CN110540578A - Preparation of main antigen epitope region recombinant protein of pseudorabies virus GE gene and colloidal gold immunochromatographic test strip - Google Patents

Abstract

the invention provides a pseudorabies virus GE protein and preparation of a colloidal gold immunochromatographic test strip thereof. The expression recombinant protein of the invention exists in the application, is convenient to purify, and the detection method established by using the recombinant protein purified by affinity chromatography as a diagnostic antigen has lower cost and simple and convenient detection method. Particularly, the main epitope of the gE antigen is concentrated as a diagnostic antigen, and the detection sensitivity and specificity are better than those of the complete gE protein.

Description

preparation of main antigen epitope region recombinant protein of pseudorabies virus GE gene and colloidal gold immunochromatographic test strip

Technical Field

The invention relates to the technical field of animal virology and immunology detection, in particular to construction of virus gE protein for detecting a pseudorabies antibody in pig serum, a method for preparing immune colloidal gold test paper by using the protein, and application of the immune colloidal gold test paper of the virus gE protein for detecting the pseudorabies in the pig serum.

background

Pseudorabies Virus (PRV) is a herpes Virus that causes fever, itching (except in pigs) and acute encephalomyelitis of various domestic animals and wild animals such as cattle, sheep, pigs, dogs and cats as main symptoms, and the disease symptoms caused by the Virus are similar to rabies, so it is called Pseudorabies. Pigs are the reservoir and source of infection for PRV, and healthy pigs can be infected by direct contact with sick and virulent pigs. Mainly causes sow breeding disorder, abortion, mummy miscarriage, stillbirth and weak farrowing, and newborn piglets show diarrhea and nervous symptoms, the infection rate and the death rate are as high as 100 percent, and serious economic loss is caused to the pig industry. Viruses can be transmitted by bodily fluids, semen, milk, feces, and the like, as well as by air, and can often cause seasonal outbreaks in swine farms (Lisa e. Pomeranz et al, 2005).

currently, the serology commonly used for diagnosing PRV comprises Latex Agglutination Test (LAT), agar immunodiffusion test (AGID), hemagglutination test (HA), colloidal gold Immunochromatography (ICG) and the like, wherein the colloidal gold immunochromatography is convenient and rapid to use, convenient for basic layer use and field use, and all reactions can be completed within 15 minutes; the cost is low, and special instruments and equipment are not needed; the application range is wide, and the method can adapt to various detection conditions; multiple detections can be carried out, if positive samples are difficult to obtain, multiple detection markers are stable, and the marked samples are stored at 4 ℃ for more than two years without signal attenuation; the colloidal gold is red, and a color development reagent is not required to be added, so that the steps of an enzyme-labeled carcinogenic substrate and a stopping solution are omitted, and the colloidal gold is harmless to a human body.

The neutralization test is the most standard pseudorabies detection method generally considered by the academia at present, but in the actual detection work, the neutralization test can generate a positive reaction on nonspecific viruses, the false positive rate is increased for the detection result, and the neutralization test needs 24h for incubation. There is an urgent need for simple and rapid detection of porcine pseudorabies virus in clinic.

Disclosure of Invention

The invention mainly solves the technical problem of providing a quick and high-sensitivity immunoassay product and method, and realizing efficient and high-density immunoassay.

The invention aims to provide a pseudorabies virus GE gene with antigenicity, a continuous epitope at the N end of a gE protein, namely an amino acid sequence from 52 to 238 sites, is selected, a gene sequence of the gE protein is reconstructed according to the bias of a bacterial codon of a prokaryotic expression system, a novel base sequence is constructed, and the N-end epitope of the gE protein with the same amino acid sequence is expressed by prokaryotic bacteria.

Furthermore, the invention provides application of the GE protein epitope in preparation of diagnostic reagents or medicines for diagnosing, preventing or treating pseudorabies virus.

Furthermore, the invention provides a pseudorabies virus colloidal gold immunochromatographic test strip prepared by utilizing the GE recombinant protein

When the product exists in a test strip form, the GE recombinant protein can be placed in a detection line for capturing target molecules, and gold-labeled nanoparticles and the like are adopted for detection; furthermore, when the complex is fused with different functional ligands, the simultaneous detection of multiple target molecules can be realized.

