CN114316038B - Rapid detection test strip for porcine reproductive and respiratory syndrome virus, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a monoclonal antibody of porcine reproductive and respiratory syndrome virus, wherein the heavy chain variable region sequence of the monoclonal antibody 3 is shown as SEQ ID NO.2, and the light chain variable region sequence is shown as SEQ ID NO. 3; the heavy chain variable region sequence of the monoclonal antibody 8 is shown as SEQ ID NO.4, and the light chain variable region sequence is shown as SEQ ID NO. 5; the antigen epitope bound by the monoclonal antibody 3 is positioned at aa34-aa48 of the optimized PRRSV GP5 protein; the epitope of the monoclonal antibody 8 is positioned at aa96-aa109 of the optimized PRRSV GP5 protein. The invention also discloses a kit for detecting porcine reproductive and respiratory syndrome virus, which comprises an effective amount of monoclonal antibody 3, monoclonal antibody 8 and a matched detection reagent. The monoclonal antibody prepared by the invention has good specificity and sensibility, and is suitable for preparing different porcine reproductive and respiratory syndrome virus diagnostic reagents, such as colloidal gold, ELISA, chemiluminescence and other detection kits.

Description

Rapid detection test strip for porcine reproductive and respiratory syndrome virus, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of diagnosis of viral epidemic diseases, and particularly relates to a rapid detection test strip for porcine reproductive and respiratory syndrome viruses, a preparation method and application thereof.

Background

Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS for short), commonly known as Blue Ear Disease, is an infectious Disease that severely harms the pig industry caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). PRRS can cause reproductive dysfunction in sows and severe respiratory diseases in piglets, and is a swine infectious disease which seriously affects economic benefits.

PRRSV is a single-stranded positive-strand RNA (about 15kb in size) that is not segmented into viruses of the genus arteriviridae arterivirus. PRRSV is a enveloped virus that is spherical, 45-65 nm in diameter, and has fibers on the surface of the envelope that are relatively smooth. PRRSV can grow on porcine alveolar macrophages (PAM cells), and obvious Cytopathy (CPE) such as cell shrinkage, aggregation and disintegration can occur when cultured on MARC-145 cells. The RNA of PRRSV has a Cap structure (5 '-Cap) at the 5' end and a polyadenylation tail structure (3 '-polyA) at the 3' end, and contains 9 Open Reading Frames (ORFs) in total, 1a, 1b, 2a, 2b, 3, 4, 5, 6 and 7, respectively. There is an overlap region between adjacent ORFs. The major structural proteins of PRRSV include GP5 protein (envelope glycoprotein), M protein (matrix protein), N protein (nucleocapsid protein), and the minor structural proteins include GP2, GP3, GP4, etc.

The GP5 protein is encoded by ORF5, about 26-30 kD, a glycosylated protein, containing 4 glycosylation sites and 6 antigenic determinants. There is evidence that GP5 plays a key role in the antibody recognition process of PRRSV, is a major structural protein that induces the body to produce neutralizing antibodies, is a recognized major protective antigen of PRRSV, and is also one of the target antigens for PRRSV diagnosis.

Disclosure of Invention

In order to make up for the defects of the prior art, one of the purposes of the invention is to provide a monoclonal antibody of PRRSV GP5 protein and a preparation method thereof; the invention also provides a kit for detecting porcine reproductive and respiratory syndrome virus by using the GP5 protein and the monoclonal antibody prepared by the invention.

Therefore, in one aspect, the invention discloses a monoclonal antibody of porcine reproductive and respiratory syndrome virus, wherein the monoclonal antibodies can specifically bind PRRSV GP5 protein; the heavy chain variable region sequence of the monoclonal antibody 3 is shown as SEQ ID NO.2, and the light chain variable region sequence of the monoclonal antibody 3 is shown as SEQ ID NO. 3; the heavy chain variable region sequence of the monoclonal antibody 8 is shown as SEQ ID NO.4, and the light chain variable region sequence of the monoclonal antibody 8 is shown as SEQ ID NO. 5.

