CN116444625A - Duplex colloidal gold immunochromatography test strip for simultaneously detecting antibodies of CD2v and p54 of African swine fever virus and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of virus immune detection, and in particular relates to a double colloidal gold immunochromatographic test strip for simultaneously detecting African swine fever virus CD2v and p54 antibodies and a preparation method thereof. In the present invention, the CD2v recombinant protein and p54 recombinant protein of African swine fever virus are firstly obtained, and are used for the construction of a colloidal gold immunochromatographic detection system. The present invention further uses Staphylococcus aureus A to carry out colloidal gold labeling, and colloidal gold is used as label tracer, has prepared antibody detection colloidal gold immunochromatography test strip, and described test strip comprises support base plate, is pressed on support base plate The sample pad, the gold standard protein pad, the chromatography detection membrane and the water-absorbing pad are sequentially fixed. The test strip uses antigen-antibody reaction and lateral flow chromatography to detect samples. The labeled complex and antibody are stable in combination, bright in color and visible to the naked eye. It has great advantages in antibody detection and improves the detection efficiency. Sensitivity, reducing non-specific reactions and missed detection rates.

Description

A double colloidal gold immunoassay for simultaneous detection of African swine fever virus CD2v and p54 antibodies Immunochromatographic test strip and preparation method thereof

technical field

The invention belongs to the technical field of virus immune detection, and in particular relates to a double colloidal gold immunochromatographic test strip for simultaneously detecting African swine fever virus CD2v and p54 antibodies and a preparation method thereof.

Background technique

African swine fever (African swine fever, ASF) is a highly pathogenic, high mortality, and highly contagious viral disease caused by African swine fever virus (ASFV). It can occur in both domestic pigs and wild boars. ASF was first discovered in Kenya in 1920. Since the first African swine fever case occurred in my country in August 2018, it has spread rapidly to various provinces, municipalities and autonomous regions across the country. ASF is ubiquitous in sub-Saharan Africa, Eastern Europe and the Caucasus. ASF is also prevalent in Sardinia, Italy, and poses a serious threat to any country/region with a pig industry in the world. The clinical features of pigs infected with ASFV are high fever, diarrhea, hemorrhage and skin redness. Some clinical symptoms and autopsy results are mainly splenomegaly, lymph nodes and kidney hemorrhage, etc., which are basically consistent with the symptoms of classical swine fever virus infection in pigs.

Since 2018, China's annual pig population has accounted for 50% of the world's total. The spread of ASFV in China has seriously threatened the world's breeding industry. There is no vaccine and effective treatment for ASFV in the prior art, and the control methods mainly rely on the implementation of strict sanitation and epidemic prevention measures and the method of burning sick pigs. Therefore, the rapid and reliable detection of ASFV is very important. At present, the diagnostic methods for detecting African swine fever include red blood cell adsorption test, direct immunofluorescence test, animal inoculation test, ELISA and so on.

ASFV is an enveloped double-stranded DNA virus, which belongs to the African swine fever virus family, the only member of the African swine fever virus genus, and the only insect-borne DNA virus. Domestic pigs, wild boars and soft ticks are all hosts of ASFV, and pig monocyte-macrophages are the main target cells of ASFV. The ASFV virion has an icosahedral structure with a diameter of about 200 nm, and is assembled from the inside out by the virus core, the inner core shell, the inner envelope, the capsid and the outer envelope. The full length of the ASFV genome is about 170-193kb, containing 150-167 open reading frames (ORFs), encoding 150-200 proteins, of which more than 60 are structural proteins. Structural proteins, as the main components of virions, play a role in the infection process of ASFV adsorption, invasion and replication. Among them, the p54 protein encoded by the ASFV E183L gene is one of the main antigenic proteins of ASFV, which appears in the late stage of infection and has a good Immunogenicity of pigs; one week after pigs were infected with ASFV, antibodies against p54 protein could be detected in serum. The CD2v protein encoded by the ASFV EP402R gene is a transmembrane protein expressed in the late stage of ASFV, which plays an important role in the spread and replication of the virus, and can be used for the detection of ASFV and the development of vaccines.

Compared with conventional techniques such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography has the advantages of simple operation, high detection sensitivity, good specificity, easy storage, stable detection, and comprehensive Low cost, less affected by external factors, etc., can be applied to field and on-site diagnosis, grassroots units, and has developed rapidly in recent years. p30, p54, and p72 proteins are commonly used targets for detecting ASFV. At present, there are few colloidal gold test strips for ASFV diagnosis. The common African swine fever p30 protein and p72 protein colloidal gold test strips can only be used alone The detection of p30 protein and p72 protein cannot realize the whole monitoring of African swine swine fever antibody, and there is no related report on the double antibody detection test strip based on African swine fever virus protein p54 and CD2v in the prior art.

Since the joint detection of two commonly used detection targets can improve the specificity, accuracy, and increase the versatility of use, it plays a vital role in the rapid and accurate diagnosis and control of the spread of ASFV and the reduction of economic losses.

Contents of the invention

In order to solve the problems existing in the detection of African swine fever virus and improve the specificity and accuracy of detection, the present invention firstly obtained African swine fever virus CD2v recombinant protein and p54 recombinant protein, and used them in the construction of a colloidal gold immunochromatographic detection system.

Secondly, the present invention provides a double-linked colloidal gold immunochromatographic test strip for detecting African swine fever virus antibody, which has the advantages of strong specificity, good sensitivity, high repetition rate, and accurate detection results, and is suitable for clinical use. Rapid detection of samples.

