CN108752471B - Preparation method and application of anti-PCV 2 monoclonal antibody - Google Patents

Abstract

The invention discloses a preparation method and application of an anti-PCV 2 monoclonal antibody. The DNA sequence and amino acid sequence of the anti-PCV 2 monoclonal antibody and its heavy chain variable region and light chain variable region are disclosed. The preparation method of the anti-PCV 2 monoclonal antibody is designed. The monoclonal antibody is applied to an antigen/antibody detection kit, an antigen/antibody immunochromatography test paper and an immunoaffinity column for neutralizing PCV2 or PCV2 Cap by IFA and IPMA. The monoclonal antibody can simultaneously recognize two main epidemic subtypes, namely PCV2a and PCV2b, has good reactogenicity with PCV2 whole virus and PCV2 Cap protein, and has no cross reaction with PCV1, PCV3 subtype and swine-derived virus; lays a foundation for the research of PCV2 etiology and pathogenesis and the clinical detection research of PCV2 etiology.

Description

Preparation method and application of anti-PCV 2 monoclonal antibody

Technical Field

The invention relates to the technical field of biological immunity, in particular to a preparation method and application of an anti-PCV 2 monoclonal antibody.

Background

Porcine Circovirus (PCV) belongs to members of the Circovirus family (Circovirus) and the Circovirus genus (Circovirus), is the smallest DNA virus discovered so far, causes Porcine infectious diseases widely existing in global swinery, and is an internationally recognized important immunosuppressive pathogen which jeopardizes the large-scale pig industry of all countries in the world.

Among them, PCV2 is a major pathogen causing diseases such as PMWS, Porcine Dermatitis and Nephrotic Syndrome (PDNS), Porcine respiratory disease syndrome (PRDC), etc., resulting in severe losses in the swine industry. At present, the main epidemic strain in China is PCV2, PCV2 infection causes the immunity of swinery to be reduced, and the subsequent mixed or secondary infection of other various pathogens brings serious threats to the pig industry.

The genome of PCV2 includes two major Open Reading Frames (ORFs). ORF1 encodes two replication-related proteins (Rep and Rep') that are involved in the replication process of PCV 2. ORF2 encodes the viral structural protein Cap protein, which forms the capsid of the virus by multimeric binding. The Cap protein is used as the only structural protein of the porcine circovirus, has high immunogenicity, and is a main target for vaccine development and epidemic disease detection.

The current laboratory diagnosis method for PCV2 mainly comprises 3 major categories of virus separation and identification technology, molecular biological diagnosis method and immunological diagnosis method.

In the process of implementing the invention, the inventor finds that at least the following problems exist in the prior art:

the virus isolation and identification are the most accurate method for diagnosing PCV2 infection, but PCV2 is propagated in cells and does not produce obvious cytopathic effect, and other technologies are needed for subsequent identification, the whole process is complicated in technology, needs to be operated by professionals, is time-consuming and labor-consuming, and is not suitable for rapid disease diagnosis;

the molecular biological diagnosis method mainly comprises Polymerase Chain Reaction (PCR) detection technology, gene chip detection technology, nucleic acid hybridization detection technology and the like. With the continuous development of molecular biology, PCR is widely applied to detection of disease pathogens in laboratories due to its advantages of rapidness, high sensitivity, etc., but its operation is time-consuming and labor-consuming, and has high requirements for technology and equipment, so it cannot be popularized and applied in actual production and cultivation.

Commonly used immunological diagnostic methods mainly include Immunohistochemistry (IHC), IPMA, IFA, enzyme linked immunosorbent assay (ELISA), and immunochromatographic strip rapid test (ILFST). The immunological detection methods have the advantages of high sensitivity and specificity, good repeatability and the like, and have high practicability in epidemiological general survey and vaccine immune effect evaluation.

The monoclonal antibody is a core reagent for detecting pathogen by using an immunological detection method, and the specificity and the affinity of the monoclonal antibody are important factors for determining the specificity and the sensitivity of the immunological detection method, so that the key for establishing the pathogenic immunological detection method is to obtain the specific antibody aiming at a detection target. With the large-area usage of porcine circovirus vaccines, the serological detection data no longer reflect the infection status of porcine circovirus, and therefore, the etiologic detection becomes more and more important.

