CN112694530A

CN112694530A - Porcine circovirus type 4 Cap protein monoclonal antibody, and preparation method and application thereof

Info

Publication number: CN112694530A
Application number: CN202110317203.3A
Authority: CN
Inventors: 何召庆; 朱晓玮; 唐青海; 周永银; 尹秀凤; 王婷; 高云飞; 顾玉美; 吴晓; 张渊魁
Original assignee: Zhaofenghua Biotechnology Nanjing Co Ltd
Current assignee: Zhaofenghua Biotechnology Nanjing Co Ltd
Priority date: 2021-03-25
Filing date: 2021-03-25
Publication date: 2021-04-23
Also published as: CN113637069A

Abstract

The invention relates to the field of biological vaccines, in particular to a porcine circovirus type 4 Cap protein monoclonal antibody, a preparation method and application thereof, wherein immunogen used for preparing the monoclonal antibody is virus-like particles formed by porcine circovirus type 4 Cap protein; the porcine circovirus type 4 Cap protein comprises 3 core antigen epitopes which are respectively epitope 1: RRRSRWRRKNGIFHARFMREVTLSVSSFST, respectively; epitope 2: NAYYRIRKIKVEFLPLI, respectively; epitope 3: SIQNSNFVQVWTVRFTL, as shown in SEQ ID NO: 3-5. The invention prepares the porcine circovirus type 4 Cap protein monoclonal antibody, establishes a monomolecular imaging enzyme-linked immunosorbent assay iELISA method of PCV4 Cap protein VLP by taking the 6D5 monoclonal antibody as a capture antibody, and has the advantages of high sensitivity, strong specificity, good stability, high flux and the like.

Description

Porcine circovirus type 4 Cap protein monoclonal antibody, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological vaccines, in particular to a porcine circovirus type 4 Cap protein monoclonal antibody and a single-molecule imaging enzyme-linked immunosorbent assay (iELISA) method established by using the antibody and used for antigen quantification.

Background

Porcine Circovirus (PCV) is a non-enveloped circular DNA virus belonging to the genus Circovirus of the family Circovirridae. PCV is a minimal DNA animal virus with a genome of approximately 1.7 kb. Since the discovery of PCV1 in the 70's of the 20 th century, PCV's have been discovered primarily as three types, PCV1, PCV2 and PCV 3. Based on the gene sequence and antigenic differences, PCV1 has been identified as a renal cell contaminant that does not cause clinical disease in pigs. PCV2 and PCV3 infections cause various diseases in pigs and are distributed globally. PCV2 is a major causative agent of many diseases, such as Postweaning Multisystemic Wasting Syndrome (PMWS), porcine dermatitis and nephrotic syndrome (PNDS), reproductive disorders and congenital tremors in piglets, which severely compromise the swine industry. By 2019, only the circoviruses PCV1, PCV2 and PCV33 belong to the circoviridae of known pigs. PCV3 is also closely related to various diseases including postweaning multisystemic wasting syndrome, dermatitis and nephritis syndrome, myocarditis, respiratory syndrome, neurological diseases, and the like. In 2019, PCV4 was first identified in China as a novel circovirus. In 2019, 4 months, a novel CV is identified on a pig with serious clinical symptoms (respiratory tract, enteritis symptom and PDNS) in Hunan, and the correlation with other CVs is greatly different. The viral genome was 1770 nucleotides (nt) in size and was identified as porcine circovirus type 4 (PCV 4).

The PCV4 gene sequence has the highest homology (66.9%) with the mink circovirus, and the genome homology with other porcine circovirus is 43.2% -51.5%. The PCV4 genome contains 12 Open Reading Frames (ORFs), and two major genes predicted include, a replicase (ORF) ((r))rep) Gene (891 nt), encoding a protein consisting of 296 amino acids, and a capsid (cap) Gene (687 nt), encoding a protein consisting of 228 amino acids.

Attempts to isolate PCV4 using both PK-15 and ST cell lines were unsuccessful, while PCV2 could be isolated using the cell culture method described above, indicating that the biological properties of PCV4 are different from PCV 2. In the future, it may be necessary to establish cell lines more sensitive to PCV4 using cell engineering techniques, or to facilitate the in vitro culture of PCV4 by optimization of culture conditions. PCVs are generally difficult to grow in vitro, e.g. PCV2 replicates very slowly, requiring the addition of glucosamine, and infectious clones were also successfully constructed until 2019 for PCV3 and used for pathogenicity studies.

The current epidemiological investigation technology for PCV4 is mainly PCR technology, and real-time fluorescent quantitative PCR is most commonly used. PCV4 clinical sample analysis was performed by SYBR Green-based real-time fluorescent quantitative PCR method, which showed a positive detection rate of 10.71% (18/168) for PCV 4. The TaqMan real-time fluorescent quantitative PCR detection is adopted, and the result shows that the prevalence rate of PCV4 in Hunan province is 12.8%. The highest positive rates were nasal swabs (28.5%) and serum samples (13.4%), indicating a moderate infection rate of PCV4 in the herd in the area. At present, the pathogenicity of PCV1 and PCV2 is well-defined, the clinical manifestations of PCV3 infection are various and are detected even in a herd without obvious clinical symptoms, and PCV4 is detected in both a sick herd and a healthy herd.

