CN113150126A - Rabbit-derived porcine parvovirus 6-type VP2 protein antibody and preparation method thereof - Google Patents

Abstract

The invention discloses a rabbit-derived porcine parvovirus 6-type VP2 protein antibody and a preparation method thereof, wherein 1 pair of specific primers are designed according to a PPV6 epidemic strain genome sequence published by GenBank, a pET30a-PPV6-VP2 recombinant expression vector is constructed, and IPTG inducesExpression, Ni-NTA resin affinity chromatography purification, different gradient urea dialysis renaturation, and mixing with Freund's adjuvant 1:1 uniformly and emulsifying to prepare PPV6 recombinant VP2 protein immunogen. Injecting New Zealand white rabbit with big ear subcutaneously at multiple points via back to prepare hyperimmune serum, adopting saturation (NH4)₂SO₄Antibody was purified by salting out. The rabbit source porcine parvovirus 6-type VP2 protein antibody prepared by the method has good purity, and is sterile, free of mycoplasma and exogenous virus pollution; the specificity is good, and the porcine parvovirus type 6VP2 protein antibody does not contain other porcine common pathogenic antibodies except the porcine parvovirus type 6VP2 protein antibody. The characteristics fully meet the requirements of the positive serum for exogenous virus detection, and have important significance for the healthy development of the veterinary biological product industry.

Description

Rabbit-derived porcine parvovirus 6-type VP2 protein antibody and preparation method thereof

Technical Field

The invention belongs to the field of biological products for livestock, and particularly relates to a preparation method of a rabbit-derived porcine parvovirus 6-type VP2 protein antibody.

Background

Porcine Parvovirus (PPV) belongs to the genus parvovirus of the family parvoviridae, is an autonomously replicating virus and is one of the main pathogens causing sow reproductive disturbance diseases; after the sows are infected with the virus, abortion, mummy fetus, dead fetus and the like can be caused, which causes serious economic loss to the pig industry at home and abroad; mary and Mahncl discovered the virus for the first time in 1966, and then developed to multiple countries and regions in the world; in the 80 s of the 20 th century, porcine parvovirus diseases were found separately in various places of China. At present, with the development of molecular biological diagnostic technology, several novel porcine parvoviruses, such as PPV2, PPV3, PPV4, PPV5 and PPV6, have been discovered, and these several novel PPVs have great differences in genome, especially in VP2 gene. In 2014, Ni et al discovered a novel parvovirus in aborted pig fetuses in China by using a sequence-independent single primer amplification (SISPA) method, named as Port parvovirus type6, namely PPV 6.

PPV is a single stranded linear DNA virus with virions in an icosahedral symmetric structure with a total genome length of about 5kb containing 2 major Open Reading Frames (ORFs), of which ORF1 encodes 3 non-structural proteins NS1, NS2, NS 3; ORF2 encodes 2 structural proteins VP1, VP2, and VP2 can be further hydrolyzed to VP 3. The PPV6 has 20.5-42.6% of gene sequence homology with other parvovirus subfamily members, and is most closely related to PPV 4. The total length of the genome of the PPV6 is about 6136bp, the content of G + C is 46.7-47.1%, the genome structure is similar to that of the traditional parvovirus, the ORF1 codes NS protein consisting of 622 amino acids, and the ORF2 codes VP protein consisting of 1189 amino acids. The VP protein is approximately 132kD in size, larger than most parvoviruses. The main structural protein constituting the nucleocapsid is VP2, the molecular size of which is about 64kD, and which is composed of 579 amino acids, VP2 has good antigenicity, plays an important role in the pathogenicity of viruses, and can induce animal organism to produce protective immune response.

At present, researchers adopt eukaryotic expression systems, baculovirus systems and the like to perform inducible expression on VP2 protein, but the protein expression amount is low, and the culture is inconvenient. The escherichia coli expression vector has unique technical advantages as a very mature expression vector. The monoclonal antibody has only one cloning site, can only recognize a certain specific epitope, and has high specificity and sensitivity. The polyclonal antibody is used as an important immunodiagnosis material, has unique advantages compared with the monoclonal antibody, can identify a plurality of antigen epitopes, and even if a few of the antigen epitopes are destroyed or the antigen conformation is changed, the experimental result is not influenced; and the preparation method is convenient and the cost is low.

