] As an embodiment of the present invention, in the kit of the present invention, the colloidal gold-labeled recombinant African swine fever virus P30 protein and the recombinant African swine fever virus P30 protein fixed on the detection line are expressed by a baculovirus expression system or a CHO cell expression system.

[0012] The present invention uses recombinant African swine fever virus P30 protein expressed by a baculovirus expression system and a CHO cell expression system as a double antigen sandwich to detect African swine fever virus antibodies, which can achieve high-sensitivity clinical detection and early detection of infection.

[0013] As a preferred embodiment of the present invention, in the kit of the present invention, the colloidal gold-labeled recombinant African swine fever virus P30 protein is expressed by a baculovirus expression system, and the recombinant African swine fever virus P30 protein immobilized on the detection line is expressed by a CHO cell expression system, or the colloidal gold-labeled recombinant African swine fever virus P30 protein is expressed by a CHO cell expression system, and the recombinant African swine fever virus P30 protein immobilized on the detection line is expressed by a baculovirus expression system.

[0014] The present invention uses recombinant African swine fever virus P30 protein expressed by a baculovirus expression system and a CHO cell expression system respectively as a double antigen sandwich to detect African swine fever virus antibodies, which can achieve high-sensitivity clinical detection, far higher than the test kits in the prior art, and can detect early infection and monitor the initial immune status.

[0015] As an embodiment of the present invention, in the kit of the present invention, the concentration of the colloidal gold-labeled recombinant African swine fever virus P30 protein is 5 μg / ml to 20 μg / ml, the concentration of the recombinant African swine fever virus P30 protein fixed on the detection line is 0.05 mg / ml to 0.25 mg / ml, and the concentration of the African swine fever virus P30 protein polyclonal antibody is 1 mg / ml to 2 mg / ml.

[0016] In some embodiments of the present invention, the concentration of the colloidal gold-labeled recombinant African swine fever virus P30 protein labeling concentration can be 5μg / ml, 6μg / ml, 7μg / ml, 8μg / ml, 9μg / ml, 10μg / ml, 11μg / ml, 12μg / ml, 13μg / ml, 14μg / ml, 15μg / ml, 16μg / ml, 17μg / ml, 18μg / ml, 19μg / ml, and 20μg / ml.

[0017] In some embodiments of the present invention, the concentration of the immobilized recombinant African swine fever virus P30 protein can be 0.05 mg / ml, 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.10 mg / ml, 0.11 mg / ml, 0.12 mg / ml, 0.13 mg / ml, 0.14 mg / ml, 0.15 mg / ml, 0.16 mg / ml, 0.17 mg / ml, 0.18 mg / ml, 0.19 mg / ml, 0.20 mg / ml, 0.21 mg / ml, 0.22 mg / ml, 0.23 mg / ml, 0.24 mg / ml, and 0.25 mg / ml.

[0018] In some embodiments of the present invention, the concentration of the immobilized African swine fever virus P30 protein polyclonal antibody can be 1 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml, or 2 mg / ml.

[0019] As a preferred embodiment of the present invention, in the kit of the present invention, the concentration of the colloidal gold-labeled recombinant African swine fever virus P30 protein is 12 μg / ml, the concentration of the recombinant African swine fever virus P30 protein fixed on the detection line is 0.2 mg / ml, and the concentration of the African swine fever virus P30 protein polyclonal antibody is 2 mg / ml.

[0020] As an embodiment of the present invention, in the kit of the present invention, the African swine fever virus double antigen sandwich antibody detection test strip includes a base plate, the base plate having a first end and a second end, and a sample pad, a gold label pad, a nitrocellulose membrane and a water-absorbing pad in sequence along the direction from the first end to the second end, the nitrocellulose membrane is in contact with the gold label pad or with the sample pad and the gold label pad, so that the complex of the African swine fever virus P30 antibody and the colloidal gold-labeled recombinant African swine fever virus P30 protein can migrate on it toward the second end of the base plate.

[0021] As an embodiment of the present invention, in the kit of the present invention, the kit further comprises a sample processing solution, and the sample processing solution is a 0.02M PB solution with a pH of 7.4 and containing 0.3% V / V Tween-20.

[0022] The present invention also provides a method for preparing the kit, wherein the method comprises: step (1) expressing and purifying a recombinant African swine fever virus P30 protein labeled with colloidal gold, expressing and purifying the recombinant African swine fever virus P30 protein to be fixed on a detection line, and preparing the African swine fever virus P30 protein polyclonal antibody; step (2) labeling the recombinant African swine fever virus P30 protein expressed and purified in step (1) with colloidal gold, adsorbing the colloidal gold-labeled recombinant African swine fever virus P30 protein on the gold label pad, fixing the recombinant African swine fever virus P30 protein expressed and purified to be fixed on the detection line on the detection line, and fixing the prepared African swine fever virus P30 protein polyclonal antibody on the quality control line; Step (3) assembling the sample pad, the gold label pad adsorbed with the colloidal gold-labeled recombinant African swine fever virus P30 protein in step (2), the nitrocellulose membrane and the absorbent pad to the bottom plate in sequence, the nitrocellulose membrane contacts the gold label pad or contacts the sample pad and the gold label pad, so that the combination of the African swine fever virus P30 antibody and the colloidal gold-labeled recombinant African swine fever virus P30 protein can migrate on it toward the second end of the bottom plate, thereby obtaining the African swine fever virus double antigen sandwich antibody detection test strip; step (4) preparing a sample processing liquid; and step (5) assembling the African swine fever virus double antigen sandwich antibody detection test strip prepared in step (3) and the sample processing liquid prepared in step (4) into a test kit.