However, there is a need to prepare a PCV2 monoclonal antibody that has no cross-reaction with PCV1, PCV3 and other porcine viruses and can be used in multiple immunoassays simultaneously, and further determine the heavy chain variable region sequence and the light chain variable region sequence thereof to modify the antibody variable region sequences to prepare genetically engineered antibodies in different combinations, so as to further improve the specificity and affinity of the antibodies.

Disclosure of Invention

The invention aims to solve the technical problem of providing a preparation method and application of an anti-PCV 2 monoclonal antibody.

In order to solve the technical problems, the invention adopts the following technical scheme:

screening to obtain an anti-PCV 2 monoclonal antibody, wherein the DNA sequence of the heavy chain variable region of the monoclonal antibody is the sequence shown in SEQ ID NO. 1; the heavy chain variable region amino acid sequence is the sequence shown as SEQ ID NO.2, or the sequence of active fragment or conservative variant is obtained by adding, deleting and replacing one or more amino acids on the basis of the SEQ ID NO.2 sequence; and/or

The light chain variable region DNA sequence is shown as SEQ ID NO. 3; the light chain variable region amino acid sequence is shown as SEQ ID NO.4, or the sequence of active fragment or conservative variant is obtained by adding, deleting and replacing one or more amino acids on the basis of the SEQ ID NO.4 sequence.

Preferably, the light chain type of the monoclonal antibody is Kappa and the subtype is IgG2 a.

Preferably, the ELISA titer of the monoclonal antibody is not less than 1: 2.56 × 10⁵。

Preferably, the IPMA titer of the monoclonal antibody is 1: 8 × 10⁴。

Preferably, the monoclonal antibody has an affinity of not less than 1.26 × 10^-10mol/L。

The preparation method of the anti-PCV 2 monoclonal antibody comprises the following steps:

(1) immunizing mice with a subunit vaccine of PCV2a Cap protein;

(2) fusing the immune spleen cells of the mice and myeloma cells of the mice to obtain hybridoma cells;

(3) obtaining positive hybridoma cells by adopting an IPMA detection combination and multi-round subcloning method;

(4) extracting positive monoclonal hybridoma cell strain RNA, performing reverse transcription to obtain cDNA, and performing PCR amplification to obtain heavy chain variable region sequence and light chain variable region sequence of monoclonal antibody;

(5) cloning and culturing the positive clone to obtain a PCV2 monoclonal antibody hybridoma cell strain;

(6) and injecting the hybridoma cell strain into the abdominal cavity of a mouse to produce the monoclonal antibody.

The anti-PCV 2 monoclonal antibody is applied to an antigen/antibody detection kit, an antigen/antibody immunochromatographic test paper, IFA and IPMA.

The anti-PCV 2 monoclonal antibody is applied to an immunoaffinity column of PCV2 or PCV2 Cap.

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

1. the monoclonal antibody is produced by a single cell strain, has the characteristics of high specificity, uniformity and the like, has high purity, strong specificity and good repeatability, can be continuously supplied in an unlimited manner, and has wider research application value and commercial use value in immunoassay.

2. The monoclonal antibody of the invention can be simultaneously used for ELISA, IPMA, Western Blot and other detection means.

3. The monoclonal antibody has high specificity, has no cross reaction with PCV1 and PCV3, has no cross reaction with other swine viruses such as CSFV, PRRSV, PRV and the like, can be used for the antigen detection of porcine circovirus type 2, and reduces false positive results.

4. The monoclonal antibody has high sensitivity, only binds to PCV2 specifically, and has the subtype of IgG1, the light chain type of Kappa type and the affinity of not less than 1.26 × 10^-10mol/L，ELISA

The titer is not less than 1: 2.56 × 10⁵The IPMA titer is 1: 8 × 10⁴。

5. The monoclonal antibody obtained by screening has stable antibody secretion capacity, can quickly and specifically recognize PCV2 and Cap protein thereof, lays a good foundation for solving the technical problem of quick detection of PCV2 antigen, and has important value in immunodetection related to PCV 2.

6. On the basis of the heavy chain variable region sequence and the light chain variable region sequence of the monoclonal antibody, one or more amino acids can be added, deleted, substituted and the like through conventional genetic engineering and protein engineering to obtain an active fragment or a conservative variant thereof, thereby laying a foundation for further improving the specificity and the affinity of the antibody.

