CN108752471A - The preparation method and applications of anti-PCV2 monoclonal antibodies - Google Patents

The preparation method and applications of anti-PCV2 monoclonal antibodies Download PDF

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CN108752471A
CN108752471A CN201810332092.1A CN201810332092A CN108752471A CN 108752471 A CN108752471 A CN 108752471A CN 201810332092 A CN201810332092 A CN 201810332092A CN 108752471 A CN108752471 A CN 108752471A
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monoclonal antibodies
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张改平
王爱萍
陈玉梅
蒋敏
刘东民
刘运超
周景明
刘燕凯
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Henan Ze Ze Biological Engineering Co Ltd
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Abstract

The invention discloses a kind of preparation method and applications of anti-PCV2 monoclonal antibodies.The DNA sequence dna and amino acid sequence of the anti-PCV2 monoclonal antibodies and its heavy chain variable region and light chain variable region are disclosed.Devise the preparation method of the anti-PCV2 monoclonal antibodies.The monoclonal antibody is applied in antigen/antibody detection kit, antigen/antibody immune chromatography test paper and IFA, IPMA and in the immune affinity column of PCV2 or PCV2 Cap.The monoclonal antibody of the present invention can identify both main popular hypotypes of PCV2a and PCV2b simultaneously, all have a good reactionogenicity with PCV2 totivirus and PCV2 Cap proteins, and with PCV1, PCV3 hypotype and the equal no cross reaction of pig source virus；It lays a good foundation to carry out PCV2 aetologies and the research of pathogenesis and the clinical detection research of PCV2 cause of diseases.

Description

The preparation method and applications of anti-PCV2 monoclonal antibodies
Technical field
The present invention relates to biological immune technical fields, and in particular to a kind of preparation method of anti-PCV2 monoclonal antibodies and its Using.
Background technology
Pig circular ring virus（Porcine circovirus, PCV）Belong to circovirus section（Circoviridae）, annulus disease Poison belongs to（Circovirus）Member is the minimum DNA virus found so far, and the pig transmissible disease being induced by it is in global pig It is widely present in group, is the internationally recognized important immunosupress cause of disease for endangering countries in the world large scale of pig farm industry.
Wherein PCV2 is to cause PMWS, pigskin inflammation and nephrotic syndrome（Porcinedermatitisan Dnephropathy syndrome, PDNS）, porcine respiratory disease syndrome（Porcine respiratory disease Complex, PRDC）Etc. diseases main pathogen, cause pig breeding industry to sustain losses severely.At present China's Major Epidemic strain according to It is so PCV2, PCV2 infection leads to the reduction of Swinery immunity power, the mixing or secondary infection of other a variety of cause of diseases of the thing followed are more It is to bring serious threat to pig breeding industry.
The genome of PCV2 includes two main open reading frame（Open Reading Frame, ORFs）.ORF1 is compiled Code two replicates relevant albumen（Rep and Rep '）, the reproduction process of the two albumen participation PCV2.ORF2 encoding viral structurals PROTEIN C ap albumen, the albumen combine the capsid of rear shape virus by poly.Cap protein is as the unique structure of pig circular ring virus Albumen has very high immunogenicity, is the major target of vaccine development and epidemic disease detection.
The main virulent separation of the method for laboratory diagnosis PCV2 at present and identification technology, molecular biological testing And 3 major class of amynologic diagnostic method.
In realizing process of the present invention, inventor has found that at least there are the following problems in the prior art：
The separation of virus is the method for the most accurately diagnosing PCV2 infection with identification, but PCV2 is proliferated in cell and does not generate Apparent cytopathy, follow-up identification also need to other technologies, and whole process technology is complicated, and professional person is needed to operate, And time and effort consuming, it is unsuitable for disease quick diagnosis；
Molecular biological testing includes mainly PCR（polymerase chain reaction, PCR） Detection technique, gene chip detecting technique, nucleic acid hybridisationdetection technology etc..With the continuous development of molecular biology, PCR is because of it Quickly, the advantages that sensibility is high is widely used in detecting disease pathogen in the lab, but its operation takes time and effort, to technology It is high with equipment requirement, therefore can not be promoted and applied in actual production cultivation.
Common amynologic diagnostic method includes mainly immunohistochemistry（Immunohistochemistry, IHC）, IPMA, IFA, enzyme-linked immunosorbent assay（Enzyme linked immunosorbent assay, ELISA）And immunochromatography Test paper Fast Detection Technique（Immuno- chromatographic lateral flow strip test, ILFST）Deng.This A little immunological detection methods have many advantages, such as the sensitivity of height and specificity, reproducible, in epidemiology generaI investigation and vaccine Also there is very high practicability in Efficacy evaluation.