Since PCV4 belongs to a new pathogen and is combined with a complex clinical phenomenon caused by PCV4 infection, the diagnosis and vaccine development of porcine circovirus in the future are more complex, PCV4 cannot be cultured in cells, and a pure pathogen culture is difficult to obtain in vitro, so that the development of a more accurate and comprehensive serological detection and antigen detection method and an antigen quantification method is urgent.

Since no serological diagnostic reagent and corresponding method for PCV4 exist at present, an effective serological diagnostic technique is urgently needed. The future vaccine development also needs an antigen quantitative method, so that the invention prepares the porcine circovirus type 4 (PCV 4) Cap protein monoclonal antibody and establishes a single molecule imaging enzyme-linked immunosorbent assay (iELISA) method for PCV4 Cap protein VLP antigen quantification.

Disclosure of Invention

Based on PCV4, the method can not be cultured in cells, pure pathogen culture is difficult to obtain in vitro, and based on the current research and development situations of previous circovirus vaccines and diagnostic products, under the condition of no complete virus, the method for preparing the Cap protein virus-like particle (VLP) vaccine of the circovirus and the corresponding detection and evaluation method are the fastest, the most mature and the most commercial prospect. In addition, the lack of effective serological and pathogenic antigen detection means results in the lack of effective understanding of the pathogenicity and prevalence of PCV 4.

Therefore, the invention aims to provide a porcine circovirus type 4 Cap protein monoclonal antibody, and a preparation method and application thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

a monoclonal antibody of porcine circovirus type 4 Cap protein, wherein the immunogen used for preparing the monoclonal antibody is a virus-like particle formed by the porcine circovirus type 4 Cap protein;

the porcine circovirus type 4 Cap protein comprises 3 core antigen epitopes which are respectively an epitope 1: RRRSRWRRKNGIFHARFMREVTLSVSSFST, as shown in SEQ ID NO: 3 is shown in the specification; epitope 2: NAYYRIRKIKVEFLPLI, as shown in SEQ ID NO: 4 is shown in the specification; epitope 3: SIQNSNFVQVWTVRFTL, as shown in SEQ ID NO: 5, respectively.

Wherein the immunoglobulin class of the monoclonal antibody comprises monoclonal antibodies 3E6, 4a7, and 6D 5; the monoclonal antibody 3E6 is IgG2b subtype, the monoclonal antibodies 4A7 and 6D5 are IgG1 subtype, and the light chain types are kappa.

Wherein the virus-like particle is formed by recombinant porcine circovirus type 4 Cap protein and has a diameter of 10-20 nm.

The preparation method of the porcine circovirus type 4 Cap protein monoclonal antibody comprises the following steps:

(1) immunization procedure

Emulsifying virus-like particles formed by purified porcine circovirus type 4 Cap protein with equivalent volume of Freund's complete adjuvant, and carrying out intraperitoneal multipoint injection on 6-8 weeks-old BALB/c mice, wherein the immunization dose is 100 mu g/mouse; emulsifying virus-like particle protein formed by the purified porcine circovirus type 4 Cap protein and Freund's incomplete adjuvant and then immunizing; 3d before cell fusion, strengthening immunity, and injecting 500 micrograms of purified recombinant porcine circovirus type 4 Cap protein into the abdominal cavity;

(2) hybridoma cell fusion

Fusion of SP2/0 cells with splenocytes using PEG1450, as 1: 5-1: taking splenocytes and SP2/0 myeloma cells according to a proportion of 2, mixing, centrifuging and then removing supernatant; slowly adding 50% PEG1400 for fusion; resuspending the fused cells in 1% HAT selection medium, adding to a 96-well cell culture plate, and incubating at 37 ℃ and 5% CO₂Culturing in an incubator, and observing the growth condition of the cells every day; adopting an indirect ELISA method to screen MAb, selecting hybridoma cells which only have positive reaction with porcine circovirus type 4 Cap protein for cell cloning and purification;

(3) preparation of monoclonal antibody ascites

Taking a healthy BALB/c mouse of 10 weeks old, injecting 0.5mL of sterilized liquid paraffin into the abdominal cavity, and injecting 0.5mL of hybridoma cells 5 multiplied by 10 into the abdominal cavity after 7-14 days⁵～1×10⁶And (4) extracting ascites when the abdomen of the mouse is obviously enlarged, and centrifuging to remove grease and sediment to obtain the monoclonal antibody ascites.

The application of the porcine circovirus type 4 Cap protein monoclonal antibody in the quantification of PCV4 virus-like particles is characterized in that: based on the monoclonal antibody 6D5, a single-molecule imaging enzyme-linked immunosorbent assay iELISA method is established for quantitatively detecting the target protein.

The using method of the core solution of the unimolecular imaging enzyme-linked immunosorbent assay iELISA method comprises the following steps: (1) the working concentration of the streptavidin solution is 0.1-0.3 mg/mL; (2) the working concentration of the biotinylated goat anti-mouse secondary antibody solution is 20-30 ng/mL; (3) the working concentration of the mouse monoclonal antibody 6D5 monoclonal antibody solution for resisting PCV4 Cap protein VLP is 10-30 ng/mL; (4) the working concentration of the polyclonal antibody of the rabbit anti-PCV 4 Cap protein VLP is 20-30 ng/mL; (5) the working concentration of the FITC labeled goat anti-rabbit secondary antibody solution is 0.5-2 ng/mL.

