CN105044368B - The indirect Elisa detection kits of pig circular ring virus PCV 2 antibody level and its detection method and application in immune rabbit anteserum - Google Patents
The indirect Elisa detection kits of pig circular ring virus PCV 2 antibody level and its detection method and application in immune rabbit anteserum Download PDFInfo
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Abstract
The present invention provides the indirect Elisa detection kits of pig circular ring virus PCV 2 antibody level in immune rabbit anteserum, and the recombinant protein that the kit is encoded using PCV2 viral genome ORFs ORF2 is envelope antigen.The present invention also provides its detection method and application.The indirect ELISA method of the immune rabbit anteserum PCV2 antibody levels of detection of the present invention can be used for fully evaluating the antibody level for being directed to PCV2 in immune rabbit anteserum.
Description
Technical field
The present invention relates to the indirect Elisa detection kits of pig circular ring virus PCV 2 antibody level in immune rabbit anteserum and its inspection
Survey methods and applications.
Background technology
Pig circular ring virus (Porcine circovirus, PCV) is a kind of minimum animal virus found so far.
Know that PCV has two serotypes, i.e. PCV1 and PCV2.PCV1 is the virus of non-pathogenic.PCV2 is pathogenic virus, it
It is the main pathogen of pmws.PCV2 is a kind of DNA virus, at present the mechanism of causing a disease on PCV2
It is not clear.Quickly diagnose significant to effectively control epidemic disease.Antibody is analysis PCV2 mechanisms of causing a disease and built
The most basic material of corresponding diagnostic method is found, immune rabbit anteserum is a kind of most common type antibodies, it is anti-by restructuring
The validity of the immune rabbit anteserum antibody of original detection, to fully ensureing antibody or even the other detection methods set up on this basis
Reliability is significant.
PCV2 virion diameter 14-17nm, in 20 face body symmetrical structures, no cyst membrane contains covalence closed sub-thread ring
Shape minus-strand dna.Full-length genome is that the nucleotide sequence homology between 1768bp or 1767bp, strain is more than 96%.Genome
There are two main ORFs (open reading frame, ORF), i.e. ORF1 and ORF2.ORF1 codings are viral to answer
Enzyme processed, the duplication transcription with virus is relevant.The structural proteins of ORF2 coding viruses are capsid protein Cap, can be anti-with PCV2
Seroreaction.So, Cap recombinant proteins are frequently utilized for the diagnosis and detection of antibody.
The method of detection PCV2 antibody levels is mainly ELISA adsorption analysis method (enzyme-linked at present
Immunoadsorbent assay, ELISA), this method due to it is easy, quick, sensitive, special the advantages of gradually replace tradition
Agar diffusion method.Rabbit is to prepare one of primary animal of immune serum, and the anti-of high-titer can be produced by being immunized repeatedly
Body, these antiserums after purifying and further mark, can be used for ELISA, SABC, flow cytometry and
The immune analysis such as laser co-focusing.So, the ELISA method that can fully evaluate immune rabbit anteserum has been initially set up with weight
Want meaning.
The content of the invention
The invention provides the indirect Elisa detection kits of pig circular ring virus PCV 2 antibody level in immune rabbit anteserum, energy
Antibody level in enough immune rabbit anteserums of detection well, therefore, the present invention also provides its detection method and application.
The present invention provides the indirect Elisa detection kits of pig circular ring virus PCV 2 antibody level in immune rabbit anteserum, described
Kit develops the recombinant protein of reading frame ORF2 codings as envelope antigen using PCV2 viral genomes.
Preferably, the amino acid sequence of the recombinant protein is referring to SEQ ID No.2.
Further, the preparation method of the recombinant protein is：PCV2 Cap encoding genes are cloned into pET30a (+)
At the multiple cloning sites of expression vector, recombinant expression plasmid is obtained, recombinant plasmid is transferred in host's competent cell, through screening
Identification obtains recombinant strains, under IPTG inductions, expresses PCV2-rCap, by affinity chromatography, obtains purifying
PCV2-rCap。
Preferably, the kit includes primary antibody antibody diluent, and the primary antibody antibody diluent is：Contain 2%
PET30a-E.Coli cellular lysate liquid, 5% skimmed milk power and 5%BSA PBST.The percentage is mass volume ratio.