[0023] As an embodiment of the present invention, in the method described in the present invention, the recombinant African swine fever virus P30 protein prepared by colloidal gold labeling in step (1) and the recombinant African swine fever virus P30 protein prepared to be fixed on the detection line are expressed by a baculovirus expression system or a CHO cell expression system.

[0024] As a preferred embodiment of the present invention, in the method described in the present invention, the recombinant African swine fever virus P30 protein labeled with colloidal gold is expressed by a baculovirus expression system, and the recombinant African swine fever virus P30 protein fixed on the prepared detection line is expressed by a CHO cell expression system, or the recombinant African swine fever virus P30 protein labeled with colloidal gold is expressed by a CHO cell expression system, and the recombinant African swine fever virus P30 protein fixed on the prepared detection line is expressed by a baculovirus expression system.

[0025] As an embodiment of the present invention, in the method described in the present invention, the concentration of the colloidal gold-labeled recombinant African swine fever virus P30 protein in step (2) is 5 μg / ml to 20 μg / ml, the concentration of the recombinant African swine fever virus P30 protein fixed on the detection line is 0.05 mg / ml to 0.25 mg / ml, and the concentration of the African swine fever virus P30 protein polyclonal antibody is 1 mg / ml to 2 mg / ml.

[0026] As an embodiment of the present invention, in the method described in the present invention, the concentration of the colloidal gold-labeled recombinant African swine fever virus P30 protein in step (2) is 12 μg / ml, the concentration of the recombinant African swine fever virus P30 protein fixed on the detection line is 0.2 mg / ml, and the concentration of the African swine fever virus P30 protein polyclonal antibody is 2 mg / ml.

[0027] As an embodiment of the present invention, in the method of the present invention, the sample treatment solution in step (4) is a 0.02M PB solution with a pH of 7.4 and containing 0.3% V / V Tween-20.

[0028] The kit prepared by the present invention is used to detect African swine fever virus P30 antibodies in serum, has high sensitivity and good specificity, and can detect antibodies early, which is convenient for timely and accurate clinical monitoring and early warning. DETAILED DESCRIPTION

[0029] Hereinafter, embodiments of the present invention will be described.

[0030] definition

[0031] The term "African swine fever virus P30 protein" or "ASFV P30 protein", also known as CP204L or P32, is a 30kD protein encoded by the ORFCP204L gene. It is one of the main structural proteins and strong immunogenic proteins of ASFV. It is expressed and secreted in the early stage of viral infection and plays a role in the viral invasion process.

[0032] The terms "control line" and "reference line" are used interchangeably.

[0033] The term "prokaryotic expression system" refers to a method in which an exogenous target gene is expressed in a specific prokaryotic organism or cell by constructing an expression vector and introducing the vector into an expression strain through gene cloning technology.

[0034] The term "eukaryotic expression system" refers to a method in which an exogenous target gene is expressed in a specific eukaryotic organism or cell by constructing an expression vector and introducing it into eukaryotic cells through gene cloning technology.

[0035] The term "mammalian cell expression system" refers to the expression of exogenous target genes in mammalian cells through gene cloning technology. The mammalian cell expression system can guide the correct folding of proteins and provide multiple post-translational processing functions such as complex N-glycosylation and accurate O-glycosylation. Therefore, the expression products are closest to natural higher biological protein molecules in terms of molecular structure, physicochemical properties and biological functions.

[0036] The term "insect cell expression system" refers to the expression of exogenous target genes in insect cells through gene cloning technology. It has a series of protein translation processing and modification systems such as glycosylation, acetylation, phosphorylation, and can correctly fold proteins and form disulfide bonds, making the recombinant protein closer to the natural protein in structure and function, and facilitating the formation of natural higher-order structures of the expression products.

[0037] The term "yeast expression system" refers to the expression of exogenous target genes in yeast cells through gene cloning technology. Yeast reproduces quickly, can ferment at high density, and can perform post-translational modification and processing of proteins.

[0038] The term "chemical protein synthesis" refers to the formation of a polypeptide chain by multiple amino acids through peptide bonds under certain conditions. Before synthesis, each amino acid undergoes group protection, amino and carboxyl activation treatments, and then peptide grafting to finally form a polypeptide chain. Chemical synthesis provides a fast and efficient way to prepare proteins. At the same time, it can easily introduce non-natural amino acids, change the carbon chain skeleton, and other chemical modifications to improve protein activity and construct new proteins.

[0039] The term "baculovirus expression system" refers to the use of baculovirus as a vector to efficiently express foreign genes and prepare proteins.

[0040] The term "CHO ​​cell expression system", CHO cells, namely Chinese Hamster Ovary cells (Chinese Hamster Ovary Cell), are the most representative animal cell expression system and are widely used in the field of biopharmaceuticals. CHO cells are the most important expression system for the production of protein biological products.

[0041] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0042] All chemical reagents used in the examples of the present invention were of analytical grade and purchased from Sinopharm Group. The experimental methods described in the present invention, unless otherwise specified, are conventional methods; the biological materials described, unless otherwise specified, can be obtained from commercial channels.

