BLV p24 recombinant antigen protein for detecting bovine leukemia antibody, test strip and application of BLV p24 recombinant antigen protein

Abstract

The invention discloses a BLV p24 recombinant antigen protein for detecting a bovine leukemia antibody, a test strip and application of the BLV p24 recombinant antigen protein. The amino acid sequence of the BLV p24 recombinant antigen protein is as shown in SEQ ID NO. 2. The eukaryotic recombinant p24 protein is prepared by adopting a baculovirus expression system, and the recombinant p24 protein expressed by the system can be correctly folded and modified after translation, better simulates native conformation and has higher sensitivity. And the His tag on the recombinant protein is beneficial to one-step purification to achieve higher purity, and better activity can be shown without enzyme digestion. The formed colloidal gold test strip is applied to detection of bovine leukemia, and the accuracy, the sensitivity and the detection are well improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular, to a BLV p24 recombinant antigen protein for detecting bovine leukemia antibody, a test strip and application thereof. BACKGROUND

[0002] Bovine leukemia virus (BLV) is the pathogen of bovine endemic leukemia and belongs to the family of retroviruses. BLV infection not only persistently inhibits the immune response ability of infected cattle, making them more susceptible to other diseases, but also significantly reduces milk production in dairy cattle, growth performance in beef cattle and reproductive efficiency, and causes restrictions on import and export trade due to quarantine requirements, causing huge economic losses to the global cattle industry. At present, there is no effective vaccine or treatment method for the disease, and the core prevention and control measure is to eliminate or isolate infected cattle in time, therefore, it is crucial to develop a rapid, accurate and suitable on-site BLV detection technology.

[0003] Common serological methods include enzyme-linked immunosorbent assay (ELISA), agar diffusion test (AGID) and polymerase chain reaction (PCR). ELISA and AGID are recognized as the "gold standard", and the commonly used BLV detection kit is mainly a commercial ELISA antibody kit produced by foreign countries. However, this method has high requirements for blood samples, and improper handling or storage can lead to high false positive results; and it is expensive, with complicated operation steps, long time consumption, the need for expensive instruments and professional operators, and is not suitable for on-site rapid screening. AGID has low sensitivity. PCR is high in cost and requires harsh laboratory conditions. Our company independently develops and produces a colloidal gold test strip for detecting bovine leukemia antibody, which is simple to operate, low in cost, rapid in detection and stable, and can promote the standardized management of scattered farmers, small and medium-sized cattle farms or breeding farms without ELISA test conditions.

[0004] Gold immunochromatography assay (GICA) is widely used in rapid on-site diagnosis due to its rapidity, simplicity, low cost and no need for special equipment. The existing BLV antibody test strips mostly use indirect methods, and the existing BLV immunochromatography products on the market mainly focus on gp51 antigen, while the rapid test kit for another key antigen of BLV, p24 protein, is still blank in the domestic market, which limits the diversity and complementarity of detection strategies. The College of Veterinary Medicine of Yangzhou University prepared a bovine leukemia virus gp51 antibody test strip, and 22 samples were detected by test strip and ELISA test, and the positive coincidence rate of the two methods was 88.9%(8 / 9). By designing specific DNA probe Det-DNA, capture DNA probe Cap-DNA and quality control DNA probe Con-DNA, Lian Shuai et al. of Heilongjiang Bayi Agricultural and Cultivation University prepared a nucleic acid test strip for detecting BLV, and the sensitivity visual detection threshold of the nucleic acid test strip was improved to 5.0pM, and the detection limit was improved to 50pM. By comparing and analyzing the detection results with the fluorescence quantitative PCR method, it was found that the detection results of the nucleic acid test strip had high consistency with them, and the repeatability was good, which could be applied to rapid detection of bovine leukemia virus at bedside. As can be seen, most of the effect evaluation tests show that the test strip mainly has the problems of low sensitivity and accuracy. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the deficiencies and defects mentioned in the above background art, and to provide a BLV p24 recombinant antigen protein for detecting bovine leukemia antibody, a colloidal gold test strip and a preparation method and application thereof.

[0006] To solve the above technical problems, the technical solution provided by the present application is:

[0007] In a first aspect, the present application provides a BLV p24 recombinant antigen protein for detecting bovine leukemia antibody, and the amino acid sequence of the BLV p24 recombinant antigen protein is shown in SEQ ID NO. 2.

[0008] In a second aspect, the present application further relates to a vector capable of expressing the above-mentioned LV p24 recombinant antigen protein, and the vector has a coding gene of the BLV p24 recombinant antigen protein, and the nucleotide sequence of the coding gene is shown in SEQ ID NO. 1.

[0009] In a third aspect, the present application provides a colloidal gold test strip for detecting bovine leukemia antibody, which comprises a chromatography membrane pasted on a PVC bottom plate, a water absorption pad pasted on one end of the chromatography membrane, and a gold label pad and a sample pad pasted on the other end in sequence.

