CN113698474B - African swine fever polyclonal antibody and African swine fever antigen detection test strip - Google Patents
African swine fever polyclonal antibody and African swine fever antigen detection test strip Download PDFInfo
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Abstract
The invention belongs to the technical field of animal virus detection, and particularly relates to an African swine fever polyclonal antibody and an African swine fever antigen detection test strip. According to the invention, the polyclonal antibody is prepared by immunizing mice and rabbits with the African swine fever antigen, the polyclonal antibody is purified, a double-antibody sandwich colloidal gold immunochromatography test paper is established, and whether an animal is infected with ASFV or not can be determined as early as possible by detecting the ASF virus antigens p72, p54 and p 30; the African swine fever antigen detection test strip has better specificity, does not need to prepare polyclonal antibodies for resisting p30, p54 and p72 antigens respectively, and has simpler preparation method.
Description
Technical Field
The invention belongs to the technical field of animal virus detection, and particularly relates to an African swine fever polyclonal antibody and an African swine fever antigen detection test strip.
Background
African Swine Fever (ASF) is an acute, febrile, highly contagious disease of infected pigs and wild pigs caused by African Swine Fever Virus (ASFV) infection characterized by severe bleeding from hyperpyrexia, lymph and internal organs. ASF is one of the most complex infectious diseases of pigs, and is classified as a legal report animal infectious disease by the world animal health Organization (OIE), which is classified as a class of animal epidemic disease in our country. ASFV does not infect people and does not cause direct harm to public health. However, ASFV severely threatens swine health and live swine production, drastically reduces swine product trade, causes economic losses in infected and nearby countries, and causes serious social and economic impacts on food safety, particularly in countries where pork is the primary protein source. In 8 months 2018, ASF is found in China for the first time, and forms a great threat to society and economy in China.
ASFV is a complex icosahedral DAN virus, the only member of the genus African swine fever virus of the family African swine fever virus, consisting of four concentric axis structures and a hexagonal outer membrane. The viral genome varies in length from 170kb to 193kb and contains 150-167 open reading frames. The genome consists of a conserved central region of around 125kb and two variable ends encoding five polygenic families (MGFs). ASFV is classified into 24 different genotypes according to the B646L gene encoding its capsid protein p 72. The major structural component (P72) of the ASFV capsid and the membrane proteins (P54, P30 and P12) proteins are highly antigenic and highly conserved in sequence, being preferred proteins for genetically engineered vaccines and diagnostic reagents.
Although ASF has been reported a century ago, there is no effective commercial vaccine until now due to its complexity. At the same time, control of this disease without vaccine or treatment has proven to be a serious challenge. The transmission of ASFV can only be prevented and controlled by early discovery and strict adherence to traditional disease control methods. Therefore, sufficient technical support must be provided for accurate, timely, and rapid diagnosis and detection. Virus isolation and red blood cell adsorption (HAD) are considered as a definitive method for detecting ASFV in the first burst, but ASFV strains may not always be isolated from each field sample, greatly limiting their use; PCR is considered to be a gold standard for early detection of ASFV due to its high sensitivity, specificity, stability and high throughput, and can detect ASFV genome in any clinical sample of domestic pigs, wild boars and soft ticks, but requires expensive instrumentation and specialized technicians to prevent contamination (aerosols), and also limits its popularity, especially in remote areas; an enzyme-linked immunosorbent assay (ELISA) method for detecting specific antibodies has the problems of low sensitivity, poor specificity, difficult preparation of enzyme-labeled antigen by a sandwich method and the like; the double-antibody sandwich ELISA for detecting the viral self-protein cannot realize rapid and simple diagnosis. The colloidal gold immunochromatographic test strip is quick, simple and high in flux, does not need professional equipment and personnel, and provides possibility for quick and accurate on-site screening of ASFV.
Currently, there are some researches on African swine fever antigen detection test strips. For example, chinese patent document CN109254155a discloses a colloidal gold immunochromatographic test paper for detecting african swine fever virus antigen, and a preparation method and application thereof, wherein a monoclonal antibody Mab1 of an epitope of an anti-ASFV p30 antigen is labeled with colloidal gold in a colloidal gold pad of the colloidal gold immunochromatographic test paper; the nitrocellulose membrane surface is marked with a detection line and a quality control line, the detection line is a monoclonal antibody Mab2 aiming at the other epitope of the ASFV p30 antigen, and the quality control line is a goat anti-mouse IgG antibody. The colloidal gold immunochromatographic test paper for detecting the African swine fever virus antigen can only be used for detecting ASFV p30 antigen, and has poor specificity.