The pseudorabies virus colloidal gold immunochromatographic test strip is provided with a carrier plate, a sample pad, a colloidal gold binding pad, a laminated plate film, a detection line, a quality control line and an absorption pad; the sample pad, the colloidal gold combined pad, the laminated film and the absorption pad are sequentially adhered to the upper surface of the carrier plate, one end of the sample pad is arranged at one end of the colloidal gold combined pad, the other end of the colloidal gold combined pad is arranged at one end of the laminated film, one end of the absorption pad is arranged at the other end of the laminated film, and the detection line and the quality control line are sequentially arranged on the laminated film; the detection line is coated with GE recombinant protein, and the quality control line is coated with selected streptococcus recombinant protein (rSPG).

the carrier plate may be a PVC plate.

The invention provides a rapid detection reagent for bow-shaped by adopting a colloidal gold immunochromatography technology, which can be used for detecting pseudorabies viruses in specimens such as whole blood, serum, plasma, cerebrospinal fluid and the like. The amount of the sample required in the detection is very small, no special instrument is needed, the result is directly interpreted by naked eyes, and the detection is simple, convenient and quick, the specificity is strong, the sensitivity is high, the accuracy and the reliability are high, the cost is low, and the application is wide.

Advantageous effects

The pseudorabies virus structural protein GE epitope is the core epitope of GE protein. The purity of the protein obtained by affinity chromatography can reach 85%. Western-blot results prove that the protein has certain immunogenicity and can be used as a diagnostic antigen.

the recombinant GE protein is used as a detection antigen, colloidal gold particles are prepared by using the most classical trisodium citrate reduction method, the research and the development of a pseudorabies virus immunochromatography test strip are completed, the pseudorabies virus positive serum can be specifically combined, and a rapid diagnosis method for diagnosing the pseudorabies virus is initially established.

Drawings

FIG. 1 shows the PCR identification electrophoresis of recombinant plasmid 1M DNA molecular mass standard (DL 2000) and A recombinant pET-32a (+) -rGE gene PCR product

FIG. 2 shows a double restriction enzyme analysis of recombinant plasmid, M is DNA molecular mass standard (DL 5000); a, double enzyme digestion products;

FIG. 3 shows the induced expression phase of GE protein, M is the molecular mass standard of the protein; 1: no induction of bacterial lysate; 2-7 inducing the bacterial lysate for 1h,2h,3h,4h,6h and 8 h;

FIG. 4 GE protein solubility identification, M protein molecular mass standard; a is the sediment of 8M urea which is cracked after the recombinant pET-28a (+) -rGE/BL21 (the supernatant B after the DE3 transfectant bacteria: the recombinant pET-28a (+) -rGE/BL21(DE3) transfectant bacteria;

FIG. 5 Western Blotting result of GE protein immunogenicity, wherein M is protein molecular mass standard; a is recombinant protein.

fig. 6 is a schematic structural diagram of a colloidal gold labeled test strip, wherein 1 is a sample to be tested, 2 is a sample absorption pad, 3 is a gold labeled pad, 4 is an NC film, 5 is an absorption pad, 6 is a PVC base plate, 7 is a detection line (T line), and 8 is a control line (C line).

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1 construction expression and Activity identification of Pseudorabies Virus GE recombinant Gene

According to the published gene sequence of the main antigen domain of the GE glycoprotein of the classical swine fever virus given in GeneBank, a region A, a region B, a region C and a region D of the main antigen domain are found out to obtain the gene sequence of the GE, and finally, a termination sequence of TAA is added at the 3' end. The gene sequence is optimized and synthesized by Suzhou Jinzhi Biotechnology limited, 5 'BamH I and 3' Hind III are added, then the optimized gene is cloned to expression vector pET-32a (+), so as to obtain recombinant plasmid, named pET-32a (+) -rGE, the gene sequence of GE is shown in SEQ ID NO. 1.