[0010] The chromatography membrane is provided with a quality control line and a detection line, the detection line is arranged near the sample pad side, and the quality control line is arranged near the water absorption pad side;

[0011] The detection line is formed by spraying BLV p24 recombinant antigen protein, and the quality control line is formed by spraying IgY antibody.

[0012] The amino acid sequence of the BLV p24 recombinant antigen protein is shown as SEQ ID NO. 2.

[0013] The colloidal gold test strip, preferably, the substrate of the bottom plate is a PVC bottom plate, the substrate of the chromatography membrane is a nitrocellulose membrane, the substrate of the water absorption pad is a water absorption filter paper, the substrates of the gold label pad and the sample pad are glass cellulose membranes, and the IgY antibody is chicken IgY antibody or sheep anti-chicken IgY antibody.

[0014] In a third aspect, the present application provides a preparation method of the colloidal gold test strip, comprising the following steps:

[0015] (1) respectively label colloidal gold with BLV p24 recombinant antigen protein and IgY antibody to prepare colloidal gold labeled antigen protein and colloidal gold labeled antibody;

[0016] (2) mix the colloidal gold labeled antigen protein and the colloidal gold labeled antibody, spray them on the substrate of the gold label pad, and dry to obtain the gold label pad;

[0017] (3) paste the substrate of the chromatography membrane on the bottom plate, spray the BLV p24 recombinant antigen protein and the IgY antibody on the substrate of the chromatography membrane to form a detection line and a quality control line respectively, and dry to obtain the chromatography membrane;

[0018] (4) soak the substrate of the sample pad in a blocking solution, and dry to obtain the sample pad;

[0019] (5) sequentially assemble the bottom plate, the chromatography membrane, the water absorption pad, the gold label pad and the sample pad to obtain the colloidal gold test strip.

[0020] The preparation method of the colloidal gold test strip, preferably, in step (1), the preparation method of the colloidal gold labeled antigen or the colloidal gold labeled antibody comprises the following steps in sequence: adding K2CO3 solution in colloidal gold solution and mixing; adding BLV p24 recombinant antigen protein or IgY antibody, stirring uniformly, then adding PEG20000 solution to carry out blocking at room temperature; removing unbound colloidal gold particles and supernatant by centrifugation to obtain colloidal gold labeled precipitate, and thus the colloidal gold labeled antigen protein or the colloidal gold labeled antibody is obtained.

[0021] More preferably, the concentration of the K2CO3 solution is 0.1 mol / L, the adding amount is 5-80 μL / mL; the BLV p24 recombinant antigen protein or IgY antibody is diluted with PBS; the labeling amount of the BLV p24 recombinant antigen protein is 6.25-100 μg / mL, and the labeling amount of the IgY antibody is 5-50 μg / mL; the reaction time of the blocking is 10-30 min; and the parameter conditions for removing the supernatant by centrifugation are 4 ℃, 12000 rpm centrifugation for 10-60 min.

[0022] Preferably, the specific operation of step (2) is as follows: the colloidal gold labeled antigen protein, colloidal gold labeled antibody and binding pad treatment solution are mixed to prepare a gold spraying solution, the gold spraying solution is sprayed on the substrate of the gold labeled pad at a spraying amount of 1-10 μL / cm, and the gold labeled pad is obtained after drying; the binding pad treatment solution is: 10 mM PB, 1% BSA, 0.3% PVP-K30, 0.5% Tween-20 and 2.5% sucrose.

[0023] Preferably, in step (3), the coating amount of the BLV p24 recombinant antigen protein detection line and the IgY antibody quality control line on the nitrocellulose membrane is 1-10 μg / cm.

[0024] In a fourth aspect, the application provides a use of the above-mentioned BLV p24 recombinant antigen protein or the above-mentioned colloidal gold test strip in the preparation of a bovine leukemia antibody detection product.

[0025] Preferably, the bovine leukemia antibody detection product is a product for detecting bovine leukemia antibody by a double-antigen sandwich method.

[0026] The colloidal gold test strip of the application uses the BLV p24 recombinant antigen protein as a solid-phase coated antigen, and uses the colloidal gold labeled African swine fever recombinant antigen protein (i.e. the BLV p24 recombinant antigen protein) and IgY antibody. The colloidal gold test strip and the product thereof of the application can be used for detecting bovine leukemia antibody by a double-antigen sandwich method. The immunodetection method includes but is not limited to: lateral flow immunochromatography, enzyme-linked immunosorbent assay and chemiluminescence method, etc.

[0027] Compared with the prior art, the application has the following advantages:

[0028] 1. The baculovirus expression system is used to prepare the eukaryotic recombinant p24 protein in the application, the recombinant p24 protein expressed by the system can be correctly folded and post-translationally modified, better simulates the natural conformation, has high sensitivity, and the His tag on the recombinant protein is beneficial to one-step purification to achieve high purity, and better activity can be exhibited without enzyme cutting.