Drawings

FIG. 1 is a statistical chart of ELISA and IPMA determination of serum titer of immunized mice;

FIG. 2 is a graph comparing the IPMA titer determination in mouse No. 1;

FIG. 3 is a graph showing the determination of affinity constants of mAb 3B 6;

FIG. 4 is a specific Western Blot electrophoresis chart of monoclonal antibody 3B 6;

wherein, the Lane M is a protein standard molecular weight Marker; lane 1 is a prokaryotic expression of PCV2 Cap protein; lane 2 is BL21 negative control;

FIG. 5 is a comparison of the specificity detection of IPMA mab 3B 6;

FIG. 6 is a cross-reactivity comparison graph of IPMA detection monoclonal antibody 3B6 and porcine viruses such as PCV1, PCV3, CSFV, PRRSV, PRV and PEDV.

Detailed Description

The following examples are intended to illustrate the present invention in detail and should not be construed as limiting the scope of the present invention in any way.

The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the related reagents are all conventional reagents in the market, if not specifically indicated; the test methods involved are conventional methods unless otherwise specified.

The first embodiment is as follows: preparation and identification of hybridoma cell strain secreting anti-PCV 2 monoclonal antibody

1. Principal material

PCV2a Cap protein subunit vaccine is commercially available, BALB/c mouse is purchased from Zhengzhou university college of medicine, myeloma cell SP2/0 is purchased from Zhengzhou university college of Life sciences molecular immunology laboratory, porcine circovirus type 2 Cap protein is purchased from Zhengzhou university college of life sciences molecular immunology laboratory, PCV2 KFPRRS strain, porcine circovirus type 1 PCV1, PCV3, classical swine fever virus CSFV phylum strain, porcine reproductive and respiratory syndrome virus V BJ-4 strain, porcine pseudorabies virus PRV HN-JZ-16, and porcine epidemic diarrhea virus PEDV is purchased from animal focus immunology laboratory of agricultural academy of Henan province.

2. Primary reagent

Freund's complete adjuvant, Freund's incomplete adjuvant, HAT, HT, PEG-1500, RPMl-1640 cell culture medium, fetal bovine serum were purchased from Gibco, HRP-labeled goat anti-mouse IgG was purchased from Sigma, liquid AEC enzyme substrate kit was purchased from China fir Jinqiao, BCA protein concentration assay kit was purchased from Solambio, and monoclonal antibody subtype assay kit was purchased from Beijing Yi Qiao, Biotechnology, Inc.

3. Immunization of BALB/c mice

(1) Respectively adding a subunit vaccine of which the antigen component is PCV2a Cap protein into Freund's complete adjuvant and Freund's incomplete adjuvant, and emulsifying to prepare Freund's complete adjuvant immunogen and Freund's incomplete adjuvant immunogen, wherein the volume ratio of PCV2a Cap protein to Freund's complete adjuvant and Freund's incomplete adjuvant is 1: 1;

(2) 3 female BALB/c mice 8 weeks old were immunized with Freund's complete adjuvant immunogen at an immunization dose of 100. mu.l/mouse by means of dorsal subcutaneous multi-point injection;

(3) respectively carrying out boosting immunization on BALB/c mice by using Freund's incomplete adjuvant immunogen in the same method and dose 14 days and 28 days after the first immunization, taking blood from the tail part after two weeks of immunization, and measuring the serum titer of the mice;

(4) 3-30 weeks after the boosting immunization and 3-4 days before cell fusion, a mouse with the highest serum titer is selected to perform super-strong immunization on a BALB/c mouse by a tail vein injection method and a subunit vaccine of PCV2a Cap protein without adjuvant, wherein the immunization dose is 50 mu l/mouse.