Then, the present invention also provides a method for double detection of CD2v and p54 antibodies using the double colloidal gold immunochromatographic test strip, which can quickly and accurately detect African swine fever virus antibodies, and is very suitable for grassroots and on-site rapid Detection and diagnosis.

For realizing the purpose of the present invention, the technical scheme that the present invention creates is realized like this:

The invention provides a double colloidal gold immunochromatographic test strip for simultaneously detecting African swine fever virus CD2v and p54 antibodies, comprising a support base plate, on which a sample pad, a gold standard protein pad, and a chromatography Detection membranes and absorbent pads;

Among them, the gold-labeled protein pad is coated with glass fiber cotton, and colloidal gold-labeled Staphylococcal Protein A (Staphylococcal Protein A, SPA) is immobilized; the chromatographic detection membrane is a nitrocellulose membrane, and a detection line and a quality Control line, in which the purified ASFV p54 recombinant protein and CD2v recombinant protein are used as the detection line to imprint T1 and T2 respectively, and the complex of the capture-labeled recombinant protein antigen and virus antibody is used as the detection line; the quality control line (C) is immobilized with rabbit anti-mouse IgG polyclonal antibody.

The test strip can effectively distinguish the infection of African swine fever virus wild strain or CD2v deletion strain infection. When the T1 line and T2 line develop color at the same time, it means that the sample contains antibodies against CD2v and p54 at the same time, indicating that it is African swine fever virus. Infection with wild strains of the virus, when the T1 line develops color, but the T2 line does not, it means that the sample only contains antibodies against p54 protein, but no antibody against CD2v protein, indicating that it is CD2v-deficient strain infection, when the T1 line When the T2 and T2 lines are not colored, it means that there is no ASFV infection.

Specifically, the double colloidal gold immunochromatographic test strip for simultaneously detecting African swine fever virus CD2v and p54 antibodies is prepared through the following steps:

1) Preparation of chromatographic detection membrane

Cut the nitrocellulose detection membrane into long strips, place it on the platform of the spray dot instrument, and fix it; spray the diluted ASFV p54 recombinant protein and CD2v recombinant protein on the nitrocellulose membrane, which is the detection line T1 and T2 blots; The diluted rabbit anti-mouse IgG polyclonal antibody is sprayed on the nitrocellulose membrane, which is the quality control line C blot, dried and sealed for future use;

2) Preparation of sample pads

Cut glass wool into long strips, use PBS solution containing BSA, Tween20, and _NaNO3 as the sample pad treatment solution to soak the glass wool strips, then dry to make sample pads, and store them sealed at room temperature for later use;

3) Preparation of gold-labeled protein pad

Cut the glass fiber cotton into long strips, place it on the sprayer platform, and fix it; spray the Na ₂ B ₄ O ₇ buffer solution containing BSA and NaN ₃ on the glass fiber cotton for pretreatment, and then dry it for later use;

Dilute the colloidal gold-labeled SPA solution, spray it on the pretreated glass fiber cotton, dry it, and make a gold-labeled protein pad, and store it sealed at room temperature for later use;

4) Preparation of absorbent pad

Cut absorbent paper into long strips to make absorbent pads, dry them, and store them airtight at room temperature for later use;

5) Preparation of support base plate

Paste the double-sided tape on the PVC board, cut it into a long board, and prepare the supporting base board;

6) Assembly

Assemble the sample pad, gold standard protein pad, chromatographic detection membrane, and water-absorbent pad in sequence on the support base plate; first paste the detection film on the center of the support base plate, and then paste the absorbent paper on the detection line near the quality control line from top to bottom. For the membrane end, paste the gold-labeled protein pad and the sample pad in turn on the end of the detection line near the detection membrane (2mm overlap between each layer), then cut into test strips, dry and seal for storage.

Specifically, before spraying in step 1), first use 0.1mol/L Tris-HCl buffer to dilute the ASFV p54 recombinant protein and CD2v recombinant protein to a concentration of 1mg/mL, and then spray the nitrocellulose at an amount of 0.9-1μL/cm and use PBS buffer to dilute the rabbit anti-mouse IgG polyclonal antibody to 1mg/mL, and spray it on the nitrocellulose membrane at an amount of 0.9-1μL/cm.

Specifically, in step 2), the concentration of the PBS solution containing BSA, Tween20 and _NaNO3 is 0.1mol/L, wherein the concentration of BSA is 10g/L, the concentration of Tween20 is 5g/L, and the concentration of _NaNO3 is 3g/L.

Concretely, the _{Na2B4O7 buffer} solution concentration containing BSA and _NaN3 in step 3) is 20mmol/L, wherein _BSA concentration is 1% (w/v), _NaN3 concentration is 0.1% (w/v) .

Specifically, when diluting the colloidal gold-labeled SPA solution in step 3), the colloidal gold-labeled SPA solution and the gold-labeled antibody preservation solution are mixed at a volume ratio of 1:1.

Further, the present invention obtains the African swine fever virus p54 recombinant protein and the African swine fever virus CD2v recombinant protein through codon optimization and construction of eukaryotic expression vector.

Based on this, the present invention also provides the gene encoding ASFV p54 protein after codon optimization, its nucleotide sequence is shown in SEQ ID NO.2, full length 531bp; and, the gene encoding ASFV CD2v protein after codon optimization, Its nucleotide sequence is shown in SEQ ID NO.4, and its full length is 582bp.