Compared with the prior art, the invention has the beneficial effects that:

the invention is based on clinical pathological materials, obtains the Cap protein of PCV4 by a molecular biological method, and prepares the VLP of PCV4 Cap protein, thereby having important value in the aspects of later vaccine development and diagnostic reagent development.

The invention prepares the porcine circovirus type 4 (PCV 4) Cap protein monoclonal antibody based on the good immunogenicity of PCV4 Cap protein VLP, establishes a single-molecule imaging enzyme-linked immunosorbent assay (iELISA) method of PCV4 Cap protein VLP by taking 6D5 monoclonal antibody as a capture antibody, has the advantages of high sensitivity, strong specificity, good stability, high flux and the like, and particularly can be used for the rapid quantitative detection of PCV4 virus-like particles, and the establishment of the method has important application value for the quality evaluation of PCV4 VLP vaccine, the PCV4 clinical diagnosis and the epidemiological investigation.

Drawings

FIG. 1 shows a specific band obtained by PCR amplification of PCV4 Cap gene; wherein, M: DL2000 marker; 1: PCV4 Cap.

FIG. 2 is an SDS-PAGE identification of recombinant PCV4 Cap protein; wherein, M: protein marker; 1: BL21(DE3) -pET28a supernatant; 2: BL21(DE3) -pET28 a; 3: BL21(DE3) -pET28a-PCV4-Cap supernatant; 4: BL21(DE3) -pET28aPCV4-Cap precipitate.

FIG. 3 is a Virus Like Particle (VLP) formed from PCV4 Cap protein.

FIG. 4 shows the reactivity identification of PCV4-Cap specific monoclonal antibody with PCV4-Cap protein expressed by eukaryotic cells; wherein, A: PK15 cell transfected by an empty vector pcDNA3.1; b: recombinant plasmid pcDNA-PCV4-Cap transfected PK15 cells.

FIG. 5 shows the results of the sensitivity test.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 cloning of PCV4-Cap Gene

(1) Collection and treatment of pathological material and extraction of DNA

The disease material is collected from the spleen of a weaned piglet in a certain pig farm in Jiangsu in 12 months in 2019, 10mL of PBS is added into each 1g of tissue for grinding, the mixture is repeatedly frozen and thawed at minus 20 ℃ and 37 ℃ for 3 times, shaken for 15s, centrifuged at 3000rpm for 2min, and the supernatant is taken and stored at minus 20 ℃ for later use. The extraction of the disease material DNA adopts a blood/cell/tissue genome DNA extraction kit (purchased from Beijing Tiangen Biochemical technology Co., Ltd.) to extract and prepare the DNA, and the DNA is stored at the temperature of minus 20 ℃ for standby.

(2) Primer design and Synthesis

A pair of specific primers F1 and R687 (see table 1) were designed based on a conserved region on the PCV4 Cap nucleic acid sequence published on GenBank (GenBank n 00: MK 986820), as shown in SEQ ID NO: 6-7, the amplified target DNA fragment size is 687 bp.

TABLE 1 primer name, sequence, annealing temperature and amplified fragment length

Primer name	Sequence of	Annealing temperature-^oC	Fragment Length (bp)
				F1	catggatccatgccaatcagatctaggtacagca	60	687
R687	catgtcgacttatccctgtttggggtagttaac

Note: GGATCC asBamHI cleavage site sequence, GTCGAC isSalI enzyme cutting site sequence.

(3) PCR amplification

And (2) carrying out PCR by taking the DNA extracted and prepared in the step (1) as a template, preparing a reaction solution (shown in table 2) in a PCR tube, covering the reaction solution, flicking the finger for 5 times, and carrying out PCR after instantaneous separation. The reaction procedure was as follows: pre-denaturation at 94 ℃ for 2 min; 10s at 98 ℃, 30s at 60 ℃, 2min at 68 ℃ for 30s, and 35 cycles; storing at 68 deg.C for 10min and 4 deg.C.

TABLE 2 PCR reaction System

Reaction components	Volume (μ L)
		pMD19T-PCV4 CAP	2.0
KOD Fx Neo	0.5
		2X Bufffer for KOD Fx Neo	12.5
2mM dNTPs	5.0
		Upstream primer	1.0
Downstream primer	1.0
		ddH₂O	3.0

As a result: it can be seen from FIG. 1 that there is a band between the standard molecular weights of 500-750bp that is amplified to obtain specificity.

Through sequence determination, the PCV4 Cap gene has the full length of 687bp, and the full-length nucleotide sequence is shown as SEQ ID NO: 1, and the coded amino acid sequence is shown as SEQ ID NO: 2, respectively.

Example 2 preparation and characterization of recombinant PCV4-Cap protein and Virus-like particle thereof

(1) Enzyme digestion and purification of PCV4 Cap gene and vector

The PCR-amplified PCV4 Cap gene and its truncated fragment were subjected to agarose gel electrophoresis, photographed, recorded the electrophoresis result, purified with a PCR product clean kit, and subjected to double digestion reaction (see Table 3), while placing the pET28a vector in the same reaction system (see Table 4). The digested PCV4 Cap gene and the empty vector were again purified and concentrated using the PCR clean kit.

