CN110967483B - Detection kit for malignant catarrhal fever virus, preparation method and application thereof - Google Patents

Abstract

The invention provides a detection kit for malignant catarrhal fever virus, a preparation method and application thereof, belonging to the technical field of biology, and comprising an ELISA plate coated with MCFV gB recombinant protein, wherein the MCFV gB protein is the sequence of SEQ ID NO.1, and the ELISA antibody comprises an HRP-marked MCFV gB recombinant protein polyclonal antibody. The preparation method provided by the invention can improve the reactivities and immunogenicity of the recombinant protein and improve the purity of the purified recombinant protein; the provided detection kit has better specificity, simple operation and higher detection rate.

Description

Detection kit for malignant catarrhal fever virus, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a detection kit for malignant catarrhal fever virus, a preparation method and application thereof.

Background

Malignant catarrhal fever (Maglignant Catarrhal Fever, MCF), the pathogen being a malignant catarrhal fever virus (Maglignant Catarrhal Fever virus, MCFV) of the herpesviridae, herpesviridae genus. The most fragile of the herpesviruses is difficult to preserve and to spread from cow to cow, and the malignant catarrhal fever of cows is generally infected by contact with asymptomatic, virulent sheep in most parts of the world, except continents. Since the disease usually progresses, the prevention of immune nonsensical, no specific therapy exists at present. Therefore, by detecting the malignant catarrhal fever virus, the infected asymptomatic sheep with the virus is isolated from the cattle, and is an important prevention and control method for Niu Exing catarrhal fever. The existing detection method for the malignant catarrhal fever virus is mainly a PCR method, but the method has some disadvantages: the pollution is easy to cause, and false positive occurs; special instruments and reagents are required and are not suitable for large-scale veterinary clinical tests. With the development of immunology and biotechnology, the establishment of a sensitive, specific and rapid serological detection method has important significance for the prevention and control of Niu Exing catarrh fever.

The prior art, such as Chinese patent publication No. CN 105807052B, discloses a direct competitive ELISA detection kit for O-type FMDV antibodies, which comprises an ELISA plate, a washing solution, a sample diluent, HRP-labeled rabbit antibody, a substrate A solution, a substrate B solution, a stop solution, positive control serum, negative control serum and a sealing plate film, wherein the ELISA plate is coated with O-type FMDV genetic engineering antigen. The kit adopts a one-step method for sample addition, and shortens the detection time. The HRP-marked rabbit antibody is used for replacing the enzyme-labeled secondary antibody, so that animal species difference is eliminated, and serum samples of pigs, cows, sheep and the like can be detected. The kit can overcome the defects of the existing serological diagnosis technology, has the characteristics of sensitivity, specificity, rapidness, simple preparation and the like, and can be widely applied to the monitoring of the immune effect of the O-type FMD vaccine and epidemiological investigation.

Disclosure of Invention

An object of the present invention is to provide a kit for detecting a malignant catarrhal fever virus and a preparation method thereof, which can promote the expression of a soluble recombinant protein and improve the reactivity and immunogenicity of the recombinant protein; the exposure of His Tag in the recombinant protein can be promoted, the specific combination of target protein and resin can be enhanced, and the purity of the purified recombinant protein can be improved; the detection kit has better specificity, simple operation and higher detection rate.

The technical scheme adopted by the invention for achieving the purpose is as follows:

provided is a kit for detecting a malignant catarrhal fever virus, which detects a malignant catarrhal fever virus antibody by a direct competition ELISA method, comprising:

the ELISA plate is coated with MCFV gB recombinant protein, and the MCFV gB protein is the sequence of SEQ ID NO. 1;

the enzyme-labeled antibody comprises an HRP-labeled MCFV gB recombinant protein polyclonal antibody;

the result judgment standard of the detection kit is as follows: the sample serum inhibition rate is larger than or equal to 28.3 percent, and is positive; the sample was negative when the serum inhibition rate was less than 28.3%.

Preferably, the preparation method of the MCFV gB recombinant protein comprises the following steps: the MCFV genome is used as a template to amplify the MCFV gB protein gene, the obtained PCR product is connected with an expression plasmid pET-28a and then is introduced into escherichia coli BL21 for induced expression, and then the PCR product is obtained through separation and purification.

Preferably, the primers used for amplifying the MCFV gB protein gene are SEQ ID NO.3 and SEQ ID NO.4.

Preferably, the method for inducing and expressing the MCFV gB recombinant protein comprises the following steps: inoculating the bacterial liquid containing positive plasmid into liquid LB containing Amp, culturing until bacterial liquid OD ₆₀₀ The value of (2) is 0.45-0.55, IPTG with the final concentration of 1.0mmol/L, 1,2, 4-triazole sodium with the final concentration of 0.17-0.34mmol/L and 4-hydroxy-3-methoxybenzyl alcohol with the final concentration of 1.0-1.45mmol/L are added for induction culture for 4-6h. When the expression of the recombinant protein exists in the form of inclusion bodies, the insoluble and biologically inactive inclusion bodies must be denatured and renatured to obtain the natural structure and biological activity, and these complex operations greatly increase the difficulty and cost of obtaining high-activity pure products, and easily cause the reduction of the biological activity of the recombinant protein, resulting in the reduction of immunogenicity. When the recombinant protein is induced to be expressed, 1,2, 4-sodium triazole and 4-hydroxy-3-methoxyl benzyl alcohol are added, so that the expression of the soluble recombinant protein can be promoted, complex denaturation and renaturation are not needed, the reactivity and immunogenicity of the recombinant protein can be improved, the affinity and specificity of a polyclonal antibody of the recombinant protein are improved, and the detection rate of a kit is further improved.