adding 200ul of sterile water into 4ug of the recombinant plasmid dry powder, transferring the recombinant plasmid dry powder into BL21(DE3), converting the recombinant plasmid dry powder in a water bath at 42 ℃ for 45s, adding the recombinant plasmid dry powder into an LB culture medium without antibiotics, incubating the recombinant plasmid dry powder at 37 ℃ and 250rmp for 45min, coating the recombinant plasmid dry powder on an Amp + resistant LB plate, culturing the recombinant plasmid dry powder for 12h at 37 ℃, picking a plurality of positive clone colonies into the Amp + resistant LB culture medium, and performing shake culture at 37 ℃ for 12 h.

the transformed recombinant plasmid was subjected to PCR reaction, identified by 1% agarose gel (FIG. 1), and sent for sequencing. And extracting recombinant plasmids from the correctly sequenced PCR bacterial liquid according to an Axygen plasmid miniprep kit, and performing double enzyme digestion identification (figure 2). The size of the PCR target fragment is about 1300bp (as shown in FIG. 1); the double restriction enzyme identification of BamHI and HindIII yielded 5900bp (vector fragment) and 576bp (target gene fragment) (see FIG. 2), consistent with expectations.

EXAMPLE 2 preparation and purification of proteins

expression of the recombinant plasmid

(1) The correctly sequenced host strain BL21(DE3) containing the recombinant plasmid pET-32a (+) -GE was inoculated into 250 mL LB liquid medium containing 50. mu.g/mL Amp +, and placed in an incubator at 37 ℃ and cultured with shaking at 250 r/min until the log phase of bacterial growth (OD 600 was about 0.6).

(2) adding IPTG solution with final concentration of 1 mmol/L, inducing for 8h, taking 1mL of bacterial liquid before induction and after 1h,2h,3h,4h,6h and 8h respectively, performing SDS-PAGE electrophoresis identification, and observing optimal induction expression time.

(3) After inducing for 4h, the bacterial liquid is centrifuged for 15min at 4 ℃ and 12000 r/min, and the bacterial precipitation is collected.

(4) The bacterial pellet was resuspended in 12 mL of 1 XPBS, and after repeated freezing and thawing for 3 times, it was sonicated in ice bath for 20min (sonication for 2 s, 9.9 s intervals).

(5) Centrifuging the bacteria solution after ultrasonic treatment for 15min at 4 ℃ and 12000 r/min, collecting supernatant and precipitate, dissolving the precipitate in 10mL of 8M urea, and centrifuging under the same conditions to collect supernatant.

(6) And respectively taking the supernatant after ultrasonic treatment and the precipitate dissolved supernatant, and identifying the expression form of the recombinant protein by SDS-PAGE. (FIG. 3).

(II) purification of recombinant proteins

Analyzing the expression form of the recombinant protein according to the SDS-PAGE identification result, and purifying the target protein by adopting a His resin Bind column chromatography method:

(1) Filling grease: taking an empty column, adding 3 mL of resin to perform pre-column loading, when the preservation solution is reduced to the surface of the resin, sequentially cleaning by using double distilled water with 3 times of resin volume, ionizing the resin by 1 × charge Buffer with 5 times of resin volume, and balancing a chromatographic column by 1 × binding Buffer with 3 times of resin volume.

(2) and (3) column chromatography purification:

adding a solution containing recombinant protein when the 1 binding buffer is reduced to the surface of the resin, and keeping the solution after passing through the column;

Adding 1 binding buffer of 10 times of resin volume, and reserving the solution after passing through the column;

③ adding 1 × washing buffer 6 times of the volume of the resin, eluting the foreign protein which is not combined on the resin, and reserving the eluent;

Adding 1 XElute buffer 6 times the volume of the resin, eluting the target protein bound on the resin, and sequentially retaining the eluates in an EP tube of 1.5 mL;

Adding 1 × Strip buffer of 6 times of resin volume, and washing the resin;

Sixthly, storing the resin in a proper amount of 20 percent ethanol;

(3) the effect of recombinant protein purification was identified by SDS-PAGE.