Preferably, the kit also includes ELIAS secondary antibody, and the ELIAS secondary antibody is goat antirabbit secondary antibody, and the enzyme is peppery
Root peroxidase.
Further, the dilution factor of the ELIAS secondary antibody is 1:2000.
Preferably, the coating concentration of envelope antigen is in the kit：100ng/100μL.
Second object of the present invention is to provide the indirect Elisa of pig circular ring virus PCV 2 antibody level in immune rabbit anteserum
Detection method, step is as follows：
(1) coated elisa plate：By PCV2-rCap-pET30a recombinant proteins after purification and pET30a-E.Coli thalline eggs
Microwell plate is coated with respectively in vain, is washed；
(2) ELISA Plate, washing are closed；The sealer preferably comprises the PBST of 5% skimmed milk power；
(3) the rabbit anteserum primary antibody added after dilution, washing；Dilution rabbit anteserum primary antibody used in dilution be preferably containing
2%pET30a-E.Coli cellular lysate liquid, 5% skimmed milk power and 5%BSA PBST；The percentage is mass volume ratio；
(4) ELIAS secondary antibody added after dilution, 37 DEG C of insulating boxs are incubated 1 hour, washing；Dilute dilute used in ELIAS secondary antibody
Release the PBST dilutions that liquid preferably comprises 5% skimmed milk power；Select the dilution best results of this concentration.
(5) develop the color, terminate and OD is read on ELIASA490nmAbsorbance value.
Preferably, the purification process of the recombinant protein is：The recombinant protein, tool are purified using affinity chromatography method
Body is：Recombinant protein inclusion body is first washed with inclusion body cleaning solution, centrifugation retains uses urea washes, Ran Houyong again after precipitation
BufferB dissolving inclusion body precipitations, are collected by centrifugation supernatant, the nickel post that supernatant is added after balance, room temperature is combined；First afterwards
Pillar is washed with Buffer C and Buffer D afterwards, component is collected, pillar is finally washed with Buffer E and collects component.
Third object of the present invention is to provide mentioned reagent box in the immune rabbit anteserum pig circular ring virus PCV 2 antibody of detection
Application in level.
The method of the present invention is except using the antibody diluent processing rabbit anteserum reduction nonspecific reaction for rabbit anteserum
Outside, at the same also using the pET30a-E.Coli of appropriate (consistent with the amount of PCV2-rCap coated elisa plates) cellular lysate liquid as
Rabbit anteserum antibody level is immunized in envelope antigen coated elisa plate, Parallel testing, and the non-specific of immune serum is excluded to greatest extent
Property reaction, more can fully evaluate the PCV2 antibody titers of immune rabbit anteserum.Rabbit anteserum PCV2 antibody is immunized in the detection of the present invention
The indirect ELISA method of level can be used for fully evaluating the antibody level for being directed to PCV2 in rabbit anteserum.
Brief description of the drawings
Accompanying drawing is used for providing a further understanding of the present invention, and constitutes a part for specification, the reality with the present invention
Applying example is used to explain the present invention together, is not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is that pCV2-rCap (121-669bp)-pET30a [+] recombinant plasmid double digestion identifies collection of illustrative plates.M is nucleic acid molecules
Quality standard, 1 is to use BamHI and XhoI double digestion positive recombinant plasmids.
The induced expression that Fig. 2 is PCV2-rCap is protein standard with purifying collection of illustrative plates .M, and 1 is to contain zero load
The Host Strains of body are the mycoprotein of pET30a-BL21 (DE) 3 IPTG induced expressions；2 be the weight containing PCV2-Cap genes
Group expression vector bacterial strain is pET30a-PCV2-Cap-BL21 (DE) 3 non-induced expression mycoprotein；3 be to contain PCV2-Cap
The recombinant expression carrier bacterial strain of gene is pET30a-PCV2-Cap-BL21 (DE) 3 IPTG induced expression mycoproteins；4 be to adopt
With the PCV2-rCap of affinity chromatography method after purification.