[0043] Example 1 Preparation and identification of recombinant ASFV P30 protein

[0044] 1.1 Preparation of recombinant ASFV P30 protein using baculovirus expression system

[0045] The ASFV P30 gene sequence (Genbank accession number: MH766894.10) was codon-optimized according to the codon preference of the baculovirus expression system, and six histidine residues were added to the C-terminus to obtain the ASFV P30 sequence shown in SEQ ID No. 1. This sequence was inserted into the pFastBacI vector, and the recombinant plasmid pFastBac-P30 was synthesized at GenScript Biotechnology Co., Ltd.

[0046] Transform 5 μl of the recombinant plasmid pFastBac-P30 into DH10Bac competent cells, incubate on ice for 30 minutes, in a 42°C water bath for 60 seconds, and then on ice for 2 minutes. Add 400 μl of SOC medium and incubate at 37°C at 200 rpm for 4 hours. Spread 100 μl of the bacterial suspension onto a plate containing IPTG / X-gal / kanamycin / tetracycline / gentamicin and incubate at 37°C for at least 48 hours. Once blue and white colonies are evident, streak a single white colony onto a plate containing IPTG / X-gal / kanamycin / tetracycline / gentamicin and incubate at 37°C for at least 12 hours. Then, transfer a single white colony to liquid LB medium containing kanamycin / tetracycline / gentamicin and shake overnight. The next day, sample 1 μl as a template.

[0047] PCR was performed on the bacterial suspension using the upstream primer 5'-GGATTATTCATACCGTCCCA-3' and the downstream primer 5'CAAATGTGGTATGGCTGATT-3'. The PCR reaction system consisted of 25 μl 2× PrimeSTAR GC buffer, 1 μl template, 1 μl / 1 μl primers (10 pM), 4 μl dNTPs (2.5 mM), 0.5 μl PrimeSTAR (2.5 U / μl), and 17.5 μl ddH2O. PCR amplification conditions were: initial denaturation at 98°C for 2 minutes; 30 cycles of denaturation at 98°C for 10 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 1 minute; termination extension at 72°C for 10 minutes, and then at 4°C forever. The PCR product was identified by gel electrophoresis, revealing a distinct specific band at approximately 750 bp, consistent with expectations. Sequencing by a sequencing company confirmed the product to be consistent with SEQ ID No. 1. The recombinant bacmid was extracted according to the Bac-to-Bac operating instructions and named recombinant Bac-P30.

[0048] Recombinant Bac-P30 was transfected into sf9 insect cells according to the instructions of the CellfectinⅡ Reagent transfection kit and cultured in a 27°C constant temperature incubator for approximately 72 hours. When the cell pathological changes were obvious, the cell supernatant was harvested, which was the recombinant baculovirus rBac-P30 P1 generation.

[0049] Add rBac-P30 P1 to a dish containing sf9 cells at a volume ratio of 1:20-1:40. Continue culturing at 27°C until cytopathic effects are evident for approximately 72 hours. Harvest the supernatant and label it as rBac-P30 P2. Store at 2-8°C in the dark. Repeat the above steps at a volume ratio of 1:100-1:200 to obtain rBac-P30 P3 and rBac-P30 P4, respectively.

[0050] rBac-P30 P4 was then inoculated into sf9 cells at a volume ratio of 1:10 to 1:100. The cells were cultured in a shaker at 27°C and 120 rpm. The cells were harvested 48 to 72 hours after inoculation and centrifuged at 3000 × g for 10 minutes. The infected cells were lysed with cell lysis buffer (25 mM NaHCO3, pH 8.3) and centrifuged at 10,000 × g at 4°C for 10 minutes to obtain the supernatant. SDS-PAGE gel electrophoresis and Western blotting revealed a distinct specific band at approximately 30 kD. The correctly identified supernatant was collected and used to prepare recombinant ASFV P30 protein expressed in a baculovirus expression system.

[0051] 1.2 Preparation of recombinant ASFV P30 protein using CHO cell expression system

[0052] The ASFV P30 gene sequence (Genbank accession number: MH766894.10) was codon-optimized according to the codon preference of the CHO cell expression system and six histidine residues were added to the C-terminus to obtain the ASFV P30 sequence shown in SEQ ID No. 2. A synthetic gene of ASFV P30 with a mouse IgG signal peptide added to the N-terminus of the sequence shown in SEQ ID No. 2 was obtained from GenScript Biotech Co., Ltd.

[0053] The synthetic gene was double-digested with restriction endonucleases XbaI and HindIII using the following digestion system: 2 μg synthetic gene, 5 μl 10× buffer, 2.5 μl / 2.5 μl XbaI / HindIII, and ddH2O to a total volume of 50 μl. After incubation at 37°C in a water bath for 2 hours, the target fragment was recovered by gel extraction. The target fragment was then ligated with the pCDNA3.4 vector, which had also been double-digested and gel-extracted, using the following ligation system: 1 μl 10× T4 ligation buffer, 1 μl T4 ligase, 6 μl target fragment, and 2 μl vector. The fragment was incubated in a 22°C water bath for 2 hours. The ligation product was transformed into Escherichia coli Trans 5α competent cells and plated with ampicillin to select for resistance. Single colonies were picked and the plasmids were extracted using a plasmid extraction kit. Positive recombinant plasmids were screened by double enzyme digestion. The double enzyme digestion identification system consisted of 2 μl 10× buffer, 1 μg recombinant plasmid, 1 μl / 1 μl XbaI / HindIII, and ddH2O to a total volume of 20 μl. The digestion products were identified by gel electrophoresis, and clear specific bands appeared at approximately 6000 bp in the vector position and approximately 750 bp in the target fragment position, consistent with expectations. The clone with the correct insert was selected and sent to a sequencing company for sequencing. The sequencing results were consistent with SEQ ID No. 2, and the positive plasmid identified was named pCDNA3.4-P30.