4. Determination of serum antibody titer in immunized mice

Measuring the titer by ELISA:

(1) diluting the purified prokaryotic expression porcine circovirus type 2 Cap protein to 1 mug/mL by using a coating solution, adding 50 mul of the coating solution into each hole, incubating for 2h at 37 ℃, discarding the coating solution, and washing for 3 times by using PBST;

(2) sealing with 300 μ l of sealing solution (5% skimmed milk powder + PBST) overnight at 4 ℃;

(3) adding 50 mul of each serum to be detected diluted by 2 times of dilution buffer solution (PBST) into each well (the initial dilution multiple is 1: 400), incubating for 1h at 37 ℃, discarding the supernatant, and washing for 6 times by using PBST;

(4) to each well was added a dilution buffer at 1: 50 mul of HRP-labeled goat anti-mouse IgG diluted by 5000 is kept at 37 ℃ for 0.5h, then the supernatant is discarded, and the supernatant is washed by PBST washing liquid for 6 times;

(5) adding 50 mul DAB color development liquid into the concave hole, and adding 2M H after keeping out of the sun at room temperature for 20min₂SO₄Stopping the reaction with 50 mul stop solution, and determining OD (optical density) by using an enzyme-linked immunosorbent assay (ELISA) instrument₄₅₀The value is obtained.

Measuring the potency by IPMA:

(1) freshly digested PK15 cell suspensions (2 × 10) were inoculated with PCV2 KF strain as seed virus at 5%, 10% and 15% seed doses, respectively⁵mL), the virus maintenance solution is RPMI 1640 containing 2% FCS;

(2) 100 μ l of virus culture per well was plated in 96-well cell culture plates, while non-inoculated PK15 cells were used as negative controls at 37 ℃ with 5% CO₂Culturing under the condition to form a monolayer;

(3) fixing with acetone-PBS, washing with PBS once, and treating serum with PBS according to a ratio of 1: performing 2-fold gradient dilution on 200 samples, simultaneously performing positive serum, negative serum and non-toxic cell control, loading the sample at 100 mu l/hole, and incubating l h at 37 ℃;

(4) PBS was washed 3 times, l: 1000 dilution of HRP-labeled goat anti-mouse secondary antibody, and incubation at 37 ℃ for l h;

(5) washing with PBS for 3 times, adding AEC substrate solution, developing for 30min, and observing with optical microscope to determine result.

The results are shown in fig. 1 and fig. 2, and the ELISA and IPMA results show that the ELISA titer and IPMA titer of mouse No.1 are highest, and the IPMA titer can reach 1: 6400, mouse No.1 was selected for cell fusion to prepare monoclonal antibody.

5. Cell fusion

(1) Preparation of feeder cells

a. 2 Kunming mice are taken to be led to the neck for death and then soaked in 75 percent alcohol disinfectant for body surface disinfection;

b. fixing Kunming mouse soaked in 75% alcohol on paraffin plate, and cutting off abdominal skin with sterile surgical scissors in super clean bench to expose peritoneum;

c. lifting the peritoneum by using sterile forceps, slowly injecting 5ml of HAT selective culture medium into the abdominal cavity of the mouse by using a sterile injector, slightly pressing the abdominal cavity, and then re-sucking out the injected culture medium;

d. the feeder cell concentration was adjusted to about 2 × 10⁵cells/ml, 100 mul per well, laid in 96-well cell culture plates at 37 ℃ with 5% CO₂Culturing in an incubator.

Feeder cells were prepared 1 day prior to fusion. Feeder cells may also be diluted to the desired amount with the appropriate amount of HAT selection medium and temporarily stored in a 500ml sterile screw flask, and plated into 96-well cell culture plates together with the cells to be fused.

(2) Preparation of splenocytes

a. Carrying out hyperimmunization on a No.1 BALB/c mouse with the highest titer, leading the neck to death after 4-5 days, and disinfecting the body surface with 75% alcohol;

b. aseptically taking out mouse spleen in a superclean bench, washing with GNK solution preheated at 37 deg.C for 2 times, adding HAT culture medium, and cutting with small scissors on sterile 120 mesh nylon gauze;

c. and filtering the splenocytes to a sterile beaker, transferring the splenocytes to a sterile cell centrifuge tube, centrifuging the cells at 1000r/min for 10min, and washing the cells for 1-2 times for later use.

(3) Cell fusion and culture of fused cells

a. On day 3 after the mice were hyperimmunized, splenocytes from immunized mice were combined with mouse myeloma cells SP2/0 using polyethylene glycol as a 10: 1, performing cell fusion;

b. the fused cells were suspended gently in HAT selection medium, and the fused cells were dispersed in a 96-well cell culture plate at 250. mu.l/well and placed at 37 ℃ in 5% CO₂Culturing in an incubator

c. Culturing for 3-4 days, and observing small cell clusters under a microscope;

d. and changing the HT culture medium for half amount of the solution 7 days after fusion, sucking 25 mu l of cell culture supernatant on the 10 th day, and performing primary screening by using IPMA.