Fig. 3 be using three kinds of different rabbit anteserum antibody diluent dilution standard rabbit anteserums, carry out respectively PCV2-rCap and
The coated ELISA detections of pET30a-E.Coli.Ordinate represents OD492nm absorbance value, and abscissa represents different rabbit blood
Clear antibody diluent classification, wherein " 1 ", which represents the first antibody diluent, contains 10% horse serum, 2%pET30a-E.Coli
Cellular lysate liquid, 5% skimmed milk power and 5%BSA PBST；It is to contain 2%pET30a- that " 2 ", which represent second of antibody diluent,
E.Coli cellular lysate liquid, 5% skimmed milk power and 5%BSA PBST；It is de- containing 5% that " 3 ", which represent the third antibody diluent,
The PBST of fat milk powder and 5%BSA.Column diagram of the inside with twill represents coating PCV2-rCap and carries out ELISA examination criteria rabbits
Serum (1:1000 dilutions), column diagram of the inside with grid represents coating pET30a-E.Coli and carries out ELISA examination criteria rabbits
Serum (1:1000 dilutions).
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Test material used in following embodiments, is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.
The restructuring of embodiment 1 PCV2 capsid protein is PCV2-rCap preparation
1.PCV2-rCap construction of recombinant expression plasmid
The plasmid containing complete PCV2 encoding genes (common 1767bp) preserved using laboratory as template, with
P163-F(5-cgcggatccAtgaaaaatggcatcttcaacacccgcct-3, is aggravated and attached underscore is represented
BamH I restriction enzyme sites) and P164-R (5-ccgctcgagTtctctgaattgtacatacatggt-3, is aggravated and attached underscore
Represent Xho I restriction enzyme sites) standard PCR amplification is carried out for primer, PCR reaction systems are：1μL 1:10 times dilution plasmids be
Template, each 2 μ L, EXTaq archaeal dna polymerases of 1 μ L, 2.5mmol/L dNTPs of 10 μm of ol/L upstream and downstream primers 0.2 μ L, 10 ×
The μ L of PCR buffer solutions 2, plus ddH2O to 20 μ L.PCR response procedures are as follows：95 DEG C of 5min, then 94 DEG C of 1min, 56 DEG C of 1min, 72
DEG C 1min, 30 circulations, 72 DEG C re-extend 10min.
The encoding gene segment 121-669bp for the PCV2 Cap Cap for removing signal peptide is obtained, while being introduced in upstream
BamH I restriction enzyme sites, introduce Xho I restriction enzyme sites in downstream, pure using BamH I and Xho I double digestion processing simultaneously
The PCR primer of change and pET30a (+) recombinant expression carrier of commercialization, after the connection of T4DNA ligases, connection product is imported
In e. coli bl21 (DE3) pLys competent cells, transformant even spread to the LA solid fine jades containing kalamycin resistance
On lipolysaccharide flat board, growth is stayed overnight, the bacterium colony with kanamycins antibody of picking growth, is cultivated and is identified.Shake bacterium and carry
The plasmid of candidate's bacterium colony is taken, is detected using BamH I and Xho I double digestions, the DNA fragmentation of estimated size is as a result discharged (see figure
1).The plasmid of extraction is subjected to sequencing analysis, as a result shows that recombinant plasmid (being named as PCV2-rCap-pET30a [+]) contains mesh
Gene and corresponding restriction enzyme site, specific nucleotide sequence is referring to SEQ ID No.1 in sequence table, its amino encoded
Acid sequence is referring to SEQ ID No.2 in sequence table.
2.PCV2-rCap induced expression and purifying
The positive transformant containing recombinant plasmid PCV2-rCap-pET30a [+] of identification is inoculated in containing kanamycins
LB culture mediums in, under the conditions of 37 DEG C, 200rpm carry out shaking table concussion and cultivate, as bacterium solution OD600When reaching 0.6-0.8, add
Final concentration of 1.0mM IPTG, 37 DEG C of induced expression 6-10h, collect zymotic fluid, and 12000rpm centrifugations 5min collects thalline, thalline
Use ddH2O washes settling flux twice, ultrasonic disruption thalline, centrifuge it is broken after supernatant and precipitation, with a little
ddH2O, which suspends, to be precipitated, and prepares electrophoresis detection.The sample of different piece is taken to add 2x albumen sample-loading buffers, boiling water boiling 5min takes
Supernatant after centrifugation carries out 12%SDS-PAGE protein electrophoresis detections, as a result shows that PCV2-rCap is big with inclusion bodies
Enterobacteria successful expression, is as a result shown in Fig. 2.