Disclosure of Invention

Aiming at the defects pointed out in the background technology, the invention designs 2 specific primers according to the genome sequence of PPV6 epidemic strains (GenBank: KX384821.1, MH820262.1, MH447540.1 and MK378402.1), provides a rabbit source anti-porcine parvovirus type 6VP2 protein antibody and a preparation method thereof, and aims to solve the problems in the prior art in the background technology.

The invention adopts the following technical scheme:

a preparation method of a rabbit-derived anti-porcine parvovirus type 6VP2 protein antibody comprises the following steps:

(1) preparation of immunogens

2 specific primers (P1 and P2) are designed according to the genome sequence of PPV6 epidemic strains (GenBank: KX384821.1, MH820262.1, MH447540.1 and MK378402.1), and PCR identification is carried out on the aborted fetuses of sows collected in the district and the peripheral area; constructing a pET30a-PPV6-VP2 recombinant expression vector by taking the PCR amplified fragment as a target sequence, and carrying out PCR, double digestion and sequencing identification on the positive recombinant plasmid; inducing and expressing the positive clone bacteria for 6 hours at 37 ℃ by 1mmol/LIPTG, ultrasonically crushing the expression bacteria liquid, centrifuging at 12000r/min, collecting supernatant lysate, carrying out Ni-NTA resin affinity chromatography purification, carrying out dialysis renaturation on urea with different gradients, carrying out SDS-PAGE analysis, and carrying out Westernblot identification; the purified renaturation VP2 protein is evenly mixed and emulsified with Freund's adjuvant 1:1 to prepare PPV6 recombinant VP2 protein immunogen.

(2) Preparation of hyperimmune serum

The purified renaturation VP2 protein is taken as immunogen, a new Zealand big ear white rabbit is injected subcutaneously and multiply at the back, the VP2 protein immunogen emulsified by Freund's complete adjuvant is used for the first time, and the immunizing dose is 200 mug/mouse; after the first immunization, 14d, 28d and 42d are changed into VP2 protein immunogen emulsified by Freund incomplete adjuvant for boosting immunization, and the immunization dose is 400 mu g/mouse; and 7d (namely 49d after first immunization) after the fourth immunization, bleeding the carotid artery, centrifuging at 4000r/min to separate serum, and collecting hyperimmune serum with the antibody titer being more than or equal to 1:25600 for later use.

(3) Antibody purification, desalting, concentration

Saturation (NH4)₂SO₄The salting-out method comprises the following steps:

s1, diluting the hyperimmune rabbit serum with normal saline, slowly adding dropwise precooling saturation (NH4) under magnetic stirring₂SO₄Standing the solution until the final concentration is 20%, centrifuging at 3000r/min for 20min, and discarding the precipitate;

s2, slowly adding dropwise precooled saturated (NH) into the supernatant obtained in the step S1 under magnetic stirring₄)₂SO₄Standing the solution until the final concentration is 45%, centrifuging at 3000r/min for 20min, and discarding the precipitate;

s3, adding normal saline into the precipitate obtained in the step S2 for dissolving, slowly dropwise adding precooling saturation (NH) under magnetic stirring₄)₂SO₄Standing the solution to a final concentration of 33%, centrifuging at 3000r/min for 20min, discarding the supernatant,

s4, repeating the step S3 three times;

s5, adding PBS into the precipitate obtained in the step S2 for dissolution, and desalting by ultrafiltration and concentration at 4000r/min for 30 min.

S6, taking 3-4 mL of ultrafiltration dialysate, dropwise adding 1-2 drops of Nashiner reagent, and detecting whether NH exists in the ultrafiltration dialysate₄ ⁺(appearance of brick Red with NH₄ ⁺Present).

(4) Identifying antibody, filtering, sterilizing and packaging.