6. Screening and identification of hybridoma cells

(1) PCV2 KF strain is used as the virus seed, and the virus seed dosage is respectively 5%, 10% and 15%Inoculated into a freshly digested suspension of PK15 cells (2 × 10)⁵mL), virus maintenance solution is RPMI l640 containing 2% FCS;

(2) 100 μ l of virus culture per well was plated in 96-well cell culture plates, while non-inoculated PK15 cells were used as negative controls at 37 ℃ with 5% CO₂Culturing under the condition to form a monolayer;

(3) fixing with acetone-PBS to prepare an IPMA reaction plate, drying, and storing at 20 ℃ for later use;

(4) taking the IPMA reaction plate, placing the IPMA reaction plate at room temperature for preheating, washing the IPMA reaction plate once by PBS, and mixing the IPMA reaction plate and the PBS to obtain the fusion cell culture supernatant to be detected according to the ratio of 1: 20, diluting, simultaneously taking positive serum, negative serum and non-toxic cell control, adding sample amount of 500 mu l/hole, and incubating l h at 37 ℃;

(5) PBS was washed 3 times, l: 1000 dilution of HRP-labeled goat anti-mouse secondary antibody, and incubation at 37 ℃ for l h;

(6) washing with PBS for 3 times, adding AEC substrate solution, developing for 30min, observing and judging result with optical microscope, and screening out positive hybridoma cell strain for amplification culture and further subcloning.

7. Subcloning of hybridoma cells by limiting dilution method

(1) Diluting the positive hybridoma cells to about 3 cells/ml with 1640/10 complete medium, adding 100 mul per well into a 96-well plate pre-paved with 100 mul feeder cells, placing at 37 ℃, and 5% CO₂Culturing for 6-8 days in an incubator;

(2) transferring the positive monoclonal cell strain into a 24-hole cell culture plate for amplification culture;

(3) carrying out secondary subcloning until obtaining a hybridoma cell strain which stably secretes the anti-porcine circovirus monoclonal antibody, thus obtaining a target hybridoma cell which is named as 3B 6;

(4) the positive monoclonal obtained by screening is subjected to expanded culture, and the number of cells is 1-2 × 10⁶Freezing and storing in a tube.

8. Monoclonal hybridoma cell stability identification

The obtained positive monoclonal hybridoma cells were continuously passaged to 35 times, and the culture supernatants of the respective passages were subjected to stability measurement by ELISA, and the measurement results are shown in table 1:

TABLE 1 potency of antibody secretion by hybridoma cells of different generations

。

ELISA results show that the specific monoclonal antibody can be stably secreted by the corresponding hybridoma cell strain from the cell to 35 generations, and the hybridoma cell strain is high in monoclonal degree and stable in character, and can be used as seeds for long-term storage and large-scale preparation of monoclonal antibodies.

Example two: preparation of anti-PCV 2 monoclonal antibody ascites

The monoclonal hybridoma cell line 3B6 in the first example was subjected to expansion culture, the titer of the culture supernatant was measured by ELISA method to ensure stable monoclonal cell line character, and the cells were collected for mass production of monoclonal antibodies.

1. Preparation and purification of monoclonal antibody ascites

(1) Selecting female BALB/c mice born by women, injecting 500 mul of sterilized paraffin into the abdominal cavity, and stimulating immune cells to promote the proliferation of hybridoma cells;

(2) observing the state of the mice, and after 7-10 days, carrying out the observation according to the weight ratio of 1 × 10 to each mouse⁷Injecting the amount of each cell into the prepared monoclonal positive cells, observing the state of the mouse in time, extracting ascites after about 10 days, centrifuging at 8000 r/min at 4 ℃ for 20min to remove grease and cell precipitate, collecting the supernatant of the ascites, and storing at-80 ℃ for later use;

(3) one week later, the obtained monoclonal hybridoma cells were again injected intraperitoneally in an amount of 2 × 10⁵(ii) individual cells;

(4) after one week, ascites is extracted after the abdomen of the mouse is enlarged, and the supernatant is obtained after centrifugation;

(5) ascites IgG was roughly extracted by the ammonium caprylate method, and mouse IgG was purified by DE-52 ion exchange column, and ascites titer was measured by ELISA.