Preferably, the expressed protein is in the form of a soluble protein.

Preferably, the purification method of the MCFV gB recombinant protein comprises the following steps:

a. centrifuging the induced bacterial liquid, collecting precipitate, suspending bacterial body with buffer solution (50 mmol/L Tris-HCl, pH 8.0), ultrasonic crushing, centrifuging, collecting supernatant, filtering with 0.45 μm filter membrane, adding trimethyl sulfoxide bromide with final concentration of 0.6-0.83mg/mL and 2-sulfapyridine with final concentration of 0.26-0.34 mg/mL.

b. Washing Ni-NTA His-Bind nickel ion resin with a balance buffer solution, re-suspending with the balance buffer solution, loading into a chromatographic column, naturally settling, and passing the supernatant in the step a through the column;

c. washing off the foreign proteins with a washing buffer;

d. the target protein is washed off with elution buffer. The recombinant protein contains His Tag label and recombinant eggThe white solution is added with the trimethyl sulfoxide bromide and the 2-sulfapyridine, which is favorable for exposing His Tag and promoting histidine and Ni of His Tag sequence ²⁺ The specific binding of the target protein and the resin is enhanced, and under the condition of ensuring the recovery rate, the nonspecific binding protein can be eluted by utilizing high-concentration imidazole, so that the co-purification of the nonspecific binding protein is reduced, the purity of the purified recombinant protein is improved, the affinity and the specificity of the recombinant protein polyclonal antibody are improved, and the detection rate of a detection kit is further improved.

Preferably, the preparation method of the MCFV gB recombinant protein polyclonal antibody comprises the following steps: the purified MCFV gB recombinant protein with the concentration of 1.13-1.25mg/mL is taken to immunize the rabbits, the immunization program is 0d, 3-5d, 25-28d and 32-35d, the first immunization and the second immunization adopt the complete emulsification of the recombinant protein and the equal volume Freund complete adjuvant, the third immunization and the fourth immunization adopt the complete emulsification of the recombinant protein and the equal volume Freund incomplete adjuvant, and the dosage after the emulsification is 250-280 mug/animal.

It is an object of the present invention to provide the use of sodium 1,2, 4-triazole and 4-hydroxy-3-methoxybenzyl alcohol for promoting expression of soluble recombinant proteins.

An object of the present invention is to provide a use of a detection kit for detecting sheep MCFV antibodies.

It is still another object of the present invention to provide a use of the detection kit for detecting bovine MCFV antibodies.

The invention has the advantages that:

1. according to the invention, the MCFV gB recombinant protein is used as a coating antigen, the HRP-marked MCFV gB protein polyclonal antibody is used as an enzyme-labeled antibody, and the direct competition ELISA method is adopted to detect the malignant catarrhal fever virus antibody, so that the method has the characteristics of sensitivity, simplicity and rapidness in operation and high detection rate;

2. the invention optimizes the expression condition of MCFV gB recombinant protein, promotes the expression of soluble recombinant protein, improves the reactivities and immunogens of the recombinant protein, improves the affinity and the specificity of the recombinant protein polyclonal antibody, and further improves the detection rate of a detection kit;

3. the recombinant protein is treated before purification, so that the exposure of His Tag and the histidine and Ni of His Tag sequence can be promoted ²⁺ The specific binding of the target protein and the resin is enhanced, and under the condition of ensuring the recovery rate, the non-specific binding protein can be eluted by utilizing high-concentration imidazole, so that the purity of the purified recombinant protein is improved, the affinity and the specificity of the recombinant protein polyclonal antibody are improved, and the detection rate of the detection kit is further improved.

Drawings

FIG. 1 is a SDS-PAGE electrophoresis of the supernatant in test example 1 of the present invention;

FIG. 2 is a SDS-PAGE electrophoresis of the precipitate in test example 1 according to the present invention; the measurement results of the concentration of the recombinant protein after purification are shown in FIG. 3. SDS-PAGE of the purified recombinant proteins is shown in FIG. 4.

FIG. 3 is a graph showing the results of measurement of the concentration of recombinant protein after purification in test example 1 of the present invention;

FIG. 4 is a SDS-PAGE electrophoresis of the recombinant protein purified in test example 1 according to the present invention;

FIG. 5 shows the reactivities of recombinant proteins in test example 2 according to the invention;

FIG. 6 shows the antibody levels of the recombinant protein immunized rabbit of test example 2 of the present invention;

FIG. 7 shows the blocking effect of the recombinant protein polyclonal antibody of test example 3 according to the present invention;

FIG. 8 shows the Western Blot detection results of recombinant protein polyclonal antibodies of Experimental example 3 of the present invention;

FIG. 9 shows the results of measuring the compliance rate in test example 4 of the present invention.