The purified antibody was identified by SDS-PAGE, Westernblot and IFA, sterilized by filtration through a 0.22 μm filter, and stored at-40 ℃.

Wherein the PCR amplified fragment has a size of 981 bp.

The size of the recombinant VP2 protein is 40kD, and the protein does not have cross reaction with swine fever virus, porcine parvovirus, porcine pseudorabies virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and porcine encephalitis B virus positive serum.

The immune adjuvant is Freund's complete adjuvant or Freund's incomplete adjuvant, and the detection of the antibody titer refers to the detection of the antibody titer of immune rabbit serum by adopting an indirect ELISA method.

The test animals are New Zealand big ear white rabbits with 20 days old, healthy and similar body weight.

The invention discloses a rabbit-derived anti-porcine parvovirus 6-type VP2 protein antibody and a preparation method thereof.A pair of specific primers is designed by an inventor according to a genome sequence of a PPV6 epidemic strain published by GenBank, and a pET30a-PPV6-VP2 recombinant expression vector, IPTG induced expression and Ni-NTA resin are constructedAffinity chromatography purification, dialysis renaturation with different gradient urea, mixing with Freund's adjuvant 1:1, emulsifying, preparing PPV6 recombinant VP2 protein immunogen. Injecting New Zealand white rabbit with big ear subcutaneously at multiple points via back to prepare hyperimmune serum, adopting saturation (NH4)₂SO₄Antibody was purified by salting out. The rabbit source porcine parvovirus 6-type VP2 protein antibody prepared by the method has good purity, and is sterile, free of mycoplasma and exogenous virus pollution; the specificity is good, and the porcine parvovirus type 6VP2 protein antibody does not contain other porcine common pathogenic antibodies except the porcine parvovirus type 6VP2 protein antibody. The characteristics fully meet the requirements of the positive serum for exogenous virus detection, and have important significance for the healthy development of the veterinary biological product industry.

Drawings

FIG. 1 is a diagram showing the results of PCR amplification of the PPV6VP2 gene provided in the examples of the present invention, in which M: DL2000 molecular weight Marker, 1, 2: negative ddH₂O control; 3,4: PCR amplification product of PPV6VP2 gene.

FIG. 2 is a diagram showing the result of PCR amplification of the recombinant plasmid pET30a-PPV6-VP2 provided in the example of the present invention, in which M: DL2000 molecular weight Marker, 1-3: PCR amplification products of the pET30a-PPV6-VP2 recombinant plasmid; 4: negative ddH₂And (4) performing O control.

FIG. 3 is a diagram showing the result of double-restriction enzyme identification of the recombinant plasmid pET30a-PPV6-VP2 provided by the embodiment of the present invention, wherein M1: DL2000 molecular weight Marker; 1: pET30a (+) empty vector; 2: pET30a-PPV6-VP2 recombinant plasmid; m2: DL10000 molecular weight Marker.

FIG. 4 is a SDS-PAGE analysis result of PPV6VP2 protein induced expression and purification provided by the embodiment of the invention, wherein M: protein marker; 1: pET30a (+) empty vector; 2: recombining bacteria before induction; 3: recombining bacteria after induction; 4: cracking and precipitating the induced recombinant bacteria; 5: purified renatured recombinant VP2 protein.

FIG. 5 is a diagram showing the result of Western blot analysis of PPV6VP2 protein, in which 1: pET30a (+) empty vector control; 2: recombinant VP2 protein.

FIG. 6 is a diagram showing the result of SDS-PAGE analysis of the purification of rabbit-derived anti-PPV 6VP2 antibody provided in the examples of the present invention, in which M: protein Marker, 1, 2: PPV6VP2 antibody; 3: a non-immunized rabbit seronegative control.

Fig. 7 is a graph showing IFA analysis results of rabbit-derived anti-PPV 6VP2 antibody provided in the example of the present invention, in which fig. 7A is a non-immune rabbit seronegative control, and fig. 7B is PPV6VP2 antibody.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments.