TABLE 3 restriction enzyme System for Gene of interest

Components	Volume/. mu.L
		PCR cleaning product	30
BamH I	2
		Sal I	2
10xBuffer	5
		ddH₂O	11

TABLE 4 vector cleavage System

Components	Volume/. mu.L
		Plasmid pET28a	10
BamH I	1
		Sal I	1
10xBuffer	5
		ddH₂O	33

(2) Construction and identification of recombinant expression strain BL21(DE3) -pET28a-PCV4 Cap

The purified and concentrated target gene of step (1) and the vector were added to the PCR tube according to the following reaction system (see Table 5). Flicking the bottom of the tube, and mixing. And (5) performing instantaneous centrifugation to ensure that the pipe wall has no water drops. And (4) placing the obtained product in a low-temperature connector at 16 ℃ for 16-18 h. The next day, 100. mu.L of competent cells BL21(DE3) were taken, the plasmid in the ligation was transformed into competent cells, cultured in non-resistant LB medium for 60min, part of the medium was discarded, and the remaining 200. mu.L was mixed well and divided into 150. mu.L and 50. mu.L portions, which were applied to antibiotic-containing plate medium, respectively. Culturing in a constant temperature incubator for 16-10 hr, selecting single colony, performing amplification culture under the same conditions, and extracting plasmid. The prepared plasmids were loaded according to the reaction systems in the following table (see table 6), the bottom of the tube was flicked 6-8 times to mix the reactants uniformly, centrifuged instantaneously, and digested in a 37 ℃ low temperature ligase for 15 min. And (3) uniformly mixing 5 mu L of each double enzyme digestion product with loading Buffer, carrying out agarose electrophoresis, sending the recombinant plasmid with positive enzyme digestion result to China Dagen sequencing, and analyzing the spliced sequence by using DNAStar software.

TABLE 5 Carrier and purified PCV4 CAP ligation System

Components	Volume/. mu.L
		Vector pET28a	1.0
Purification of Cap	4.0
		Solution I	5.0

TABLE 6 recombinant plasmid double restriction enzyme identification System

Components	Volume/. mu.L
		Recombinant plasmid DNA	7.0
Sal Ⅰ	0.5
		BamHⅠ	0.5
10×Buffer	2.0
		ddH₂O	10.0

(3) Induced expression of recombinant expression strain BL21(DE3) -pET28a-PCV4-Cap and SDS-PAGE detection of target protein

Small-scale expression: 10 mu L of positive recombinant expression strain and corresponding empty vector strain liquid are inoculated into 2mL of LB culture medium (the final concentration of antibiotics is 100 mu g^-1). The next day, 0.8mL of the bacterial suspension was inoculated to 4mL of a suspension containing antibiotics (the final concentrations of the antibiotics below were all 100 μ g.mL)^-1) The liquid LB medium of (1) is placed at 220 r.min^-1Shaking-culturing at 37 deg.C for 2.5 h. Adding IPTG (final concentration of 1 mmol. L.) into group 1^-1) The rest culture medium is not added with inducer IPTG as control group, and is placed at 37 deg.C for 220 r.min^-1Was induced in a shaker for 4 h. Extracting the protein of the strain by using a bacterial protein extraction kit, and storing at-80 ℃. And taking 20 muL samples of the protein samples respectively, and carrying out polyacrylamide gel electrophoresis. Taking out the electrophoresis tank after electrophoresis, discarding the concentrated gel, cutting the separation gel into appropriate size, placing in a staining dish containing Coomassie brilliant blue R250, and standing for 45 r.min^-1Dyeing for 3h, washing off the dye solution with water after successful dyeing, adding a decolorizing solution, and carrying out dyeing at 45 r.min^-1Eluting for 3h, and replacing the decolorizing solution every 1 h. Until the band is clearly visible. And (5) placing the film on a film observer, and recording the experimental result.

And (3) large-scale expression: respectively adding 50 muL of the preserved BL21(DE3) -pET28a-PCV4-Cap recombinant strain bacterial liquid into 50mL of 3 bottles of liquid LB culture medium added with 50 muL Kana, and putting the culture medium into a constant temperature oscillator, wherein the set parameters are 37 ℃ and 230 r.min^-1And culturing overnight for about 16-20 h. The next day, respectively pouring the activated 3 bottles of 50mL bacterial liquid into 200mL liquid LB culture medium added with 200 muL Kana, and performing 230 r.min at 37 DEG C^-1The constant temperature oscillator of (1) oscillates for 2 h. To each Erlenmeyer flask was added a final concentration of1mmoL•L^-1The IPTG was placed in a constant temperature oscillator with the set parameters of 37 ℃ and 230 r.min^-1And inducing expression for 6 h. Concentrating 750mL bacterial liquid and 1 centrifugal tube of 100mL by centrifugation method, wherein the centrifugation parameter is 8000 r.min^-1And 10 min. 4 ℃ is prepared. 30mL of sterile PBS Buffer was added into the tube, and the cells were blown up using a pipette gun to suspend the cells at 8000 r.min^-1Centrifuge for 10min and remove the supernatant. 20mL of sterile PBS Buffer was added to each centrifuge tube, and the cells were suspended by pipetting with a pipette and sonicated on ice for 5s at 5s intervals for a total of 1 h. The ultrasonic cracking product is 10000 r.min^-1Centrifuge for 15min at 4 ℃ and store the primary ultrasound supernatant in 1 100mL centrifuge tube. The precipitate after the first sonication was washed with 20mL of inclusion body washing buffer containing TritonX-100 at a final concentration of 1%, and the cells were suspended by pipetting with a pipette and sonicated on ice for 5s at 5s intervals for a total of 1 h. The ultrasonic cracking product is 10000 r.min^-1Centrifuging for 15min at 4 ℃, storing the secondary ultrasonic supernatant in 1 centrifugal tube of 100mL, and obtaining the remaining precipitate as the inclusion body protein to be collected. Dissolving the obtained inclusion body protein with a proper amount of inclusion body dissolving solution, performing ultrasonic treatment for 10min to completely dissolve the inclusion body protein until the liquid is clear, and storing at-20 ℃. And taking 20 muL samples of the protein samples respectively, and carrying out polyacrylamide gel electrophoresis. Taking out the electrophoresis tank after electrophoresis, discarding the concentrated gel, cutting the separation gel into appropriate size, placing in a staining dish containing Coomassie brilliant blue R250, and standing for 45 r.min^-1Dyeing for 3h, washing off the dye solution with water after successful dyeing, adding a decolorizing solution, and carrying out dyeing at 45 r.min^-1Eluting for 3h, and replacing the decolorizing solution every 1 h. Until the band is clearly visible. And (5) placing the film on a film observer, and recording the experimental result.