Patent document CN 110456038A discloses a duplex detection reagent for african swine fever virus antigen and a preparation method thereof, and specifically discloses a colloidal gold-labeled murine anti-p 30 protein monoclonal antibody 1 and a murine anti-p 72 protein monoclonal antibody 2 coated on a colloidal gold-labeled pad of the detection reagent; the inner surface of the nitrocellulose membrane detection pad is sequentially provided with a p30 protein detection line (coated with a mouse p30 protein monoclonal antibody 3), a p72 protein detection line, a quality control line (coated with a mouse p72 protein monoclonal antibody 4) and a quality control line (coated with a sheep anti-mouse IgG polyclonal antibody) from left to right. The detection reagent can be used for detecting the African swine fever p30 and p72 antigens, and although the detection specificity can be improved to a certain extent, a plurality of monoclonal antibodies for resisting the African swine fever p30 and p72 antigens are required to be prepared respectively, and the preparation method is complicated.
Patent document CN 109781980a discloses a fast detection card for african swine fever virus and application thereof, wherein colloidal gold-labeled polyclonal antibodies PoAbI against african swine fever virus p30, p54 and p72 proteins are adsorbed on a gold-labeled pad; the detection membrane is provided with a quality control line C printed by goat or rabbit anti-mouse IgG antibody or staphylococcus aureus SPA, and also comprises a detection line T of polyclonal antibodies PoAbII of anti-African swine fever virus p30, p54 and p72 proteins. Although the detection card can be used for detecting the p30, p54 and p72 proteins of African swine fever at the same time, corresponding polyclonal antibodies are required to be prepared respectively, and the preparation method is complex.
Disclosure of Invention
The invention aims to provide an African swine fever polyclonal antibody, which is prepared by immunizing experimental animals with an African swine fever antigen. The African swine fever antigen has an epitope selected from p72 protein, p54 protein and p30 protein, and after the African swine fever antigen is adopted to immunize animals, a polyclonal antibody which specifically recognizes any one or any combination of the p72 antigen, the p54 antigen and the p30 antigen of the African swine fever can be obtained in a separated way, so that the detection and recognition specificity of the African swine fever antigen can be improved.
The second object of the present invention is to provide a method for preparing the african swine fever polyclonal antibody.
The third objective of the invention is to provide an African swine fever antigen detection test strip.
The invention also aims to provide an African swine fever antigen detection card/kit.
The African swine fever polyclonal antibody adopts the following technical scheme: an african swine fever polyclonal antibody prepared by immunizing an experimental animal with an african swine fever antigen, the amino acid sequence of the african swine fever antigen comprising the following fragments: at least one epitope selected from the group consisting of p72 protein, at least one epitope selected from the group consisting of p54 protein and at least one epitope selected from the group consisting of p30 protein.
Preferably, the epitope selected from p72 protein is shown in SEQ ID NO. 1-5; the antigen epitope selected from p54 protein is shown in SEQ ID NO. 6-10; the epitope selected from p30 protein is shown in SEQ ID NO. 11-15; the antigen epitope selected from the p72 protein, the antigen epitope selected from the p54 protein and the antigen epitope selected from the p30 protein are connected through a connector, and the amino acid sequence of the connector is GSGS and/or KK; the preparation method of the African swine fever antigen comprises the following steps: (1) Synthesizing a nucleotide sequence for encoding the African swine fever antigen and connecting the nucleotide sequence with a vector to obtain a recombinant vector; (2) Transforming the recombinant vector into an expression strain to obtain an engineering strain; (3) Inducing the engineering strain to enable the engineering strain to express the African swine fever antigen, and purifying to obtain the African swine fever antigen.
Preferably, the African swine fever antigen has an amino acid sequence as shown in SEQ ID NO. 16.
Preferably, the experimental animal is a mouse or a rabbit; preferably, the mouse is a Kunming mouse and the rabbit is New Zealand white rabbit.
The preparation method of the African swine fever polyclonal antibody adopts the following technical scheme: the method for preparing the african swine fever polyclonal antibody according to any one of the above, comprising the steps of: mixing the African swine fever antigen and an adjuvant, performing subcutaneous multipoint injection on experimental animals, and detecting serum titer by ELISA (enzyme-Linked immuno sorbent assay) until the titer reaches 1:1×10 7 Then, taking blood by heart, separating serum and purifying to obtain the African swine fever polyclonal antibody; preferably, the african swine fever antigen is mixed with an adjuvant in equal volumes; preferably, freund's complete adjuvant is used for the first time, and Freund's incomplete adjuvant is used for the rest.