Because restructuring PCV2-rCap albumen contain it is histidine-tagged, using affinity chromatography method purify this restructuring
Albumen.Process is as follows：First washed with inclusion body cleaning solution ultrasonic disruption collection inclusion body, wash every time using centrifuge from
The heart, 10 minutes, abandons supernatant and retains precipitation by 10000 revs/min, then with 2M urea washes 2 times, then with buffer B dissolving bags
Contain body precipitation, the supernatant of dissolving, then the nickel post that supernatant is added after being balanced with buffer B is collected by centrifugation, room temperature is combined
40min, allows albumen and resin hatching combination.Pillar is washed with the Buffer C of 5 times of applied sample amount volumes afterwards, is washed altogether 3 times；Again
Pillar is washed with 500 μ l Buffer D, 8-10 collection component is washed, finally washes pillar with 500 μ l Buffer E, typically wash 10 times
Component is collected, as a result 12%SDS-PAGE gel detection purifying proteins are shown in Fig. 2.Solution compound method is referred to《Molecular cloning
Experiment guide》.
Inclusion body lavation buffer solution：50mM Tris-Cl(pH8.0),10mM EDTA(pH8.0),100mM NaCl,
0.05%Triton X-100；
Buffer B (lysate/combination buffer):8M urea, 0.1M sodium phosphate buffers, 0.01M Tris-Cl,
pH8.0；
Buffer C (lavation buffer solution):8M urea, 0.1M sodium phosphate buffers, 0.01M Tris-Cl pH6.3；
Buffer D (elution buffer):8M urea, 0.1M sodium phosphate buffers, 0.01M Tris-Cl pH5.9；
Buffer E (elution buffer):8M urea, 0.1M sodium phosphate buffers, 0.01M Tris-Cl pH4.5.
The Immunofluorescent antibody analysis method (ELISA) of the immune rabbit anteserum PCV2 antibody levels of the detection of embodiment 2
Set up
On the basis of the PCV2-rCap of above-mentioned purifying, the PCV2 rabbit anteserums using commercialization is standard positive serums, with rather
Epidemic disease rabbit anteserum is standard female serum, by optimizing reaction condition, sets up the indirect of the immune rabbit anteserum PCV2 antibody levels of detection
ELISA method.
1. determine rabbit anteserum antibody diluent
The antibody diluent of antibody diluent, especially primary antibody, is setting up the immune rabbit anteserum PCV2 antibody levels of detection
Played an important role in indirect ELISA method, diluted first by using a different antiantibodys, PCV2- is being coated with respectively
On rCap and pET30a-E.Coli ELISA Plate, by the detection to standard positive rabbit anteserum, screening determines that suitable primary antibody resists
Body dilution.It is final to determine to contain 2%pET30a-E.Coli cellular lysates by the com-parison and analysis to different antibodies dilution
Liquid, 5% skimmed milk power and 5%BSA PBST are optimum rabbit anteserum antibody diluents, using this antibody diluent greatly
Reduce the nonspecific reaction of PCV2 rabbit anteserums and pET30a-E.Coli mycoproteins (see Fig. 3).