Monoclonal antibody is using the core reagent of immunological detection method detection cause of disease, and specificity and affinity are to determine An important factor for determining specificity and the sensitivity of immunological detection method, therefore acquisition is for the specific antibody of detection target Establish the key of cause of disease immunological detection method.As pig circular ring virus vaccine uses in large area, serological detection data The no longer infection state of the wild poison of reaction pig circular ring virus, therefore, pathogeny detection becomes more and more important.
However, a kind of with PCV1, PCV3 and the equal no cross reaction of other pig sources virus and can be same there is an urgent need for preparing at present When PCV2 monoclonal antibodies for panimmunity detection means, and then determine its weight chain variabl area sequence and light chain variable region sequence Row are to be transformed the genetic engineering antibody that its antibody variable sequences prepares different combinations, to further promote the spy of antibody Anisotropic and affinity.
Invention content
The technical problem to be solved in the present invention is to provide a kind of preparation method and applications of anti-PCV2 monoclonal antibodies.
In order to solve the above technical problems, the present invention adopts the following technical scheme that：
A kind of anti-PCV2 monoclonal antibodies are screened to obtain, heavy chain variable region DNA sequence dna is the sequence as shown in SEQ ID NO.1； Its heavy chain variable amino acid sequence is the sequence as shown in SEQ ID NO.2, or on the basis of the SEQ ID NO.2 sequences The upper addition for carrying out one or more amino acid, deletion, replacement and obtain active fragment or the sequence of conservative variant；With/ Or
Its light chain variable region DNA sequence dna is the sequence as shown in SEQ ID NO.3；Its chain variable region amino acid sequence is such as Sequence shown in SEQ ID NO.4, or adding for one or more amino acid is carried out on the basis of the SEQ ID NO.4 sequences Add, delete, replace and obtain active fragment or the sequence of conservative variant.
Preferably, the light chain type of the monoclonal antibody is Kappa, hypotype IgG2a.
Preferably, the ELISA potency of the monoclonal antibody is not less than 1：2.56×105。
Preferably, the IPMA potency of the monoclonal antibody is 1：8×104。
Preferably, the affinity of the monoclonal antibody is not less than 1.26 × 10-10 mol/L。
A kind of preparation method of the anti-PCV2 monoclonal antibodies is designed, is included the following steps：
（1）Mouse is immunized with the subunit vaccine of PCV2a Cap proteins；
（2）The mouse immune splenocyte and murine myeloma cell are merged, hybridoma is obtained；
（3）The method for combining and taking turns subclone more is detected using IPMA obtains positive hybridoma cell；
（4）Positive monoclonal hybridoma cell strain RNA reverse transcriptions are extracted into cDNA, the weight of monoclonal antibody is gone out by PCR amplification Chain variable region sequence and light-chain variable sequence；
（5）Colonized culture is carried out to positive colony, obtains PCV2 monoclonal antibody hybridoma cell strains；
（6）The hybridoma cell strain is injected in mouse peritoneal and produces monoclonal antibody.
The anti-PCV2 monoclonal antibodies antigen/antibody detection kit, antigen/antibody immune chromatography test paper and Application in IFA, IPMA.
Application of the anti-PCV2 monoclonal antibodies in the immune affinity column of PCV2 or PCV2 Cap.
Compared with prior art, the beneficial technical effect of the present invention lies in：
1. the monoclonal antibody of the present invention is generated by individual cells strain, there is the characteristics such as high degree of specificity, homogeneity, monoclonal antibody Purity height, high specificity, it is reproducible and can constantly endless supply, in immune detection have widely research answer With value and business use value.
2. the monoclonal antibody of the present invention can be used for a variety of detection hands such as ELISA, IPMA, Western Blot simultaneously Section.
3. the monoclonal antibody of the present invention has compared with high specific, and the equal no cross reaction of PCV1, PCV3, with other pig sources The virus equal no cross reaction of such as CSFV, PRRSV, PRV can be used for the antigen detection of porcine circovirus 2 type, reduce false positive As a result.