Specifically, the method of codon optimization is as follows:

According to China/2018/Anhui XCGQ strain (GenBank: AYW34030.1) obtain the sequence of p54 protein (amino acid sequence as shown in SEQ ID NO.1), and the sequence of CD2v protein (amino acid sequence as shown in SEQ ID NO.3), from Codon preference, balanced GC content, forward and reverse repeat sequence, mRNA secondary structure, expression vector construction restriction site and other factors are considered, referring to the codon usage frequency of Cricetulus griseus (Mamalian/CHO cells), using codon optimization software The nucleotide sequence encoding the p54 protein and the nucleotide sequence encoding the CD2v protein were optimized for better expression in the CHO expression system; the optimized nucleotide sequence was analyzed using EMBOSS (European Molecular Biology Open Software Suite) bioinformatics software The sequence codon adaptation index CAI value.

Specifically, the method for obtaining the ASFV p54 recombinant protein is as follows:

Referring to the codon-optimized gene encoding ASFV p54 protein, it was synthesized to construct the pCAGGS vector, and the His tag was added to the C segment of the p54 protein to obtain a recombinant plasmid, which was named pCAGGS-p54; the recombinant plasmid was transfected into HEK293F cells and cultured for 24 After -48h, centrifuge to get the cell supernatant, filter, use Ni-NAT affinity chromatography column, remove impurities, elute and purify, and concentrate through ultrafiltration centrifuge tube to obtain African swine fever virus p54 recombinant protein, which will be packed in- Store at 80°C for later use.

Specifically, the method for obtaining the ASFV CD2v recombinant protein is as follows:

Referring to the codon-optimized gene encoding the ASFV CD2v protein, synthesize it, construct the pCAGGS vector, add a His tag to the C segment of the CD2v protein, and obtain a recombinant plasmid, named pCAGGS-CD2v; transfect the recombinant plasmid into HEK293F cells, and culture for 24 After -48h, centrifuge to get the cell supernatant, filter, use Ni-NAT affinity chromatography column, remove impurities, elute and purify, and concentrate through ultrafiltration centrifuge tube to obtain African swine fever virus CD2v recombinant protein, which will be packed in- Store at 80°C for later use.

Further, the preparation method of the rabbit anti-mouse IgG polyclonal antibody is:

Rabbits were immunized with mouse serum IgG as the immunogen, and the antigen was emulsified with Freund's complete adjuvant for the first immunization, and the dose of 150-200 μg/rat was injected subcutaneously at multiple points; the interval between each booster immunization was 2-3 weeks, and Freund's incomplete The adjuvant emulsifies the antigen for injection; 1-2 weeks after the last booster immunization, mouse serum IgG is coated with an enzyme-linked immunosorbent plate at a concentration of 1-2 μg/mL and 20-50 μL per well, and measured by indirect ELISA Immune serum titer, when the titer reaches the expected value or above, collect high-immune rabbit whole blood, place it at 35-37°C for 1-2h, centrifuge for 5-10min, collect the supernatant, and extract the immune serum by ammonium sulfate precipitation IgG was purified to obtain rabbit anti-mouse IgG polyclonal antibody, which was stored at -80°C after aliquoting.

Specifically, healthy New Zealand white rabbits aged 3 months to 6 months were selected as experimental animals.

Specifically, the expected value of the potency is 1:102400.

Further, the preparation method of described colloidal gold solution is:

Add chloroauric acid to ultrapure water and boil, then quickly add trisodium citrate solution under heating and stirring, observe the color from colorless→blue→dark red→bright red until the color no longer changes, and wait for the solution to cool to At room temperature, dilute to volume with ultrapure water, and store at 4°C in the dark.

Specifically, the volume ratio of chloroauric acid and ultrapure water is 1:(100-120), preferably 1:100, and the concentration of chloroauric acid is 8-10g/L, preferably 10g/L; chloroauric acid and lemon The volume ratio of trisodium citrate solution is 1:(1.5-1.8), preferably 1:1.6, and the concentration of trisodium citrate solution is 8-10g/L, preferably 10g/L.

Specifically, the total volume of the prepared colloidal gold solution is 100 mL.

Further, when the colloidal gold-labeled SPA solution was prepared, colloidal gold was prepared by trisodium citrate reduction chloroauric acid method, and labeled Staphylococcus aureus protein A (SPA), specifically, the colloidal gold-labeled SPA solution The preparation method is:

Add K ₂ CO ₃ to the colloidal gold solution to adjust the pH to 6.0, then add Staphylococcus aureus protein A (SPA) solution, react at room temperature for 5-10 minutes, add casein solution, block at room temperature for 10-20 minutes, centrifuge for 5-10 minutes, The supernatant was discarded, and PBS buffer containing BSA and sodium azide was added to resuspend to obtain a colloidal gold-labeled SPA solution.

Specifically, the volume ratio of Staphylococcus aureus protein A to the colloidal gold solution is 1:(50-100), preferably 1:100.

Specifically, the concentration of the casein solution is 2-3%; the volume ratio of the casein solution to the colloidal gold solution is 1:(5-10), preferably 1:10.

Specifically, in the PBS buffer solution containing BSA and sodium azide, the BSA content is 1.2 wt%, and the sodium azide content is 0.05 wt%; the volume ratio of the PBS buffer solution containing BSA and sodium azide to the colloidal gold solution is 1 :(5-10), preferably 1:10.

Further, based on a general inventive concept, the present invention also provides the application of the double colloidal gold immunochromatographic test strip for simultaneously detecting African swine fever virus CD2v and p54 antibodies in the preparation of diagnostic reagents for African swine fever virus infection .

Further, based on a general inventive concept, the present invention also provides the application of the double colloidal gold immunochromatographic test strip for simultaneously detecting African swine fever virus CD2v and p54 antibodies in the preparation of quarantine inspection reagents.