It is determined that the process of most suitable antibody diluent is as follows：(1) coated elisa plate：First use the BCA albumen of Pierce companies
Quantification assay kit determines the PCV2-rCap-pET30a recombinant proteins and pET30a-E.Coli mycoprotein concentration of purifying,
With the coating buffer diluted protein of Sigma companies, microwell plate is coated with 200ng/100 μ L/ pore systems, 4 DEG C of coatings are stayed overnight；(2) wash
Wash ELISA Plate：The ELISA Plate being coated with is taken out, supernatant is abandoned, PBST liquid is washed 2 times, hole, micro concussion instrument concussion are filled it up with for the third time
5min, dries liquid in hole, relaunders twice, hole is filled it up with for the third time, is stored at room temperature 5min, dries liquid in hole；(3)
Close ELISA Plate：The PBST containing 5% skimmed milk power is added, 300 μ l/ holes are incubated at room temperature 1 hour；(4) ELISA Plate is washed：Such as
Step 2 method is washed；(5) primary antibody is added：Following three kinds of different antibody diluents are used with 1:1000 dilution Abcam companies
PCV2 rabbit anteserums, 100 μ L/ holes, are added on the ELISA Plate closed, and 37 DEG C of insulating boxs are incubated 1 hour.Three kinds of antibody diluents
Respectively：The first is that 2%pET30a-E.Coli cellular lysates liquid is (i.e. using IPTG induced expressions containing 10% horse serum
The coli somatic ultrasonic treatment liquid of BL21 (DE3) pLys containing pET30a (+) empty carrier), 5% skimmed milk power and
5%BSA PBST, is for second the PBST of the liquid of cellular lysate containing 2%pET30a-E.Coli, 5% skimmed milk power and 5%BSA,
The third is the PBST containing 5% skimmed milk power and 5%BSA；(6) microwell plate is washed：Such as the washing of step 2 method；(7) enzyme mark is added
Secondary antibody：With the PBST dilutions containing 5% skimmed milk power with 1:The goat antirabbit two of 2000 dilution horseradish peroxidase-labeleds
Anti- (Sigma companies), 100 μ L/ holes, 37 DEG C of insulating boxs are incubated 1 hour；(8) microwell plate is washed：Such as the washing of step 2 method；(9)
Colour developing：Adding the o-phenylenediamine OPD nitrite ions containing hydrogen peroxide, (6ml OPD liquid adds 10 μ L H2O2), 100 μ L/ holes, 37 DEG C incubate
Educate 30min；(10) terminating reaction and readings：Colour developing ELISA Plate is taken out, 1.5M sulfuric acid terminate liquid terminating reaction, 50 is directly added into
μ L/ holes, that is, be engraved on ELIASA and read OD490Nm absorbance value.4 repetitions of each sample, take its average.
By comparing the normal rabbit serum for using different rabbit anteserum antibody diluents to dilute simultaneously respectively with PCV2-rCap
Reacted with pET30a-E.Coli for the ELISA of envelope antigen, it can be seen that second of antibody diluent is containing 2%
PET30a-E.Coli cellular lysate liquid, 5% skimmed milk power and 5%BSA PBST are optimum rabbit anteserum antibody diluents,
Because to greatly reduce the non-specific of PCV2 rabbit anteserums and pET30a-E.Coli mycoproteins after this liquid diluting rabbit anteserum
Property reaction, response value stills remain in higher level 0.1 or so with PCV2-rCap reaction, and response value is 1.0 or so
(see Fig. 3).Non-immune rabbit anteserum is resisted with after this antibody dilute and by coating of PCV2-rCap and pET30a-E.Coli
Former ELISA response values are equal to or less than 0.1.With other two kinds of antibody diluents dilute normal rabbit serum with
PET30a-E.Coli is higher for the ELISA response values of envelope antigen, close to and above 0.3, disturbs to rabbit anteserum PCV2
The judgement of antibody level.
2. determine PCV2-rCap package amount
It is determined that after most suitable rabbit anteserum antibody diluent, further optimizing PCV2-rCap coating concentration, by PCV2-
RCap carries out ELISA reactions according to 800,400,200,100,50,25ng/100 μ L/ holes coated elisa plate holes by above-mentioned flow,
As a result the optimum coating concentration for showing PCV2-rCap is 100ng/100 μ L/ holes.
3. determine the concentration of ELIAS secondary antibody
It is determined that after most suitable rabbit anteserum antibody diluent and PCV2-rCap coating concentration, further optimizing ELIAS secondary antibody
Reaction density, by the goat antirabbit secondary antibody (Sigma companies) of horseradish peroxidase-labeled according to 1:8000,1:4000；1:
2000,1:1000 and 1:Reacting hole is added after 500 dilutions, ELISA reactions is carried out by above-mentioned flow, as a result shows ELIAS secondary antibody
Optimum dilution degree is 1:2000.