Materials and methods

1. Material

American type-Porcine Reproductive and Respiratory Syndrome Virus (NA-Porcine Reproductive and Respiratory Syndrome Virus, NA-PRRSV) positive serum, Porcine circovirus type 2 (PCV 2) positive serum were purchased from VMRD company; classical Swine Fever Virus (CSFV) positive serum, Japanese Encephalitis Virus (JEV) positive serum and Porcine pseudorabies virus (PRV) positive serum are prepared and stored by the animal epidemic prevention control center in Kanjiang county.

The clinical samples are 50 aborted fetuses of sows, collected from 4 provinces of different pig farms in the southwest region of China in 2017-year 2020, and stored at-40 ℃.

The small-dose nucleic acid extraction kit is purchased from Boehringer bioengineering (Dalian) Co., Ltd; pET30a (+) vector was purchased from Novagen; the PCR amplification kit is purchased from Beijing Meilaibo medical science and technology Limited; the plasmid extraction kit is purchased from AxyPrep company; isopropyl-beta-D-thiogalactopyranoside (IPTG) and horseradish peroxidase (HRP) labeled rabbit anti-porcine IgG were purchased from SIGMA; Ni-NTA resin was purchased from QIAGEN.

2. Method of producing a composite material

(1) Primer design and Synthesis

The genome sequence of the registered PPV6 epidemic strain in Gen Bank is subjected to multi-sequence alignment by Megalign software to find out a highly conserved sequence, specific primers P1 and P2 are designed by using Primer Premier 6.0 software according to the highly conserved gene sequence of the registered PPV6 (registration numbers: KX384821.1, MH820262.1, MH447540.1 and MK378402.1) in GenBank, the size of an amplified target fragment is expected to be 981bp, and the gene sequences of the primers are as follows:

P1：5'-CAGGATCCAGACCCCTTGGTGCTATT-3', the BamHI cleavage site is underlined

P2：5'-GCCTCGAGCACTCCAGGGTTCCAGAT-3', XhoI cleavage sites are underlined

The primers were committed to be synthesized by Scout Biotechnology, Inc.

(2) Sample processing and nucleic acid extraction

DNA extraction was performed on 50 aborted fetus samples according to the nucleic acid extraction kit.

(3) PCR amplification

P1 and P2 are used as primers, the nucleic acid of the aborted fetus sample is used as a template, the size of a target fragment is 981bp, and the reaction system is as follows: Ex-Taq enzyme 25. mu.L, upstream and downstream primers 1. mu.L each, template 2.5. mu.L, and water to 50. mu.L. The reaction procedure is as follows: pre-denaturation at 94 ℃ for 5 min; 35 cycles of 94 ℃ for 30s, 58 ℃ for 30s and 72 ℃ for 1 min; extension at 72 ℃ for 10 min. 10 μ L of the amplification product was subjected to 1% agarose gel electrophoresis, and the target fragment was recovered by using a DNA gel recovery kit and stored at-20 ℃.

(4) Construction and identification of pET30a-PPV6-VP2 recombinant expression vector

BamHI and XhoI are adopted to carry out enzyme digestion reaction on pET30a (+) vector and DNA gel recovery product respectively, and the reaction system is as follows: the PCR gel recovered product or pET30a (+) vector 20. mu.L, 10 XK Buffer 3. mu.L, BamH I2. mu.L, Xho I2. mu.L, ddH2O 3. mu.L, in a total volume of 30. mu.L. Shaking and mixing, putting into 30 deg.C water bath for 3h, and then 37 deg.C water bath for 3 h. The enzyme digestion products are connected overnight at 16 ℃ by T4 ligase, the connection products are taken to be transformed into BL21(DE3) escherichia coli competent cells, the cells are coated on a LA plate containing kanamycin resistance, the cells are cultured for 12h at 37 ℃, single colony is selected for overnight culture, bacteria are collected by centrifugation at 12000r/min, plasmids are extracted, PCR and double enzyme digestion identification are carried out on the plasmids, the reaction products are electrophoresed in 1% agarose gel, and simultaneously the positive recombinant plasmids are sent to Ducheng Scienke biotechnology Limited company for sequencing.