4. there is the monoclonal antibody of the present invention sensitivity of height, the monoclonal antibody only to be specifically bound with PCV2, Its hypotype is IgG1, and light chain type is Kappa types, and affinity is not less than 1.26 × 10-10Mol/L, ELISA
Potency is not less than 1：2.56×105, IPMA potency is 1：8×104。
5. the obtained monoclonal antibody of present invention screening has stable antibody-secreting ability, can quickly special identification PCV2 and its Cap protein, excellent basis has been established in the solution for the technical barrier of PCV2 rapid antigen detections, in the relevant immune inspections of PCV2 There is important value in survey.
6. on the basis of the weight chain variabl area sequence of monoclonal antibody of the present invention and light-chain variable sequence, can pass through Conventional gene engineering and protein engineering carry out the modifications such as addition, deletion, the replacement of one or more amino acid, obtain its activity Segment or conservative variant, specificity and affinity further to promote antibody lay the foundation.
Description of the drawings
Fig. 1 is ELISA and IPMA to immune serum titration statistical chart；
Fig. 2 is the IPMA titration comparison diagrams of No. 1 mouse；
The affinity costant that Fig. 3 is monoclonal antibody 3B6 measures curve graph；
Fig. 4 is the specific Western Blot electrophoretograms of monoclonal antibody 3B6；
Wherein, swimming lane M is protein standard molecular weight Marker；Swimming lane 1 is the PCV2 Cap proteins of prokaryotic expression；Swimming lane 2 is BL21 negative controls；
Fig. 5 is the specific detection comparison diagram of IPMA monoclonal antibodies 3B6；
Fig. 6 is the cross reaction that IPMA detects the pigs such as monoclonal antibody 3B6 and PCV1, PCV3, CSFV, PRRSV, PRV and PEDV source virus Property comparison diagram.
Specific implementation mode
Illustrate the specific implementation mode of the present invention with reference to the accompanying drawings and examples, but following embodiment is used only in detail It describes the bright present invention in detail, does not limit the scope of the invention in any way.
Involved instrument and equipment is routine instrument device unless otherwise instructed in the examples below；Involved Reagent is commercially available conventional reagent unless otherwise instructed；Involved test method is unless otherwise instructed conventional method.
Embodiment one：Secrete the preparation and identification of the hybridoma cell strain of anti-PCV2 monoclonal antibodies
1. main material
PCV2a Cap protein subunit vaccines are commercially available, and BALB/c mouse is purchased from Medical School of Zhengzhou University, myeloma cell SP2/ 0 is purchased from Zhengzhou University's Life Science College molecular immunology laboratory, and carrying Cap gene of porcine circovirus type 2 is given birth to purchased from Zhengzhou University Life science institute's molecular immunology laboratory, KF plants of PCV2,1 type PCV1, PCV3 of pig circular ring virus, swine fever virus CSFV crossdrifts Strain, BJ-4 plants of porcine reproductive and respiratory syndrome virus PRRSV, porcine pseudorabies virus PRV HN-JZ-16, Porcine Epidemic Diarrhea Malicious PEDV is purchased from animal immunology key lab of Henan Academy of Agricultural Sciences.
2. main agents
Freund's complete adjuvant, incomplete Freund's adjuvant, HAT, HT, PEG -1500, RPMl-1640 cell culture medium, fetal calf serum Purchased from Gibco companies, HRP marks sheep anti-mouse igg to be purchased from Sigma companies, and liquid A EC zymolyte kits are public purchased from Zhong Shan Golden Bridge Department, BCA determination of protein concentration kits are purchased from Solarbio companies, and monoclonal antibody hypotype assay kit is given birth to purchased from Divine Land Yi Qiao, Beijing Object Technology Co., Ltd..
3. immune BALB/c mouse
（1）The subunit vaccine that antigenic component is PCV2a Cap proteins is separately added into Freund's complete adjuvant and Freund is incomplete Freund's complete adjuvant immunogene and incomplete Freund's adjuvant immunogene, wherein PCV2a Cap proteins and Freund is made in adjuvant emulsion Freund's complete adjuvant, incomplete Freund's adjuvant volume ratio are 1：1；
（2）By the method for dorsal sc multi-point injection, female BAl BIc/c with 8 week old of Freund's complete adjuvant immunogen immune is small Mouse 3, immunizing dose are 100 μ l/；
（3）Used respectively after first immunisation 14 days and 28 days incomplete Freund's adjuvant immunogene in the same way with dosage pair BALB/c mouse carries out booster immunization, is immunized after two weeks, and mice serum potency is surveyed in tail portion blood sampling；
（4）3~30 weeks after booster immunization, 3~4 days before cell fusion, chooses the highest mouse of serum titer and noted by tail vein The method penetrated carries out BALB/c mouse superpower immune, immunizing agent with the subunit vaccine of the PCV2a Cap proteins without adjuvant Amount be 50 μ l/only.