[0029] 2. The colloidal gold test strip of the present application adopts colloidal gold immunochromatography technology, and the same BLV p24 recombinant antigen protein is coated on the gold label pad and detection line containing colloidal gold, so that the double-antigen sandwich method can be used for rapid detection of bovine leukemia antibody; in view of the defects of the current immunocolloidal chromatography technology applied to bovine leukemia detection, the accuracy, sensitivity and detection are improved.

[0030] 3. The colloidal gold test strip of the present application adopts the double-antigen sandwich method to detect the antibody, so that the whole process monitoring of the antibody produced in the early, middle and late stages of BLV infection in pigs can be realized, and the current problem of early detection difficulty is solved; the colloidal gold test strip is suitable for bovine leukemia antibody detection by grass-roots livestock breeders, is convenient to carry, does not need to be prepared by professional detection personnel, and is easy to operate; and has important significance for promoting animal quarantine work, standardizing bovine leukemia prevention and control in breeding and slaughter fields, and maintaining the quality and safety of raw beef products.

[0031] 4. The colloidal gold test strip of the present application has a detection limit of 1:128 for BLV positive serum sensitivity test; the ELISA comparison test is performed on 138 clinical test samples, the negative coincidence rate is 100%, and the positive coincidence rate can reach 93.1%. The accuracy and sensitivity can achieve good clinical detection effect. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments, and it should be understood that the following drawings only show some embodiments of the present application, and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0033] Figure 1 It is a structural schematic diagram of the immunochromatography test strip for detecting bovine leukemia antibody of the present application.

[0034] Figure 2 It is the construction of pFastBacDual-2p24 plasmid in the embodiment 1 of the present application, wherein Figure 2 (a) is the amplification of gene P10-p24 and Ph-p24, Figure 2 (b) is the double enzyme digestion identification of recombinant baculovirus expression vector, Figure 2 (c) is the plasmid map.

[0035] Figure 3 It is the construction of rB-2p24 bacmid in the embodiment 2 of the present application, wherein Figure 3 (a) is the blue-white spot screening result, Figure 3 (b) is the double enzyme digestion identification of recombinant shuttle bacmid.

[0036] Figure 4 IFA identification of BLV p24 eukaryotic expression in Example 3 of the present application, Figure 4 (a) for Sf9 cells infected with rBv-p24, Figure 4 (b) for blank Sf9 cells.

[0037] Figure 5 Identification of BLV p24 expression purification in Example 3 of the present application, wherein, Figure 5 (a) for eukaryotic expression, Figure 5 (b) for protein purification, Figure 5 (c) for WB identification.

[0038] Figure 6 is a schematic diagram of the principle of determining the negative and positive of a blood sample by a test strip.

[0039] Figure 7 is a specific test result diagram of the antibody test strip of the present application.

[0040] Figure 8 is a sensitivity test result diagram of the antibody test strip of the present application.

[0041] Figure 9 is a repeatability test result diagram of the antibody test strip of the present application. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not indicated in the embodiments, the conventional conditions or the conditions suggested by the manufacturer are used. If the reagents or instruments used are not indicated by the manufacturer, they are all conventional products that can be purchased in the market.

[0043] The features and performances of the present application are further described in detail below in combination with the embodiments.

[0044] Example 1

[0045] Construction of baculovirus expression vector pFastBacDual-2p24:

[0046] The published p24 gene sequence of NCBI (https: / / www.ncbi.nlm.nih.gov / ) was aligned, the base combination with the highest frequency of occurrence in the variable site was selected, the sequence was determined as p24 KF154971, a eukaryotic Kozak (GCCACC) was added at the 5' end, and 6 consecutive histidine tags (6× His) were added at the 3' end. The codon optimization was carried out in the insect cell expression system, the amplification primers were designed and inserted into the BamH I and Hind III enzyme cutting sites, the gene was synthesized by Shanghai Generay Biotech Co., Ltd., and was inserted into the pUC57 vector. The recombinant plasmid was pUC57-p24.

[0047] The specific sequence is:

[0048] ATGGGTTTGCCAATCATCTCAGAGGGTAACAGAAACCGTCACAGAGCTTGGGCTCTGCGTGAGTTGCAAGACATTAAGAAGGAAATCGAGAACAAGGCTCCTGGTTCTCAGGTGTGGATTCAGACTCTGAGACTGGCTATCTTGCAGGCTGACCCAACACCAGCTGACCTGGAACAATTGTGCCAGTACATTGCTTCTCCAGTGGACCAAACTGCTCACATGACTAGCTTGACAGCTGCTATCGCTGCTGCTGAAGCTGCTAACACTCTGCAAGGCTTCAATCCTCAGAACGGTACTCTGACCCAACAATCAGCTCAGCCCAACGCTGGTGACTTGAGATCTCAGTACCAAAATTTGTGGTTGCAGGCTTGGAAGAACTTGCCTACAAGACCTTCTGTCCAACCATGGTCTACAATCGTGCAGGGTCCTGCTGAATCATACGTGGAATTCGTGAACAGATTGCAAATCTCTCTGGCTGACAACTTACCTGACGGAGTGCCTAAGGAACCTATCATTGATTCTCTGAGTTACGCTAACGCTAACAAGGAGTGTCAACAGATCTTGCAAGGTAGAGGTCTGGTTGCTGCTCCTGTGGGTCAGAAGCTGCAGGCTTGCGCTCACTGGGCTCCTAAGATCAAGCAGCCTGCTATCCTGCACCATCACCACCACCACTAA