Compared with the prior art, the beneficial effects of the present invention are:

1. The present invention uses ASFV p54 and CD2v genes to obtain two correctly folded recombinant proteins, further uses Staphylococcus aureus protein A to carry out colloidal gold labeling, and uses colloidal gold as a labeling tracer to prepare antibody detection colloidal gold immunochromatography The test strip uses antigen-antibody reaction and lateral flow chromatography to detect samples. The labeled complex and antibody are stable in combination, bright in color and visible to the naked eye. It has great advantages in antibody detection and improves the sensitivity of detection. Reduced non-specific reactions and missed detection rates.

2. The present invention selects ASFV-p54/CD2v recombinant protein as the detection target, and ASFV-p54/CD2v is the most immunogenic antigen of African swine fever virus. Different from proteins obtained from other systems such as prokaryotic expression systems in the past, the recombinant proteins used in the present invention have similar antigenicity, immunogenicity and high biological activity to viral proteins in mammals, and are equally effective in terms of immunity and colloidal gold labeling. Advantages.

3. The present invention utilizes ASFV-p54/CD2v recombinant protein as the colloidal gold immunochromatographic test strip for rapid detection of African swine fever virus antibody prepared by antigen, which can accurately and quickly detect whether there is African swine fever virus antibody in the serum to be tested , with short detection time, strong specificity, and high sensitivity, it is a product that is easy to carry, fast, and accurately detects African swine fever virus antibodies; simultaneous detection of multiple targets greatly reduces the missed detection rate and improves the accuracy of detection. In addition, the detection of CD2v antibodies can also be used to monitor the quality of CD2v-deficient vaccines. The test strip can be used for the preparation of ASFV infection diagnostic reagents, live pig trade quarantine inspection reagents, and epidemiological investigation and research, and has popularization and application value and great economic and social benefits.

Description of drawings

Fig. 1 is the structural representation of test paper strip of the present invention; Among the figure: 1PVC bottom plate, 2 sample pads, 3 colloidal gold mark pads, 4 nitrocellulose membrane detection pads, 5 water absorption pads, 6p54 protein detection line, 7CD2v protein detection line, 8 quality control lines;

Figure 2 is a graph of Cricetulus griseus codon usage frequency (Mamalian/CHO cells);

Fig. 3 is the GC content analysis of the sequence of the P54 protein coding region after optimization;

Figure 4 is an analysis of the GC content of the CD2v protein coding region sequence after optimization;

Figure 5 is the purification diagram of ASFV p54 recombinant protein; among them, M: Marker; a: SDS-PAGE of p54 recombinant protein purification; b: Western Blot identification result of p54 recombinant protein and His monoclonal antibody reaction; c: Western Blot result of p54 and ASFV positive serum ;

Figure 6 is the analysis of the glycosylation level of recombinant ASFV p54 protein; among them, M: Marker; a: SDS-PAGE of purified p54 protein sample treated with PNGase F enzyme; b: Western Blot of reaction between p54 and His monoclonal antibody after removal of glycosylation modification Identification results; c: Western Blot results of p54 and ASFV positive serum after removal of glycosylation modification;

Fig. 7 is the picture of purification of ASFV CD2v recombinant protein; Wherein A picture is SDS-PAGE identification result; B picture is WesternBlot identification result; Among the figure, M is Marker; 1 is the CD2v recombinant protein of purification;

Figure 8 is the SDS-PAGE identification of CD2v recombinant protein before and after PNGase F treatment; in the figure, M is Marker; 1 is the purified CD2v recombinant protein; SDS-PAGE identification, 1: purified recombinant ASFV-CD2v protein; 2: PNGase F Treated CD2v protein;

Figure 9 is a schematic diagram of the judgment of the detection results of ASFV standard positive serum samples; wherein A-B is the double positive detection result of CD2v and p54 antibody; C is the positive detection result of CD2v antibody; Figure D is the positive detection result of p54 antibody; Figure E is the negative serum Test results.

Detailed ways

The specific implementation of the present invention will be described below in conjunction with the accompanying drawings and examples, but the following examples are only used to describe the present invention in detail, and do not limit the scope of the present invention in any way. In addition, regarding the numerical ranges in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents are described. In case of conflict with any incorporated document, the contents of this specification control.

Materials and reagents used in the present invention:

1. Competent cells: E.coli DH5α was purchased from Bomed Biotechnology Co., Ltd.; 2. Plasmid: the pCAGGS plasmid used was purchased from Wuhan Miaoling Biotechnology Co., Ltd.; 3. Cells and medium: HEK293F cells, medium and transfection reagents were purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.; 4. Experimental animals: clean grade New Zealand female white rabbits, weighing 2.5kg, purchased from Zhengzhou Dingguo Biotechnology Co., Ltd.; 5. Kit: endotoxin-free The large extraction kit mentioned in the plasmid was purchased from Kangwei Century Biotechnology Co., Ltd. 6. Mouse serum IgG (10 mg/bottle, product number B1102) was purchased from Beijing Boersi Technology Co., Ltd.

The instruments and equipment used in the present invention are commercially available conventional instruments and equipment unless otherwise specified; other reagent materials used are commercially available conventional reagent materials unless otherwise specified; the identification of the recombinant plasmid is provided by Shanghai Sangong Bioengineering Co., Ltd. conduct.

The preparation of embodiment 1ASFV CD2v and p54 recombinant protein

Codon optimization and construction of eukaryotic expression vectors

Since the usage frequency of synonymous codons is different between species of different species, when the usage frequency of synonymous codons of the foreign gene matches the expression host, the expression level of the foreign gene will be significantly increased, Commonly used codon adaptation index (Codon Adaptation Index) to represent.