[0054] Take a stock of pCDNA3.4-P30 plasmid and inoculate it into 50 ml of LB liquid medium containing kanamycin resistance. Incubate at 37°C and 220 rpm for 12–16 hours. Then, use the OMEGA Endotoxin-Free Plasmid Extraction Kit to extract the plasmid. Determine the plasmid concentration to 1 μg / μl. Linearize the extracted pCDNA3.4-P30 plasmid using BstZ17I endonuclease. The linearization system consists of 50 μl of 10× buffer, 50 μg of pCDNA3.4-P30, 10 μl of BstZ17I, and 390 μl of ddH2O. Digestion conditions are 37°C for 1–2 hours.

[0055] Plasmid transfection was performed based on the liposome method. 24 hours before transfection, 5×10 5~6×10 5 CHO-S cells were passaged at a cell density of 10 viable cells / ml. The culture flasks were placed in a shaking incubator at 37°C, 8% CO2, and a relative humidity of 70% to 80% at a speed of 130 rpm. On the day of transfection, viable cells were counted using Countstar. The cell viability should be greater than 95%. 6 Inoculate the cells at a density of 100 viable cells / ml into a new 125ml culture flask, add medium to 30ml, and place the culture flask on a shaking platform until transfection.

[0056] The transfection process is as follows: Add 50 μg of linearized plasmid to a centrifuge tube containing OptiPRO SFM medium to a final volume of 1.5 ml and gently mix to prepare the plasmid mixture. Add 50 μl of the mixed transfection reagent FreeStyle MAX to 1.45 ml of OptiPRO SFM, mix gently, and immediately add it to the plasmid mixture. After mixing, incubate at room temperature for 10 minutes to allow the transfection reagent and plasmid to form a DNA-lipid complex. Add 3 ml of the DNA-lipid complex dropwise to the 125 ml culture flask containing cells, gently shaking the flask. Incubate the flask on a shaking incubator at 130 rpm at 37°C, 8% CO2, and 70-80% relative humidity to obtain transfected cells.

[0057] 48 hours after transfection, pressure screening was started. The screening process was as follows: (1) the transfected cells were centrifuged and resuspended in selection medium (CD FortiCHO medium containing 8 mM L-glutamine and 1% anti-clumping agent) and the cells were plated at 5×10 5 (2) Add G418 to the first T-150 culture flask to a final concentration of 0.6 mg / ml; add G418 to the second T-150 culture flask to a final concentration of 0.8 mg / ml, and then culture the culture flasks in an incubator at 37°C, 8% CO2, and 70% to 80% relative humidity. (3) Count the cells in the culture flasks from the 7th day after culture. When the survival rate is not less than 30% and there are signs of recovery, transfer the cells to a 125 ml shake flask at a seeding density of 3×10 5 The cells were cultured in a shaking incubator at 37°C, 8% CO2, and 70% to 80% relative humidity at a speed of 130 rpm. (4) The cells in the culture flask were subcultured every 3 to 4 days, with a seeding density of 3 × 10 5 viable cells / ml, add an appropriate amount of G418 according to the volume of the culture medium to maintain the selection pressure. (5) When the cell survival rate is greater than 85% and the viable cell density is greater than 1×10 6When the concentration of viable cells / ml is reached, pressure screening is completed and the cells are frozen.

[0058] The cell pool obtained by pressure screening was subjected to limiting dilution using a G418-resistant selection medium and then seeded into a 96-well plate, with the cell count per well being 0.5 to 1 cell / well. The cells were then cultured statically at 37°C and 5% CO2. After approximately 20 days of culture, positive cell lines with high expression levels were selected using dot blot detection and expanded. Single cell colonies selected were transferred from the 96-well plate to a 24-well plate containing cloning growth medium (CD FortiCHO complete medium and an anti-clumping agent diluted 1:100). Colonies were transferred every 2-5 days to expand the cultured cells.

[0059] The expression levels of different clones were identified by fed-batch culture, and EfficientFeed TM C+AGT TM The different cell clones obtained by screening were cultured with a combination of additives and Dynamis medium. The specific operation was as follows: each clone was revived and placed in a 125ml culture flask containing Dynamis medium. The cells were cultured in a shaking incubator at 37°C, 8% CO2, and a relative humidity of 70% to 80% at a speed of 150 rpm. The cells were passaged every 2 to 3 days. After two passages, each clone was inoculated into 60ml Dynamis medium. TM 250ml culture flask of culture medium, 3×10 5 Cells / ml were inoculated. Samples were taken daily from the third day of culture to determine glucose levels and perform cell counts. When the glucose level was lower than 2 g / L, glucose was added to 6 g / L. Feeds were performed on the third, fifth, seventh, and tenth days after culture, taking 2 × EfficientFeed each time. TM C+AGT TM , respectively, adding up to 5% of the total cell culture volume. Cultivate until cell viability reaches 80%. Harvest the culture and centrifuge at 1000g for 5 minutes. Collect the supernatant, which will reveal a distinct specific band at approximately 30 kD by SDS-PAGE gel electrophoresis and Western blot analysis. The culture supernatant from the highest-yielding positive cell line was used to prepare a CHO cell expression system for expressing recombinant ASFV P30 protein.