The African swine fever antigen detection test strip adopts the following technical scheme: the African swine fever antigen detection test strip comprises a sample pad, a marking pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped, wherein the nitrocellulose membrane is provided with a detection line and a quality control line, the marking pad is coated with an African swine fever polyclonal antibody-marking compound, the detection line is coated with an African swine fever rabbit polyclonal antibody, and the quality control line is coated with goat anti-mouse IgG; the african swine fever murine polyclonal antibody and the african swine fever rabbit polyclonal antibody are independently selected from the african swine fever polyclonal antibodies as described in any one of the above, which differ only in that the experimental animals are mice and rabbits, respectively.
Preferably, the african swine fever mouse polyclonal antibody-labeled complex is an african swine fever mouse polyclonal antibody-colloidal gold complex, and the preparation method of the african swine fever mouse polyclonal antibody-colloidal gold complex is as follows: adding double distilled water into 1% chloroauric acid to a volume of 1000mL, heating to boil, and adding 1.0% trisodium citrate solution to adjust pH to change into wine red; cooling at room temperature after 4-8min, filtering to obtain colloidal gold solution, and preserving at 4deg.C for use; adding african swine fever mouse polyclonal antibody solution into the above colloidal gold solution, mixing, standing at room temperature for 30min, adding 1% BSA for blocking, mixing, standing at room temperature for 10min,12000r/min, centrifuging at 4deg.C for 30min, removing supernatant, precipitating with 10mL 20mM Na 2 B 4 O 7 Re-suspending the heavy suspension of the (2) to obtain the African swine fever mouse polyclonal antibody-colloidal gold complex.
Preferably, the dosage of the African swine fever murine polyclonal antibody is 0.06-0.24mg, and the concentration of the African swine fever rabbit polyclonal antibody is 1.0-2.0mg/mL; the particle size of the colloidal gold in the colloidal gold solution is 30-40nm; the nitrocellulose membrane is any one of HF1800425, HF1200425 or HF 0900425.
Preferably, the concentration of the African swine fever rabbit polyclonal antibody is 1.5mg/mL; the particle size of the colloidal gold in the colloidal gold solution is 30nm; the nitrocellulose membrane was HF1200425.
The African swine fever antigen detection card/kit adopts the following technical scheme: an african swine fever antigen detection card/kit comprising an african swine fever polyclonal antibody as described in any one of the above and/or an african swine fever antigen detection test strip as described in any one of the above.
The beneficial effects of the invention are as follows:
the African swine fever antigen contains p72, p54 and p30 tandem dominant epitopes, and the polyclonal antibody prepared by immunizing a mouse can specifically identify any one or any combination of the antigens containing the African swine fever p72, p54 and p30, so that the detection and identification specificity of the African swine fever antigen can be improved.
According to the invention, the polyclonal antibody is prepared by immunizing mice and rabbits with the African swine fever antigen, the polyclonal antibody is purified, a double-antibody sandwich ELISA immunochromatography test paper is established, and whether an animal is infected with ASFV or not can be determined as early as possible by detecting the ASF virus antigens p72, p54 and p 30; the African swine fever antigen detection test strip has better specificity, does not need to prepare polyclonal antibodies for resisting p30, p54 and p72 antigens respectively, and has simpler preparation method.