Detailed Description

The invention is described in further detail below with reference to examples:

example 1: the technical route of the invention is that firstly, the total RNA of OHV-2 virus is extracted, reverse transcribed into cDNA, and the gene of gB protein is amplified by PCR. Then constructing pET28a-gB recombinant plasmid. And transforming the plasmid to obtain a prokaryotic expression system of the gE protein, and obtaining the recombinant gE protein through induced expression, separation and purification. The recombinant gE protein is used for immunizing rabbits, and after the immune success, the polyclonal antibody is obtained through heart blood sampling, serum separation and purification, and then the enzyme-labeled polyclonal antibody is prepared. Finally, the detection kit of the malignant catarrhal fever virus is assembled.

The cells and cell lines were as follows: e.coli BL21 for selection of pET28a-gB positive plasmid and expression of recombinant gB protein.

Axyprep humoral virus DNA/RNA minikit, M-MLV reverse transcriptase, RNase inhibitor were purchased from Invitrogen company. TaqDNA polymerase, T4DNA ligase and endonucleases were all purchased from NEB company. DNA gel recovery kit was purchased from Tiangen. The primer sequences for the amplification of the gE protein gene are as follows:

gB-XF：CTGGATCCCATCTATTGGTAATGATACAGCCAC；

gB-XR：TGAAGCTTATGGCAAGGTTACCGTATGCTACGT。

1.1. extraction of OHV-2 viral RNA

200 mu L of virus liquid after the OHV-2 strain amplification is taken, and the OHV-2 total RNA is extracted according to the operation procedure of an Axyprep humoral virus DNA/RNA small-scale kit.

2. Construction of prokaryotic expression System for gB protein

2.1 PCR amplification of OHV-2-gB protein Gene:

the extracted OHV-2RNA was used as a template, which was rapidly dissolved in 17. Mu.L DEPC H ₂ O was used for the reverse transcription reaction, gB-XF and gB-XR were added in an amount of 0.5. Mu.L each, allowed to act at 75℃for 5 minutes, and then, after 3 minutes in an ice bath, the following reaction system was added:

5×RT Buffer 5μL

dNTPs(10mmol/L) 1.5μL

M-MLV 0.5μL

42 ℃ 45min and 95 ℃ 5min to obtain cDNA.

The cDNA obtained by reverse transcription is used as a template to amplify the gB protein gene by PCR. The reaction system (30. Mu.L) was as follows:

mixing, placing in a PCR amplification instrument, pre-denaturing at 94 ℃ for 3min, and circulating according to the following procedures: denaturation at 94℃for 40s, annealing at 52℃for 40s, extension at 72℃for 1min, and extension at 72℃for 10min after 32 cycles.

After electrophoresis on a 1% agarose gel, the purified target fragment was recovered according to the instructions of the OMEGA agarose gel DNA recovery kit.

2.2 Construction of recombinant pET28a-gB plasmid:

and (3) respectively carrying out double digestion on the target fragment and the pET-28a vector by using BamHI and HindIII on the PCR product of the gB protein gene, and connecting the digested target fragment and the pET-28a vector. The enzyme digestion system is as follows:

mixing uniformly, placing into a water bath kettle at 37 ℃, reacting for 3 hours, carrying out agarose gel electrophoresis at 1%, cutting a target strip, and purifying an enzyme-cut product by using an OMEGA agarose gel DNA recovery kit. The connection system is as follows:

the mixture was placed in a PCR apparatus and reacted at 16℃for 12 hours, and after the reaction was completed, the EP tube was placed in an ice box.

10. Mu.L of ligation product was added to 60. Mu.L of E.coli BL21 competent cells and left on ice for 25min; heat shock at 42 ℃ for 90s, and immediately placing on ice for 2min; adding 500 mu L of LB liquid medium, and shake culturing at 37 ℃ for 1h at 200 r/min; evenly spread on LB plate containing ampicillin, and cultured overnight in an incubator at 37℃upside down. Extracting plasmid by a small amount method, performing enzyme digestion and PCR analysis and identification, converting positive expression plasmid into BL21 competent cells again, plating, extracting plasmid, performing PCR and enzyme digestion identification, and delivering positive clone to Shanghai Boya sequencing. The obtained gB gene is 523bp, the measured sequence is shown as SEQ ID NO.2, and the coded amino acid sequence is shown as SEQ ID NO.1, and the specific steps are as follows:

gB gene, SEQ ID NO.2:

ACACAGGGCTTCCCTAATGTGGAATGAGTTAAGTAAAATTAATCCTACTAGTGTGATGAGCTCTATTTACGGACGGCCAGTCTCAGCCAAGCGAATTGGTGACGTAATATCTGTTTCCCACTGTGTAGTGGTGGACCAGCAAAGTGTGTCTCTACACAGGAACATGCGCGTTCCTGGGCGAGACCACGCGCACGAGTGCTACTCTAGGCCGCCTGTGACCTTCAAGTTTATTAATGACAGTCACTTGTACAAAGGCCAACTGGGCGTAAACAATGAAATTCTGCTTACAACAACCGCCCTGGAAGTGTGCCATGAGAACACCGAACATTATTTTCAAGGGGGGAACAACATGTACTTTTACAAAAACTACAGACACGTAAAAACCATACCTGTGAGCGCCATCGCTACCCTAGATACCTTTATTGTGCTTAACGTAACCCTAGTAGAAAACATTGGCTTTCAGGTCATAGAACTGTACTCTAGAGAAGAAAAGCGCATGAGCACGGTTTTTGACATTGAGACC；

amino acid sequence of gB gene, SEQ ID No.1:

HRASLMWNELSKINPTSVMSSIYGRPVSAKRIGDVISVSHCVVVDQQSVSLHRNMRVPGRDHAHECYSRPPVTFKFINDSHLYKGQLGVNNEILLTTTALEVCHENTEHYFQGGNNMYFYKNYRHVKTIPVSAIATLDTFIVLNVTLVENIGFQVIELYSREEKRMSTVFDIET；

SEQ ID NO.2 and SEQ ID NO.1 correspond as follows:

3. inducible expression of recombinant proteins

Inoculating bacterial liquid containing pET28a-gB plasmid into liquid LB containing Amp, shake culturing at 37deg.C and 180rpm until bacterial liquid OD ₆₀₀ IPTG with a final concentration of 1.0mmol/L and sodium 1,2, 4-triazole with a final concentration of 0.18mmol/L were added, and the culture was induced for 6 hours at 37℃and 150rpm with a final concentration of 1.2mmol/L of 4-hydroxy-3-methoxybenzyl alcohol.

4. Separation and purification of recombinant proteins

Centrifuging 1L of the induced bacterial liquid at 10000rpm for 5min, collecting precipitate, suspending bacterial cells with 20mL of buffer (50 mmol/L Tris-HCl, pH 8.0), ultrasonically crushing for 5min, centrifuging at 10000rpm for 5min, collecting supernatant, filtering with a 0.45 μm filter membrane, and adding trimethyl sulfoxide bromide with a final concentration of 0.6mg/mL and 2-sulfapyridine with a final concentration of 0.28 mg/mL; with equilibration buffer (46 mM Na ₃ PO ₄ 3.8g，500mM NaCl 14.61g，500mL H ₂ O, pH 7.4) washing Ni-NTA His-Bind nickel ion resin, re-suspending with balancing buffer solution, loading into chromatographic column,naturally settling, and passing the supernatant through a column; with washing buffer (50 mM NaH) ₂ PO ₄ 300mM NaCl,50mM imidazole, pH 8.0) to wash out contaminating proteins; with elution buffer (50 mM NaH) ₂ PO ₄ 300mM NaCl,250mM imidazole, pH 8.0), and centrifuging to collect the supernatant.

5. Identification of recombinant proteins

The protein characteristic band of about 25kDa is detected by SDS-PEG electrophoresis, and is the target protein with expected molecular weight.

6. Preparation of HRP-labeled gB recombinant protein polyclonal antibody

Taking purified MCFV gB recombinant protein with the concentration of 1.15mg/mL, performing subcutaneous multipoint immunization on two sides of the spine, the abdomen, armpits and the like of the back of an adult large-ear white rabbit, performing immunization procedures of 0d, 3d, 26d and 33d, completely emulsifying the recombinant protein with an equal volume Freund complete adjuvant for the first immunization and the second immunization, completely emulsifying the recombinant protein with an equal volume Freund incomplete adjuvant for the third immunization and the fourth immunization, performing heart blood sampling and serum separation after the immunization is completed, and purifying by using an octanoic acid-ammonium sulfate method and a G protein column method. Using NaIO ₄ Oxidizing sugar molecules on the surface of HRP into aldehyde groups, and then combining with amino groups of serum antibodies to obtain the HRP-labeled gB recombinant protein polyclonal antibody.

7. Kit for detecting malignant catarrhal fever virus

After ELISA screening out the appropriate gB recombinant protein antigen, a detection kit for malignant catarrhal fever virus is assembled. The kit comprises an antibody coating slat, negative control serum, positive control serum, reagent diluent, HRP-marked gB protein polyclonal antibody, 10 multiplied by concentrated washing liquid, a color developing agent and a stop solution. The specific composition is shown in Table 1.

TABLE 1 detection kit Components of malignant catarrhal fever Virus

The preparation of the antigen coated strip comprises the following steps:

diluting gB recombinant protein with coating solution (0.05M, pH9.6 carbonate buffer solution) to 18 μg/mL, adding 96-well ELISA plate according to 100 μl per well, coating at 37deg.C for 2h, continuously coating at 4deg.C for 12h, washing with PBST for 3 times, washing for 5min each time, and drying after washing; PBS blocking solution containing 1% BSA (0.25 g BSA was added to 50mL PBS) was added to the ELISA plate in an amount of 200. Mu.L per well, blocking was performed at 37℃for 3 hours, washing was performed with PBS-T3 times for 5 minutes, and the plate was dried after washing was completed.