SDS-PAGE analysis shows that pET-32a (+) -rGE is induced by IPTG in Escherichia coli BL21(DE3) and is expressed in large quantity. There is a clear band of interest at about 37kDa, consistent with the expected molecular mass size of the recombinant protein (see FIG. 3). After IPTG induction for 1h,2h,3h,4h,6h and 8h, the expression level reaches the highest after 3h, but after 8h of expression, the expression level is reduced, and the target protein band does not appear in the non-induced bacterial liquid (0 h) (figure 3), so that the bacterial liquid for inducing expression for 3h is taken for carrying out soluble expression identification. SDS-PAGE analysis showed that the protein of interest was expressed as inclusion bodies and the inclusion bodies were lysed in 8M urea (FIG. 4).

(III) renaturation of recombinant proteins

The purified recombinant protein was renatured by dialysis with a gradient urea solution (PBS solution containing 5 mol/L, 4 mol/L, 3 mol/L, 2 mol/L, 1 mol/L urea and 1 XPBS in this order), and the solutions at each concentration were dialyzed at low temperature for 2 hours. The inclusion body of the recombinant protein rGE is purified to obtain a clear target band with the size of about 37kDa which is consistent with the size of the target protein, and the renaturation of the recombinant protein is completed by gradually dialyzing through urea gradient.

(IV) determination of protein concentration

And (3) determining the protein concentration of the renatured protein by using a TaKaRa BCA protein detection kit according to the operation instructions:

Preparing a working solution: mixing according to the proportion of BCA Reagent A to BCA Reagent B =100:1 to prepare working solution

BSA standard curve preparation: diluting BSA Standard solution, adding 2 mg/mL BSA Standard into pore plates according to the ratio of 24ul, 18ul, 12ul, 9ul, 6ul, 3ul, 2ul and 0ul, supplementing each pore plate with 1 × PBS until the final volume is 25ul, adding 200ul of working solution, and immediately mixing;

Determination of the detection protein: adding 25ul of dialyzed protein into a pore plate, adding 200ul of working solution, and immediately mixing uniformly;

After 30 minutes of reaction in a 37 ℃ water bath, it was cooled to room temperature. The absorbance at 562nm was measured using a microplate reader.

Drawing a standard curve and analyzing the concentration of the protein to be detected.

EXAMPLE 3 immunogenicity identification of recombinant proteins

The immunogenicity of the recombinant protein was tested as follows:

(1) Carrying out SDS-PAGE electrophoresis on the purified and dialyzed recombinant protein, transferring for 1h by using 260 mA current under the ice bath condition after the electrophoresis is finished, and electrically transferring the recombinant protein onto a nitrocellulose membrane;

(2) Sealing of the nitrocellulose membrane: weighing 5 g of skimmed milk powder, dissolving in 100mL of 1 xPBST, soaking the nitrocellulose membrane in the solution, and sealing at 37 ℃ for 2 h;

(3) Primary antibody incubation: after blocking, unbound skim milk powder was washed off with PBST (10 min each time, 4 times), and then incubated at 37 ℃ for 2h with PRV Ab (+) (1: 100 dilution) as primary antibody;

(4) And (3) secondary antibody incubation: washing away unbound primary antibody with PBST (10 min each time, 4 times), taking goat anti-pig IgG (diluted 1: 3000) as secondary antibody, and incubating at 37 deg.C for 1 h;

(5) color development: unbound secondary antibodies were washed away with PBST (10 min each, 4 times), and developed with a precipitation-type HRP-DAB color development kit, and when a clear protein band appeared, the development was stopped immediately with clear water, confirming that the obtained recombinant protein had better immunogenicity (fig. 5).

Example 4 preparation of colloidal gold immunochromatographic test strip

4.1 protein expression purified recombinant protein prepared as in example 2

4.2 spotting of nitrocellulose membranes: the recombinant GE protein was diluted to 0.5mg/ml for detection line (line T) streaking, and the mouse anti-His tag monoclonal antibody was diluted to 0.5mg/ml for quality control line (line C) streaking. Attaching an NC film to a bottom plate, then marking by using a colloidal gold sample application system, wherein the marking amount is 1 mu l/cm, placing the bottom plate in a 37 ℃ oven for drying for 2 hours after marking, sealing in a tin foil bag, and internally arranging a drying agent with the validity period of 15 months.