4. the indirect ELISA method of the immune rabbit anteserum PCV2 antibody levels of detection is set up
By the optimal PCV2-rCap coating concentration of the screening to rabbit anteserum antibody diluent and determination and ELIAS secondary antibody
Dilution factor after, establish the indirect ELISA method of the immune rabbit anteserum PCV2 antibody levels of detection, its program is as follows：
(1) coated elisa plate：PCV2-rCap-pET30a recombinant proteins and pET30a-E.Coli mycoproteins are coated with
Liquid is diluted, and is coated with microwell plate respectively with the system in 100ng/100 μ L/ holes, and 4 DEG C of coatings are stayed overnight；
(2) ELISA Plate is washed：The ELISA Plate being coated with is taken out, supernatant is abandoned, PBST liquid is washed 2 times, and hole is filled it up with for the third time, micro
Concussion instrument shakes 5min, dries liquid in hole, relaunders twice, hole is filled it up with for the third time, is stored at room temperature 5min, dries liquid in hole
Body；
(3) ELISA Plate is closed：The PBST containing 5% skimmed milk power is added, 300 μ l/ holes are incubated at room temperature 1 hour；
(4) ELISA Plate is washed：Such as the washing of step 2 method；
(5) rabbit anteserum primary antibody is added：Using containing 2%pET30a-E.Coli cellular lysate liquid, 5% skimmed milk power and 5%
BSA PBST dilution rabbit anteserums, 100 μ L/ holes are added on the ELISA Plate closed, and 37 DEG C of insulating boxs are incubated 1 hour；
(6) microwell plate is washed：Such as the washing of step 2 method；
(7) ELIAS secondary antibody is added：With the PBST dilutions containing 5% skimmed milk power with 1:2000 dilution horseradish peroxidases
The goat antirabbit secondary antibody (Sigma companies) of enzyme mark, 100 μ L/ holes, 37 DEG C of insulating boxs are incubated 1 hour；
(8) microwell plate is washed：Such as the washing of step 2 method；
(9) show：Adding the o-phenylenediamine OPD nitrite ions containing hydrogen peroxide, (6ml OPD liquid adds 10 μ L H2O2), 100 μ
L/ holes, 37 DEG C of incubation 30min；
(10) terminating reaction and readings：Colour developing ELISA Plate is taken out, 1.5M sulfuric acid terminate liquid terminating reaction, 50 is directly added into
μ L/ holes, that is, be engraved on ELIASA and read OD490nmAbsorbance value.
After the indirect ELISA method of the immune rabbit anteserum PCV2 antibody levels of detection is established as described above, it can use
The PCV2 antibody levels of rabbit anteserum are immunized in this method evaluation.
Embodiment three：The application of the indirect ELISA method of the immune rabbit anteserum PCV2 antibody levels of detection of foundation
1. it is prepared by immune rabbit anteserum
Adult male New Zealand White Rabbit 3 is immunized using PCV2 inactivated vaccines circle Bi Jing (production of Shanghai Hai Li companies),
Every rabbit is immune 1 milliliter every time, point multi-point injection in dorsal sc, every two weeks booster immunization once, continuous immunity 3 months,
In immune preceding collection ear source venous blood every time after one month, serum, -20 DEG C of preservations are collected.
2. the detection of immune rabbit anteserum antibody titer
The indirect ELISA method detection for the immune rabbit anteserum PCV2 antibody levels of detection set up by embodiment 2 is immune
Inactivate the rabbit anteserum antibody level of PCV2 vaccines：PCV2-rCap and pET30a-E.Coli mycoproteins are pressed into 100ng/ respectively
100 μ L/ holes coated elisa plates, are added after closing with containing 2%pET30a-E.Coli cellular lysate liquid, 5% skimmed milk power and
The immune rabbit anteserum that 5%BSA PBST antibody diluents are serially diluted, incubation adds 1 after terminating:2000 ELIAS secondary antibodies, are added
Zymolyte is shown, reads OD490nmAbsorbance value.3 repetitions of each dilution factor, take its average, the testing result such as institute of table 1- tables 3
Show.