The negative control serum was negative serum from sheep not infected with malignant catarrhal fever virus. The positive control serum is the positive serum of sheep infected with malignant catarrhal fever virus. The reagent diluent is phosphate buffer solution with the pH value of 7.2 and the bovine serum albumin content of 1% (W/V). The concentrated washing solution is phosphate buffer solution with pH value of 7.2 and Tween 20 content of 0.1% (V/V). The color-developing agent is tetramethyl benzidine. The stop solution was 1N HCl (1N hydrochloric acid). The sealing plate film is a transparent plastic film with the same size as the plate surface of the ELISA plate.

The kit is used for detecting bovine vesicular stomatitis virus, and a direct competition ELISA detection method is adopted. The gB recombinant protein antigen is coated on an ELISA plate, serum to be detected, negative control serum and positive control serum are respectively added, then an HRP-marked gB recombinant protein polyclonal antibody is added, incubation is carried out for 30min at 37 ℃, and the HRP-marked specific rabbit source antibody competes with MCFV antibody in a serum sample to be detected for binding with the gB recombinant protein antigen coated in the micro-holes of the ELISA plate. And adding a color development liquid, catalyzing a color development reaction by using HRP, measuring an OD value at 450nm, and inversely correlating the color development depth with the content of the antibody in the sample to be measured. The method specifically comprises the following steps:

self-contained test equipment required for the test:

1) A microplate reader (450 nm detection wavelength filter, 570nm or 690nm correction wavelength filter);

2) High-precision liquid feeder and disposable suction head: 0.5-10 mu L,2-20 mu L,20-200 mu L and 200-1000 mu L;

3) Microplate shaker, double distilled water or deionized water.

And (3) sample collection:

1) The test tube for collecting blood should be disposable, pyrogen-free, endotoxin-free;

2) EDTA is recommended for the plasma anticoagulant, so that hemolysis and hyperlipidemia specimens are avoided;

3) The standard should be clear and transparent, and suspended matters should be removed by centrifugation;

4) If the sample is not detected in time after collection, the sample is sub-packaged according to the usage amount, and the sample is frozen in a refrigerator at the temperature of minus 20 ℃ or minus 70 ℃ to avoid repeated freezing and thawing;

5) Can be diluted by proper times according to the actual condition of the specimen.

Notice that:

1) The kit is stored at 2-8deg.C before use;

2) The positive control serum, the negative control serum and the HRP-marked gB recombinant protein polyclonal antibody are small in volume, and the liquid can be adhered to a pipe wall or a bottle cap due to jolt and possible inversion in transportation. Therefore, before use, the liquid attached to the pipe wall or the bottle cap is deposited on the bottom of the pipe by hand throwing or centrifuging at 1000rpm for 1 minute;

3) The concentrated washing liquid taken out of the refrigerator may have crystals, which are normal phenomena, and the washing liquid is prepared after the crystals are heated to 40 ℃ slightly to be completely dissolved;

4) The kit components of different batches cannot be mixed (except the washing solution and the reaction stopping solution);

5) The full slight mixing is particularly important to the reaction result, and a micro-oscillator (with the lowest frequency) is preferably used, for example, a micro-oscillator is not used, and the ELISA plate can be manually and gently shaken before the reaction to mix the reaction solution in the inlet hole;

6) The positive control serum, negative control serum and sample in the enzyme immunoassay are recommended to be re-plated.

Preparation work before detection:

1) Taking the kit out of the refrigerator 20 minutes in advance to equilibrate to room temperature;

2) Diluting the concentrated washing solution with double distilled water (1:20), and returning the unused washing solution to a refrigerator at 4 ℃;

3) Positive control serum, negative control serum: diluting according to the requirement, and diluting according to the ratio of 1:16;

4) HRP-labeled gB protein polyclonal antibody working solution: HRP-labeled gB recombinant protein polyclonal antibody (1:500) was diluted with reagent diluent to prepare biotinylated antibody working solution in the amounts required for the current assay. Prepared 30 minutes before use. For use only on the same day.

The washing method comprises the following steps:

1) Automatic plate washing machine: the washing solution to be injected is required to be 350. Mu.L, and the interval between injection and aspiration is 20-30 seconds. Washing the plate for 4 times;

2) Manual plate washing: adding 350 mu L of washing liquid into each hole, standing for 30 seconds, throwing out the liquid in the holes, beating the holes on thick overlapped absorbent paper, and washing the plate for 5 times.