4. immune serum antibody titer measures
ELISA surveys potency：
（1）50 μ l are added per hole to 1 μ g/mL for the carrying Cap gene of porcine circovirus type 2 of the prokaryotic expression purified with coating buffer dilution Coating buffer, 37 DEG C of incubation 2h, abandons coating buffer, is washed 3 times with PBST；
（2）With 300 μ l confining liquids（5% skimmed milk power+PBST）In 4 DEG C of closings overnight；
（3）Per hole addition dilution buffer（PBST）With each 50 μ l of the serum to be checked of 2 times of doubling dilutions（Initial dilution multiple is 1：400）, after 37 DEG C is incubated 1h, supernatant is abandoned, is washed 6 times with PBST；
（4）To every hole addition dilution buffer with 1：Goat anti-mouse igg each 50 μ l of 5000 diluted HRP labels, 37 DEG C Supernatant is abandoned after heat preservation 0.5h, is washed 6 times with PBST cleaning solutions；
（5）50 μ l DAB developing solutions are added into shrinkage pool, room temperature is protected from light after effect 20min plus 2M H2SO450 μ l terminate liquids are whole It only reacts, microplate reader is used in combination to measure OD450Value.
IPMA surveys potency：
（1）Use 2 KF strains of PCV as seed culture of viruses, it is thin in the PK15 newly digested by 5%, 10% and 15% seed culture of viruses dose inoculation respectively Born of the same parents' suspension (2 × 105/ mL), viral maintaining liquid is the RPMI 1640 containing 2% FCS；
（2）It is laid in 96 porocyte culture plates by 100 μ l viral cultures per hole, while the PK15 cells for setting non-virus inoculation are made For negative control, in 37 DEG C of 5% CO2Under the conditions of culture form single layer；
（3）It is fixed with acetone-PBS, is embathed once with PBS, serum to be checked presses 1 with PBS：200 progress, 2 times of gradient dilutions, together When make positive serum, negative serum and do not connect poison cell control, sample-adding amount is 100 μ l/hole, sets 37 DEG C and is incubated l h；
（4）PBS is washed 3 times, and l is added：1000 diluted HRP mark sheep anti mouse secondary antibody, set 37 DEG C and are incubated l h；
（5）PBS is washed 3 times, and AEC substrate solutions colour developing 30min is added, and judgement result is observed with light microscope.
As a result as depicted in figs. 1 and 2, ELISA and IPMA results show that No. 1 mouse ELISA potency and IPMA potency are Highest, IPMA potency can reach 1：6400, it chooses No. 1 mouse and prepares monoclonal antibody for cell fusion.
5. cell fusion
（1）The preparation of feeder cells
A. take 2 Kunming mouses crane one it is lethal after be immersed in 75% alcohol disinfection solution body surface and sterilize；
B. the Kunming mouse that 75% alcohol impregnated is fixed in paraffin plate, abdominal cut skin is cut with aseptic operation in super-clean bench Skin, exposure peritonaeum；
C. lift peritonaeum with aseptic nipper, 5ml HAT Selective agar mediums are slowly injected into mouse peritoneal with asepsis injector, gently The culture medium of injection is sucked out again again for light press abdominal cavity；
D. feeder cells concentration is adjusted to about 2 × 105Cells/ml is spread per 100 μ l of hole into 96 porocyte culture plates, 37 DEG C, 5% CO2It is cultivated in incubator.
Feeder cells need to be prepared for 1 day before fusion.Feeder cells can also be diluted to institute with appropriate HAT Selective agar mediums Requirement is temporarily stored in the sterile screw socket bottles of 500ml, and is spread together into 96 porocyte culture plates after cell to be fused.
（2）The preparation of splenocyte
A. take highest No. 1 BALB/c mouse of potency carry out it is super exempt from, crane one after 4~5 days lethal, sterilized with 75% alcohol body surface；
B. mouse spleen is taken out in sterile working in superclean bench, and the GNK solution preheated with 37 DEG C is washed 2 times, and a little HAT is added Culture medium shreds spleen on 120 sterile mesh nylon gauzes with small scissors；
C. splenocyte is filtered to sterile beaker and is gone in steril cell centrifuge tube, 1000r/min centrifuges 10min and washs cell 1 ~2 times spare.