[0049] According to the pUC57-p24 gene sequence and pFastBac Dual vector design PCR primer (Table 1), the primer of Table 1 is used to amplify the target gene in the pUC57-p24 plasmid, and the obtained target fragment is named P10-p24 and Ph-p24. The amplification conditions are: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, cycle for 30 times; 72℃ extension for 10 min; 12℃ storage. After the reaction, agarose gel electrophoresis is carried out to verify the correct band, and the gel is cut and recovered, and the concentration is measured. The target fragment Ph-p24 and pFastBacDual vector are double digested by using BamH I and Hind III two endonucleases, and the program is 37℃ water bath reaction for 3 h. After enzyme digestion, the gel is run according to the above method to verify and recover the gel. The enzyme digestion product is connected with T4 ligase, and then the connected product is transferred to DH5α competent cells, and is coated on LB agar plate with ampicillin resistance. The next day, single colonies are picked and colony PCR is carried out with the universal primers pFastBacDual-F and pFastBacDual-R in Table 1 to screen out positive clones containing the target recombinant plasmid, and Shanghai Shengong Biotechnology Co., Ltd. is sent for sequencing. The bacterial liquid completely correct in sequencing is inoculated into 20 mL LB medium containing ampicillin for overnight culture, and the next day, the plasmid DNA is extracted and purified according to the operation steps of the plasmid DNA extraction kit, and the extracted plasmid is named pFastBacDual-p24, which is stored at -20℃.

[0050] The target fragment P10-p24 and pFastBacDual-p24 vector are double digested by using Xho I and Kpn I two endonucleases, and the plasmid is extracted according to the above method and named pFastBacDual-2p24. Figure 2 For the construction of pFastBacDual-2p24 plasmid in Example 1 of the application, wherein Figure 2 (a) is the amplification of genes P10-p24 and Ph-p24, Figure 2 (b) is the double enzyme digestion identification of the recombinant baculovirus expression vector, Figure 2 (c) is the plasmid map.

[0051] Table 1

[0052]

[0053] Example 2

[0054] Construction of recombinant shuttle baculovirus rB-2p24:

[0055] The pFastBacDual-2p24 plasmid is transformed into DH10Bac competent cells and uniformly coated on blue-white spot screening plates, and placed in a 37°C constant temperature incubator in the dark for 24-36 h. White single colonies are selected and shaken, and colony PCR is performed with the universal primers pUC / M13-F and pUC / M13-R of Bacmid in Table 2 to screen for positive clones containing the recombinant plasmid of interest. The PCR program is: 98°C pre-denaturation for 5 min; 98°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 3 min, 30 cycles; 72°C extension for 10 min; 12°C storage. The PCR product is subjected to agarose gel electrophoresis. If the foreign DNA fragment is successfully transposed to the specific site of the Bacmid plasmid, the electrophoresis result will present a 3642 bp (2280 bp+1362 bp) target gene band; if no transposition occurs, only a band of about 300 bp will be shown in the electrophoretogram. If both the expected size 3642 bp band (2280 bp+1362 bp) and the 300 bp band appear in the PCR product, it indicates that the corresponding white colony needs to be further cultured and purified. That is, repeat the above steps until the PCR result only shows the expected size 3942 bp band, confirming successful purification. After successful purification, the colony is cultured in large quantities, and the recombinant bacmid rB-2p24 is extracted using the Biyun Tian baculovirus shuttle vector bacmid small-scale extraction kit, and stored at -20°C after determining the concentration. Figure 3 For the construction of rB-2p24 bacmid in Example 2 of the present application, Figure 3 (a) is the blue-white spot screening result, Figure 3 (b) is the double enzyme digestion identification of the recombinant shuttle bacmid.

[0056] Table 2

[0057]

[0058] Example 3

[0059] Expression and purification of recombinant p24 protein:

[0060] 1. Rescue and identification of recombinant baculovirus

[0061] Take Sf9 cells in the logarithmic growth phase, centrifuge at 500 rpm for 3 minutes, and discard the supernatant. Resuspend the cells with SF-900™ medium without double antibody, and adjust the density to 1×10 6Add 500ul cell suspension to each well of 12-well plate, shake the plate in '8' shape to make the cells evenly distributed. You can put the plate in 27°C incubator for 15-30 minutes to make the cells more stable. Take two sterile 1.5 mL centrifuge tubes, label them as A and B. Tube A: add 40ul double-antibody-free medium and 3.2ul transfection reagent, mix gently. Tube B: add 40ul double-antibody-free medium and 6.4ug recombinant bacmid DNA, mix gently. Add all the DNA dilution in tube B to tube A, mix gently, and let it stand at room temperature for 20-30 minutes to form DNA-transfection reagent complex. Slowly and evenly add the mixture to the cell culture plate, and continue to culture in 27°C incubator for 4-6 hours. Then remove the transfection solution from the culture plate, add fresh SF-900™ medium containing double-antibody, and continue to culture the cells. After 96 hours, successfully transfected Sf9 cells will show larger, rounder, or cytopathic cells. Collect the cells and supernatant, centrifuge at 12000r / min for 10 minutes at 4°C, collect the supernatant, which is P1 generation virus rBv-2p24, add FBS to a final concentration of 2% under light protection and store at -80°C. Meanwhile, transfect rB-pFastBac-Dual as a negative control. Use P1 generation virus to infect Sf9 cells for 4 days, collect the cells and supernatant, and centrifuge as above to obtain P2 generation virus for later use.

[0062] 2Indirect immunofluorescence test

[0063] Sf9 cells were evenly plated in 24-well plates and infected with P2 generation recombinant baculovirus rBv-2p24 at a MOI of 1. The cells were cultured in a 27°C incubator for 72 hours, then the culture medium was discarded, the cells were fixed with 4% paraformaldehyde at room temperature for 45 minutes, and washed with PBS solution 3 times; then 5% BSA blocking solution was added to each well at 37°C for 30 minutes, and washed with PBS solution 3 times; then 200ul of mouse anti-His primary antibody (1:1000 dilution) was added to each well, incubated overnight for 12-16 hours, then washed with PBS solution 3 times; then 200ul of FITC-labeled goat anti-mouse secondary antibody (1:500 dilution) was added to each well, incubated in a 37°C oven for 60 minutes in the dark, washed with PBS solution 3 times in the dark, and then the fluorescence signal of the sample was observed using an inverted fluorescence microscope. The results are shown in Figure 4 Figure 4 (a) rBv-2p24 infected Sf9 cells have specific green fluorescence, Figure 4 (b) rBv-pFastBac-Dual infected Sf9 cells have no fluorescence.

[0064] ​3 SDS-PAGE electrophoresis and Coomassie brilliant blue staining and Western blot identification

[0065] The P2 generation virus of rBv-2 p24 was used to infect the cultured Sf9 cells, and the cell precipitate was harvested after 4 days of infection, and after crushing treatment, SDS-PAGE was performed. The harvested protein was about 25 kDa, as shown in Figure 5 To further prove the expression of the recombinant p24 protein, WB test was performed using anti-His-tag monoclonal antibody and goat anti-mouse IgG HRP secondary antibody, and the results are shown in Figure 5 As shown, specific bands can be detected at about 25 kDa in the supernatant of the lysate of Sf9 cells infected with rBv-p24, which is consistent with the expected size of the recombinant p24 protein; and no specific bands were detected in the supernatant of the lysate of blank Sf9 cells. The results show that the Sf9 cells infected with rBv-p24 can express the p24 protein and mainly express in the cells.

[0066] 4 Expression and purification of recombinant protein

[0067] First, the Sf9 cells were transferred to a T75 cell culture flask. When the cells were attached and reached 80% density, the Sf9 cells were inoculated with P2 generation recombinant baculovirus rBv-p24 at 1 MOI, and after 72 h of culture, the cells showed significant cytopathic characteristics. Then the cells were collected by centrifugation at 12000 r / min for 10 min at 4°C, and the cells were resuspended with 30-40 mL of PBS, and then the cells were ultrasonically lysed on ice. Subsequently, the protein was purified by affinity chromatography. First, the protein was combined with HisSep Ni-NTA Agarose filler in a 4°C environment overnight, and the next day, 20 mmol / L low-concentration imidazole solution was used to elute the impurities, and finally 150 mmol / L high-concentration imidazole solution was used to elute the target protein. Finally, SDS-PAGE electrophoresis was used to detect the protein samples in the flow-through liquid, washing liquid and eluate to test the protein purification effect. The results are shown in Figure 5 As shown, the purified recombinant p24 protein was obtained at about 25 kDa, and the protein was quantified by BSA method, and the concentration of the purified recombinant p24 protein was 1.43 mg / mL. The sequencing result shows that the amino acid sequence is SEQ ID No. 2, and the specific is:

[0068] ATMG LPII SEGNRNRHRAWALRELQDIKKEIENKAPGSQVWIQTLRLAILQADPTPADLEQLCQYIASPVDQTAHMTSLTAAIAAAEAANTLQGFNPQNGTLTQQSAQPNAGDLRSQYQNLWLQAWKNLPTRPSVQPWSTIVQGPAESYVEFVNRLQISLADNLPDGVPKEPIIDSLSYANANKECQQILQGRGLVAAPVGQKLQACAHWAPKIKQPAILHHHHHH