(5) Recombinant VP2 protein induction expression, purification, concentration and identification

The positive clone bacteria were cultured at 37 ℃ for about 3 hours with shaking to OD₆₀₀When the concentration is 0.6-1.0, adding IPTG with the final concentration of 1mmol/L, inducing at 37 ℃ for 6h, centrifuging at 12000r/min, collecting bacteria, and ddH₂O suspension washing, ultrasonic crushing of the expression bacteria liquid, and centrifuging at 12000r/min to collect supernatant lysate; washing with inclusion body washing Buffer (pH 8.0) and 2M urea for 3 times, centrifuging, collecting precipitate, dissolving the precipitate with Buffer B (pH 8.0), and centrifuging to collect supernatant; the supernatant was bound to Ni-NTA resin at room temperature for 40min, and then the column was washed with Buffer C (pH 6.3), Buffer D (pH 5.9), and Buffer E (pH 4.5), respectively; putting the purified protein solution in 8M urea, 4M urea, 2M urea, 1M urea and PBS solution in sequence for dialysis renaturation, 6h each time, and carrying out ultrafiltration concentration at 5000 r/min; the purified renaturated VP2 protein is analyzed by 12 percent SDS-PAGE, and the size of the protein is 40 kD. Separating the purified recombinant VP2 protein by SDS-PAGE, transferring to a PVDF membrane, and sealing with 5% skimmed milk powder at room temperature for 1 h; adding 1: 100 dilution of PPV6 positive pig serum, room temperature incubation for 1h, PBST buffer washing membrane, adding HRP labeled rabbit anti-pig IgG (1: 5000 dilution), room temperature incubation for 1 h; and (3) washing the membrane by PBST buffer solution, adding HRP-DAB substrate chromogenic reagent, carrying out chromogenic exposure, carrying out Western blot analysis, identifying the reactogenicity of the recombinant VP2 protein, and setting a pET30a (+) empty vector as a negative control.

(6) Preparation of hyperimmune serum

Uniformly mixing and emulsifying the purified renaturated VP2 protein and Freund's adjuvant 1:1 according to the immune dose to prepare immunogen, injecting the new Zealand big ear white rabbits with 20 days old, health and similar body weight subcutaneously and in multiple points at the back, and using the VP2 protein immunogen emulsified by Freund's complete adjuvant for the first time, wherein the immune dose is 200 mu g/rabbit; after the first immunization, 14d, 28d and 42d are changed into VP2 protein immunogen emulsified by Freund incomplete adjuvant, and the second, third and fourth immunization are carried out, wherein the immunization dose is 400 mu g/mouse; 1mL of blood is collected from the ear marginal vein before each immunization, carotid bleeding is performed 7d (namely 49d after first immunization) after the fourth immunization, serum is separated, the specific antibody titer of the VP2 protein in the immune serum is determined by adopting an indirect ELISA method, and high-immune serum with the antibody titer of more than 1:25600 is collected and is used for preparing the VP2 protein antibody.

The indirect ELISA method for immune rabbit serum is as follows: the ELISA method was used to detect the rabbit serum titers at 0, 14, 28, 42 and 49d after each group immunization. Coating the antigen on a 96-well ELISA reaction plate at 100. mu.L/well, and standing overnight at 4 ℃; PBST washing plate for 3 times, sealing with 5% skimmed milk powder at 37 deg.C for 1 h; PBST washing plate 3 times, adding diluted rabbit serum (1: 200, 1: 400, 1: 800, 1: 1600, 1: 3200, 1: 6400, 1: 12800, 1: 25600) at a multiple ratio, 100 μ L/well, and incubating at 37 deg.C for 1h with non-immune rabbit serum as negative control; washing the plate 3 times with PBST, adding HRP-labeled goat anti-rabbit IgG (diluted 1: 6000), incubating at 100 μ L/well for 1h at 37 ℃; washing the PBST for 3 times, adding 100 mu L of TMB substrate color development solution, and incubating for 20min at room temperature in a dark place; 50 μ L of 1.5moL/L H₂SO₄The reaction was terminated and OD was measured₄₅₀The value is obtained. If the hole OD is to be measured₄₅₀The value was 2.1 times that of the negative control well, and the test was positive.