The African swine fever antigen detection test strip has good specificity and sensitivity.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a diagram showing purification and Western blotting of rASFV-EP recombinant protein prepared in example 1 of the present invention;
FIG. 2 is a schematic diagram of a test strip assembly;
FIG. 3 is a chart of the sensitivity test results of the African swine fever antigen test strip of the present invention, wherein FIG. 3A is a chart of the qualitative detection results of the sensitivity by visual inspection, and FIG. 3B is a chart of the detection results of a TSR 3000 reader; in fig. 3: 1-9 represent sample dilution factors of 1-1:20, 2-1:200, 3-1:400, 4-1:800, 5-1:1600, 6-1:3200, 7-1:6400, 8-1:12800, 9-1 respectively: 25600;
FIG. 4 is a diagram showing the specific detection result of the African swine fever antigen detection test strip of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 construction of antigen for the preparation of African swine fever monoclonal antibody
The major structural component (P72) of the ASFV capsid and the membrane protein (P54 and P30) protein sequences are highly conserved and can be used as candidate target proteins for ASFV virus antigens. The invention designs a tandem combination candidate epitope as immunogen for preparing antibodies by analyzing protein sequences of African swine fever viruses P72, P52, P12, P30 and CDv2 published by NCBI through bioinformatics and combining dominant epitopes published and confirmed by a binding literature. In order to achieve the above object, the present invention provides a technical solution comprising:
the method comprises the steps of utilizing an online free biological information database http:// tools.iedb.org/bcell/, https:// blast.ncbi.nlm.nih.gov/blast.cgi, biological information software DNASTAR and the like, combining existing literature analysis, selecting dominant epitopes of ASF virus coat proteins P72, P54 and P30, and combining the dominant epitopes through flexible connectors to obtain the dominant epitope of the tandem combination;
after the tandem dominant antigen epitope is subjected to escherichia coli codon optimization, the nucleotide sequence of the tandem dominant antigen epitope is synthesized by a biological engineering (Shanghai) stock company, and is recombined on a vector pET-22b (+). The recombinant vector with tandem dominant epitope is used for transforming an escherichia coli expression strain Ecoli.BL21 (DE 3) to obtain an engineering strain. The engineering strain is induced, expressed and purified to obtain a tandem recombination dominant epitope antigen, which is named rASFV-EP;
the method comprises the following specific steps:
1) The dominant epitopes of ASF virus coat proteins P72, P54 and P30 are screened, and the epitope sequences are as follows:
P72：
E1：MASGGAFCLIANDGKADKI(SEQ ID NO.1)
E2：SYGKPDPEPTLSQIEETHLVHFNAHFKPYVPVGFEYNKVRPHTGTPTLGNK(SEQ ID NO.2)
E3：RNGYDWDNQTPLEGAVYTLVDPFGRPIVPGTKNAYRNLVYYCEYPGERL(SEQ ID NO.3)
E4：HKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPI(SEQ ID NO.4)
E5：SSWQDAPIQGTSQMGAHG(SEQ ID NO.5)
P54：
E6：MDSEFFQPVYPRHYGECLSPVTTPSFFSTHMY(SEQ ID NO.6)
E7：PYQDQQWVEVTPQPGTSKPAGATTASVGKPVTGRPATNRPA(SEQ ID NO.7)
E8：TNKPVTDNPVTDRLVMATGGPAAAPA(SEQ ID NO.8)
E9：LRQRNTYTHKDLENSL(SEQ ID NO.9)
E10：YLFSSRKKKAAAIEE(SEQ ID NO.10)
P30：
E11：MEVIFKTDLRSSSQVVFHAG(SEQ ID NO.11)
E12：QEEWNMILHVLFEEETESSASSEN(SEQ ID NO.12)
E13：QYGKAPDF(SEQ ID NO.13)
E14：TIYGTPLKEEEKEV(SEQ ID NO.14)
E15：GQGYTEHQAQE(SEQ ID NO.15)
2) The direct tandem of different epitopes may easily form new epitopes at the junction of the epitopes, thereby affecting or masking the original epitope. Therefore, the flexible connector GSGS is added between the two connecting epitopes, so that the novel epitope is avoided. Meanwhile, in order to improve antigen presentation, different epitopes are connected by using 2 lysines-Lysine-sine (KK), wherein KK is an important protease recognition site and a cleavage site in cells, and different cell epitopes are connected by KK so as to facilitate correct cleavage of each epitope by protease, so that each epitope can be better recognized and presented by presentation cells, and the effects can be better and independently exerted. The linker between the different epitopes is therefore: "KKGGS", the tandem recombination sequence is as follows:
rASFV-EP：
YLFSSRKKKAAAIEEKKGSGS GQGYTEHQAQE KKGSGS SSWQDAPIQGTSQMGAHG
3) Preparation of tandem recombinant dominant antigen (rASFV-EP)
The rASFV-EP amino acid sequence was subjected to E.coli codon optimization, and then a recombinant plasmid was synthesized and constructed by the division of biological engineering (Shanghai) Co. Preparing competent escherichia coli BL21 (DE 3) cells, taking an escherichia coli BL21 (DE 3) single colony to be cultured in 2ml of LB culture medium at 37 ℃ at 200rpm for 12-16 hours, taking 1ml of culture to be inoculated in 100ml of LB culture medium, and culturing at 37 ℃ at 200rpm for 3 hours; ice-bath the bacterial liquid for 2 hours, then centrifuging 2500g at a low temperature for 20 minutes, and collecting bacterial bodies; 100ml ice-cold Tritures buffer (100 mmol/L CaCl2,70mmol/L MgCl2,40mmol/L sodium acetate, pH 5.5) was added, mixed well, placed in an ice bath for 45 min, centrifuged at 1800g for 15 min, the supernatant was discarded, 10ml ice-cold Tritures buffer was added to suspend the cells, and 200 μl of each was dispensed and stored at-80℃for further use.