Preferably, the epitope bound by the monoclonal antibody 3 is positioned at aa34-aa48 of the optimized PRRSV GP5 protein, and the amino acid sequence of aa34-aa48 of the optimized PRRSV GP5 protein is ETFVIFPVLTHIVSY.

Preferably, the epitope of the monoclonal antibody 8 is positioned at aa96-aa109 of the optimized PRRSV GP5 protein, and the amino acid sequence of aa96-aa109 of the optimized PRRSV GP5 protein is LAKNCMSWRYSCTR.

In another aspect, the invention also discloses a method for preparing the monoclonal antibody, which comprises the following steps: 1) Preparing PRRSV GP5 protein; 2) Preparing hybridoma cells for specifically expressing the anti-PRRSV GP5 protein monoclonal antibody; 3) Preparing ascites fluid of the mouse by using the hybridoma prepared in the step 2), and purifying the anti-PRRSV GP5 protein monoclonal antibody from the ascites fluid.

Preferably, when PRRSV GP5 protein is prepared in step 1) of the invention, the nucleotide sequence of the expressed GP5 protein is shown in SEQ ID No. 1.

In yet another aspect, the invention also discloses a kit for detecting porcine reproductive and respiratory syndrome virus, the kit comprising an effective amount of monoclonal antibody 3 and monoclonal antibody 8 of claim 1; and a matched detection reagent.

Preferably, the kit is a colloidal gold detection test strip, and the colloidal gold detection test strip comprises a test strip containing monoclonal antibodies 3 and 8 and a sample diluent.

Preferably, the test strip in the kit comprises a sample pad, a latex microsphere pad, a detection line, a quality control line and an absorption pad.

Preferably, the emulsion microsphere pad in the test strip of the kit comprises a monoclonal antibody 3, and the detection line comprises a monoclonal antibody 8.

The PRRSV GP5 protein prepared by the invention is suitable for preparing different diagnostic reagents for porcine reproductive and respiratory syndrome virus, such as colloidal gold, chemiluminescence detection kit and the like. The monoclonal antibodies 3 and 8 of the PRRSV GP5 protein prepared by the invention have good specificity and sensibility, and are suitable for preparing different diagnostic reagents for porcine reproductive and respiratory syndrome virus, such as detection kits of colloidal gold, ELISA, chemiluminescence and the like.

The detection kit (colloidal gold detection test strip) for the porcine reproductive and respiratory syndrome virus provided by the invention is suitable for detecting the porcine reproductive and respiratory syndrome virus in porcine serum, tissues, whole blood and anticoagulation, has strong specificity, high sensitivity and good stability, is high in detection speed (completed within 10 min), can be used for early screening of the porcine reproductive and respiratory syndrome virus, and is particularly suitable for on-site porcine reproductive and respiratory syndrome infection diagnosis, epidemiological investigation and the like.

Drawings

FIG. 1 SDS-PAGE of PRRSV GP5 protein after purification, 1 is the PRRSV GP5 protein after purification.

FIG. 2 is an assembly schematic diagram of the test strip, wherein A: sample pad, B: latex microsphere mat, C: detection line, D: quality control line, E: an absorbent pad.

FIG. 3 is a diagram of a test strip detection result judging mode.

FIG. 4 shows the results of the specificity test, and 1-7 show the results of detecting the porcine foot-and-mouth disease type O virus antigen, the porcine pseudorabies virus, the swine fever virus, the porcine parvovirus, the porcine circovirus type 2, the porcine epidemic diarrhea virus and the porcine transmissible gastroenteritis virus by using 21101 batches of test strips.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

Example 1: preparation of porcine reproductive and respiratory syndrome virus GP5 protein