(7) Antibody purification and desalting

Saturation (NH4)₂SO₄The salting-out method comprises the following steps:

s1, diluting the hyperimmune rabbit serum with normal saline, slowly adding dropwise precooling saturation (NH4) under magnetic stirring₂SO₄Standing the solution until the final concentration is 20%, centrifuging at 3000r/min for 20min, and discarding the precipitate;

s2, slowly adding dropwise precooled saturated (NH) into the supernatant obtained in the step S1 under magnetic stirring₄)₂SO₄Standing the solution until the final concentration is 45%, centrifuging at 3000r/min for 20min, and discarding the precipitate;

s3, adding normal saline into the precipitate obtained in the step S2 for dissolving, slowly dropwise adding precooling saturation (NH) under magnetic stirring₄)₂SO₄Standing the solution until the final concentration is 33%, centrifuging at 3000r/min for 20min, and removing the supernatant;

s4, repeating the step S3 three times;

s5, adding PBS into the precipitate obtained in the step S2 for dissolution, and desalting by ultrafiltration and concentration at 4000r/min for 30 min.

S6, taking 3-4 mL of ultrafiltration dialysate, dropwise adding 1-2 drops of Nashiner reagent, and detecting whether NH exists in the ultrafiltration dialysate₄ ⁺(go outBrick red with NH₄ ⁺Present).

(8) Identifying antibody, filtering, sterilizing and packaging.

And (3) identifying the purified antibody by SDS-PAGE and Western blot (see the step 5 for reaction conditions). Simultaneously, inoculating a PPV6 strain separated and identified in a laboratory into PK-15 cells growing into a 70% monolayer, and culturing for 48 h at 37 ℃ in an incubator; fixing the cells with 4% paraformaldehyde at 4 deg.C for 30 min; washing with PBS for 5min for 3 times, sealing with 5% skimmed milk powder at 37 deg.C for 1 hr; the prepared anti-recombinant VP2 protein antibody and non-immune rabbit serum (diluted by 3% BSA 1: 100) are used as primary antibodies, FITC-labeled goat anti-rabbit IgG (diluted by PBST 1: 300) is used as a secondary antibody, incubation is carried out at 37 ℃, PBST is washed, and indirect Immunofluorescence (IFA) identification is carried out. Finally, the prepared antibody is filtered and sterilized by a 0.22 mu m filter membrane, and subpackaged at-40 ℃.

Second, results and analysis

1. PCR amplification and pET30a-PPV6-VP2 recombinant expression vector construction result

The target fragment with the size of 981bp is obtained by amplification by using P1 and P2 as primers and abortive fetal sample nucleic acid as a template, and the result is shown in figure 1 as shown in sequence 1. PCR and double restriction enzyme identification of the pET30a-PPV6-VP2 recombinant expression vector show that target fragments appear at 981bp, and the results are shown in FIGS. 2 and 3. The homology of the sequencing result and NCBI published accession numbers KX384821.1, MH820262.1, MH447540.1 and MK378402.1 is 100 percent, which indicates that the constructed pET30a-PPV6-VP2 recombinant plasmid is correct.

2. Recombinant VP2 protein expression and identification result

The expressed recombinant protein is analyzed by 12% SDS-PAGE and exists in the form of inclusion body, the relative molecular mass is about 40kD, the size is consistent with a theoretical value, the purified recombinant protein has higher purity, the yield can reach 4.1mg/mL, and the result is shown in figure 4. The recombinant VP2 protein reacts specifically with PPV6 positive pig serum, while pET30a empty vector has no specific band, and the result is shown in figure 5, which shows that the recombinant VP2 protein has good reactogenicity.

3. Immune rabbit serum antibody titer

The indirect ELISA detection result shows that 14d after the first immunization, the serum antibody titer of the recombinant VP2 protein is 1: 400, respectively; antibody titers were 1: 6400; after priming 42d, the antibody titer was 1: 25600; antibody titers were 1:25600, results are shown in table 1.