The present invention obtains the p54 protein sequence (the amino acid sequence of the p54 protein such as SEQ ID NO.1) according to China/2018/Anhui XCGQ strain (GenBank: AYW34030.1), from codon preference, balanced GC content, forward and reverse repetition Considering factors such as sequence, mRNA secondary structure, and restriction site for expression vector construction, referring to the codon usage frequency of Cricetulus griseus (Mamalian/CHO cells) (as shown in Figure 2), the p54 protein encoding p54 protein nuclear The nucleotide sequence was optimized for better expression in the CHO expression system.

Using EMBOSS (European Molecular Biology Open Software Suite) bioinformatics software to analyze and optimize the sequence codon adaptation index CAI value (Codon Adaptation Index, CAI) is CAI: 0.914; average GC content is adjusted to 61.14% (as shown in Figure 3 ); the optimized nucleic acid sequence is shown in SEQ ID NO.2, with a full length of 531bp. Referring to the codon optimized sequence above, it was synthesized by Shanghai Sangon Bioengineering Co., Ltd., and a His tag was introduced into the protein C segment to construct the pCAGGS vector . The recombinant plasmid was obtained and named pCAGGS-p54; the recombinant plasmid was transfected into HEK293F cells, the density of HEK293F cells was 3.0×10 ⁶ cells/mL, after 48 hours of culture, centrifuge at 1000g/min for 10 minutes to collect the cell supernatant; the supernatant After filtering through a 0.45um filter membrane, use a Ni-NAT affinity chromatography column to wash away impurities with WashingBuffer (PBS+20mM imidazole, pH 7.4), and then elute with Elution Buffer (PBS+100mM imidazole, pH7.4) Purify and concentrate 5-10 times in an ultrafiltration centrifuge tube to obtain the African swine fever virus p54 recombinant protein, and store it at -80°C after aliquoting for subsequent preparation of immunochromatographic test strips.

According to China/2018/Anhui XCGQ strain (GenBank: AYW34030.1), obtain the sequence of CD2v protein (amino acid sequence is shown in SEQ ID NO.3), refer to Cricetulus griseus codon usage frequency (Mamalian/CHO cells) (Figure 2 shown), the nucleotide sequence encoding the CD2v protein was optimized using codon optimization software, so that it could be better expressed in the CHO expression system.

EMBOSS bioinformatics software was used to analyze and optimize the codon adaptation index CAI value of the sequence CAI: 0.940; the average GC content was adjusted to 45% (as shown in Figure 4). The optimized nucleic acid sequence is shown as SEQ ID NO.4, with a full length of 582bp. Referring to the above codon optimized sequence, it was synthesized by Shanghai Sangon Bioengineering Co., Ltd., a His tag was added to the protein C segment, and the pCAGGS vector was constructed to obtain a recombinant plasmid, which was named pCAGGS-CD2v; the recombinant plasmid was transfected into HEK293F cells, HEK293F The cell density was 3.0×10 ⁶ cells/mL. After 48 hours of culture, centrifuge at 1000g/min for 10 minutes to collect the cell supernatant; Buffer (PBS+20mM imidazole, pH 7.4) to wash away impurities, and then eluted and purified by Elution Buffer (PBS+100mM imidazole, pH 7.4), then concentrated 5-10 times in an ultrafiltration centrifuge tube to obtain African swine fever virus CD2v recombinant protein , and stored at -80°C after aliquoting for subsequent preparation of immunochromatographic test strips.

2. Results determination

Take an appropriate amount of ASFV-p54 recombinant protein expression for SDS-PAGE and Western blot detection. The results are shown in Figure 5, and the results in Figure 5 indicate the successful expression of the recombinant protein. The eluted p54 protein has a diffuse target band at about 25-35KDa. The p54 protein was digested with the glycosidase PNGase F at 37°C for 1 h under non-denaturing conditions. The results of SDS-PAGE and Western-Blot analysis showed that the p54 protein had different degrees of glycosylation, and the results are shown in Figure 6. The diffuse target bands around 25-35 KDa are all p54 protein, the purity is greater than 90%, and the concentration is about 1.8 mg/ml.

Take an appropriate amount of cell supernatant samples expressing ASFV-CD2v recombinant protein for Western blot detection. The results are shown in Figure 7, and the results in Figure 7 indicate the successful expression of the recombinant protein. The eluted CD2v protein has a diffuse target band at about 55-110KDa. The CD2v protein was digested with glycosidase PNGase F at 37°C for 1 h under non-denaturing conditions. SDS-PAGE results showed that the CD2v protein had different degrees of glycosylation. As shown in Figure 8, the protein after deglycosylation was 23KDa About 55-110KDa, indicating that the target band diffused around 55-110KDa is CD2v protein, its purity is greater than 90%, and its concentration is about 1.6mg/ml.

The preparation of embodiment 2 rabbit anti-mouse IgG polyclonal antibody

Use mouse serum IgG as the immunogen to immunize healthy New Zealand white rabbits aged 3 to 6 months. For the first immunization, the antigen is emulsified with Freund's complete adjuvant, and the dose is 200 μg/rat for subcutaneous injection at multiple points; each booster immunization interval is 3 weeks , injected with the emulsified antigen in Freund's incomplete adjuvant; 2 weeks after the last booster immunization (three immunizations), the mouse serum IgG was coated with an enzyme-linked immunosorbent plate at a concentration of 2 μg/mL and 50 μL per well. Use the indirect ELISA method to measure the titer of the immune serum. When the titer reaches the expected value (1:102400) and above, collect hyperimmune rabbit whole blood, place it at 37°C for 2h, centrifuge at 4000rpm/min for 5min, and collect the clear. Immune serum IgG was extracted and purified by ammonium sulfate precipitation method to obtain a rabbit anti-mouse IgG polyclonal antibody with a final concentration of 3.8 mg/ml, and the titer was determined to be 1:204800 by indirect ELISA method, and stored at -80°C after aliquoting , for the preparation of quality control lines.