4.3 firing of colloidal gold

Sintering the colloidal gold according to a sodium citrate reduction method: and putting all glassware used for firing and storing the colloidal gold into an acid jar, soaking for 24 hours, taking out, washing, and drying for later use. 100mL of deionized water and 1mL of 1% chloroauric acid were heated to boiling. 1.5mL of 1% trisodium citrate was added dropwise until the color stabilized to red. Thereafter boiling was continued for 15min and cooling to room temperature.

4.4 preparation of colloidal gold Probe

Adjusting the pH of the prepared colloidal gold solution to 5.0 by using 0.2% K2CO3, dropwise adding the recombinant protein rSPG, shaking for 15min, and standing for 15 min. Adding 10mL of 10% PEG20000 stabilizing solution, shaking at room temperature for 15min, and standing for 15 min. The solution was centrifuged at 2000rpm for 20min, the supernatant was centrifuged at 10000rpm for 30min, and the precipitate was resuspended in 20ml of PBS. The colloidal gold probe is evenly covered on a gold label pad, and the gold label pad is frozen for 2h at the temperature of-20 ℃ and 2h at the temperature of-80 ℃ and then is put into a multifunctional freeze dryer for vacuum freeze drying.

4.5 preparation of colloidal gold immunochromatographic test strip

Test strips were prepared as shown in FIG. 6, with recombinant GE protein diluted to three concentrations of 1.5mg/mL, 0.75mg/mL, and 0.375 mg/mL. The NC membrane is pasted on a bottom plate, recombinant GE protein is taken as a detection line (T line), protein rSPG is taken as a quality control line (C line), and point sample is applied on a colloidal gold point sample system, wherein the line drawing amount is 1 mu L/cm. And placing the marked NC membrane bottom plate in a 37 ℃ oven for overnight drying. The gold label pad, the sample pad and the absorbent paper are sequentially stuck on the bottom plate, and a gold label slitting machine is used for slitting, wherein the width is 3 mm. The test paper strip is put into a sealed test paper cylinder, and a drying agent is added for sealed preservation at 4 ℃.

4.6 establishment of test strip judgment standard

The pseudorabies antibody detection test strip adopts a colloidal gold immunochromatographic technique, after the test strip is inserted into a positive sample, IgG in the sample is combined with gold-labeled r-SPG protein with a His label on a gold-labeled pad to form a compound, in the process of the compound migrating by a coating film, specific IgG in the sample and recombinant GE protein on a detection line (T) form a gold-labeled His-r-SPG protein-IgG-antigen compound, the compound is developed at the detection line, then the redundant gold-labeled His-r-SPG protein-IgG compound at a quality control line and a mouse anti-His label monoclonal antibody at a quality control line form a mouse anti-His label monoclonal antibody-gold-labeled His-r-SPG protein-IgG compound, so that the quality control line (C) is developed, otherwise, when the test strip is negative sample, the detection line (T) has no antigen, the gold-labeled His-r-SPG protein-IgG-antigen complex cannot be formed, the detection line (T) does not develop color, and only can be combined with the mouse anti-His-tag monoclonal antibody on the quality control line to form the mouse anti-His-tag monoclonal antibody-gold-labeled His-r-SPG protein complex for color development. Therefore, the test strip result judgment standard is formulated as follows: a purple red strip appears at the positive detection line (T) and the quality control line (C) respectively, and the result is judged to be positive; and (4) judging the test result to be negative if a mauve strip appears at the quality control line (C) and a mauve strip does not appear at the detection line (T) after the test result is negative. And (4) judging that the invalidation is invalid because no obvious strip appears at the quality control line (C).

4.7, test strip sensitivity.

3 batches of trial-produced pseudorabies antibody detection test strips are adopted to detect known negative samples and serum samples of pathogenic animals, and the results show that 10 known negative serum samples are detected, and the results are negative; the detection rate of 20 serum samples of known pathogenic animals is 100% (20/20).

4.8, test strip specificity.