Conversion: melting 200 μl of competent cells in 5 tubes on ice, adding 3 μl DMSO, mixing, adding 1 μl of V1, V2, V3, V4, and V5 respectively, mixing again, placing on ice for 30min, standing at 42deg.C for 45 seconds, rapidly placing back into ice bath for 1.5 min, adding 2ml LB culture solution, shaking and culturing at 37deg.C for 1 hr at 200rpm, centrifuging at 4000g at low temperature for 10 seconds, discarding supernatant, and re-suspending thallus with 200 μl LB culture medium; the bacterial liquid is spread on an LB agar plate containing ampicillin resistance, evenly spread, placed for 30 minutes at room temperature, and placed in a 37 ℃ incubator for culturing for 12-16 hours.
Induction of expression: i.e. overnight culture of the clone, transferring in 1:100 in the morning the next day, adding 0.5mM IPTG after 3 hours of culture, and continuing to culture for 3 hours to prepare a sample; detecting the expression condition of target protein by conventional SDS-PAGE, and finding that the specific band is correctly cloned; and taking correct clones, and culturing in an amplifying way.
Purifying: the collected cells were subjected to ultrasonic lysis after being suspended with inclusion body washing liquid I (1% Triton X-100, 20mM Tris-HCl pH 8.0) per 100g, 200W ultrasonic lysis was performed for 1 hour, stirred at room temperature for 4 hours, centrifuged at 8000 rpm for 30 minutes at low temperature, and the pellet was discarded. The supernatant was retained and the protein concentration was measured using a BCAprotein assay kit commercial kit (PIERCE). Renaturation of the denatured protein by dilution renaturation method is carried out, and the final concentration of the protein is 100 mug/ml. The renaturation solution adopts a Tris (pH 8.0) buffer system, 0.3M arginine is added, and stirring renaturation is carried out at 4 ℃ for 48 hours. Renaturation solution was equilibrated with 20mM Tris-cl buffer pH=8.0 for anion exchange chromatography, purification and Western blotting (His antibody) detection as shown in FIG. 1.
EXAMPLE 2 preparation of polyclonal antibodies Using the tandem recombinant dominant antigen (rASFV-EP) prepared in example 1 as an immunogen
Preparing and purifying the polyclonal antibody: mixing the purified rASFV-EP with an equal volume of adjuvant, subcutaneously injecting into 5 Kunming mice and New Zealand white rabbits at a dose of 150 μg/mouse, using Freund's complete adjuvant for the first time, using Freund's incomplete adjuvant for the rest, detecting serum titer after immunization by ELISA, and obtaining a titer of 1:1X10 7 And then taking blood from the heart, separating serum, purifying the polyclonal antibody (firstly, adopting an ammonium octoate sulfate precipitation method for preliminary purification, then adopting Pierce Protein G Columns for further purification), and obtaining the African swine fever mouse anti-polyclonal antibody and the African swine fever rabbit anti-polyclonal antibody respectively after dialysis and desalination, and preserving at the temperature of minus 20 ℃ for later use.
Example 3 preparation of African swine fever antigen detection test strip
3.1 preparation of colloidal gold: adding double distilled water into 1.0% chloroauric acid to 1000mL, heating to boil, adding 1.0% trisodium citrate solution at a certain ratio, adjusting pH to change into wine red, starting timing, cooling at room temperature after about 5min, filtering, and preserving at 4deg.C for use. And then, the quality of the colloidal gold is identified by observing the particle size, shape, granularity, uniformity and the like of the colloidal gold by using a transmission electron microscope.
3.2 preparation of the african swine fever mouse anti-rastv-EP polyclonal antibody-labeled complex and labeled pad of the present invention: slowly adding appropriate proportion of anti-rASFV-EP polyclonal antibody solution of African swine fever mouse into the colloidal gold solution which is prepared and has been subjected to quality identification, mixing, standing at room temperature for 30min, slowly adding BSA with final concentration of 1% (W/V) for blocking, mixing, standing at room temperature for 10min,12000rAfter centrifugation at 4℃for 30min, the supernatant was discarded and the pellet was resuspended in 10mL (20 mM Na 2 B 4 O 7 ) Re-suspending and storing at 4 ℃ for standby.