The preparation of embodiment 3 colloidal gold solution and the colloidal gold mark of SPA

1. Preparation of Colloidal Gold Solution

Take 100mL ultrapure water in a 500mL clean beaker, add 1mL 10g/L chloroauric acid and boil; add freshly prepared 1.6mL 10g/L trisodium citrate solution quickly under heating and stirring, observe the color from colorless to blue Color→dark red→bright red until the color does not change any more. After the solution is cooled to room temperature, dilute to 100mL with ultrapure water and store at 4°C in the dark.

2. Colloidal Gold Labeling of Staphylococcus aureus Protein A (SPA)

Taking 1mL colloidal gold solution as an example, add 2μL 0.2mol/L K ₂ CO ₃ to adjust the pH of the colloidal gold solution to about 6.0, add 10μL Staphylococcus aureus protein A solution (1mg/ml, purchased from Sigma Company), and react at room temperature for 5min , add 100 μL of 3% casein solution, block at room temperature for 10 min, centrifuge at 12000 r/min for 10 min, discard the supernatant, add 100 μL of PBS buffer containing 1.2 wt% BSA and 0.05 wt% sodium azide to resuspend, and obtain colloidal gold labeling SPA solution.

The preparation of embodiment 4 colloidal gold immunochromatography test strips

1. The structure of the test strip:

The double colloidal gold immunochromatography test strip (double gold standard detection test strip) of the present invention comprises a support base plate (made of PVC board), on which a sample pad, a gold standard protein, and a sample pad are sequentially fixed in sequence. Pad, chromatographic detection membrane and water-absorbent pad; the gold-labeled protein pad is coated with glass fiber cotton, and colloidal gold-labeled Staphylococcus aureus protein A is immobilized; the chromatographic detection membrane is a nitrocellulose membrane, and a detection Line and quality control line, in which the purified p54 recombinant protein and CD2v recombinant protein were used to prepare detection line imprints T1 and T2 respectively, and the distance between the two lines was 5mm, and the complex of recombinant protein antigen and virus antibody was captured as the detection line, quality control A rabbit anti-mouse IgG polyclonal antibody is immobilized on the line.

2. Preparation method

1) Preparation of NC detection membrane (chromatographic detection membrane)

The nitrocellulose detection membrane was cut into strips of 2.5 × 30 cm, placed on the platform of XYZ3000 spray dot instrument, and fixed with layering; use 0.1mol/L Tris-HCl buffer (pH8.0) to dilute ASFV p54 recombinant protein and CD2v recombinant protein to a concentration of 1mg/mL, using Biojet Quanti 3000 to spray 0.9μL/cm on the nitrocellulose membrane, which is the detection line T1 and T2 blots;

Dilute the rabbit anti-mouse IgG polyclonal antibody concentration (the original concentration prepared in Example 2 is 3.8 mg/ml) to 1 mg/mL with PBS buffer, and use Biojet Quanti 3000 to spray on nitrocellulose in an amount of 0.9 μL/cm Membrane, which is the C imprint of the quality control line; the shortest distance between the detection line and the quality control line is 0.5cm; dry at 42°C for 4 hours; put the detection membrane in a ziplock bag, add a desiccant and seal it for storage for later use.

2) Preparation of sample pads

Cut the glass wool into strips of 1.8×30cm, use 0.1mol/L PBS solution containing BSA 10g/L, Tween20 5g/L, and NaNO ₃ 3g/L as the sample pad treatment solution to soak the glass wool for 10min, Then put it in a drying oven at 45°C for 30 minutes to make a sample pad, put the sample pad in a plastic bag, add a desiccant and store it airtight at room temperature for later use.

3) Preparation of gold-labeled protein pad

Cut the glass fiber wool into strips of 0.75×30cm ² , place it on the XYZ 3060 sprayer platform, and fix it with beading strips; The Na ₂ B ₄ O ₇ buffer solution of NaN ₃ is evenly sprayed on the glass fiber sliver for pretreatment, dried in a 42°C drying oven for 50 minutes, and taken out for use;

Take 4.5ml colloidal gold-labeled SPA solution, and add 4.5ml gold-labeled antibody preservation solution (containing 2% (w/v) BSA, 3% (w/v) sucrose, 0.2% Tween 20 (v/v) Mix and dilute with 0.1% (w/v) sodium azide 20mmol/L sodium borate solution (pH value 8.0)) to obtain the colloidal gold marker solution; Spray the solution on the pretreated glass fiber sliver; dry it in a 42°C drying oven for 1 hour to make a gold-labeled protein pad, then put it in a plastic bag, add a desiccant and store it airtight at room temperature (15-25°C) for later use .

4) Preparation of absorbent pad

Cut absorbent paper into strips of 2.5×30 cm to make absorbent pads, add a desiccant and store them airtight at room temperature for later use.

5) Preparation of support base plate

Paste the double-sided tape on the PVC board and cut it into 7.5×30cm long boards to prepare the support base board.