The nucleotide sequence of the PRRSV GP5 protein of GenBank MH579772.1, from which the N-terminal 31 amino acids (signal peptide sequence) are removed, is subjected to codon optimization, and the nucleotide sequence of the PRRSV GP5 protein (shown as SEQ ID NO. 1) after the codon optimization is cloned into a prokaryotic expression vector, and the protein is expressed and purified. The specific method is briefly described as follows: the nucleotide sequence of the codon optimized PRRSV GP5 protein is used as a template, 6 His tags are added at the C end of the template by EcoRI and BamHI respectively, and the template is cloned into a vector pET-30 a. E.coli BL21 (DE 3) was transformed with the recombinant plasmid, single colonies were picked up in LB medium containing kanamycin (final concentration 50. Mu.g/ml), and when shaking culture was performed at 37℃and 225r/min to logarithmic growth phase (OD 600 was about 0.6), IPTG was added to transfer to 16℃overnight to induce expression of the target protein. The expressed recombinant protein is purified by a conventional nickel column purification method, finally, the target protein is eluted by 300mM imidazole, the eluent is concentrated by a10 kD ultrafiltration tube, the protein is dialyzed and changed into PBS, 20 μl of GP5 protein is taken and subjected to electrophoresis inspection by using SDS-PAGE protein gel, and a clear band is arranged at about 19.8kDa, and the purity is more than or equal to 95% (figure 1). Protein content was measured using BCA kit at a protein concentration of 1.3mg/ml.

Example 2: preparation and purification of PRRSV GP5 protein monoclonal antibody

Vaccine preparation and animal immunization: after the prepared PRRSV GP5 protein was emulsified with an equal mass of Freund's complete adjuvant, BALB/c mice (100. Mu.g/mouse) of 6-8 weeks of age were subcutaneously injected at multiple points. After two weeks, the mixture was emulsified with incomplete Freund's adjuvant, subjected to subcutaneous multipoint immunization (100. Mu.g/dose), repeated once at two-week intervals, and after the third immunization, the antibody titer was determined. The boost was given once (100 μg/dose) 3 days prior to cell fusion by intraperitoneal injection.

Determination of mouse serum ELISA titers at 1:10 ⁵ Spleen cells of the mice were collected. The method for collecting spleen cells is as follows: BALB/c mice were boosted for 3 days, neck removed and killed, after 5min of cadaver was soaked in 75% alcohol, BALB/c mice spleens were aseptically collected, placed in sterile dishes containing RPMI1640 (containing three antibodies 100U), washed twice with RPMI1640, transferred spleens onto 200 mesh copper wire, and spleens were rolled with a sterile pestle, gradually squeezing the splenocytes into the liquid phase. Repeatedly blowing and beating dispersed cells, filtering with 200 mesh copper mesh, transferring into 50ml sterile centrifuge tube, centrifuging at 1000rpm at room temperature for 10min, discarding supernatant, re-suspending with RPMI1640, and cleaning once with trypan blue staining count (number of living cells)>90%) for standby.

The cell fusion method is as follows: the myeloma cells in the logarithmic growth phase were placed in a 50ml centrifuge tube, centrifuged at 1000rpm for 10min at room temperature, and the supernatant was discarded, and the cells were washed once with RPMI1640 for counting. Myeloma cells and spleen cells were mixed at a ratio of 1:10, centrifuged at 1000rpm for 10min at room temperature, the supernatant was aspirated, RPMI1640 was washed once, and a 50ml centrifuge tube was placed in a 37℃water bath. 1ml of 50% PEG 1450 solution preheated at 37℃was added for 1min to perform fusion, RPMI1640 was added dropwise after standing for 1min to terminate fusion, centrifugation was performed at 1000rpm for 10min at room temperature, the supernatant was discarded, 50ml of complete RPMI1640 containing 20% HAT neonatal bovine serum was added, and 100. Mu.l of fused cells were added to a 96-well plate per well. Placing the culture plate at 37deg.C and 5% CO ₂ Culturing in incubator for 15 days, and changing to complete RPMI1640 containing HT 20% calf serum.

Monoclonal screening: and (3) cloning and screening the hybridoma cells detected to be positive by ELISA (enzyme-linked immunosorbent assay) by a limiting dilution method, and continuously cloning the screened cell lines with high antibody titer and good morphology by the limiting dilution method until monoclonal is obtained, and performing amplification culture and preservation. Finally, 4 positive hybridoma cells were obtained, which were numbered 3, 8, 11, and 18.