After obtaining the anti-rASFV-EP polyclonal antibody of the African swine fever mouse marked by the colloidal gold, the polyclonal antibody is sprayed on the surface of a glass fiber membrane, and the marking pad (the colloidal gold pad) is obtained after drying
3.3 preparation of coated sheets according to the invention: preparing a detection line on a nitrocellulose membrane by taking African swine fever rabbit anti-rASFV-EP polyclonal antibody with a final concentration of 1.5mg/ml and a spray amount of 1.0 mu l/cm and a moving speed of a membrane drawing instrument platform of 100 mm/s; the quality control line is prepared by the same method with the final concentration of sheep anti-mouse of 2 mg/ml. The distance between the detection line and the quality control line on the NC film is about 4.0+/-0.1 mm, and the width of the line is about 0.8-1.0 mm; the front and the back are uniform; the juxtaposition temperature is 37 ℃; humidity is less than or equal to 30%; drying for more than 4 hours.
3.4 test strip assembly: the test paper was assembled according to the conventional procedure (fig. 2), NC membrane (polyclonal antibody of rabbit anti-rasev-EP scored on NC membrane as detection line and quality control line of goat anti-mouse IgG) was adhered to the middle of the support plate, absorbent paper was fixed at one end thereof, gold pad (polyclonal antibody of mouse anti-rasev-EP sprayed with gold label) was attached to the other end, and then the sample pad was attached to the gold pad to assemble the long test paper. Cutting the assembled test paper into test paper with the width of 4.0mm by using a slitter, fixing the test paper into a test paper card drying box, drying, packaging by using an aluminum foil bag, writing a date and a batch, and preserving for later use.
Example 4 examination of factors affecting the Performance of the African swine fever antigen test strip of the present invention
4.1 determination of the optimal labelling amount of the anti-rASFV-EP polyclonal antibody of African swine fever mouse: concentrating the purified African swine fever mouse anti-rASFV-EP polyclonal antibody to a concentration of 2mg/mL, sequentially adding a specified volume of polyclonal antibody into an ELISA plate except for a blank control according to the sequence shown in the following table 1, sequentially adding 125 mu L of colloidal gold into each hole, uniformly mixing, and reacting at room temperature for 10min; then, 125 mu L of 100g/L NaCl solution is added into each hole and evenly mixed, and after reaction is carried out for 10 minutes at room temperature, the result is observed according to the influence of the NaCl solution with high ion concentration on the stability of the colloidal gold. The wells with antibodies had different levels of effect on the stability of the gold, depending on the concentration of the antibodies, thus changing the color of the gold, whereas the control wells without antibodies, the gold was unstable and precipitated very quickly, and the color changed from red to blue very quickly (table 1). The color is unchanged when the concentration of the antibody is high and reaches or exceeds the minimum stabilizing amount, and the concentration of the antibody at this time is the optimal antibody labeling amount.
TABLE 1 colloidal gold labeled monoclonal antibody assay
As is clear from Table 1, the optimal labelling amount of the anti-rASFV-EP polyclonal antibody was 30. Mu.L.
4.2 selection of colloidal gold particle size: the size of the colloid Jin Lijing is different along with the different proportions of chloroauric acid and citric acid, so that a series of marks for polyclonal antibodies, such as 10nm,20nm,30nm and 40nm, of colloidal gold with different particle sizes are prepared. The test results of the effect of colloidal gold with different particle sizes on the test strips are shown in the following table 2:
TABLE 2
| Colloid Jin Lijing | Degree of color development |
| 10nm | － |
| 20nm | ± |
| 30nm | ++ |
| 40m | + |
4.3 selection of nitrocellulose membranes: four nitrocellulose membranes of different specifications and different flow rates are selected, and the specifications are HF1800425, HF1200425, HF0900425 and HF0650425 (the flow rates corresponding to the nitrocellulose membranes are 45s/cm, 30s/cm, 22.5s/cm and 12.75s/cm respectively), and after the nitrocellulose membranes are assembled into four specifications of test papers, the influences of the four specifications on the samples are compared (see Table 3 below for details).
TABLE 3 Table 3
4.4 selection of rabbit anti-rASFV polyclonal antibody on detection line: four concentrations of IgG, 0.5mg/mL, 1.0mg/mL, 1.5mg/mL and 2.0mg/mL, were selected for the preparation of detection lines (T lines), the influence of detection lines of different concentrations of IgG on test paper sensitivity and specificity was observed, and the optimal IgG concentration was finally selected (see Table 4 below for details).