6) Assembly of test strips

Assemble the above materials into test paper boards manually or by using LM5000 test paper assembly machine, and assemble the sample pad, gold standard protein pad, chromatographic detection membrane, and water-absorbent pad in sequence on the support base plate. First paste the detection membrane on the center of the support base plate, then paste the absorbent paper on the end of the detection membrane near the quality control line from top to bottom, paste the gold standard protein pad and the sample pad on the end of the detection line near the detection membrane in turn, each layer overlap by 2mm. The assembled test paper board was cut into test paper strips with a width of 0.25 cm using a CM4000 cutting instrument, and stored dry and sealed. The product structure is shown in Figure 1.

3. Detection principle and judgment method

When the sample end of the test strip is added to the sample solution to be tested, the solution to be tested will drive the antibody in the sample to be tested and the gold-labeled SPA protein complex to diffuse to the nitrocellulose membrane through siphon action, and finally penetrate into the filter paper layer. If the sample to be tested contains any corresponding antibody of ASFV-p54(T1)/CD2v(T2), the ASFV-specific antibody in the serum will combine with the antigen protein sprayed on the detection line during the diffusion process to form an antigen-antibody-colloidal gold The complex is intercepted by the detection line, and the corresponding detection line (T1 or T2) is red, and the quality control line is intercepted by the gold standard SPA, and the red quality control line (C) appears; when the tested serum does not contain ASFV specific antibodies, the corresponding detection line The line does not show color, only the red quality control line appears. If the quality control line on the detection membrane does not develop color, it indicates that the test strip has expired and the test result is invalid.

When in use, place the diluted suspicious pig serum (or anticoagulant) on the sample hole of the test strip sample pad, observe the result within 5 minutes, and judge the serum (or anticoagulant) sample that appears at the same time as the detection line and the quality control line. If the ASFV antibody is positive and only the quality control line appears, it is judged as ASFV antibody negative. The test strip can effectively distinguish the infection of African swine fever virus wild strain or CD2v deletion strain infection. When the T1 line and T2 line develop color at the same time, it means that the sample contains antibodies against CD2v and p54 at the same time, indicating that it is African swine fever virus. Infection with wild strains of the virus, when the T1 line develops color, but the T2 line does not, it means that the sample only contains antibodies against p54 protein, but no antibody against CD2v protein, indicating that it is CD2v-deficient strain infection, when the T1 line When the T2 and T2 lines are not colored, it means that there is no ASFV infection.

The present invention detects two main and quite conservative proteins (p54 and CD2v) that AFSV has antigenicity, and can obtain two results at the same time by adding a sample, and if any one of the two is positive, it can prompt AFSV infection and improve The detection rate is high, and it can quickly detect whether the early pig fever is caused by African swine fever virus infection, detect the epidemic early and take effective prevention and control measures.

The double colloidal gold immunochromatographic test strip prepared by the invention has the advantages of high sensitivity, strong specificity, fast speed (5-10min), easy operation, good repeatability, stable structure, good performance, etc., and is especially suitable for on-site ASFV Serological diagnosis, epidemiological investigation, self-examination by farmers or epidemic investigation by professional institutions.

Embodiment 5 test strip specificity test, sensitivity test and repeatability test measure

In this example, the specificity and sensitivity of the test strips prepared under the optimal conditions in Example 4 were determined, and the final sample volume was 60 μL. Select PRRSV positive serum, CSFV positive serum, PCV2 positive serum, PRV positive serum, enterotoxigenic Escherichia coli ETEC, ASFV positive serum and ASFV negative serum as samples to determine the specificity of the test strip, and the test results are shown in Figure 9 .

First, to detect specificity, the serum to be tested (the ASFV standard positive serum sample purchased from the China Veterinary Microbiological Culture Collection Management Center) was diluted with the sample diluent at a ratio of 1:100. Add it to the sample hole, and observe the test results after 5 minutes. The results of the specificity test showed that the method had no cross-reaction with other major swine pathogen antibody-positive sera, indicating that the method had good specificity.

Secondly, ASFV standard positive serum was selected and diluted in different gradients (1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1: 102400) as a sample, and use this test strip for sensitivity determination. The results show that the minimum detection limit of the antibody sensitivity of the African swine fever virus standard positive serum samples to p54 and CD2v is 1:51200; the indirect ELISA antibody detection method is used, and the specific operation judgment method refers to the national standard of the People's Republic of China "African swine According to the indirect ELISA antibody detection method in "Plague Diagnostic Technology" (GB/T 18648-2020), the antibody titer determined by ELISA according to the national standard detection method is about 1:12800. The detection results are shown in Table 1, and the results show that the sensitivity of the dual detection test paper of the present invention to detect AFSV antibodies is about 4 times higher than that of the national standard ELISA method.

Table 1 ASFV standard positive serum samples for p54 and CD2v antibody sensitivity test results

Finally, the ASFV strong positive serum, weak positive serum and negative serum were selected for three repeated tests between batches and three replicates within a batch to determine the detection repeatability of the test strips. The reproducibility results showed that the results of negative and positive sera were consistent after 5 repeated determinations, indicating that the method had good reproducibility.

In summary, the double colloidal gold immunochromatographic test strip prepared by the present invention has the advantages of strong specificity, good sensitivity, high repetition rate, and accurate detection results, and can quickly and accurately detect African swine fever virus The antibody can be applied to the detection of clinical ASFV serum antibody.