[0060] 1.3 Purification and identification of recombinant ASFV P30 protein

[0061] The recombinant ASFV P30 protein expressed in the baculovirus expression system in Example 1.1 and the recombinant ASFV P30 protein expressed in the CHO cell expression system in Example 1.2 were purified using nickel column affinity chromatography and molecular sieve purification, respectively, and labeled as recombinant ASFV P30① and recombinant ASFV P30②. SDS-PAGE gel electrophoresis and Western blot analysis revealed a single band of the target protein, with a purity of ≥95%. The protein content was 1 mg / ml using a BCA protein concentration assay kit.

[0062] Example 2 Preparation and identification of recombinant ASFV P30 protein polyclonal antibodies

[0063] 2.1 Preparation of recombinant ASFV P30 protein polyclonal antibodies

[0064] The recombinant ASFV P30 ① prepared in Example 1 was mixed with mineral oil 206 adjuvant at a volume ratio of 23:27 and then emulsified. The mixture was used as an immunogen and injected into the neck muscle to immunize pigs that were negative for antigen and antibody as identified by the ASFV PCR method and the ASFV ELISA antibody kit (prepared by indirect method) disclosed in the patent CN111929433A, 2 ml / head; after an interval of 21 days, the same dose was used for booster immunization. After the second immunization, blood was collected regularly to separate the serum, and IFA detection was performed using the indirect immunofluorescence IFA antigen plate prepared with the recombinant baculovirus rBac-P30 in Example 1.1. When the serum IFA antibody titer was ≥1:1280, the carotid artery was bled and the serum was collected. The serum was then purified by Protein G affinity chromatography and aliquoted to obtain the recombinant ASFV P30 protein polyclonal antibody, which was stored below -70°C for future use.

[0065] 2.2 Identification of recombinant ASFV P30 protein polyclonal antibodies

[0066] The protein content of the recombinant ASFV P30 protein polyclonal antibody was determined to be 5 mg / ml using a BCA protein concentration assay kit.

[0067] The recombinant ASFV P30 protein polyclonal antibody was tested by IFA using the indirect immunofluorescence IFA antigen plate prepared with the recombinant baculovirus rBac-P30 in Example 1.1. The result showed that the IFA titer of the recombinant ASFV P30 protein polyclonal antibody was 1:1280.

[0068] Example 3 Preparation of Colloidal Gold Test Strips

[0069] 3.1 Preparation and identification of gold-labeled antigens

[0070] Prepare colloidal gold by boiling 100 ml of 0.01% v / v chloroauric acid solution and then adding 1.8 ml of 1% w / v trisodium citrate solution. Cool to room temperature and bring the volume back to the original volume with distilled water to obtain a colloidal gold solution. UV scanning reveals a maximum absorption peak of 521-523 nm and an OD value of 0.9-1.0.

[0071] The pH value of the colloidal gold solution was adjusted to 8.4 with 0.2 mol / L K2CO3 solution. After uniform stirring for 30 minutes, the recombinant ASFV P30① and recombinant ASFV P30② (working concentration 12 μg / ml) prepared in Example 1 were respectively added to the colloidal gold solution for labeling. After uniform stirring for 30 minutes, 10% W / V BSA was added dropwise to a final concentration of 30 μl / ml. After uniform stirring for 30 minutes, the mixture was centrifuged at 2-8°C and 11000 rpm for 30 minutes. The supernatant was discarded and the precipitate was dissolved with 1 / 20 volume of gold label recovery solution to obtain gold-labeled antigens, thereby obtaining gold-labeled antigens ASFV P30① and ASFV P30②. The gold label recovery solution is a solution containing 0.58% W / V Na2HPO4·12H2O, 0.059% W / V NaH2PO4·2H2O, 0.01% W / V BSA, 0.05% W / V sucrose, 0.01% W / VPVP, and 0.5% V / V Tween 20. It is filtered with a 0.22 μm filter membrane and stored at 2-8°C.

[0072] 3.2 Preparation of gold label pad

[0073] The preparation of the gold label pad includes the following steps:

[0074] A1) Preparation: The gold-labeled antigen prepared in Example 3.1 was diluted 3-fold with the gold-labeled recovery solution in Example 3.1, and then sprayed onto a glass cellulose membrane using a three-dimensional gold streaking sprayer at a spray volume of 5 μl / cm.

[0075] A2) Drying: Place the sprayed gold label pad in a 37°C constant temperature drying oven and dry for 4-6 hours. Store at room temperature, dry, and sealed.

[0076] 3.3 Preparation of nitrocellulose membrane (NC membrane)

[0077] The preparation of nitrocellulose membrane (NC membrane) includes the following steps:

[0078] B1) Coating: The recombinant ASFV P30 protein prepared in Example 1 was diluted to 0.2 mg / ml with coating buffer (containing 0.58% W / V Na2HPO4·12H2O, 0.059% W / V NaH2PO4·2H2O, 0.85% W / V NaCl, and 1% W / V sucrose, filtered through a 0.22 μm filter membrane, and stored at 2-8°C) as the test line (T line) coating solution; the recombinant ASFV P30 protein polyclonal antibody prepared in Example 2 was diluted to 2 mg / ml with coating buffer as the control line (C line) coating solution; the T line and C line coating solutions were coated on the T line and C line of the NC membrane using a three-dimensional film gold spraying instrument at an amount of 1.0 μl / cm.

[0079] B2) Drying: Place the coated NC membrane in a 37°C constant temperature drying oven and dry for 16 to 20 hours. Store at room temperature, dry, and sealed.