Preparing ascites: taking 6-8 week old BALB/c mice, injecting 1ml sterilized liquid paraffin into the abdominal cavity, and injecting hybridoma cells (3, 8, 11, 18) 1×10 into each mice at 7 days later ⁶ And after 7 days, extracting the ascites of the mice, centrifuging at 1000rpm for 10 minutes at a temperature of 2-8 ℃, collecting supernatant, sub-packaging the ascites into 5 ml/tube, and preserving at-20 ℃ for later use.

Antibody purification, octanoic acid-ammonium sulfate precipitation, was briefly described as follows: taking 10ml of ascites, centrifuging at 1000rpm at 4 ℃ for 10 minutes after melting, collecting supernatant, adding 40ml of acetate buffer (0.06 mol/L, pH value is 4.5), adding 330 mu L of octanoic acid at room temperature after uniformly mixing by magnetic stirring, dripping and stirring at the same time, mixing at room temperature for 30 minutes, standing at 2-8 ℃ for 2 hours, centrifuging at 10000rpm for 30 minutes at 2-8 ℃, collecting supernatant, recording the volume of the supernatant, slowly adding the same volume of saturated ammonium sulfate solution stored at 2-8 ℃ for 30 minutes under ice bath, standing at 2-8 ℃ for 16 hours, centrifuging at 5000rpm for 30 minutes at 2-8 ℃, and collecting precipitate. After dissolving the pellet with 5ml of PBS, it was dialyzed against PBS 1L for 16 hours, during which the dialysate was changed at least 3 times. Filtering and sterilizing the dialyzed monoclonal antibody by a microporous membrane with the size of 0.22 mu m, and subpackaging the monoclonal antibody into centrifuge tubes with the size of 0.1 ml/tube. The concentration of the 4 monoclonal antibodies was detected by using BCA kit and the detection results were 1.82mg/ml, 1.76mg/ml, 0.98mg/ml and 1.55mg/ml, respectively.

EXAMPLE 3 systematic identification of monoclonal antibodies

Antibody specific detection: monoclonal antibodies are respectively taken for detecting CSFV, PRV, PCV2, JEV and PPV to judge the specificity, and the result shows that the detection of 4 monoclonal antibodies is negative, which indicates that the specificity of the monoclonal antibodies is good

Class and subclass determination: monoclonal antibodies were taken and tested according to the instructions using ELISA kit for mouse monoclonal Ig class/subclass identification (available from Beijing Boolone immunotechnology Co., ltd.). Through detection, the subclasses of the 4 monoclonal antibodies are IgG ₁ 。

Reactivity determination: ELISA titers (gradient dilution of monoclonal antibodies) of 4 monoclonal antibodies (3, 8, 11, 18) were determined by indirect ELISA (coating amount of coating original PRRSV GP5 protein is 1. Mu.g/ml, 100. Mu.l/well), and the results were 1:10 in order ⁶ 、1:10 ⁶ 、1:10 ⁷ 、1:10 ⁵ (OD 450nm value greater than 0.1 positive). The titers of the 4 monoclonal antibodies are good, and the monoclonal antibodies can be used for subsequent reagent development. The specific results are shown in the following table.

HRP-labeled titer determination: the 4 monoclonal antibodies were labeled using an HRP coupling kit (ab 102890), and the labeling titers of the 4 monoclonal antibodies (positive when OD value is not less than 1.0) were determined by a direct ELISA method (coating amount of the original PRRSV GP5 protein is 1. Mu.g/ml, 100. Mu.l/well), and the results were 1:10000, 1:5000, 1:20000 in order. The labeling titers of the 4 monoclonal antibodies are good, and the 4 monoclonal antibodies can be used for subsequent reagent development.