[0069] Example 4

[0070] Colloidal gold test strip for detecting bovine leukemia antibody and preparation method thereof (colloidal gold double antigen sandwich method)

[0071] A colloidal gold test strip for detecting bovine leukemia antibody of the present application, the method used by the test strip is a double antigen sandwich method, colloidal gold and the detection line are all coated with BLV p24 recombinant antigen protein (the coding gene sequence is shown as SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 2). The specific structure is shown in Figure 1 The test strip is pasted on a 60mm long PVC bottom plate; the test strip is assembled by pasting a 25mm long chromatography membrane (nitrocellulose membrane), an 18mm water absorption pad, a 10mm gold label pad, and an 8mm glass cellulose membrane sample pad in turn, and is cut into a 3mm wide strip by a high-speed strip cutting machine; the solid-phase nitrocellulose membrane (chromatography membrane) is provided with a quality control line and a detection line; the detection line is arranged close to the sample pad, and the detection line is formed by spraying BLV p24 recombinant antigen protein; the quality control line is arranged close to the water absorption pad, and the quality control line is formed by spraying goat anti-chicken IgY antibody. The gold label pad is sprayed with BLV p24 recombinant antigen protein combined with colloidal gold, and can be used for bovine leukemia antibody detection.

[0072] The above-mentioned reagent strip for rapidly detecting BLV antibody is prepared by the following steps:

[0073] 1. Preparation of colloidal gold labeled antigen

[0074] 1.5ml of 1% trisodium citrate solution is added to 250mL of boiling 0.5% chloroauric acid aqueous solution and heated for 15min to obtain a colloidal gold solution; the solution is red, the obtained colloidal gold particles have a diameter of about 15nm, and after stopping heating, it is left to room temperature, and is diluted to 250mL, which can be stored at 4°C for standby.

[0075] Take 25 1.5 mL centrifuge tubes, add 1 mL of the above prepared colloidal gold solution and 5 μL of 10% PEG20000, mix well, take 5 of each to add 5 μL, 10 μL, 20 μL, 40 μL and 60 μL of 0.1 mol / L K2CO3 solution, mix well, to obtain colloidal gold solutions of different pH. Under different pH conditions, add 50 μL of BLV p24 recombinant antigen protein diluted with PBS, the dilution concentration is 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL and 6.25 μg / mL, slowly stir for 30 min; add 10 μL of 10% PEG20000, mix well, and rotate on a mixer for 30 min for blocking. Centrifuge the centrifuge tubes at 12000 rpm at 4°C for 20 min, remove the supernatant, and obtain colloidal gold-labeled antigen precipitate; suspend with 100 μL of reconstitution solution, which is 20 mM Tris and 0.1% PEG20000.

[0076] After adding K2CO3 solution and BLV p24 recombinant antigen protein or chicken IgY, the color change of colloidal gold and the gold spraying test were observed to determine that the addition amount of 0.1 mol / L K2CO3 20 μL / mL was used to adjust the pH of colloidal gold, and the optimal labeling amount of colloidal gold-labeled antigen protein was 50 μg / mL, and the optimal labeling amount of colloidal gold-labeled chicken IgY antibody was 6.25 μg / mL.

[0077] 2. Preparation and assembly of test paper

[0078] Preparation of chromatography membrane: nitrocellulose membrane was pasted on PVC bottom plate, 0.5 mg / mL BLV p24 recombinant antigen protein was pumped into sample A pool of membrane marker, and 1 mg / mL goat anti-chicken IgG antibody was pumped into sample B pool; set the coating amount to 1 μL / cm, and spray BLV p24 recombinant antigen protein detection line (T line) and goat anti-chicken IgG antibody control line (C line) on the nitrocellulose membrane, the two lines were parallel and the interval was 10 mm; set the temperature of electric heating constant temperature drying oven to 37°C, and the membrane marker after marking was placed overnight; vacuum sealing, and stored at 4°C for standby;

[0079] Preparation of gold-labeled pad: the obtained colloidal gold-labeled p24 antigen protein (1.5 mg / mL), colloidal gold-labeled chicken IgY antibody (24.1 mg / mL) and binding pad treatment solution were mixed according to the ratio of 2:1:10 to prepare gold spraying solution, and the binding pad treatment solution was: 10 mM PB, 1% BSA, 0.3% PVP-K30, 0.5% Tween-20 and 2.5% sucrose. Set the parameters of gold spraying instrument, control the gold spraying solution to be sprayed on the glass cellulose membrane at a spraying amount of 1 μL / cm, and then immediately placed in a 37°C drying oven for 16-18 h to obtain the gold-labeled pad; vacuum sealing, and stored at 4°C for standby;

[0080] Preparation of sample pad: the sample pad was soaked in the blocking solution for 2 h, and then dried at 37℃ for 16-18 h, to obtain the sample pad; the sample pad was sealed after vacuumizing, and stored at 4℃ for standby; the blocking solution was 10 mM PB, 0.5% Tween-20, 0.5% BSA, 1% PEG20000 and 1% PVP-K40;

[0081] Assembly and cutting: the test strip was assembled as shown in Figure 1 , and the assembly sequence was PVC bottom plate, nitrocellulose membrane (chromatographic membrane), water absorption pad, gold label pad and sample pad. The specific operation was as follows: the chromatographic membrane was pasted on the bottom plate; the water absorption pad was pasted on the chromatographic membrane with an overlap of 2 mm at the upper end; then the gold label pad was pasted on the other end of the chromatographic membrane with an overlap of 2 mm; finally, the sample pad was pasted at a position 3 mm overlapped with the gold label pad; the assembled test strip was placed in an automatic cutting machine, and cut into test strips with a width of 3 mm.