The operation steps are as follows:

1) Taking out the required strips for test from the sealed bag which is balanced to room temperature, and placing unused strips and drying agent back into the aluminum foil bag for sealing at 4 ℃;

2) Leaving blank holes (if a dual wavelength reading plate is used, the blank holes can be omitted);

3) Preparing a sample, positive control serum and negative control serum in advance, and preparing an HRP-labeled gB recombinant protein polyclonal antibody working solution;

4) Firstly, positive control serum and negative control serum are respectively added into corresponding holes (100 mu L/hole); to each sample well was added 50. Mu.L of sample dilution and 50. Mu.L of sample (at which time the sample had been diluted 1:2 times, the sample content was multiplied by 2 at the time of calculation), and HRP-labeled gB recombinant protein polyclonal antibody working solution (100. Mu.L/well) was added in addition to the blank wells. Sealing the reaction hole by using sealing plate gummed paper;

5) Incubate at 37℃for 30min. Preferably, a micro-oscillator (minimum frequency, 100 rpm) is used;

6) Washing the plate for 5 times;

7) Adding a chromogenic substrate (comprising blank holes) at 100 mu L/hole, and incubating for 15 minutes at 37 ℃ in a dark place;

8) Adding stop solution (including blank hole) 100 μl/hole, mixing, and measuring OD immediately ₄₅₀ Value (within 10 minutes).

And (3) judging results:

and calculating the inhibition rate. The sample serum inhibition rate is larger than or equal to 28.3 percent, and is positive; the sample was negative when the serum inhibition rate was less than 28.3%. The inhibition rate calculation formula:

inhibition = [ (negative control OD) ₄₅₀ Sample serum OD ₄₅₀ ) /(negative control OD) ₄₅₀ Positive control OD ₄₅₀ )]×100％

Comparative example 1:

when the recombinant protein was induced to express, sodium 1,2, 4-triazole was not added, and the remainder was completely identical to example 1.

Comparative example 2:

when the recombinant protein was induced to express, 4-hydroxy-3-methoxybenzyl alcohol was not added, and the remainder was completely identical to example 1.

Comparative example 3:

when the recombinant protein was induced to express, 1,2, 4-sodium triazole and 4-hydroxy-3-methoxybenzyl alcohol were not added, and the rest was completely identical to example 1.

Comparative example 4:

centrifuging 1L of the induced bacterial liquid at 10000rpm for 5min, collecting precipitate, suspending bacterial cells with 20mL of buffer (50 mmol/L Tris-HCl, pH value 8.0), ultrasonically crushing for 5min, centrifuging at 10000rpm for 5min, collecting precipitate, dissolving in Tris-HCl buffer containing 6M guanidine hydrochloride, standing on ice for 1h, intermittently mixing and dissolving, centrifuging and collecting supernatant; to the collected supernatant was added Ni-NTA His.Bind resin, followed by addition of binding buffer (0.1M NaH ₄ PO ₄ Gently mixing with 0.01M Tris-Cl pH8.0, suspending for 30min, eluting with 25. Mu.L of elution buffer (0.1M NaH) ₄ PO ₄ 0.01M Tris-Cl pH 4.5) to wash off the target protein, and collecting the supernatant by centrifugation. The remainder is identical to comparative example 3.

Comparative example 5:

before purification of the recombinant protein by column, no trimethylsulfoxide bromide was added, and the concentration of imidazole in the washing buffer used was 25mM, the remainder being exactly identical to example 1.

Comparative example 6:

before purification of the recombinant protein by column, no 2-sulfapyridine was added, the concentration of imidazole in the wash buffer used was 25mM, the remainder being exactly identical to example 1.

Comparative example 7:

before purification of the recombinant protein by column, no trimethylsulfoxide bromide and no 2-sulfapyridine were added, and the concentration of imidazole in the washing buffer used was 25mM, the remainder being exactly identical to example 1.

Comparative example 8:

before purification of the recombinant protein by column, no trimethylsulfoxide bromide was added, the remainder being exactly identical to example 1.

Comparative example 9:

before purification by column, 2-sulfapyridine was not added, and the remainder was identical to example 1.

Comparative example 10:

before purification of the recombinant protein by column, no trimethylsulfoxide bromide and 2-sulfapyridine were added, and the remainder were identical to example 1.

Test example 1:

1. identification of the expression form of the recombinant protein:

1L of the induced bacterial liquid was centrifuged at 10000rpm for 5min, the precipitate was collected, bacterial cells were suspended in 20mL of buffer (50 mmol/L Tris-HCl, pH 8.0), sonicated for 5min, centrifuged at 10000rpm for 5min, and the precipitate and supernatant were collected, respectively, for SDS-PAGE analysis.

SDS-PAGE of the supernatants is shown in FIG. 1. The SDS-PAGE pattern of the pellet is shown in FIG. 2.

As can be seen from FIGS. 1 and 2, example 1, comparative example 2 and comparative example 3 successfully induced expression of a recombinant protein having a molecular weight of about 25 kDa. Example 1 the recombinant proteins induced to be expressed were present in the form of soluble proteins, and the recombinant proteins induced to be expressed in comparative examples 1,2 and 3 were present in the form of inclusion bodies, which suggests that the expression of soluble recombinant proteins can be promoted when the recombinant proteins were induced to be expressed with sodium 1,2, 4-triazole and 4-hydroxy-3-methoxybenzyl alcohol.