TABLE 1 immune Rabbit serum antibody titers

4. Immune rabbit serum SDS-PAGE and Western blot analysis result

SDS-PAGE analysis showed that the desired band appeared at 40kD, as shown in FIG. 6; the purified immune rabbit serum reacts specifically with the recombinant VP2 protein, while the negative control rabbit serum has no specific band, and the result is shown in 3 in figure 6, which shows that the purified rabbit anti-PPV 6VP2 antibody has good specificity.

5. IFA analysis result of rabbit-derived anti-PPV 6VP2 antibody

The prepared rabbit anti-PPV 6VP2 antibody can specifically react with PPV6 whole virus, while the negative control rabbit serum has no fluorescence signal, and the result is shown in FIG. 7.

The invention discloses a rabbit-derived anti-porcine parvovirus 6-type VP2 protein antibody and a preparation method thereof, wherein an inventor designs 1 pair of specific primers according to a PPV6 epidemic strain genome sequence published by GenBank, constructs a pET30a-PPV6-VP2 recombinant expression vector, performs IPTG induced expression, Ni-NTA resin affinity chromatography purification, different gradient urea dialysis renaturation, and uniformly mixes and emulsifies the recombinant expression vector with Freund's adjuvant 1:1 to prepare PPV6 recombinant VP2 protein immunogen. Injecting New Zealand white rabbit with big ear subcutaneously at multiple points via back to prepare hyperimmune serum, adopting saturation (NH4)₂SO₄Antibody was purified by salting out. The rabbit source porcine parvovirus 6-type VP2 protein antibody prepared by the method has good purity, and is sterile, free of mycoplasma and exogenous virus pollution; the specificity is good, and the porcine parvovirus type 6VP2 protein antibody does not contain other porcine common pathogenic antibodies except the porcine parvovirus type 6VP2 protein antibody. The characteristics fully meet the requirements of the positive serum for exogenous virus detection, and have important significance for the healthy development of the veterinary biological product industry.

Sequence listing

<110> prevention and control center for animal epidemic diseases in Kaijiang county

<120> rabbit-derived porcine parvovirus 6-type VP2 protein antibody and preparation method thereof

<130> 20210419

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 981

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ggatccagac cccttggtgc tattatgatc tgaacgtcat ctcagcacat ttttctcctt 60

ctgcttggca gaggcttttg gaggattatg atgcctttcg acctaaatcc cttaaagtta 120

ccatccagtc tttagttttt aaagatgtct gtcaaggtgc agaaaaacaa actacagttc 180

acgattccca gtcagccacc attgctatct ttgaggataa agactatgac tacccctatg 240

tgatgggagg gggtcaaaaa acagttccgg gtcacttgcc cggtcaacct tataatcttc 300

ccaagtattc ttacagaacc cttggttcag tcaaagaaag taatagggcc agtatgggcg 360

gttcagggta cactttcaaa tccaatcaag atacggaatt gtttctgctt gaaacacatg 420

atgccactct tattcgaggc gggggtactt ttgagcagta ctatgaattt ccaaacgatc 480

ttcctttcga aaatttgact cagtatcctt gggatatccg ccgtcaggat aacccccttt 540

atcagcagag gatcactgtc atgtcaggtt ctgacagaga tacggtaggc attctagatg 600

gagattttta ctctcctttt cggttcaaag gtcatgatag acccgccatg tggctgccag 660

gacagaggtt gattcagggc aaattcatag atacgcaccc aatacccaat acagggagga 720

gtggggttca tcctaatgat tttcacacaa ggggcgatgg tcatggtgac acccatagaa 780

cacatgaaga gaggatctac agtctagata caggtcttgc tgctatgcca cgtgccgctc 840

atagacccac ccttcagccc ggacctagga ctctgtctca tgccgtacgc agacccgatg 900

gttccaccgt ggtcacggct aatgcgtgtg cttacgctta cacccaggag aatcctcatc 960

aggaaccctg gagtgctcga g 981

Claims (6)

1. A preparation method of a rabbit-derived anti-porcine parvovirus type 6VP2 protein antibody is characterized by comprising the following steps:

(1) primer design and Synthesis

(2) Construction and identification of recombinant expression vector

(3) Inducible expression, purification, identification and concentration of recombinant VP2 protein

(4) Preparation of hyperimmune serum

(5) Antibody purification, desalting and identification

(6) Antibody concentration, filtering sterilization and subpackaging.