TABLE 4 Table 4
| Rabbit anti rASFV polyclonal antibody concentration | Test paper sensitivity | Specificity (specificity) |
| 0.5mg/mL | － | + |
| 1.0mg/mL | + | + |
| 1.5mg/mL | +++ | ++ |
| 2.0mg/mL | + | + |
EXAMPLE 5 Performance test of African swine fever antigen test strip of the present invention (African swine fever mouse anti-rASFV-EP polyclonal antibody 30. Mu.l, colloidal gold 30nm, HF1200425, rabbit anti-rASFV polyclonal antibody 1.5 mg/mL)
5.1 reactivity of test paper with rASFV-EP recombinant protein
Taking rASFV-EP protein (0.8 g/mL) and carrying out gradient dilution according to the proportion of 1:20-1:25600; 70 μl of the gradient dilution and a blank (PBS solution) are respectively dripped on a sample pad of the African swine fever antigen detection test strip of the invention, and the detection result can be obtained by naked eye observation after 10 min.
Visual observation: obvious specific bands are seen when rASFV-EP protein (0.8 g/mL) is diluted in a gradient of 1:20-1:3200 (see FIG. 3A for details);
TSR 3000 reader detection results: the sample detection line was scanned with a TSR 3000 reader, which showed a dilution of 1:25600, which showed a difference from the negative control, but not so much, and a final assay sensitivity of 1:12800 (see FIG. 3B for details).
5.2 reactivity comparison of reagents with P72, P54, P30 and rASFV-EP recombinant proteins
Taking the standard concentrations of P72, P54, P30 and rASFV-EP protein to be 0.8g/ml, and respectively mixing the concentrations according to the ratio of 1:20 and 1: 100. 1: 400. 1: 800. 1: 1600. 1:3200 and 70 μl of the sample pad of the African swine fever antigen detection test strip of the present invention were added dropwise, and the detection result was obtained by visual observation after 10min (see Table 5 for details)
TABLE 5
5.2 specificity of the reaction of the test paper with the Virus
The assembled strips were chromogenic with recombinant rASFV-EP proteins but not with CSFV attenuated vaccine, sonicated PRRSV, PRV virus antigen (FIG. 4). The test paper containing the gold-labeled 4A8 monoclonal antibody and the rabbit polyclonal antibody has strong specificity. (CSFV, PRRSV, BVDV, PRV antigen from Beijing Nanogen)
The African swine fever antigen detection test strip is suitable for detecting virus protein antigens P72, P54 and P30 such as nasal discharge, saliva, feces, urine, conjunctival exudates, genital excretions and the like of pigs, can rapidly, conveniently and accurately detect and diagnose whether animals are infected with ASFV on the field, and is particularly suitable for customs trade inspection, field epidemiological investigation, food safety detection, large-scale field detection of pig farms and the like.
The African swine fever antigen detection test strip can be matched with other necessary components/reagents to prepare a corresponding kit/detection card, and is more convenient to use.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Sequence listing
Organism name: artificial sequence sequence type: PRT (PRT)
SEQ ID NO.1
MASGGAFCLIANDGKADKI
SEQ ID NO.2
SYGKPDPEPTLSQIEETHLVHFNAHFKPYVPVGFEYNKVRPHTGTPTLGNK
SEQ ID NO.3
RNGYDWDNQTPLEGAVYTLVDPFGRPIVPGTKNAYRNLVYYCEYPGERL
SEQ ID NO.4
HKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPI
SEQ ID NO.5
SSWQDAPIQGTSQMGAHG
SEQ ID NO.6
MDSEFFQPVYPRHYGECLSPVTTPSFFSTHMY
SEQ ID NO.7
PYQDQQWVEVTPQPGTSKPAGATTASVGKPVTGRPATNRPA
SEQ ID NO.8
TNKPVTDNPVTDRLVMATGGPAAAPA
SEQ ID NO.9
LRQRNTYTHKDLENSL
SEQ ID NO.10
YLFSSRKKKAAAIEE
SEQ ID NO.11
MEVIFKTDLRSSSQVVFHAG
SEQ ID NO.12
QEEWNMILHVLFEEETESSASSEN
SEQ ID NO.13
QYGKAPDF
SEQ ID NO.14
TIYGTPLKEEEKEV
SEQ ID NO.15
GQGYTEHQAQE
SEQ ID NO.16(rASFV-EP)
YLFSSRKKKAAAIEEKKGSGS GQGYTEHQAQE KKGSGS SSWQDAPIQGTSQMGAHG
Claims (9)
1. The African swine fever polyclonal antibody is characterized in that the African swine fever polyclonal antibody is prepared by immunizing experimental animals with an African swine fever antigen, and the amino acid sequence of the African swine fever antigen is shown as SEQ ID NO. 16.