The detection of 10 pseudorabies positive serum, 36 pseudorabies negative serum, 10 swine fever positive serum and 10 porcine reproductive and respiratory syndrome positive serum is carried out by adopting 3 pseudorabies antibody detection test strips, and the result shows that the positive detection rate of the research on 10 known pseudorabies positive serum is 100 percent (10/10); the test results of 36 known pseudorabies negative sera are all negative (36/36), and no cross reaction exists in the test of other non-pseudorabies positive sera, which indicates that the test strip has good specificity.

The preferred embodiments of the present invention have been fully described above, but various substitutions and modifications may be made thereto. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents. Any feature, whether preferred or not, may be combined with any other feature, whether preferred or not. The claims hereof are not to be read as having method + functional limitations unless such limitations are expressly recited in a claim by the term "method of …".

<110> Shanghai institute of veterinary medicine (Shanghai center of animal health and epidemiology, China) of agricultural and scientific colleges, Shanghai Jie-Biotechnology Co., Ltd

<120> preparation of recombinant protein in main antigen epitope region of pseudorabies virus GE gene and colloidal gold immunochromatographic test strip

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 498

<212> DNA

<213> full length sequence of GE protein

<400>GGATCCGGTCTGACCACCACCTGGAAAGAATATAGCCACGACCTGCAGCTGAACGATGGTGGTGGTG GTACCAATCCGAGCACCGAAGAAATGGGCGACGATTTTGGCTTTGGCCTGTGCCCGTTTGATACCAGCCCGGTGGTG AAAGGCAAAGGCAGCGTGAGTCCGACCACCCTGGGTGGTCGAGAAAAACCGTTTCCGGGTAGCACCACCGTGGAAGG TGGTACCTGCGTGAAAGGCGAACCGGTGGTGTATACCGGTGGTCAGGTGGGTAGCTTTAATGAACCGGATGGCCTGC CGCATTATGGCGGTTACCGCATCGTGGATAGCACCGATTGCAATCGCGGCAGCGAAGGCAGCGGTGGTAAAGTGCAT GCAAGCGATGAACGCCTGGGTCCGATGCCGTGTCGTCCGAAAGAAATCGTGAGCAGCGCAGGTCCGGTGCGCAAAGG CAGCAAGAACAAGTACTACGAGCCGCGCGATAGCTATTAAAAGCTT

Claims (6)

1. a recombinant protein of a main antigen epitope region of a pseudorabies virus GE gene is characterized in that the gene sequence of the recombinant protein GE is shown in SEQ ID NO. 1.

2. The use of the recombinant protein of claim 1, in the preparation of a diagnostic reagent or a medicament for the diagnosis, prevention or treatment of pseudorabies.

3. A product for use in an immunoassay, said product comprising the recombinant protein of claim 1.

4. The product of claim 3, which can be in the form of a probe (sensor), a test strip, a chip, a kit, etc., and when used, the fusion protein of the present invention can be mixed with other existing commercial reagents (e.g., enzyme-labeled antibody, fluorescent-labeled antibody, color-developing agent, substrate, etc.) to be used in various forms of immunoassays, such as antibody detection, antibody screening, antigen detection, pathogen detection, protein interaction screening, high-throughput target protein detection, protein-nucleic acid interaction analysis, drug screening, etc.

5. a colloidal gold immunochromatographic strip comprising the recombinant protein of the major epitope region of the GE gene of pseudorabies virus of claim 1, said colloidal gold immunochromatographic strip comprising a carrier plate, a sample pad, a colloidal gold binding pad, a laminate film, a detection line, a quality control line and an absorption pad; the sample pad, the colloidal gold combined pad, the laminated film and the absorption pad are sequentially adhered to the upper surface of the carrier plate, one end of the sample pad is arranged at one end of the colloidal gold combined pad, the other end of the colloidal gold combined pad is arranged at one end of the laminated film, one end of the absorption pad is arranged at the other end of the laminated film, and the detection line and the quality control line are sequentially arranged on the laminated film; the detection line is coated with GE recombinant protein, and the quality control line is coated with selected streptococcus recombinant protein (rSPG).