（3）The culture of cell fusion and fused cell
A. mouse surpass and be exempted from the 3rd day afterwards, using the method for polyethylene glycol, by the splenocyte and mouse bone marrow cells of immune mouse Oncocyte SP2/0 presses cell quantity 10：1 ratio carries out cell fusion；
B. the cell after merging selects culture solution to be gently suspended with HAT, in Dispersion Fusion cell to 96 porocyte culture plates, 250 37 DEG C, 5% CO are set in the holes μ l/2Culture in incubator
C. small cell cluster can be observed with microscope by cultivating 3~4 days；
D. it uses within 7 days half amount of HT culture mediums after merging instead and changes liquid, draw 25 μ l cells and supernatants within the 10th day, carried out just with IPMA Sieve.
6. the screening and identification of hybridoma
（1）Use 2 KF strains of PCV as seed culture of viruses, it is thin in the PK15 newly digested by 5%, 10% and 15% seed culture of viruses dose inoculation respectively Born of the same parents' suspension（2×105/mL）, viral maintaining liquid is the RPMI l640 containing 2% FCS；
（2）It is laid in 96 porocyte culture plates by 100 μ l viral cultures per hole, while the PK15 cells for setting non-virus inoculation are made For negative control, in 37 DEG C of 5% CO2Under the conditions of culture form single layer；
（3）It is fixed with acetone-PBS and prepares IPMA reaction plates, set 20 DEG C after drying and save backup；
（4）It takes IPMA reaction plates to set room temperature preheating, is washed once with PBS, fused cell culture supernatant to be checked presses 1 with PBS：20 carry out Dilution, while making positive serum, negative serum and not connecing poison cell control, sample-adding amount is 500 μ l/hole, sets 37 DEG C and is incubated l h；
（5）PBS is washed 3 times, and l is added：1000 diluted HRP mark sheep anti mouse secondary antibody, set 37 DEG C and are incubated l h；
（6）PBS is washed 3 times, and AEC substrate solutions colour developing 30min is added, with light microscope observation judgement as a result, therefrom filtering out sun Property hybridoma cell strain be enlarged culture and further subclone.
7. being subcloned to hybridoma by limiting dilution assay
（1）Above-mentioned positive hybridoma cell is diluted to about 3 cells/ml with 1640/10 complete medium, is added per 100 μ l of hole To being covered in advance in 96 orifice plates of 100 μ l feeder cells, 37 DEG C are placed in, 5% CO2It is cultivated 6 ~ 8 days in incubator；
（2）Positive monoclonal cell strain is transferred to 24 porocyte culture plates and is enlarged culture；
（3）Secondary subclone is carried out, until the hybridoma until obtaining stably excreting resisting porcine circovirus monoclonal antibody Strain, you can obtain purpose hybridoma, be named as 3B6；
（4）The positive monoclonal that screening is obtained expands culture, and cell number presses 1~2 × 106/ pipe is frozen.
8. monoclonal hybridoma repeated pruning
The positive monoclonal hybridoma of acquisition is subjected to continuous passage to 35 times, the culture supernatant of different generations is taken to use respectively ELISA carries out Stability Determination, and measurement result is as shown in table 1：
The potency of the hybridoma secretory antibody of the different generations of table 1
。
ELISA can steadily secrete specific Dan Ke the result shows that cell reaches 35 generations corresponding hybridoma cell strain Grand antibody illustrates that the monoclonal degree of this strain of hybridoma strain is relatively high, and character is more stable, can be used as seed Long-term preservation and a large amount of preparation monoclonal antibodies.
Embodiment two：The preparation of anti-PCV2 monoclonal antibodies ascites
Monoclonal hybridoma strain 3B6 in embodiment one is expanded into culture, ELISA method surveys culture supernatant potency, it is ensured that Dan Ke Grand cell strain character is stablized, and collects cell for largely preparing monoclonal antibody.
1. the preparation and purifying of monoclonal antibody ascites
（1）The female BAl BIc through production/c mouse, 500 μ l sterilizing paraffin of intraperitoneal injection is selected to stimulate immunocyte to promote The proliferation of hybridoma；
（2）Mouse state is observed, according to every about 1 × 10 after 7~10 days7The amount injection of a cell shifts to an earlier date ready monoclonal Positive cell observes mouse state, extracts ascites after about 10 days in time, 8000 r/min, 4 DEG C of 20 min of centrifugation removal greases and Cell precipitation is collected -80 DEG C of ascites supernatant and is saved backup；
（3）After a week, the monoclonal hybridoma that intraperitoneal injection obtains again, injection volume are 2 × 105A cell；
（4）After after a week, extracting ascites after mouse web portion expands, supernatant is taken after centrifugation；
（5）Ascites IgG is slightly carried using sad ammonium sulfate method, DE-52 ion exchange column purification mouse IgGs are used in combination, with ELISA to abdomen Water potency is measured.