2. The method of claim 1, wherein: the PCR amplification primers in the step (1) are 1 pair, namely P1 and P2, and the size of the amplified fragment is 981 bp;

the gene sequences of the primers are as follows:

P1：5'-CAGGATCCAGACCCCTTGGTGCTATT-3', the BamH I cleavage site is underlined;

P2：5'-GCCTCGAGCACTCCAGGGTTCCAGAT-3', the Xho I cleavage site is underlined.

3. The method of claim 1, wherein: the process for preparing the porcine parvovirus type 6VP2 protein immunogen comprises the following steps:

(a) constructing a pET30a-PPV6-VP2 recombinant expression vector, and identifying a recombinant plasmid through PCR, double enzyme digestion and sequencing;

(b) inducing and expressing the positive clone bacteria for 6h at 37 ℃ by 1mmol/L IPTG, ultrasonically crushing the expression bacteria liquid, centrifuging at 12000r/min, collecting supernatant lysate, carrying out affinity chromatography and purification on expression protein Ni-NTA resin, carrying out SDS-PAGE analysis, carrying out Westernblot identification, and determining that the size of the recombinant VP2 protein is 40 kD;

(c) dialyzing the purified protein liquid in 8M urea, 4M urea, 2M urea, 1M urea and PBS liquid in sequence, wherein the dialysis time is 6h each time, and performing ultrafiltration concentration at 5000 r/min;

(d) and (3) uniformly mixing and emulsifying the purified renaturated VP2 protein and Freund's adjuvant 1:1 according to the immune dose to prepare the immunogen.

4. The method of claim 1, wherein: the method for preparing the hyperimmune serum in the step (4) comprises the following steps: the purified renaturation VP2 protein is used as immunogen, a new Zealand big ear white rabbit with 20 days old, healthy and similar body weight is injected subcutaneously at multiple points at the back, VP2 protein immunogen emulsified by Freund complete adjuvant is used for the first time, and the immunizing dose is 200 mug/mouse; after the first immunization, 14d, 28d and 42d are changed into VP2 protein immunogen emulsified by Freund incomplete adjuvant, and the second, third and fourth immunization are carried out, wherein the immunization dose is 400 mu g/mouse; bleeding the carotid artery 7d after the fourth immunization, separating serum, measuring the specific antibody titer of the VP2 protein in the immune serum, collecting hyperimmune serum with the antibody titer of more than 1:25600, and preparing the VP2 protein antibody.

5. The method of claim 1, wherein: the antibody purification steps were as follows:

s1, diluting the serum with normal saline, slowly adding dropwise precooling saturation (NH4) under magnetic stirring₂SO₄Standing the solution until the final concentration is 20%, centrifuging at 3000r/min for 20min, and discarding the precipitate;

s2, slowly adding dropwise precooled saturated (NH) into the supernatant obtained in the step S1 under magnetic stirring₄)₂SO₄Standing the solution until the final concentration is 45%, centrifuging at 3000r/min for 20min, and discarding the precipitate;

s3, adding normal saline into the precipitate obtained in the step S2 for dissolving, slowly dropwise adding precooling saturation (NH) under magnetic stirring₄)₂SO₄Standing the solution to a final concentration of 33%, centrifuging at 3000r/min for 20min, discarding the supernatant,

s4, repeating the step S3 three times;

s5, adding PBS into the precipitate obtained in the step S2 for dissolving, and carrying out ultrafiltration concentration at 4000r/min for 30 min.

6. An antibody against porcine parvovirus type 6VP2 protein, produced by the method of any one of claims 1 to 5.

Images