As can be seen from FIG. 2, compared with the empty vector strain BL21(DE3) -pET28a (lanes 1 and 2), the recombinant expression strain BL21(DE3) -pET28a-PCV4-Cap has a distinct band between 26 and 34kDa, indicating that the recombinant PCV4 Cap protein is successfully expressed.

(4) Affinity chromatography purification of proteins

The test was carried out using a kit of one-stop His-labeled protein micro-purification kit (CAT #: 100102A-20) from Beijing Tianengze Gene technology Co., Ltd.

(5) Identification of Virus-like particles

Whether VLPs are formed by the recombinant PCV4 Cap protein was observed by Transmission Electron Microscopy (TEM) and a photograph was taken. The results in FIG. 3 show that the purified PCV4 Cap protein forms virus-like particles with diameters between 10 and 20nm, and the particles are uniform in size and high in purity.

Example 3 preparation and Activity identification of monoclonal antibody (MAb) of PCV4-Cap protein

(1) Immunization procedure

Emulsifying virus-like particles formed by purified PCV4 Cap protein with equivalent volume of Freund complete adjuvant, and carrying out intraperitoneal multi-point injection on 6-8 week-old BALB/c mice, wherein the immunization dose is about 100 mu g/mouse; 14d, emulsifying the virus-like particle protein formed by the purified PCV4 Cap protein with Freund incomplete adjuvant, and then immunizing; cell fusion pre-3 d boost, intraperitoneal injection of 500. mu.g of purified recombinant protein PCV4 Cap.

(2) Hybridoma cell fusion

Fusion of SP2/0 cells with splenocytes using PEG1450, as 1: 5-1: splenocytes were taken at ratio 2 and mixed with SP2/0 myeloma cells, centrifuged and the supernatant discarded. Fusion was performed by slowly adding 50% PEG 1400. Resuspending the fused cells in 1% HAT selection medium, adding to a 96-well cell culture plate, and incubating at 37 ℃ and 5% CO₂Culturing in an incubator, and observing the growth condition of the cells every day. MAb screening is carried out by adopting an indirect ELISA method, and hybridoma cells which only positively react with PCV4 Cap protein are selected for cell cloning and purification.

(3) Preparation of monoclonal antibody ascites

Taking a healthy BALB/c mouse of 10 weeks old, injecting 0.5mL of sterilized liquid paraffin into the abdominal cavity, and injecting 0.5mL of hybridoma cells (5 multiplied by 10) into the abdominal cavity after 7-14 days⁵～1×10⁶One mouse or only mouse), when the abdomen of the mouse is obviously enlarged, extracting ascites, centrifuging to remove grease and precipitate to obtain the monoclonal antibody ascites, and freezing and storing at-80 ℃ for later use.

(4) Identification of MAb against PCV4 Cap

With mouse sheetClone antibody Ig class/subclass identification kit (Pierce)^@Rapid ELISA Mouse mAb Isotyping Kit) identifies the immunoglobulin class of the monoclonal antibody. The identification result shows that 3E6 is IgG2b subtype, the rest 2 monoclonal antibodies (4A 7 and 6D 5) are all IgG1 subtype, and the light chain types are all K.

(5) Construction of PCV4 Cap eukaryotic expression recombinant plasmid and immunoreaction of expression protein and MAb

Cloning PCV4 Cap gene into pcDNA3.1 vector to construct recombinant plasmid pcDNA-PCV4-Cap, transfecting pig kidney cell (PK 15 cell), comprising the following steps:

5.1 preparation of cells:

adherent porcine kidney cells (PK 15 cells): 1 day before transfection, cells were seeded in complete medium to allow full growth and cell density at transfection would reach 50-70% confluency.

5.2 preparation of transfection reagents: the Xfet Polymer was fully vortexed.

5.3 dilution of plasmid DNA: in a sterilized 1.5mL EP tube, 5 μ g of plasmid DNA (pcDNA-PCV 4-Cap) was diluted with Xfect Reaction Buffer to a final volume of 100 μ L. Vortex at high speed for 5sec to mix well.