Monoclonal antibody pairing validation: the 4 monoclonal antibodies were screened and coated on ELISA plates at 1. Mu.g/ml and 100. Mu.l/well, respectively, and the prepared PRRSV GP5 protein was used as a sample (diluted to 50ng/ml and 100. Mu.l per well) and incubated at 37℃for 30min; after washing, the matched HRP-labeled monoclonal antibody (1:10000 times dilution) was added, incubated at 37℃for 30min, after washing TMB developer (10 min incubation at 37 ℃) and stop solution were added, and then OD450 values were detected. The results showed that the OD450 value was highest when antibody No.3 was coated and antibody No. 8 was detected, indicating that the reaction was best at this time, so that antibody No.3 coated and detection No. 8 were selected as the paired antibodies for subsequent PRRSV detection. The specific results are shown in the following table:

determination of the sequence of the variable region of the monoclonal antibody: the method of Chinese patent invention (CN 111393525B, CN 113354734A) is used for determining the heavy chain variable region and the light chain variable region of the monoclonal antibodies (No. 3 and No. 8), and the sequences of the heavy chain variable region and the light chain variable region of the No.3 antibody are shown as SEQ ID NO.2 and SEQ ID NO.3 after sequencing; the sequences of the heavy chain variable region and the light chain variable region of the antibody No. 8 are shown as SEQ ID NO.4 and SEQ ID NO. 5. The amino acid sequence information for antibodies No.3 and No. 8 are shown in the following table:

determination of antigen recognition site: the monoclonal antibodies (No. 3 and No. 8) are detected by the Nanjing Jinsri biotechnology Co., ltd. And the result shows that the antigen epitope combined by the No.3 antibody is positioned at aa34-aa48 (amino acid sequence concrete ETFVIFPVLTHIVSY) of the optimized PRRSV GP5 protein, and the antigen epitope of the No. 8 antibody is positioned at aa96-aa109 (amino acid sequence concrete LAKNCMSWRYSCTR) of the optimized PRRSV GP5 protein. Wherein the amino acid sequence of the optimized PRRSV GP5 protein is shown below (wherein the antigen recognition site is bolded and inclined):

example 4 detection of PRRSV (latex method)

1 preparation of test paper strip

Preparation of nitrocellulose membrane: the nitrocellulose membrane (purchased from Saidolite) is stuck on the corresponding position of a PVC base plate (Hangzhou Ruijin), monoclonal antibody 8 is diluted to 1mg/ml by coating buffer solution (PBS buffer solution), the scribing position and the height of a membrane scribing machine are adjusted, the scribing is a T line which is a detection line, the T line is close to the end of a latex microsphere mat, sheep anti-mouse IgG antibody (purchased from Soy treasure company) is diluted to 1mg/ml by coating buffer solution, the scribing position and the height of the membrane scribing machine are adjusted, the scribing is a C line which is a quality control line, the C line is close to an absorption mat, and the distance between two lines is 5-8 mm. Drying in a 37 deg.C oven for 20 hr, sealing in aluminum foil bag containing desiccant, and storing at normal temperature.

Preparation of a latex pad: monoclonal antibody 3 was added to the latex microspheres (purchased from Shanghai Hui) in an amount of 1mg/ml latex microspheres and labeled for 2 hours. BSA was added to a final concentration of 1% and blocked for 1 hour. Centrifugation at 12000rpm at 4℃for 30min, removal of supernatant, collection of pellet, resuspension of pellet with 1ml of stock (0.05M Tris buffer with 1% BSA, pH 8.0) for 1 min. Uniformly spreading the re-suspended latex microsphere marked antibody on the treated latex microsphere mat, drying in a baking oven at 37 ℃ for 20 hours, packaging in an aluminum foil bag, and preserving at room temperature for later use.

Sample pad treatment: sample pads (from general paper industry, 300 mm. Times.20 mm) were immersed in a blocking solution (10% BSA solution) for 30 minutes, then oven dried at 37℃for 20 hours, packaged in aluminum foil bags, and stored at room temperature for use.

And (3) assembling: as shown in fig. 2, the sample pad, the latex microsphere pad, and the absorbent pad were sequentially attached to the corresponding positions of the PVC base plate to which the nitrocellulose membrane had been attached, so that the latex microsphere pad and the absorbent pad were respectively brought into contact with the nitrocellulose membrane portion, and the sample pad was brought into contact with the latex microsphere pad portion, to thereby produce a large plate. .