2. Detection of recombinant protein concentration: the purified protein was assayed for concentration using the BCA protein concentration kit from Thermo company, according to the procedures described herein.

3. Detection of recombinant protein purity: the purified recombinant protein was detected by SDS-PAGE electrophoresis.

The measurement results of the concentration of the recombinant protein after purification are shown in FIG. 3. SDS-PAGE of the purified recombinant proteins is shown in FIG. 4.

As can be seen from FIG. 3, the concentration of recombinant protein after purification in example 1 is significantly higher than that in comparative examples 8, 9 and 10, and as can be seen from FIG. 4, the presence of a distinct, single band of target protein at about 25kDa in example 1, the presence of a blurry band at 25kDa in comparative examples 5, 6 and 7, and the presence of a hetero band, indicates that the addition of trimethylsulfoxide bromide and 2-sulfapyridine to the recombinant protein solution facilitates exposure of His Tag, and promotes histidine and Ni in His Tag sequence ²⁺ The specific binding of the target protein and the resin is enhanced, and the non-specific binding protein can be eluted by using imidazole with higher concentration under the condition of ensuring the recovery rate, so that the purity of the purified recombinant protein is improved.

Test example 2:

detection of the reactivities of recombinant proteins: the antigen was coated at 0.25. Mu.g/well of recombinant protein, and an indirect ELISA was performed using the serum from sheep infected with the malignant catarrhal fever virus and the serum from sheep not infected with the malignant catarrhal fever virus, and the absorbance was read with an ELISA reader at a wavelength of 450 nm. And positive serum OD/negative serum OD (P/N) was calculated.

Immunogenicity detection of recombinant proteins:

taking purified MCFV gB recombinant protein with the concentration of 1.15mg/mL, performing immunization on subcutaneous multiple points such as two sides of the back vertebral column, the abdomen, armpits and the like of an adult large-ear white rabbit, wherein immunization programs are 0d, 3d, 26d and 33d, completely emulsifying the recombinant protein with an equal volume Freund complete adjuvant, completely emulsifying the recombinant protein with an equal volume Freund incomplete adjuvant after three times of immunization and four times of immunization, taking the emulsified doses of 250 mug/PBS as negative control, taking blood for 18d, 33d, 56d and 72d respectively, separating serum, and purifying by using an octanoic acid-ammonium sulfate method and a G protein column method. The level of polyclonal antibodies (1:100 dilution) was measured at different immunization times by indirect ELISA with 0.25. Mu.g of coated antigen per well of recombinant protein. The reactivities of the recombinant proteins are shown in FIG. 5. The antibody level of the recombinant protein immunized rabbit is shown in FIG. 6.

As can be seen from FIG. 5, the recombinant proteins in the soluble form of example 1 and the recombinant proteins in the inclusion body form of comparative example 4 reacted with positive serum from infected sheep, but compared with comparative example 4, example 1 reacted more strongly with positive serum, had a higher absorbance value, and reacted less with negative serum, had a lower absorbance value, and had a larger P/N; as can be seen from FIG. 6, the absorbance value is significantly higher than that of the negative control, and the recombinant protein in the soluble form and the recombinant protein in the inclusion body form can be used for producing specific antibodies of the recombinant protein after immunization of rabbits, and can last for 72 days after the first time, but compared with comparative example 4, the antibody level induced by the recombinant protein in the soluble form in example 1 is higher, and the immune response is stronger, which indicates that when the recombinant protein is induced to express by using 1,2, 4-sodium triazole and 4-hydroxy-3-methoxybenzyl alcohol, the expressed recombinant protein exists in the form of the soluble protein, and the reactivity and immunogenicity of the recombinant protein can be improved.

Test example 3:

specific detection of recombinant protein polyclonal antibody:

and taking the polyclonal antibody obtained after the immunization for 33d for specific detection.

1. Blocking effect of recombinant protein polyclonal antibodies on malignant catarrhal fever virus:

cell culture: inoculating NA cells into a cell bottle, pouring out a culture medium when the cells grow into the cell bottle, and washing with PBS for 3 times, wherein each time is 5mL; adding proper amount of pancreatin to digest the cells, slowly shaking the cell bottle, and digesting for about 1.5min; pouring out pancreatin, blowing off cells with culture medium, and centrifuging at 1000rpm for 2min; fresh medium was added and transferred to new cell flasks. The next day after the passage of the cells, when the cells grow to a monolayer, the culture medium is poured out, the cells are washed for 3 times by PBS, and diluted OHV-2 virus mixed solution is added; after culturing for 1h, pouring out the mixed solution, washing with PBS, and adding a cell maintenance culture medium; after 48 hours of inoculation, pouring out the culture medium, washing 3 times with PBS, adding a proper amount of cell lysate and 10% protease inhibitor, and placing at 4 ℃ for 20 minutes of lysis; the lysate was transferred to an EP tube, centrifuged at 12000rpm at 4℃for 15min, the pellet was discarded, the supernatant was taken, and after penetration with 0.5% Triton X100, freeze-thawing was performed 2 times at-70℃and ELISA plates (0.25. Mu.g/well) were coated to detect blocking by recombinant protein polyclonal antibodies (1:100 dilution), PBS immunized as a negative control.