2. Monoclonal antibody ascites ELISA potency assay

(1) Diluting PCV2 Cap protein into coating liquid with the concentration of 2 mug/mL by CBS liquid to coat an enzyme label plate, sealing at the temperature of 4 ℃ for overnight;

(2) diluting PCV2 monoclonal antibody (primary antibody) by 5% of skimmed milk in a multiple ratio, sequentially adding the diluted PCV2 monoclonal antibody (primary antibody) into an ELISA plate with 100 mu l/hole, wherein the positive control is PCV2 pig positive serum, the negative control is PRRSV monoclonal antibody ascites, and incubating for 30min at 37 ℃;

(3) discarding the primary antibody, washing the plate by PBST, cleaning and drying;

(4) diluted HRP-labeled goat anti-mouse IgG (secondary antibody) was added to the reaction wells at 100 μ l/well. Incubating at 37 ℃ for 30 min;

(5) discarding the secondary antibody, washing with PBST, and patting dry;

(6) adding 100 mul of TMB color developing solution prepared on site into each hole, and carrying out a dark room reaction for 15 min;

(7) adding 50 mul 2M H into each hole₂SO₄Terminating the reaction;

(8) microplate reader for reading OD of each well₄₅₀The value is obtained.

The ELISA detection result shows that the ascites titer of the monoclonal antibody is 1: 1.024 × 10⁶。

3. Determination of monoclonal antibody ascites IPMA titer

The potency of the monoclonal antibody 3B6 was determined according to the IPMA method in penwuping et al (establishment of the porcine circovirus type 2 IPMA and IFA detection methods, first chinese veterinary drug congress and chinese animal society of veterinary medicine, division 2008 academic annual meeting).

Initial concentration of mab was from 1: 1000, 10-fold dilution with 5% skim milk, PK15 cell wells not infected with PCV2 as negative control, dilution of HRP-labeled secondary antibody 1: 1000, adding 50 mul of AEC color developing solution into each hole for color development.

IPMA results show 3B6 titers of 1: 5.12 × 10⁵。

Example three: identification of anti-PCV 2 monoclonal antibody

1. Subtype identification

The subclasses and types of mAbs were identified according to the instructions for use of the Mouse Monoclonal Antibody Isotyping Kit.

The identification of the subclasses and subtypes of the monoclonal antibody shows that the subtype of monoclonal antibody 3B6 is IgG2a, and as shown in Table 2, the light chain type is Kappa type.

TABLE 2 monoclonal antibody subtype identification

。

2. Affinity identification

Diluting PCV2 Cap protein into coating solutions with concentrations of 0.5 mug/mL and 1 mug/mL by CBS solution, respectively coating enzyme label plates, and determining the titer of monoclonal antibody ascites by indirect ELISA method, wherein the concentration of monoclonal antibody is horizontal coordinate, OD₄₅₀Values are plotted on the ordinate and the corresponding 2 indirect ELISA reaction curves are plotted.

The OD450 value of the upper flat portion of each curve was taken as 100%, and the 50% OD was calculated on the curve₄₅₀The antibody concentration at value was determined according to the formula Kaff = (n-1)/2(n [ Ab']t-[Ab]t) calculating the affinity constant of the monoclonal antibody, wherein n = [ Ag%]t/[Ag′]t，[Ag]t、[Ag′]t is 2 different concentrations of coating antigen, [ Ab ]]t、[Ab′]t is 50% OD at each original concentration₄₅₀Value corresponds to antibody concentration.

As shown in FIG. 3, the affinity constant K value of 3B6 monoclonal antibody was calculated to be 3.6 × 10^-9mol/L。

3. Identification of specificity

The specificity of the monoclonal antibody 3B6 is identified by ELISA, Western blot and IPMA experiments respectively, and the result of ELISA detection is carried out on the purified PCV2 virus, which shows that the monoclonal antibody 3B6 can generate specific reaction with the virus but not react with PK15 cell contrast.