[0080] 3.4 Test strip assembly

[0081] Attach the sample pad, the gold-labeled pad prepared in Example 3.2, the nitrocellulose membrane prepared in Example 3.3, and the absorbent pad to the base plate to create a colloidal gold test strip for African swine fever virus. Combine the test strips into test strips 1 through 4 as follows, depending on the gold-labeled antigen and the antigen used in the T-line coating solution.

[0082] Table 1 Combination of test strips 1 to 4

[0083] Kit number Test strip number Gold-labeled antigen T-line coating with antigen Kit 1 Test strip 1 ASFV P30① ASFV P30② Kit 2 Test strip 2 ASFV P30② ASFV P30① Kit 3 Test strip 3 ASFV P30① ASFV P30① Kit 4 Test strips 4 ASFV P30② ASFV P30②

[0084] Together with the sample processing tube containing the sample processing liquid A2, kits 1 to 4 are assembled.

[0085] 3.5 Establishment of the detection method of the African swine fever virus double antigen sandwich antibody detection kit

[0086] The detection method is as follows:

[0087] Step 1) Sample treatment: Use a pipette to draw up the serum, add 4 drops to 1 ml of sample treatment solution, and mix well; or use a pipette to dilute the serum 10-fold (e.g., 15 μl of sample plus 135 μl of sample treatment solution) and mix well.

[0088] Step 2) Add sample: At room temperature, use a pipette to draw up the treated sample and add 4 drops to the sample well of the test strip; or use a pipette to take 100 μl of the treated sample and add it to the sample well of the test strip.

[0089] Step 3) Let the sample stand horizontally and observe the results within 10 minutes. Result determination: if both the control line and the test line show color, the result is positive; if only the control line shows color, the result is negative; if the control line does not show color, the result is invalid.

[0090] 3.5 Sample treatment solution formula

[0091] Test strip 1 was selected and sample treatment solutions A1 to A6 (see Table 2) were used. Kits A1 to A6 were prepared respectively under the same conditions. African swine fever (CD2v deletion) positive serum (purchased from the China Veterinary Drug Administration, with a P30 protein antibody titer of 1:16 as determined by indirect ELISA) and specific serum (10 negative sera) were tested. The results (see Table 2) showed that when the sample diluent was A2, the kit A2 prepared had the highest detection sensitivity, the highest dilution factor, the highest positive detection rate, and better specificity.

[0092] Table 2 Components of sample treatment solution and corresponding test kit test results

[0093]

[0094] Example 4 Evaluation of the kit

[0095] 4.1 Sensitivity

[0096] The sample processing solution A2 of Example 3 of African swine fever (CD2v deletion) positive serum was diluted by 2 times, 4 times, 8 times, 16 times, 32 times, 64 times, 128 times, and 256 times the volume, and tested with kits 1 to 4 according to Example 3.4. The results showed that the positive serum detected by kits 1 and 2 was positive at 2 to 64 times the volume, and negative at 128 to 256 times the volume; the positive serum detected by kits 3 and 4 was positive at 2 to 32 times the volume, and negative at 64 to 256 times the volume. This shows that the sensitivity of the detection of kits 1 and 2 corresponds to a dilution factor of 64 times, which is better than the sensitivity of the detection of kits 3 and 4, which corresponds to a dilution factor of 32 times.

[0097] 4.2 Specificity

[0098] 30 samples with clear background and negative detection by the ASFV ELISA antibody kit (prepared by indirect method) disclosed in the CN111929433A patent (including 6 other porcine virus serum samples: PRRSV, PRV, PCV2, CSFV, PEDV, TGEV and 24 porcine sera collected before 2018) were tested respectively by the detection method using kits 1 to 4 prepared in Example 3. The results showed that all 30 samples were negative when tested by kits 1 to 4, with good specificity of 100% (30 / 30).

[0099] 4.3 Repeatability

[0100] Three batches of reagent kits 1 to 4 were randomly selected to test 15 samples repeatedly for 5 times, and the consistency of the test results between batches and within batches was statistically analyzed. The results showed that the consistency rates between the test results of the three batches of homemade test strips and within batches were all 100%, indicating that the three batches of reagent kits 1 to 4 had good reproducibility between batches and within batches.

[0101] 4.4 Antibody growth and decline test

[0102] Five healthy pigs that were negative for African swine fever antigen and antibody as identified by the ASFV PCR method and the ASFV ELISA antibody kit (prepared by indirect method) disclosed in patent CN111929433A were selected and immunized with the recombinant ASFVP30① protein prepared in Example 1. Serum was collected on the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, 15th, 21st and 28th day after vaccination. Serum was tested using the ASFV ELISA antibody kit (prepared by indirect method, referred to as comparison) disclosed in patent CN111929433A and kits 1 to 4 prepared by the present invention, respectively. The results showed that kits 1 and 2 tested positive on days 7 to 9 after vaccination, kits 3 and 4 tested positive on days 9 to 11 after vaccination, while the comparison kits only tested positive on the 11th day and all were positive on the 15th day. The results showed that the serum converted to positive earlier after immunization by kits 1, 2, 3, and 4, and the detection effects of kits 1 and 2 were the best, which were better than those of the ASFV ELISA antibody kit (prepared by indirect method).

[0103] Table 3 Summary of test results of different kits

[0104]

[0105] 4.5 Shelf Life

[0106] Three batches of test kits, batches 1-4, were stored at 37°C for 6 and 9 days for thermal stability testing. Sterility, sensitivity, and specificity tests were performed on test kits 1-4 after they were stored at 2-30°C for 3, 7, 10, 12, and 15 months, respectively, to evaluate the real-time shelf life of test kits 1-4. Results: Test kits 1-4 were stable at 37°C for 6 or 9 days or at 2-30°C for 15 months.