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principle and spirit of the present invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

Claims (10)

1. The gene encoding the ASFV p54 protein after codon optimization is characterized in that its nucleotide sequence is as shown in SEQ ID NO.2. 2. The method for obtaining the ASFV p54 recombinant protein is characterized in that, referring to the codon-optimized gene of the ASFV p54 protein, it is synthesized, the pCAGGS vector is constructed, and a His tag is added to the C segment of the p54 protein to obtain a recombinant plasmid; the recombinant plasmid Transfected into HEK293F cells, cultured for 24-48 hours, centrifuged to get the cell supernatant, filtered, using Ni-NAT affinity chromatography column to remove impurities, eluted and purified, and concentrated in an ultrafiltration centrifuge tube to obtain ASFV p54 recombinant protein , save for later use. 3. The gene encoding ASFV CD2v protein after codon optimization is characterized in that its nucleotide sequence is as shown in SEQ ID NO.4. 4. The method for obtaining the ASFV CD2v recombinant protein is characterized in that, with reference to the codon-optimized gene of the ASFV CD2v protein, it is synthesized to construct a pCAGGS carrier, and a His tag is added to the C segment of the CD2v protein to obtain a recombinant plasmid; Transfected into HEK293F cells, cultured for 24-48 hours, centrifuged to get the cell supernatant, filtered, using Ni-NAT affinity chromatography column to remove impurities, eluted and purified, and concentrated in an ultrafiltration centrifuge tube to obtain ASFV p54 recombinant protein , save for later use. 5. A double colloidal gold immunochromatographic test strip for simultaneously detecting African swine fever virus CD2v and p54 antibody is characterized in that it includes a support base plate, on which a sample pad and a gold-labeled protein pad are sequentially fixed on the support base plate , chromatography detection membrane and absorbent pad; Among them, the gold-labeled protein pad is coated with glass fiber cotton, and colloidal gold-labeled Staphylococcus aureus protein A is immobilized; the chromatographic detection membrane is a nitrocellulose membrane, and a detection line and a quality control line are arranged on it. ASFV p54 recombinant protein and CD2v recombinant protein were imprinted as detection lines respectively; rabbit anti-mouse IgG polyclonal antibody was immobilized on the quality control line. 6. the preparation method of the double colloidal gold immunochromatography test strip that detects African swine fever virus CD2v and p54 antibody simultaneously described in claim 5 is characterized in that, comprises the steps: 1) Preparation of chromatographic detection membrane Cut the nitrocellulose detection membrane into long strips, place it on the platform of the spray dot instrument, and fix it; spray the diluted ASFV p54 recombinant protein and CD2v recombinant protein on the nitrocellulose membrane, which is the detection line T1 and T2 blots; The diluted rabbit anti-mouse IgG polyclonal antibody is sprayed on the nitrocellulose membrane, which is the quality control line C blot, dried and sealed for future use; 2) Preparation of sample pads Cut glass wool into long strips, use PBS solution containing BSA, Tween20, and _NaNO3 as the sample pad treatment solution to soak the glass wool strips, then dry to make sample pads, and store them sealed at room temperature for later use; 3) Preparation of gold-labeled protein pad Cut the glass fiber cotton into long strips, place it on the sprayer platform, and fix it; spray the Na ₂ B ₄ O ₇ buffer solution containing BSA and NaN ₃ on the glass fiber cotton for pretreatment, and then dry it for later use; Dilute the colloidal gold-labeled SPA solution, spray it on the pretreated glass fiber cotton, dry it, and make a gold-labeled protein pad, and store it sealed at room temperature for later use; 4) Preparation of absorbent pad Cut absorbent paper into long strips to make absorbent pads, dry them, and store them airtight at room temperature for later use; 5) Preparation of support base plate Paste the double-sided tape on the PVC board, cut it into a long board, and prepare the supporting base board; 6) Assembly Assemble the sample pad, gold standard protein pad, chromatographic detection membrane, and water-absorbent pad in sequence on the support base plate; first paste the detection film on the center of the support base plate, and then paste the absorbent paper on the detection line near the quality control line from top to bottom. At the membrane end, paste the gold-labeled protein pad and the sample pad in turn on the end of the detection line close to the detection membrane, then cut into test strips, dry and seal them for storage. 7. preparation method as claimed in claim 6, is characterized in that, described rabbit anti-mouse IgG polyclonal antibody is prepared by the following steps: Rabbits were immunized with mouse serum IgG as the immunogen, and the antigen was emulsified with complete Freund's adjuvant for the first immunization, and injected subcutaneously at multiple points at a dose of 150-200 μg/rat; Complete adjuvant emulsified antigen for injection; 1-2 weeks after the last booster immunization, mouse serum IgG was coated with an enzyme-linked immunosorbent plate at a concentration of 1-2 μg/mL and 20-50 μL per well, and indirect Measure the titer of immune serum by ELISA method. When the titer reaches the expected value or above, collect hyperimmune rabbit whole blood, place it at 35-37°C for 1-2 h, centrifuge for 5-10 min, collect the supernatant, and wash with ammonium sulfate. Immune serum IgG was extracted and purified by precipitation method to obtain rabbit anti-mouse IgG polyclonal antibody, which was stored for future use. 8. preparation method as claimed in claim 6, is characterized in that, the SPA solution of described colloidal gold label is prepared through the following steps: Add K ₂ CO ₃ to the colloidal gold solution to adjust the pH to 6.0, then add Staphylococcus aureus protein A solution, react at room temperature for 5-10 min, add casein solution, block at room temperature for 10-20 min, centrifuge for 5-10 min, The supernatant was discarded, and PBS buffer containing BSA and sodium azide was added to resuspend to obtain a colloidal gold-labeled SPA solution. 9. the application of the double-linked colloidal gold immunochromatographic test strip for simultaneously detecting African swine fever virus CD2v and p54 antibody described in claim 5 in the preparation of African swine fever virus infection diagnostic reagent. 10. the application of the double-linked colloidal gold immunochromatography test strip for simultaneously detecting African swine fever virus CD2v and p54 antibody described in claim 5 in the preparation of quarantine inspection reagents.