Taking the purified PCV2-Cap protein and other irrelevant proteins of escherichia coli as controls, carrying out SDS-PAGE gel electrophoresis, then transferring the SDS-PAGE gel electrophoresis onto a nitrocellulose membrane, screening to obtain monoclonal antibody 3B6 as a primary antibody, using goat anti-mouse IgG (H + L) marked by HRP as a secondary antibody, and developing by using an AEC enzyme substrate kit.

As shown in fig. 4 and 5: western blot test results show that the monoclonal antibody 3B6 can perform specific reaction with PCV2-Cap protein, but does not perform reaction with unrelated protein; the IPMA results show that mab 3B6 reacts specifically with PCV2 virus, but not with PK15 cells.

4. Identification of Cross-reactivity with porcine Virus

Diluting the ascites fluid of the monoclonal antibody according to a certain proportion, and then respectively adding PCV2 KF strain of porcine circovirus type 2, PCV1, PCV3, CSFV Shimen strain of swine fever virus, PRRSV BJ-4 strain of porcine reproductive and respiratory syndrome virus, PRV HN-JZ-16 strain of porcine pseudorabies virus and cells infected by porcine epidemic diarrhea virus V, and determining whether PCV2 monoclonal antibodies 3B6 and 3G11, PCV1, PCV3, CSFV, PRRSV, PRV and PEDV have cross reactivity by an IPMA detection method.

The IPMA result is shown in figure 6, the monoclonal antibodies 3B6 and 3G11 both react with PCV2 to be positive results, and the monoclonal antibodies 3B6 react with other viruses (PCV 1, PCV3, CSFV, PRRSV, PRV and PEDV) to be negative results, so that the specificity of the reaction between the monoclonal antibodies 3B6 and PCV2 is good, and the reaction has no cross reaction with other common porcine viruses.

Example four: amplification of variable region Gene of anti-PCV 2 monoclonal antibody

Designing a heavy chain variable region primer sequence according to the sequence characteristics of the mouse-derived monoclonal antibody:

P1：5’-TTCTCTTGACGT TGTACTGG-3’；

P2：5’-TTTTGAGGAGACGGTGA-3’。

design of light chain variable region primer sequence:

P3：5’-TTATGGGTAGTTCATGGAGACA-3’；

P4：5’-ACACATGGTGCAGCATCAGCC-3’。

the variable region sequences of the monoclonal antibody 3B6 were obtained by molecular cloning techniques and sequenced by Shanghai Biometrics Ltd. The gene sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 3B6 are respectively shown in SEQ ID NO.1 and SEQ ID NO.3, and the deduced amino acid sequences of the heavy chain variable region and the light chain variable region of 3B6 are respectively shown in SEQ ID NO.2 and SEQ ID NO. 4.

While the present invention has been described in detail with reference to the drawings and the embodiments, those skilled in the art will understand that various specific parameters in the above embodiments can be changed without departing from the spirit of the present invention, and a plurality of specific embodiments are formed, which are common variation ranges of the present invention, and will not be described in detail herein.

SEQUENCE LISTING

<110> Henan Zhongze bioengineering, Inc

<120> preparation method and application of anti-PCV 2 monoclonal antibody

<130>2018

<160>8

<170>PatentIn version 3.2

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acacatggtg cagcatcagc c 21

Claims (6)

1. A heavy chain variable region DNA sequence of the monoclonal antibody for resisting PCV2 is shown as SEQ ID NO. 1; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2; and, its light chain variable region DNA sequence is the sequence shown in SEQ ID No. 3; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4.

2. The anti-PCV 2 monoclonal antibody according to claim 1, characterized in that the light chain type of said monoclonal antibody is Kappa and the subtype is IgG2 a.

3. The anti-PCV 2 monoclonal antibody according to claim 1, wherein the ELISA titer of the monoclonal antibody is not less than 1: 2.56 × 10⁵。

4. The anti-PCV 2 monoclonal antibody according to claim 1, wherein the IPMA titer of the monoclonal antibody is 1: 8 × 10⁴。

5. anti-PCV 2 nanogram according to claim 1A diabody, wherein said monoclonal antibody has an affinity of not less than 1.26 × 10^-10mol/L。

6. An antigen/antibody detection kit comprising the anti-PCV 2 monoclonal antibody of claim 1.

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