Note that: firstly, adding plasmids into an Xfet Reaction Buffer before adding Xfet Polymer; the volume of the Xfect Reaction Buffer in the solution is not less than 50 mu L; and thirdly, the plasmid amount used in one well of each 6-well plate is not less than 2.5 mug and can be 5-7.5 mug.

5.4 preparation of plasmid-Polymer mixture: to the diluted plasmid DNA, 1.5. mu.L of Xfect Polymer was added. High velocity vortex was oscillated for 10 sec.

Note that: the ratio of polymer to DNA was always kept constant. 0.3 μ L Xfect Polymer was used per 1 μ g plasmid DNA.

5.5 incubation: incubation was performed at room temperature for 10 minutes to allow nanoparticle complexes to form.

Note that: it is recommended that the residence time of the Xfect polymer in aqueous solution not exceed 30 min.

5.6 instantaneous separation: the tube was centrifuged instantaneously for 1sec to collect the contents of the bottom of the tube.

5.7 transfection: and dropwise adding 100 mu L of nanoparticle composite solution into a cell culture medium. Gently shake the plate back and forth and mix well.

5.8 culture: incubate at 37 ℃ for 4h, or overnight.

5.9 liquid change: the nanoparticle complexes were aspirated from the cells with a pipette gun, 2mL of fresh complete medium was added, and the plate was returned to the 37 ℃ incubator until the time of analysis. The peak of protein expression is usually about 48 h.

5.10 blank control: the transfection procedure was carried out using cells transfected with the empty vector (pcDNA3.1) as a control.

At 72h after transfection, the medium was discarded, washed 3 times with PBS pH7.4, 30% acetone-PBS was added, fixed for 15min, the fixative discarded, vacuum dried for 30min, 200. mu.L of 1:200 fold diluted PCV4 Cap protein mAb was added, 1h at 37 ℃. PBS was washed 3 times. Add 200 μ L1: goat anti-mouse HRP-IgG enzyme-labeled secondary antibody diluted by 5000 times is washed for 3 times at 37 ℃ for 1h by PBS, and 100 mu L of AEC substrate developing solution is added for 15min at 37 ℃. Discarding the reaction solution, ddH₂Wash 1 time with O and observe the result under microscope. As can be seen from FIG. 4, the prepared monoclonal antibody has specific nuclear staining reaction with the recombinant plasmid pcDNA-PCV4-Cap transfected cells, and has no specific staining reaction with the empty vector pcDNA3.1 transfected cells, which indicates that the prepared Mab has good immunogenicity.

Example 4 identification of epitopes of PCV4 Cap protein

(1) Preliminary identification of PCV4 Cap protein epitope

Detection by an indirect ELISA method: artificially synthesizing a truncated peptide segment covering the PCV4 Cap protein amino acid sequence, coating the artificially synthesized peptide segment, coating 50 ng/hole into an ELISA plate, and detecting by taking hybridoma cell supernatant as a primary antibody and goat anti-mouse HRP-IgG as a secondary antibody. The results identified 3 epitopes, specific epitope sequences as follows:

epitope 1 (SEQ ID NO: 3): RRRSRWRRKNGIFHARFMREVTLSVSSFST

Epitope 2 (SEQ ID NO: 4): NAYYRIRKIKVEFLPLI

Epitope 3 (SEQ ID NO: 5): SIQNSNFVQVWTVRFTL

Example 5 use of monoclonal antibodies in quantification of PCV4 virus-like particles (VLPs)

The prepared monoclonal antibody is utilized to establish a single-molecule imaging enzyme-linked immunosorbent assay (iELISA) method for quantitatively detecting the target protein.

The detection of target protein is a key step of molecular biology research, and has important significance in the detection of pathogeny and disease. The existing relatively mature protein quantitative detection methods comprise Western blotting, colloidal gold test paper, enzyme-linked immunosorbent assay (ELISA) and the like, and are widely applied. Western blotting has high sensitivity and is commonly used for detecting trace proteins, however, sample pretreatment and measurement operations are complex, and the detection time is often more than 5 hours.

The colloidal gold test strip is simple and rapid in target protein detection, and even the result can be directly observed by naked eyes, but the sensitivity is often poor, so that the detection result is qualitative rather than quantitative.

ELISA is another tool for detecting a protein of interest, relying on high selectivity of the capture antibody. Conventional ELISA methods typically require a long time to be spent in sample preparation, and can only detect the target protein semi-quantitatively. However, the ELISA procedures developed at present are rather fast, can detect the target protein even within a few minutes, and can also achieve quantitative detection with high precision using a compact detection device and advanced detection algorithms.

In order to realize the quantitative detection of protein without nanoparticles and complex micropore arrays, the invention provides a single-molecule imaging enzyme-linked immunosorbent assay (iELISA) method of PCV4 VLP, which comprises the following specific operations:

(1) after the glass slide was mounted, the cover plate was cleaned and aminated and pinned to the glass surface.

(2) 0.1-0.3mg/mL streptavidin solution was prepared in T50 buffer, then 10. mu.L of streptavidin-T50 mixture was injected into the chamber, incubated at room temperature for 5min, washed 2 times for 30 sec/time with 20. mu.L T50 buffer, and excess streptavidin was removed. The T50 buffer was prepared by mixing 10mM Tris-HCl (pH = 8.0) and 50mM NaCl.