2. The african swine fever polyclonal antibody according to claim 1, wherein the method for preparing the african swine fever antigen comprises the steps of: (1) Synthesizing a nucleotide sequence for encoding the African swine fever antigen and connecting the nucleotide sequence with a vector to obtain a recombinant vector; (2) Transforming the recombinant vector into an expression strain to obtain an engineering strain; (3) Inducing the engineering strain to enable the engineering strain to express the African swine fever antigen, and purifying to obtain the African swine fever antigen.
3. The african swine fever polyclonal antibody according to any one of claims 1-2, wherein the experimental animal is a Kunming mouse or a New Zealand white rabbit.
4. The method for preparing african swine fever polyclonal antibody according to any one of claims 1 to 2, comprising the steps of: mixing the African swine fever antigen and an adjuvant, performing subcutaneous multipoint injection on experimental animals, and detecting serum titer by ELISA (enzyme-Linked immuno sorbent assay) until the titer reaches 1:1×10 7 Then, taking blood by heart, separating serum and purifying to obtain the African swine fever polyclonal antibody; the African swine fever antigen is mixed with an adjuvant in an equal volume; freund's complete adjuvant is used for the first time, and Freund's incomplete adjuvant is used for the rest.
5. The African swine fever antigen detection test strip comprises a sample pad, a marking pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped, wherein the nitrocellulose membrane is provided with a detection line and a quality control line; the african swine fever murine polyclonal antibody and the african swine fever rabbit polyclonal antibody are independently selected from the african swine fever polyclonal antibodies as described in any one of claims 1 to 3, which differ only in that the experimental animals are mice and rabbits, respectively.
6. The african swine fever antigen detection test strip of claim 5, wherein the african swine fever murine polyclonal antibody-labeled complex is an african swine fever murine polyclonal antibodyThe preparation method of the body-colloidal gold complex of the African swine fever mouse polyclonal antibody-colloidal gold complex comprises the following steps: adding double distilled water into 1% chloroauric acid to a volume of 1000mL, heating to boil, and adding 1.0% trisodium citrate solution to adjust pH to change into wine red; cooling at room temperature after 4-8min, filtering to obtain colloidal gold solution, and preserving at 4deg.C for use; adding african swine fever mouse polyclonal antibody solution into the above colloidal gold solution, mixing, standing at room temperature for 30min, adding 1% BSA for blocking, mixing, standing at room temperature for 10min,12000r/min, centrifuging at 4deg.C for 30min, removing supernatant, precipitating with 10mL 20mM Na 2 B 4 O 7 Re-suspending the heavy suspension of the (2) to obtain the African swine fever mouse polyclonal antibody-colloidal gold complex.
7. The african swine fever antigen detection test strip of claim 6, wherein the amount of african swine fever murine polyclonal antibody is 0.06-0.24mg, and the concentration of african swine fever rabbit polyclonal antibody is 1.0-2.0mg/mL; the particle size of the colloidal gold in the colloidal gold solution is 30-40nm.
8. The african swine fever antigen detection test strip of claim 7, wherein the african swine fever rabbit polyclonal antibody has a concentration of 1.5mg/mL; the particle size of the colloidal gold in the colloidal gold solution is 30nm.
9. An african swine fever antigen detection card or kit, comprising the african swine fever polyclonal antibody of any one of claims 1 to 3 and/or the african swine fever antigen detection test strip of any one of claims 5 to 8.
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| CN109254155A (en) * | 2018-09-25 | 2019-01-22 | 扬州大学 | A kind of detection African swine fever virus antigen colloidal gold immune chromatography test paper and preparation method and application |
| CN109781980A (en) * | 2019-01-30 | 2019-05-21 | 河南中泽生物工程有限公司 | African swine fever virus rapid detection card and its application |
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| WO1991009313A1 (en) * | 1989-12-20 | 1991-06-27 | Universidad De Oviedo | Method for obtaining a recombinant protein and its utilization in the assay for the african swine pest virus (aspv) |
| CN108148138A (en) * | 2017-12-14 | 2018-06-12 | 石河子大学 | African swine fever virus multi-epitope fusion diagnosis antigen and its preparation method and application |
| CN109254155A (en) * | 2018-09-25 | 2019-01-22 | 扬州大学 | A kind of detection African swine fever virus antigen colloidal gold immune chromatography test paper and preparation method and application |
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