[0107] 4.6 Clinical Application

[0108] Fifteen samples of African swine fever virus-positive sera and 35 samples of African swine fever virus-negative sera were tested using kits 1 to 4 of the present invention. The results showed that when testing positive sera, the positive detection rates of kits 1 and 2 were both 93% (14 / 15), and the positive detection rates of kits 3 and 4 were both 87% (13 / 15); when testing negative sera, kits 1 to 4 all tested negative.

[0109] The above test results show that the detection effects of kits 1 and 2 prepared by the present invention are equivalent, and the detection effects of kits 3 and 4 are equivalent. They are all used to detect African swine fever virus P30 antibodies in serum, with high sensitivity and good specificity, and the antibody detection time is relatively early. The detection sensitivity is higher than that of the kits in the prior art, and positive results can be detected earlier, which is convenient for timely, accurate and convenient clinical monitoring and early warning. At the same time, it can also monitor the early antibody levels of pigs immunized with the vaccine.

[0110] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the present profession can make some changes or modifications to equivalent embodiments of the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention. SEQUENCE LISTING <110> Luoyang Putai Biotechnology Co., Ltd. <120> African swine fever virus double antigen sandwich antibody detection kit and preparation method thereof <160> 3 <170> PatentIn version 3.3 <210> 1 <211> 567 <212> DNA <213> African swine fever virus <400> 1 atggacttca tcctgaacat cagcatgaag atggaagtga tcttcaagac cgacctgcgt 60 agcagcagcc aggtggtgtt ccacgctggt agcctgtaca actggttcag cgtggagatc 120 atcaacagcg gtcgcatcgt gaccaccgct atcaagaccc tgctgagcac cgtgaagtac 180 gacatcgtga agtccgctcg tatctacgcc ggccagggtt acaccgagca ccaggcccag 240 gaagaatgga acatgatcct gcacgtgctg ttcgagaag aaccgagag ctccgcttcc 300 tccgaaaaca tccacgagaa gaacgacaac gaaaccacg agtgtaccag ctccttcgaa 360 accctgttcg aacaggagcc ttcctccgag gtgcctagg acaccaagct gtacatgctg 420 gcccagaaga ccgtgcagca catcgaacag tacggcaagg ctcctgactt caaaggtt 480 atccgcgctc acaacttcat ccagaccacatc tacggtaccc ctctgaagga agaggaaaag 540 gaagtggtgc accacacca 567 <210> 2 <211> 567 <212> DNA <213> African swine fever virus (African swine fever virus) <400> 2 atggattca tcctgaacat cagcatgaaa atggagtga tcttcagac cgacctgaga 60 agctctccc aggtggtttt ccacgccgga tctctgtaca actggttcag cgtggaatc 120 atcacagcg gcagaatcgt gaccacagcc atcagaccc tgctgagcac cgtgaagtac 180 gatacgtca aaagcgctag aatctacgcc ggccagggct acaccgagca ccaggcccag 240 gaggaatgga atatgatcct gcacgtgctg ttcgaggaag agacagagag ctccgccagc 300 tctgagaata tccacgagaa gaacgacaac gagacaacg agtgcaccag ctccttcgag 360 acacctgttg aacaggagcc tagcagcgag gtcccaagg acaccaagct gtacatgctg 420 gcccagaaaa cagtgcagca catcgagcaa tacggcaagg cccctgattt taacaagtg 480 attagagccc acaacttcat ccagaccatc tatggcaccc cactcaaga ggaagaaaag 540 gaagtggtgc slow slow 567 <210> 3 <211> 189 <212> PRT <213> African swine fever virus (African swine fever virus) <400> 3 Met Asp Phe Ile Leu Asn Ile Ser Met Lys Met Glu Val Ile Phe Lys 1 5 10 15 Thr Asp Leu Arg Ser Ser Ser Gln Val Val Phe His Ala Gly Ser Leu 20 25 30 Tyr Asn Trp Phe Ser Val Glu Ile Ile Asn Ser Gly Arg Ile Val Thr 35 40 45 Thr Ala Ile Lys Thr Leu Leu Ser Thr Val Lys Tyr Asp Ile Val Lys 50 55 60 Ser Only Arg With Tyr Only Gly Gln Gly Tyr Thr Glu His Gln Only Gln 65 70 75 80 Glu Glu Trp Asn Met Ile Leu His Val Leu Phe Glu Glu Glu Thr Glu 85 90 95 Ser Ser Ala Ser Ser Glu Asn Ile His Glu Lys Asn Asp Asn Glu Thr 100 105 110 Asn Glu Cys Thr Ser Ser Phe Glu Thr Leu Phe Glu Gln Glu Pro Ser 115 120 125 Ser Glu Val Pro Lys Asp Ser Lys Leu Tyr Met Leu Ala Gln Lys Thr 130 135 140 Val Gln His Ile Glu Gln Tyr Gly Lys Ala Pro Asp Phe Asn Lys Val 145 150 155 160 Ile Arg Ala His Asn Phe Ile Gln Thr Ile Tyr Gly Thr Pro Leu Lys 165 170 175 Glu Glu Glu Lys Glu Val Val His His His His His His 180 185