2. monoclonal antibody ascites ELISA titrations
（1）PCV2 Cap proteins are diluted to the coating buffer coated elisa plate of a concentration of 2 μ g/mL with CBS liquid, 100 holes μ l/, 4 DEG C closing overnight；
（2）By PCV2 antibody mabs（Primary antibody）Doubling dilution is carried out with 5% defatted milk, is sequentially added in ELISA Plate, 100 μ l/ Hole, positive control are PCV2 pig positive serums, and negative control is the odd contradictive hydroperitoneum of PRRSV, 37 DEG C of incubation 30min；
（3）Primary antibody is discarded, with PBST board-washings, wash clean pats dry；
（4）The sheep anti-mouse igg that the HRP diluted is marked（Secondary antibody）It is added in reacting hole, 100 holes μ l/.37 DEG C of incubation 30min；
（5）Secondary antibody is discarded, is rinsed well with PBST, is patted dry；
（6）The 100 μ l of TMB developing solutions now matched are added per hole, 15min is reacted in darkroom；
（7）50 μ l 2M H are added per hole2SO4Terminate reaction；
（8）Microplate reader reads the OD per hole450Value.
ELISA testing results show that the monoclonal antibody titer of ascites is 1：1.024×106。
3. the measurement of monoclonal antibody ascites IPMA potency
According to 5 equality of Peng（Porcine circovirus 2 type IPMA and IFA detection method establishes the veterinary drug conference of first China and China The animal drug credit of animal and veterinary association can Annual Conference in 2008）IPMA methods in document measure monoclonal antibody 3B6 potency.
Monoclonal antibody initial concentration is from 1：1000,10 times of doubling dilutions are carried out with 5% defatted milk, the PK15 for being uninfected by PCV2 is thin For hilum as negative control, it is 1 that HRP, which marks the dilution of secondary antibody,：1000,50 μ l AEC developing solutions are added per hole and develop the color.
IPMA results show 3B6 potency 1：5.12×105。
Embodiment three：The identification of anti-PCV2 monoclonal antibodies
1. subtype identification
The identification of monoclonal antibody subclass and type is grasped by Mouse Monoclonal Antibody Isotyping Kit operation instructions Make.
Monoclonal antibody subclass and the qualification result of hypotype show that monoclonal antibody 3B6 hypotypes are IgG2a, as shown in table 2, light chain Type is Kappa types.
2 monoclonal antibody subtype identification of table
。
2. affinity is identified
The coating buffer that PCV2 Cap proteins are diluted to concentration 0.5 μ g/mL and 1 μ g/mL with CBS liquid distinguishes coated elisa plate, leads to It crosses indirect ELISA method and measures odd contradictive hydroperitoneum potency, with a concentration of abscissa of monoclonal antibody, OD450Value is ordinate, is drawn corresponding 2 indirect ELISA response curves.
Using the OD450 values of every curve upper planar section as 100%, 50% OD is calculated on curve450It is corresponding when value Antibody concentration calculates the affinity costant of monoclonal antibody, wherein n=[Ag] t/ according to formula Kaff=(n-1)/2 (n [Ab '] t- [Ab] t) [Ag '] t, [Ag] t, [Ag '] t are 2 different coating original contents, and [Ab] t, [Ab '] t are 50% OD under each coating original content450 It is worth corresponding antibody concentration.
As shown in figure 3, it is 3.6 × 10 to calculate 3B6 monoclonal antibody affinity costant K values according to affinity measurement result-9 mol/L。
3. specificity identification
The specificity for using ELISA, Western blot and IPMA experimental identification monoclonal antibodies 3B6 respectively, take the PCV2 viruses of purifying into As a result row ELISA detections show that with virus specific reaction can occur for monoclonal antibody 3B6, and do not react with PK15 cell controls.