[0082] Functional verification of the colloidal gold test strip for detecting bovine leukemia antibody:

[0083] The colloidal gold test strip for detecting bovine leukemia antibody prepared in the above examples was evaluated for effect, and the specific operation method of the test strip detection was as follows: 20 μL of sample to be detected (bovine serum) was added to the sample adding position, and 30 μL of sample diluent was added to the sample adding position, and the result was determined after standing at room temperature for 10 min, and the result determination standard was as follows (as shown in Figure 6 ).

[0084] (1) Positive result: two red reaction lines, i.e. one red reaction line appeared on the detection line (T) and the control line (C) respectively, indicating that the antibody concentration in the sample was equal to or higher than the detection limit;

[0085] (2) Negative result: only one red reaction line appeared on the control line (C), indicating that the antibody concentration in the sample was lower than the detection limit;

[0086] (3) Invalid result: no red reaction line appeared on the control line (C), and the detection was invalid. When the invalid result appeared, it was necessary to determine whether the test method was correct, and retest with a new test strip, and pay attention to whether the sample amount was sufficient.

[0087] Performance evaluation and clinical specimen verification of the bovine leukemia antibody test reagent strip

[0088] 1. Specific detection of the colloidal gold test strip for detecting bovine leukemia antibody

[0089] The prepared BLV antibody test strip is used for specific detection of the serum to be detected, and the serum samples are: bovine viral diarrhea virus (BVDV) positive serum, bovine rotavirus (BRV) positive serum, bovine coronavirus (BcoV) positive serum, bovine epidemic fever virus (BEFV) positive serum, and BLV negative and positive serum. The specific detection results are shown in Figure 7 As shown in the table, the BLV positive serum appears two dark reaction bands at the detection line (T line) and the control line (C line) positions at the same time, and the result is determined to be positive; the bovine rotavirus (BRV) positive serum, the bovine coronavirus (BcoV) positive serum, the bovine epidemic fever virus (BEFV) positive serum, and the BLV negative serum only develop color at the control line (C line) position, and are negative. The detection results show that the prepared test strip can accurately detect the BLV positive serum and is not interfered by other virus positive serum, has high specificity, and has no cross reaction.

[0090] 2. Sensitivity detection of the colloidal gold test strip for detecting bovine leukemia virus antibody

[0091] The standard BLV positive serum is diluted from 1:2 times to 1:2048 times with bovine negative serum as a control, the detection operation steps of the test strip are followed, the observation results are observed, and the sensitivity of the test strip is evaluated. The specific detection results are shown in Figure 8 As shown in the table, when the BLV positive serum is detected at different dilution times, the prepared test strip appears two reaction bands at the detection line and the control line positions at the same time, and the result is determined to be positive; when the sample diluted by 512 times is detected, only one reaction band appears at the control line position, and the result is determined to be negative; therefore, the detection limit of the present application can reach 1:128, and excellent sensitivity is shown.

[0092] 3. Reproducibility detection of the colloidal gold test strip for detecting bovine leukemia virus antibody

[0093] Three BLV positive sera and three BLV negative sera are added dropwise to three test strips of the same and different batches, and the reproducibility of the test strip is judged according to the color development results of the T line and the C line. The specific detection results are shown in Figure 9 As shown in the table, Figure 9 (a) is batch-to-batch reproducibility, + indicates BLV positive serum, and - indicates BLV negative serum, Figure 9 (b) is batch-to-batch reproducibility, and the batch-to-batch test strip results are consistent, and the reproducibility is good.

[0094] 4. Stability detection of the colloidal gold test strip for detecting bovine leukemia virus antibody

[0095] The same batch of BLV antibody prepared above was stored in dry closed environment at 4°C and 37°C respectively. On the 7th, 14th, 21st, 28th, 35th day, 3 portions of BLV positive samples and 3 portions of BLV negative samples were added dropwise respectively to detect the influence of temperature on T line and C line, so as to evaluate the stability and optimal shelf life of the test strip. The test strip was stored at 37°C for 5 weeks, and the determination results of BLV positive and negative serum were lighter than before after 3 weeks. It is shown that the test strip can be stored at 37°C for 3 weeks. The determination results of BLV positive and negative serum are good when the test strip is stored at 4°C for 5 weeks, which indicates that the shelf life of colloidal gold is at least one month, and the result proves that the stability of the test strip is good.