(3) Adding mixed 10 μ L of biotinylated goat anti-mouse secondary antibody (solution concentration of 20-30 ng/mL), incubating at room temperature for 10min, washing with 20 μ L of T50 buffer solution for 2 times (30 sec/time), and removing excess reagent;

(4) adding 10 μ L of mouse monoclonal antibody (solution concentration is 10-30 ng/mL) against PCV4 Cap protein VLP, incubating for 10min at room temperature, washing for 2 times, 30 sec/time, using 20 μ L of T50 buffer solution, and removing excess reagent;

(5) adding 10 μ L of PCV4 Cap protein VLP, incubating at room temperature for 10min, washing 2 times for 30 sec/time with 20 μ L of T50 buffer solution, and removing excess reagent;

(6) adding 10 μ L polyclonal antibody (concentration of 20-30 ng/mL) of rabbit anti-PCV 4 Cap protein VLP, incubating at room temperature for 10min, washing with 20 μ L T50 buffer solution for 2 times, 30 sec/time, and removing excess reagent;

(7) adding 10 μ L FITC-labeled goat anti-rabbit secondary antibody solution (concentration of 0.5-2 ng/mL), incubating at room temperature for 10min, washing with 20 μ L T50 buffer solution for 2 times (30 sec/time), and removing excess reagent;

(8) finally, using a TIRF microscope, VLPs of the target PCV4 Cap protein were detected after excitation of FITC with light at 488nm wavelength.

The results of the preliminary experiments on the effectiveness of the three monoclonal antibodies according to the basic steps show that the stability and sensitivity of 3E6 and 4A7 are lower than those of the 6D5 monoclonal antibody, and the concentration range of the 6D5 monoclonal antibody is as follows: 10-30 ng/mL. Thus, 6D5 monoclonal antibody is determined to be used for further evaluation of specificity, sensitivity and stability tests.

(9) And (3) specificity test: the results show that 6D5 monoclonal antibody can specifically recognize VLP of PCV4 Cap protein, and has no cross reaction with VLP of Cap protein of Porcine Circovirus (PCV) type 1, 2a/2b and 3 and VLP of porcine parvovirus VP2, which indicates that the specificity is good (as shown in FIG. 5).

(10) And (3) sensitivity test: a sensitivity control test is carried out by using a traditional ELISA (a method known by the public and not repeatedly described) as a control and a single-molecule imaging enzyme-linked immunosorbent assay (iELISA) of the PCV4 Cap protein VLP, and the result shows that the detection limit value of the single-molecule imaging enzyme-linked immunosorbent assay (iELISA) of the PCV4 Cap protein VLP is 5ng/mL, while the detection limit value of the PCV4 Cap protein VLP of the traditional ELISA is 450ng/mL, which indicates that the sensitivity of the single-molecule imaging enzyme-linked immunosorbent assay (iELISA) of the PCV4 Cap protein VLP established by the invention is 90 times that of the traditional ELISA method.

(11) And (3) stability test: the reagents were prepared according to the procedure, stored at-20 deg.C, 4 deg.C, 25 deg.C and 37 deg.C, and tested every 7d sample to evaluate the stability, and the results showed that all reagents had good stability at 4 deg.C and below for 6 months, at 25 deg.C and at 37 deg.C for 4 d. The method is demonstrated for storage stability at 4 ℃.6 batches were prepared in the laboratory, stored at 4 ℃ and tested every 7 days, and the results show that the consistency between different batches is good, the difference between batches is not obvious, and the stability is good.

Therefore, the single-molecule imaging enzyme-linked immunosorbent assay (iELISA) of the PCV4 Cap protein VLP with the 6D5 monoclonal antibody as the capture antibody can effectively and rapidly quantify the PCV4 Cap protein VLP, and has important value for later-stage quantitative development of PCV4 vaccine antigen.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

<110> Mtoyowa Biotechnology (Nanjing) Ltd

<120> porcine circovirus type 4 Cap protein monoclonal antibody, and preparation method and application thereof

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 687

<212> DNA

<213> PCV4-Cap gene (Artificial Sequence)

<400> 1

atgccaatca gatctaggta cagcagacgg aggcggaacc ggcggaacca gcgcaggcgg 60

agactgtggc cccgggccaa taggcggaga tcccggtgga gaaggaagaa cggaattttc 120

catgcgcgct tcatgaggga ggtgactctc agcgtgtcaa gcttttccac gccctcttgg 180

aacgttggac attacgattt caaactgaag gactttatcc caaaaggacc gggaacgatc 240

gtaaaccttt acagcctccc aaatgcatat taccggatca gaaagatcaa agtcgaattt 300

ctgccactaa ttggcattaa cagtaatagg acttactcta gcactgctat acaactggat 360

ggagactatg tgggggaagg gaaaaaccaa acttatgatg tcctggcaaa ccacagcagc 420

aggcatggtt tcaccaatat tgctagacac agtcgctatt tcactccaaa accccaagac 480

ccatccgggg aaacccacac cctccacttc cagcctaaca acaaaagaaa ccaatggtgg 540

atcagcatgg cggaccagga cctagtccat catggcctcc aatacagtat acaaaattcc 600

aactttgtgc aggtgtggac agtgagattt actttgtatg tgcaattcag agaatttgat 660

cttgttaact accccaaaca gggataa 687

<210> 2

<211> 228

<212> PRT

<213> PCV4 Cap protein (Artificial Sequence)