The PCV2-Cap albumen and Escherichia coli other unrelated proteins for taking purifying compare, and carry out PAGE gel electricity Swimming, is then transferred on nitrocellulose filter, screens the monoclonal antibody 3B6 of acquisition as primary antibody, the sheep anti-mouse igg of HRP labels (H+L) it is used as secondary antibody, is developed the color with AEC zymolyte kits.
As shown in Figure 4 and Figure 5：It is special that Western blot test results show that monoclonal antibody 3B6 can occur with PCV2-Cap albumen Opposite sex reaction, and nothing to do with albumen does not react；It is anti-that IPMA results show that with PCV2 viruses specificity can occur for monoclonal antibody 3B6 It answers, and does not react with PK15 cells.
4. the cross reactivity with pig source virus is identified
KF plants of porcine circovirus 2 type PCV2,1 type of pig circular ring virus are separately added into after odd contradictive hydroperitoneum is diluted by a certain percentage PCV1, PCV3, swine fever virus CSFV Strain Shimens, BJ-4 plants of porcine reproductive and respiratory syndrome virus PRRSV, porcine pseudorabies virus The cell of HN-JZ-16 plants of PRV, Porcine epidemic diarrhea virus PEDV infection measure PCV2 monoclonals using IPMA detection methods Whether antibody 3B6,3G11 and PCV1, PCV3, CSFV, PRRSV, PRV, PEDV have cross reactivity.
The results are shown in Figure 6 by IPMA, and it is positive findings that monoclonal antibody 3B6 and 3G11, which are only reacted with PCV2, with other several diseases Poison（PCV1,PCV3,CSFV,PRRSV,PRV,PEDV）Reaction result be feminine gender, it was demonstrated that monoclonal antibody 3B6 and PCV2 is anti- The specificity answered is good, with other several frequently seen swine disease poison no cross reactions.
Example IV：The amplification of anti-PCV2 variable region of mab gene
According to the sequence signature of mouse resource monoclonal antibody, heavy chain variable region primer sequence is designed：
P1：5'-TTCTCTTGACGT TGTACTGG-3'；
P2：5'-TTTTGAGGAGACGGTGA-3'.
Design light chain variable region primer sequence：
P3：5'-TTATGGGTAGTTCATGGAGACA-3'；
P4：5'-ACACATGGTGCAGCATCAGCC-3'.
It is limited to serve the raw work biology in sea for the variable region sequences for obtaining monoclonal antibody 3B6 respectively by molecule clone technology Company is sequenced.Measure the heavy chain variable region of monoclonal antibody 3B6 and chain variable region gene sequence be respectively SEQ ID NO.1, It is respectively SEQ ID by the heavy chain variable region of 3B6 and the amino acid sequence of light chain variable region of its derivation shown in SEQ ID NO.3 Shown in NO.2, SEQ ID NO.4.
The present invention is described in detail above in conjunction with drawings and examples, still, those of skill in the art Member is it is understood that without departing from the purpose of the present invention, can also carry out each design parameter in above-described embodiment Change, forms multiple specific embodiments, is the common variation range of the present invention, is no longer described in detail one by one herein.
SEQUENCE LISTING
<110>Henan Zhong Ze bioengineering Co., Ltd
<120>The preparation method and applications of anti-PCV2 monoclonal antibodies
<130> 2018
<160> 8
<170> PatentIn version 3.2
<210> 1
<211> 330
<212> DNA
<213>BALB/c mouse
<400> 1
ctcttgacgt tgtactgggg ctcagtgaag atatcctgca aggcttctgg atacacattc 60
actgactaca acatgcactg ggtgaagcag agccatggaa agagccttga gtggattgga 120
tatatttatc cttacaatgg tggtactggc tacaaccaga agttcaagag caaggccaca 180
ttgactgtag acaattcctc cagcacagcc tacatggaga tccgcaggct gacatctgag 240
gactctgcag tctattactg tgcaagaccc gtctatggta actaccttga ctactggggc 300
caagggacca cggtcaccgt ctcctcaaaa 330
<210> 2
<211> 110
<212> PRT
<213>BALB/c mouse
<400> 2
Leu Leu Thr Leu Tyr Trp Gly Ser Val Lys Ile Ser Cys Lys Ala Ser
1 5 10 15
Gly Tyr Thr Phe Thr Asp Tyr Asn Met His Trp Val Lys Gln Ser His
20 25 30
Gly Lys Ser Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly
35 40 45
Thr Gly Tyr Asn Gln Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp
50 55 60
Asn Ser Ser Ser Thr Ala Tyr Met Glu Ile Arg Arg Leu Thr Ser Glu
65 70 75 80
Asp Ser Ala Val Tyr Tyr Cys Ala Arg Pro Val Tyr Gly Asn Tyr Leu