Claims

1. A double-antigen sandwich antibody detection kit for African swine fever virus, wherein, the kit contains a double-antigen sandwich antibody detection strip for African swine fever virus. Among them, the double-antigen sandwich antibody detection strip for African swine fever virus includes a bottom plate. On the bottom plate, there are successively a sample pad, a gold-labeled pad, a nitrocellulose membrane, and a water-absorbing pad. The gold-labeled pad adsorbs a colloidal gold-labeled recombinant African swine fever virus P30 protein. On the nitrocellulose membrane, there are a detection line and a quality control line. The recombinant African swine fever virus P30 protein is immobilized on the detection line, and the polyclonal antibody of African swine fever virus P30 protein is immobilized on the quality control line; among them, the recombinant African swine fever virus P30 protein is as shown in SEQ ID No. 3; the colloidal gold-labeled recombinant African swine fever virus P30 protein is expressed by a baculovirus expression system, and the recombinant African swine fever virus P30 protein immobilized on the detection line is expressed by a CHO cell expression system, or the colloidal gold-labeled recombinant African swine fever virus P30 protein is expressed by a CHO cell expression system, and the recombinant African swine fever virus P30 protein immobilized on the detection line is expressed by a baculovirus expression system; the nucleotide sequence encoding the recombinant African swine fever virus P30 protein in the baculovirus expression system is as shown in SEQ ID NO. 1, and the nucleotide sequence encoding the recombinant African swine fever virus P30 protein in the CHO cell expression system is as shown in SEQ ID NO.

2.

2. The kit according to claim 1, wherein, the labeling concentration of the colloidal gold-labeled recombinant African swine fever virus P30 protein is 5 μg / ml to 20 μg / ml, the concentration of the recombinant African swine fever virus P30 protein immobilized on the detection line is 0.05 mg / ml to 0.25 mg / ml, and the concentration of the polyclonal antibody of African swine fever virus P30 protein is 1 mg / ml to 2 mg / ml.

3. The kit according to claim 2, wherein, the labeling concentration of the colloidal gold-labeled recombinant African swine fever virus P30 protein is 12 μg / ml, the concentration of the recombinant African swine fever virus P30 protein immobilized on the detection line is 0.2 mg / ml, and the concentration of the polyclonal antibody of African swine fever virus P30 protein is 2 mg / ml.

4. The kit according to claim 1, wherein, the double-antigen sandwich antibody detection strip for African swine fever virus includes a bottom plate. The bottom plate has a first end and a second end, and successively there are a sample pad, a gold-labeled pad, a nitrocellulose membrane, and a water-absorbing pad along the direction from the first end to the second end. The nitrocellulose membrane is in contact with the gold-labeled pad, so that the binding body of African swine fever virus P30 antibody and the colloidal gold-labeled recombinant African swine fever virus P30 protein can migrate thereon towards the second end of the bottom plate.

5. The kit according to claim 1, wherein, the kit further includes a sample treatment solution, and the sample treatment solution is a 0.02 M PB solution with pH 7.4 containing 0.3% V / V Tween-20.

6. A method for preparing the kit according to claim 1, wherein, the method includes: Step (1): Express and purify the recombinant African swine fever virus P30 protein for preliminary colloidal gold labeling, express and purify the recombinant African swine fever virus P30 protein for preliminary immobilization on the test line, and prepare the polyclonal antibody against the African swine fever virus P30 protein. Step (2): Label the recombinant African swine fever virus P30 protein expressed and purified in step (1) for preliminary colloidal gold labeling with colloidal gold, and adsorb the colloidal gold-labeled recombinant African swine fever virus P30 protein onto the gold conjugate pad. Immobilize the recombinant African swine fever virus P30 protein expressed and purified in step (1) for preliminary immobilization on the test line onto the test line. Immobilize the prepared polyclonal antibody against the African swine fever virus P30 protein onto the control line. Step (3): Assemble the sample pad, the gold conjugate pad adsorbed with the colloidal gold-labeled recombinant African swine fever virus P30 protein in step (2), the nitrocellulose membrane, and the absorbent pad onto the base plate in sequence. The nitrocellulose membrane is in contact with the gold conjugate pad, so that the binding body of the African swine fever virus P30 antibody and the colloidal gold-labeled recombinant African swine fever virus P30 protein can migrate thereon towards the second end of the base plate to obtain the African swine fever virus double antigen sandwich antibody detection strip. Step (4): Prepare the sample treatment solution; and Step (5): Assemble the African swine fever virus double antigen sandwich antibody detection strip prepared in step (3) and the sample treatment solution prepared in step (4) into a kit.

7. According to the method of claim 6, wherein, in step (2), the labeling concentration of the colloidal gold-labeled recombinant African swine fever virus P30 protein is 5 μg / ml to 20 μg / ml, the concentration of the recombinant African swine fever virus P30 protein immobilized on the test line is 0.05 mg / ml to 0.25 mg / ml, and the concentration of the polyclonal antibody against the African swine fever virus P30 protein is 1 mg / ml to 2 mg / ml.

8. According to the method of claim 7, wherein, in step (2), the labeling concentration of the colloidal gold-labeled recombinant African swine fever virus P30 protein is 12 μg / ml, the concentration of the recombinant African swine fever virus P30 protein immobilized on the test line is 0.2 mg / ml, and the concentration of the polyclonal antibody against the African swine fever virus P30 protein is 2 mg / ml.

9. According to the method of claim 6, wherein, the sample treatment solution in step (4) is a 0.02 M PB solution with a pH of 7.4 containing 0.3% V / V Tween-20.

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