[0096] 5. Accuracy detection of colloidal gold test strip for detecting bovine leukemia antibody

[0097] The clinical serum samples determined as negative or positive by ELISA were detected by test strip method in the laboratory, and the detection results were compared. The coincidence rate of the two was compared to judge the accuracy of the test strip. The coincidence rate calculation method is shown in Table 3:

[0098] Table 3 Coincidence rate calculation method

[0099]

[0100] Positive coincidence rate calculation formula = 100% [a / (a+c)];

[0101] Negative coincidence rate calculation formula = 100% [d / (b+d)];

[0102] Total coincidence rate calculation formula = 100% [(a+d) / n].

[0103] The clinical serum samples determined as negative or positive by ID Screen bovine leukemia virus ELISA antibody detection kit were detected by test strip method in the laboratory, and the detection results were compared. The serum samples were 29 clinical positive samples and 109 clinical negative samples. The coincidence rate test results are shown in Table 4.

[0104] Table 4 Test strip comparison test results

[0105]

[0106] The positive coincidence rate is 93.1%, the negative coincidence rate is 100%, and the total coincidence rate is 98.55%.

[0107] The above merely describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements without departing from the principles of the present application, and these improvements should also be considered as the protection scope of the present application.

Claims

1. A BLV p24 recombinant antigen protein for detecting bovine leukemia antibodies, wherein the amino acid sequence of the BLV p24 recombinant antigen protein is shown in SEQ ID NO.

2.

2. A vector capable of expressing the BLV p24 recombinant antigen protein of claim 1, the vector having a coding gene for the BLV p24 recombinant antigen protein, the nucleotide sequence of the coding gene being shown in SEQ ID NO.

1.

3. A colloidal gold test strip for detecting bovine leukemia antibodies, comprising a chromatography membrane adhered to a PVC substrate, wherein an absorbent pad is adhered to one end of the chromatography membrane, and a gold label pad and a sample pad are sequentially adhered to the other end. The chromatography membrane is provided with a control line and a detection line. The detection line is located near the sample pad, and the control line is located near the absorbent pad. The detection line is formed by spraying BLV p24 recombinant antigen protein, and the control line is formed by spraying IgY antibody; the gold label pad is coated with BLV p24 recombinant antigen protein and IgY antibody bound to colloidal gold. The amino acid sequence of the BLV p24 recombinant antigen protein is shown in SEQ ID NO.

2.

4. The colloidal gold test strip according to claim 3, wherein the substrate of the base plate is a PVC base plate, the substrate of the chromatography membrane is a nitrocellulose membrane, the substrate of the absorbent pad is absorbent filter paper, and the substrates of the gold label pad and the sample pad are glass cellulose membranes.

5. The colloidal gold test strip according to claim 3, wherein the IgY antibody is chicken IgY antibody or goat anti-chicken IgY antibody.

6. A method for preparing a colloidal gold test strip according to any one of claims 3-5, comprising the following steps: (1) Colloidal gold labeled with BLV p24 recombinant antigen protein and IgY antibody were respectively used to label colloidal gold to prepare colloidal gold labeled antigen protein and colloidal gold labeled antibody; (2) Mix the colloidal gold-labeled antigen protein and colloidal gold-labeled antibody, spray them onto the substrate of the gold-labeled pad, and dry them to obtain the gold-labeled pad. (3) The substrate of the chromatography membrane is attached to the base plate, and the BLV p24 recombinant antigen protein and IgY antibody are sprayed onto the substrate of the chromatography membrane to form detection lines and control lines respectively. After drying, the chromatography membrane is obtained. (4) The substrate of the sample pad is fully immersed in the sealing liquid and dried to obtain the sample pad; (5) Assemble the base plate, chromatography membrane, absorbent pad, gold label pad and sample pad in sequence to obtain the colloidal gold test strip.

7. According to the preparation method of claim 6, in step (1), the preparation method of the colloidal gold-labeled antigen or colloidal gold-labeled antibody includes the following steps in sequence: adding K2CO3 solution to colloidal gold solution and mixing well; adding BLV p24 recombinant antigen protein or IgY antibody, stirring evenly, adding PEG 20000 solution and blocking at room temperature; removing unbound colloidal gold particles and supernatant by centrifugation to obtain colloidal gold-labeled precipitate, thereby obtaining the colloidal gold-labeled antigen protein or colloidal gold-labeled antibody.

8. According to the preparation method of claim 6, in step (3), when the BLV p24 recombinant antigen protein detection line and IgY antibody control line are sprayed onto the nitrocellulose membrane, the coating amount is set to 1-10 μg / cm.

9. The use of the BLVp24 recombinant antigen protein of claim 1 or the colloidal gold test strip of any one of claims 3-5 in the preparation of bovine leukemia antibody detection products.

10. The application according to claim 9, wherein the bovine leukemia antibody detection product is a product that detects bovine leukemia antibodies using a double-antigen sandwich method.

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