6. The method for preparing the colloidal gold immunochromatographic test strip of claim 5, which comprises the following steps: 1) protein expression to prepare purified recombinant protein;

2) Spotting of the nitrocellulose membrane: diluting GE recombinant protein to 0.5mg/ml for marking of a detection line (T line), and diluting the mouse anti-His tag monoclonal antibody to 0.5mg/ml for marking of a quality control line (C line); attaching an NC film to a bottom plate, then marking with a colloidal gold sample application system, wherein the marking amount is 1 mu l/cm, placing the bottom plate in a 37 ℃ drying oven for drying for 2 hours after marking, sealing in a tin foil bag, and internally arranging a drying agent with the validity period of 15 months;

Firing of the colloidal gold: sintering the colloidal gold according to a sodium citrate reduction method: putting all glassware used for firing and storing the colloidal gold into an acid jar to be soaked for 24 hours, taking out the glassware, washing the glassware clean and drying the glassware for later use; heating 100mL of deionized water and 1mL of 1% chloroauric acid to boil; 1.5mL of 1% trisodium citrate was added dropwise until the color stabilized to red; then boiling for 15min, and cooling to room temperature;

Preparing a colloidal gold probe: adjusting the pH of the prepared colloidal gold solution to 5.0 by using 0.2% K2CO3, dropwise adding the recombinant protein rSPG, shaking for 15min, and standing for 15 min; adding 10mL of 10% PEG20000 stabilizing solution, shaking at room temperature for 15min, and standing for 15 min; centrifuging the solution at 2000rpm for 20min, taking the supernatant, centrifuging at 10000rpm for 30min, taking the precipitate, and re-suspending the precipitate with 20ml LTBS; uniformly covering the colloidal gold probe on a gold label pad, freezing at-20 ℃ for 2h, freezing at-80 ℃ for 2h, and then putting the gold label pad in a multifunctional freeze dryer for vacuum freeze drying;

Preparing the colloidal gold immunochromatographic test strip: preparing a test strip, and diluting the recombinant GE protein to three concentrations of 1.5mg/mL, 0.75mg/mL and 0.375 mg/mL; attaching an NC film on a bottom plate, using the recombinant GE protein as a detection line (T line), using the protein rSPG as a quality control line (C line), and carrying out sample application on a colloidal gold sample application system, wherein the scribing amount is 1 mu L/cm; placing the marked NC membrane bottom plate in a 37 ℃ oven, and drying overnight; sequentially sticking the gold label pad, the sample pad and the absorbent paper on the bottom plate, and cutting into strips with a gold label cutting machine, wherein the width is 3 mm; putting the test strip into a sealed test paper cylinder, adding a drying agent, and sealing and storing at 4 ℃;

Establishing a test strip judgment standard: the pseudorabies antibody detection test strip adopts a colloidal gold immunochromatographic technique, after the test strip is inserted into a positive sample, IgG in the sample is combined with gold-labeled r-SPG protein with a His label on a gold-labeled pad to form a compound, in the process of the compound migrating by a coating film, specific IgG in the sample and GE recombinant protein on a detection line (T) form a gold-labeled His-r-SPG protein-IgG-antigen compound, the compound is developed at the detection line, then the redundant gold-labeled His-r-SPG protein-IgG compound at a quality control line and a mouse anti-His label monoclonal antibody at a quality control line form a mouse anti-His label monoclonal antibody-gold-labeled His-r-SPG protein-IgG compound, so that the quality control line (C) is developed, otherwise, when the test strip is negative, the detection line (T) has no antigen, the gold-labeled His-r-SPG protein-IgG-antigen complex cannot be formed, the detection line (T) does not develop color, and only can be combined with the mouse anti-His-tag monoclonal antibody on the quality control line to form the mouse anti-His-tag monoclonal antibody-gold-labeled His-r-SPG protein complex for color development; therefore, the test strip result judgment standard is formulated as follows: a purple red strip appears at the positive detection line (T) and the quality control line (C) respectively, and the result is judged to be positive; negative is judged to be negative only if a mauve strip appears at the quality control line (C) and a mauve strip does not appear at the detection line (T); and (4) judging that the invalidation is invalid because no obvious strip appears at the quality control line (C).