Western Blot: SDS-PAGE of the proteins obtained by cell lysis was converted to PVDF membrane, primary antibody was polyclonal antibody to recombinant protein (1:100 dilution), secondary antibody was HRP-labeled rabbit anti-oxygen IgG antibody, 1:5000 dilution. The blocking effect of the recombinant protein polyclonal antibody is shown in FIG. 7. The Western Blot detection results of the recombinant protein polyclonal antibody are shown in FIG. 8.

As can be seen from FIG. 7, the recombinant protein polyclonal antibody of example 1 reacted with the malignant catarrhal fever virus protein at OD ₄₅₀ The values were significantly higher than for comparative examples 4, 5, 6 and 7. As can be seen from FIG. 8, the recombinant protein polyclonal antibody of example 1 reacted specifically with the malignant catarrhal fever gB protein, with distinct, single specific bands around 21kDa, with specific bands at 21kDa being obscured for comparative example 4, comparative example 5, comparative example 6, comparative example 7, and other non-specific bands. This demonstrates that when 1,2, 4-sodium triazole and 4-hydroxy-3-methoxybenzyl alcohol are used to induce expression of recombinant protein, the expressed recombinant protein exists in the form of soluble protein, so that the affinity and specificity of the recombinant protein polyclonal antibody can be improved; the trimethyl sulfoxide bromide and the 2-sulfapyridine are added into the recombinant protein solution, so that the purity of the purified recombinant protein can be improved, and the affinity and the specificity of the recombinant protein polyclonal antibody can be improved.

Test example 4:

clinical detection: the adopted malignant catarrhal fever virus PCR diagnosis kit which has obtained production permission on the market is compared with the kit, 80 sheep serum is detected according to the kit specification and the operation method, and the positive coincidence rate, the negative coincidence rate and the total coincidence rate of the kit are calculated. The results of the measurement of the compliance rate are shown in FIG. 9.

As can be seen from fig. 9, the positive compliance rate, the negative compliance rate and the total compliance rate of example 1 were significantly higher than those of comparative examples 1, 4, 5, 6 and 7. This shows that when the recombinant protein is induced to express by using 1,2, 4-sodium triazole and 4-hydroxy-3-methoxyl benzyl alcohol, the expressed recombinant protein exists in a soluble protein form, so that the affinity and the specificity of the polyclonal antibody of the recombinant protein can be improved, and the detection rate of the kit can be further improved; the trimethyl sulfoxide bromide and the 2-sulfapyridine are added into the recombinant protein solution, so that the purity of the purified recombinant protein can be improved, the affinity and the specificity of the recombinant protein polyclonal antibody can be improved, and the detection rate of the kit can be further improved.

The conventional technology in the above embodiments is known to those skilled in the art, and thus is not described in detail herein.

The above embodiments are merely for illustrating the present invention and not for limiting the same, and various changes and modifications may be made by one of ordinary skill in the art without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions are also within the scope of the present invention, which is defined by the claims.

Claims (2)

1. A preparation method of MCFV gB recombinant protein is characterized in that the sequence of the MCFV gB protein is the sequence of SEQ ID NO. 1; the preparation method of the MCFV gB recombinant protein comprises the following steps: amplifying the MCFV gB protein gene by taking the MCFV genome as a template, connecting the obtained PCR product with an expression plasmid pET-28a, introducing the PCR product into escherichia coli BL21 for induced expression, and then separating and purifying to obtain the PCR product;

the induction expression method of the MCFV gB recombinant protein comprises the following steps: inoculating the bacterial liquid containing positive plasmid into liquid LB containing Amp, culturing until bacterial liquid OD ₆₀₀ Adding IPTG with the final concentration of 1.0mmol/L, 1,2, 4-triazole sodium with the final concentration of 0.17-0.34mmol/L and 4-hydroxy-3-methoxybenzyl alcohol with the final concentration of 1.0-1.45mmol/L into the mixture, and performing induced culture for 4-6h;

the purification method of the MCFV gB recombinant protein comprises the following steps:

a. centrifuging the induced bacterial liquid, collecting precipitate, suspending bacterial body with Tris-HCl buffer solution with pH value of 8.0 and concentration of 50mmol/L, ultrasonically crushing, centrifuging, collecting supernatant, filtering with 0.45 μm filter membrane, adding trimethyl sulfoxide bromide with final concentration of 0.6-0.83mg/mL and 2-sulfapyridine with final concentration of 0.26-0.34 mg/mL;

b. washing Ni-NTA His-Bind nickel ion resin with a balance buffer solution, re-suspending with the balance buffer solution, loading into a chromatographic column, naturally settling, and passing the supernatant in the step a through the column;

c. washing off the foreign proteins with a washing buffer;

d. the target protein is washed off with elution buffer.

2. The method for preparing the MCFV gB recombinant protein according to claim 1, wherein: the recombinant protein is in the form of a soluble protein.