And (3) packaging: cutting the large plate into test strips with the width of 3mm by using a slitter, mounting a shell, sealing and packaging by using an aluminum foil bag, wherein one test strip is contained, one desiccant is contained, and the test strips are stored at room temperature in a dark place for standby, and the storage period is 18 months.

Preparation of sample dilutions: 1 XPBS buffer solution is used for storage at normal temperature.

2 detection of test strips

Sample processing: grinding the tissue sample and PBS according to the proportion of 1:1, centrifuging, and taking the supernatant for direct detection; blood (whole blood, anticoagulated, serum and plasma containing) samples are used directly for testing.

The operation steps are as follows: a proper amount of sample (30-40 μl or 1 drop of dropper) is taken and added dropwise to the sample well, followed by adding dropwise a drop of sample diluent. And (5) after the sample is added, placing the test strip on a table top horizontally, and observing the result about 10 minutes.

Decision (as shown in fig. 3): two strips (T: detection line, C: quality control line) appear on the test strip, judge as positive; only one strip (C: quality control line) appears on the test strip, and the test strip is judged as negative; and (5) judging that no strip appears at the quality control line of the test strip to be invalid.

3 test strip performance verification

And (3) specificity test: 7 parts of pig-related specific samples and 10 parts of samples with PRRSV negative by PCR are detected by using the prepared 3 batches of test strips, and the results show that the detection results of the 3 batches of test strips are all negative. The specific results are shown in the following table and fig. 4 (partial results):

note that: "-" indicates that the test is negative.

Sensitivity test: the 3 test strips prepared are used for detecting PRRSV viruses with different titers, and the result shows that the minimum detection limit of the 3 test strips can reach 10 ² TCID ₅₀ /ml. The 3 test strips prepared are used for detecting PRRSV GP5 proteins with different concentrations, and the result shows that the minimum detection limit of the 3 test strips can reach 2.5ng/ml. The prepared test strip has good sensibility. The results are shown in the following table:

note that: "-" indicates that the test is negative, and "+" indicates that the test is positive.

Repeatability test: the prepared 3 batches of test strips are used for detecting 7 specific samples and 5 positive samples, and have good repeatability in both batch and batch. The specific results are shown in the following table:

the above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Sequence list

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Claims (7)

1. The monoclonal antibodies of the porcine reproductive and respiratory syndrome virus are a monoclonal antibody 3 and a monoclonal antibody 8, and both the monoclonal antibodies can specifically bind PRRSVGP5 protein; the sequence of the heavy chain variable region of the coded monoclonal antibody 3 is shown as SEQ ID NO.2, the sequence of the light chain variable region of the coded monoclonal antibody 3 is shown as SEQ ID NO.3, the sequence of the heavy chain variable region of the coded monoclonal antibody 8 is shown as SEQ ID NO.4, and the sequence of the light chain variable region of the coded monoclonal antibody 8 is shown as SEQ ID NO. 5.

2. The monoclonal antibody of claim 1, wherein the epitope bound by monoclonal antibody 3 is located at aa34-aa48 of the optimized prrsv gp5 protein, and the amino acid sequence at aa34-aa48 of the optimized prrsv gp5 protein is ETFVIFPVLTHIVSY.

3. The monoclonal antibody of claim 1, wherein the epitope of monoclonal antibody 8 is located at aa96-aa109 of the optimized prrsv gp5 protein, and the amino acid sequence at aa96-aa109 of the optimized prrsv gp5 protein is LAKNCMSWRYSCTR.

4. A kit for detecting porcine reproductive and respiratory syndrome virus, comprising an effective amount of monoclonal antibody 3 and monoclonal antibody 8 of claim 1; and a matched detection reagent.

5. The kit according to claim 4, wherein the kit is a colloidal gold test strip comprising a test strip containing monoclonal antibodies 3 and 8 and a sample diluent.

6. The kit of claim 5, wherein the test strip comprises a sample pad, a latex microsphere pad, a detection line, a quality control line, and an absorbent pad.

7. The kit according to claim 6, wherein the latex microsphere pad in the test strip of the kit comprises monoclonal antibody 3, and the detection line comprises monoclonal antibody 8.

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