85 90 95
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Lys
100 105 110
<210> 3
<211> 425
<212> DNA
<213>BALB/c mouse
<400> 3
ttatgggtag ttcatggaga cagacacact cctgttatgg gtactgctgc tctgggttcc 60
aggttccact ggtgacattg tgctgacaca gtctcctgct tccttagctg tatctctggg 120
gcagagggcc accatctcat acagggccag caaaagtgtc agtacatctg gctatagtta 180
tatgcactgg aaccaacaga aaccaggaca gccacccaga ctcctcatct atcttgtatc 240
caacctagaa tctggggtcc ctgccaggtt cagtggcagt gggtctggga cagacttcac 300
cctcaacatc catcctgtgg aggaggagga tgctgcaacc tattactgtc agcacattag 360
ggagcttaca cgttcggagg ggggaccaag ctggaaataa aacgggctga tgctgcacca 420
tgtgt 425
<210> 4
<211> 128
<212> PRT
<213>BALB/c mouse
<400> 4
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala
20 25 30
Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser
35 40 45
Val Ser Thr Ser Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro
50 55 60
Gly Gln Pro Pro Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser
65 70 75 80
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys
100 105 110
Gln His Ile Arg Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys
115 120 125
<210> 5
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 5
ttctcttgac gttgtactgg 20
<210> 6
<211> 17
<212> DNA
<213>It is artificial synthesized
<400> 6
ttttgaggag acggtga 17
<210> 7
<211> 22
<212> DNA
<213>It is artificial synthesized
<400> 7
ttatgggtag ttcatggaga ca 22
<210> 8
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 8
acacatggtg cagcatcagc c 21

Claims (9)

1. a kind of anti-PCV2 monoclonal antibodies, heavy chain variable region DNA sequence dna is the sequence as shown in SEQ ID NO.1；Its is heavy Chain variable region amino acid sequence be the sequence as shown in SEQ ID NO.2, or on the basis of the SEQ ID NO.2 sequences into The addition of amino acid of row one or more, deletion, replacement and obtain active fragment or the sequence of conservative variant；And/or
Its light chain variable region DNA sequence dna is the sequence as shown in SEQ ID NO.3；Its chain variable region amino acid sequence is such as Sequence shown in SEQ ID NO.4, or adding for one or more amino acid is carried out on the basis of the SEQ ID NO.4 sequences Add, delete, replace and obtain active fragment or the sequence of conservative variant.
2. anti-PCV2 monoclonal antibodies according to claim 1, which is characterized in that the light chain type of the monoclonal antibody is Kappa, hypotype IgG2a.
3. anti-PCV2 monoclonal antibodies according to claim 1, which is characterized in that the ELISA of the monoclonal antibody is imitated Valence is not less than 1：2.56×105。
4. anti-PCV2 monoclonal antibodies according to claim 1, which is characterized in that the IPMA potency of the monoclonal antibody It is 1：8×104。
5. anti-PCV2 monoclonal antibodies according to claim 1, which is characterized in that the affinity of the monoclonal antibody is not Less than 1.26 × 10-10 mol/L。
6. a kind of preparation method of anti-PCV2 monoclonal antibodies described in claim 1, includes the following steps：
（1）Mouse is immunized with the subunit vaccine of PCV2a Cap proteins；
（2）The mouse immune splenocyte and murine myeloma cell are merged, hybridoma is obtained；
（3）The method for combining and taking turns subclone more is detected using IPMA obtains positive hybridoma cell；
（4）Positive monoclonal hybridoma cell strain RNA reverse transcriptions are extracted into cDNA, the weight of monoclonal antibody is gone out by PCR amplification Chain variable region sequence and light-chain variable sequence；
（5）Colonized culture is carried out to positive colony, obtains PCV2 monoclonal antibody hybridoma cell strains；
（6）The hybridoma cell strain is injected in mouse peritoneal and produces monoclonal antibody.
7. a kind of antigen/antibody detection kit, including anti-PCV2 monoclonal antibodies described in claim 1.
8. a kind of anti-PCV2 monoclonal antibodies described in claim 1 are in antigen/antibody immune chromatography test paper, IFA or IPMA Application.
9. a kind of anti-PCV2 monoclonal antibodies answering in the immune affinity column of PCV2 or PCV2 Cap described in claim 1 With.
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