<400> 2

Met Pro Ile Arg Ser Arg Tyr Ser Arg Arg Arg Arg Asn Arg Arg Asn

1 5 10 15

Gln Arg Arg Arg Arg Leu Trp Pro Arg Ala Asn Arg Arg Arg Ser Arg

20 25 30

Trp Arg Arg Lys Asn Gly Ile Phe His Ala Arg Phe Met Arg Glu Val

35 40 45

Thr Leu Ser Val Ser Ser Phe Ser Thr Pro Ser Trp Asn Val Gly His

50 55 60

Tyr Asp Phe Lys Leu Lys Asp Phe Ile Pro Lys Gly Pro Gly Thr Ile

65 70 75 80

Val Asn Leu Tyr Ser Leu Pro Asn Ala Tyr Tyr Arg Ile Arg Lys Ile

85 90 95

Lys Val Glu Phe Leu Pro Leu Ile Gly Ile Asn Ser Asn Arg Thr Tyr

100 105 110

Ser Ser Thr Ala Ile Gln Leu Asp Gly Asp Tyr Val Gly Glu Gly Lys

115 120 125

Asn Gln Thr Tyr Asp Val Leu Ala Asn His Ser Ser Arg His Gly Phe

130 135 140

Thr Asn Ile Ala Arg His Ser Arg Tyr Phe Thr Pro Lys Pro Gln Asp

145 150 155 160

Pro Ser Gly Glu Thr His Thr Leu His Phe Gln Pro Asn Asn Lys Arg

165 170 175

Asn Gln Trp Trp Ile Ser Met Ala Asp Gln Asp Leu Val His His Gly

180 185 190

Leu Gln Tyr Ser Ile Gln Asn Ser Asn Phe Val Gln Val Trp Thr Val

195 200 205

Arg Phe Thr Leu Tyr Val Gln Phe Arg Glu Phe Asp Leu Val Asn Tyr

210 215 220

Pro Lys Gln Gly

225

<210> 3

<211> 30

<212> PRT

<213> epitope 1(Artificial Sequence)

<400> 3

Arg Arg Arg Ser Arg Trp Arg Arg Lys Asn Gly Ile Phe His Ala Arg

1 5 10 15

Phe Met Arg Glu Val Thr Leu Ser Val Ser Ser Phe Ser Thr

20 25 30

<210> 4

<211> 17

<212> PRT

<213> epitope 2(Artificial Sequence)

<400> 4

Asn Ala Tyr Tyr Arg Ile Arg Lys Ile Lys Val Glu Phe Leu Pro Leu

1 5 10 15

Ile

<210> 5

<211> 17

<212> PRT

<213> epitope 3(Artificial Sequence)

<400> 5

Ser Ile Gln Asn Ser Asn Phe Val Gln Val Trp Thr Val Arg Phe Thr

1 5 10 15

Leu

<210> 6

<211> 34

<212> DNA

<213> primer F1(Artificial Sequence)

<400> 6

catggatcca tgccaatcag atctaggtac agca 34

<210> 7

<211> 33

<212> DNA

<213> primer R687(Artificial Sequence)

<400> 7

catgtcgact tatccctgtt tggggtagtt aac 33

Claims

1. A monoclonal antibody of Cap protein of porcine circovirus type 4 is characterized in that: the immunogen used for preparing the monoclonal antibody is a virus-like particle formed by porcine circovirus type 4 Cap protein;

2. The porcine circovirus type 4 Cap protein monoclonal antibody of claim 1, which is characterized in that: the immunoglobulin types of the monoclonal antibodies include monoclonal antibodies 3E6, 4a7, and 6D 5; the monoclonal antibody 3E6 is IgG2b subtype, the monoclonal antibodies 4A7 and 6D5 are IgG1 subtype, and the light chain types are kappa.

3. The porcine circovirus type 4 Cap protein monoclonal antibody of claim 1, which is characterized in that: the virus-like particle is formed by recombinant porcine circovirus type 4 Cap protein and has a diameter of 10-20 nm.

4. The method for preparing the porcine circovirus type 4 Cap protein monoclonal antibody of any one of claims 1 to 3, which is characterized by comprising the following steps:

(1) immunization procedure

(2) hybridoma cell fusion

(3) preparation of monoclonal antibody ascites

5. The use of the monoclonal antibody against Cap protein of porcine circovirus type 4 according to any one of claims 1 to 3 for quantification of PCV4 virus-like particles, wherein the monoclonal antibody comprises: based on the monoclonal antibody 6D5, a single-molecule imaging enzyme-linked immunosorbent assay iELISA method is established for quantitatively detecting the target protein.

6. The application of the porcine circovirus type 4 Cap protein monoclonal antibody in PCV4 virus-like particle quantification according to claim 5, wherein the core solution of the single molecule imaging ELISA is used by the method comprising the following steps: (1) the working concentration of the streptavidin solution is 0.1-0.3 mg/mL; (2) the working concentration of the biotinylated goat anti-mouse secondary antibody solution is 20-30 ng/mL; (3) the working concentration of the mouse monoclonal antibody 6D5 monoclonal antibody solution for resisting PCV4 Cap protein VLP is 10-30 ng/mL; (4) the working concentration of the polyclonal antibody of the rabbit anti-PCV 4 Cap protein VLP is 20-30 ng/mL; (5) the working concentration of the FITC labeled goat anti-rabbit secondary antibody solution is 0.5-2 ng/mL.