Table 1 detects first PCV2 vaccine immunity rabbit serum antibody titer using indirect ELISA method
The result of table 1 shows that first PCV2 inactivated vaccine immune rabbits serum antibody titer eight, which exempts from rear highest, to be reached
1:6400 or so.Rabbit is immunized after exempting to terminate eight as the increase antibody level of immune time is in trend is gradually increased in the rabbit
Sub- PCV2 serum antibody titer reaches highest, with 1:During 6400 dilution, OD492nmReadings has reached more than 0.8, and with
PET30a-E.Coli response value is 0.1 or so.With antibody and target protein PCV2-rCap response value 0.8 or so, and
It is that standard is used as the antibody titer for judging immune serum to be no more than 0.2 with pET30a-E.Coli response value, then first rabbit
The PCV2 serum antibody titers highest of son can reach 1:More than 6000.
Table 2 detects second PCV2 vaccine immunity rabbit serum antibody titer using indirect ELISA method
The result of table 2 shows that second PCV2 inactivated vaccine immune rabbit serum antibody titer highest can reach 1:10000
More than.2 weeks antibody titers are significantly raised after the rabbit is immune at second, and 1:Readings can reach more than 0.8 after 3200 dilutions.It
Afterwards with the increase of immune time, exempt from three, occur in that antibody level once declines when four exempt from and exempt from five, exempt from 2 six afterwards
Start lifting again after week, immune rabbit PCV2 serum antibody titer reaches highest after exempting to terminate eight, with 1:12800 dilutions
When, OD492nmReadings has reached 1.0 or so, and with pET30a-E.Coli response value 0.1 or so.
Table 3 detects the 3rd PCV2 vaccine immunity rabbit serum antibody titer using indirect ELISA method
The result of table 3 shows that the 3rd PCV2 inactivated vaccine immune rabbit serum antibody titer can reach 1 after exempting from eight:
More than 2000.With the increase of immune time, the PCV2 of immune rabbit antibody level of serum is gradually slowly increased, before six exempt from
Antibody level is very low always, and antibody level starts significantly raised after exempting from six, and eight exempt from the serum of rear bloodletting with 1:1600 dilutions, with
PCV2-rCap response value is no more than 0.15 0.9 or so with pET30a-E.Coli response value.With antibody and target egg
White PCV2-rCap response value no more than 0.2 is that standard is used as judgement 0.8 or so, and with pET30a-E.Coli response value
The antibody titer of immune serum, then the PCV2 serum antibody titers of the 3rd rabbit can reach 1:More than 2000.
Epidemic disease is inactivated to three PCV2 using newly-established PCV2-rCap ELISA and pET30a-E.Coli ELISA methods
The serum antibody of seedling immune rabbit is tested and analyzed, and as a result shows that the immune effect of second rabbit is best, at the 8th time
After immune, serum antibody titer can reach 1:10000, and the immune effect of the 3rd rabbit is worst, eight immune rear highests
Potency is only 1:2000 or so.The immune effect of first rabbit is good, and eight, which exempt from rear antibody titer, can reach 1:6400.By right
The dynamic detection of three immune rabbit PCV2 antibody level of serum, as a result shows, rabbit anteserum PCV2 is immunized in the detection that is directed to of foundation
The indirect ELISA method of antibody level is reliable, can fully evaluate the antibody level of serum.
Finally it should be noted that：The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still may be used
To be modified to the technical scheme described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic.
Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., should be included in the present invention's
Within protection domain.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>The indirect Elisa detection kits of pig circular ring virus PCV 2 antibody level and its detection in immune rabbit anteserum
Methods and applications
<170> PatentIn version 3.5
<210> 1
<211> 561
<212> DNA
<213>Artificial sequence
<400> 1
ggatccaaaa atggcatctt caacacccgc ctctcccgca ccatcggtta tactgtcaag 60
aaaaccacag tcagaacgcc ctcctggaat gtggacatga tgagatttaa tattaatgat 120
tttcttcccc caggaggggg ctcaaacccc ctcactgtgc cctttgaata ctacagaata 180
aggaaggtta aggttgaatt ctggccctgc tccccaatca cccagggtga caggggagtg 240
ggctccactg ctgttattct agatgataac tttgtaacaa aggccaatgc cctaacctat 300
gacccctatg taaactactc ctcccgccat accataaccc agcccttctc ctaccactcc 360
cggtacttta ccccgaaacc tgtccttgat aggacaatcg attacttcca acccaataac 420
aaaagaaatc aactctggct gagactacaa actactggaa atgtagacca tgtaggcctc 480
ggcactgcgt tcgaaaacag tatatacgac caggactaca atatccgtat aaccatgtat 540
gtacaattca gagaactcga g 561
<210> 2
<211> 187
<212> PRT
<213>Artificial protein
<400> 2
Gly Ser Lys Asn Gly Ile Phe Asn Thr Arg Leu Ser Arg Thr Ile Gly
1 5 10 15
Tyr Thr Val Lys Lys Thr Thr Val Arg Thr Pro Ser Trp Asn Val Asp
20 25 30
Met Met Arg Phe Asn Ile Asn Asp Phe Leu Pro Pro Gly Gly Gly Ser
35 40 45
Asn Pro Leu Thr Val Pro Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys
50 55 60
Val Glu Phe Trp Pro Cys Ser Pro Ile Thr Gln Gly Asp Arg Gly Val
65 70 75 80
Gly Ser Thr Ala Val Ile Leu Asp Asp Asn Phe Val Thr Lys Ala Asn
85 90 95
Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser Ser Arg His Thr Ile
100 105 110
Thr Gln Pro Phe Ser Tyr His Ser Arg Tyr Phe Thr Pro Lys Pro Val
115 120 125
Leu Asp Arg Thr Ile Asp Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln
130 135 140
Leu Trp Leu Arg Leu Gln Thr Thr Gly Asn Val Asp His Val Gly Leu
145 150 155 160
Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp Gln Asp Tyr Asn Ile Arg
165 170 175
Ile Thr Met Tyr Val Gln Phe Arg Glu Leu Glu
180 185
Claims (1)
1. the indirect Elisa detection kits of pig circular ring virus PCV 2 antibody level in immune rabbit anteserum, it is characterised in that：The examination
The recombinant protein that agent box is encoded using PCV2 viral genome ORFs ORF2 is envelope antigen, with pET30a-E.Coli's
Cellular lysate liquid is control coating thing, and the amino acid sequence of the recombinant protein is coating in SEQ ID No.2, the kit
The coating concentration of antigen is：100ng/100μL；The kit also includes primary antibody antibody diluent, the antiantibody dilution
Liquid is：Contain 2% pET30a-E.ColiCellular lysate liquid, 5% skimmed milk power and 5%BSA PBST；The percentage is quality
Volume ratio；The kit also includes ELIAS secondary antibody, and the ELIAS secondary antibody is goat antirabbit secondary antibody, and the enzyme is horseradish peroxidating
Thing enzyme, the dilution factor of the ELIAS secondary antibody is 1:2000；
The preparation method of the recombinant protein is：PCV2 Cap encoding genes are cloned into many grams of pET30a (+) expression vector
Long Weidianchu, obtains recombinant expression plasmid, recombinant plasmid is transferred into e. coli bl21 (DE3) pLys host's competent cell
In, recombinant strains are obtained through Screening and Identification, under IPTG inductions, PCV2-rCap is expressed, by affinity chromatography, obtains
Obtained the PCV2-rCap of purifying.
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| CN201510141663.XA CN105044368B (en) | 2015-03-27 | 2015-03-27 | The indirect Elisa detection kits of pig circular ring virus PCV 2 antibody level and its detection method and application in immune rabbit anteserum |
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| CN109060972B (en) * | 2018-05-16 | 2021-09-24 | 苏州药明泽康生物科技有限公司 | Application of rabbit blood in preparing human disease in-vitro diagnosis kit |
| CN109001461B (en) * | 2018-06-26 | 2024-02-02 | 安阳工学院 | Immune rapid test kit for PCV-3 immune evaluation and wild virus infection diagnosis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1588076A (en) * | 2004-09-28 | 2005-03-02 | 浙江大学 | Indirect ELISA diagnostic reagent kit for pig B type circular virus antibody |
| CN200962112Y (en) * | 2006-06-07 | 2007-10-17 | 浙江大学 | Pig circovirus II type antibody detection reagent box |
| KR20080087317A (en) * | 2007-03-26 | 2008-10-01 | 전남대학교산학협력단 | Protein chip for diagnosing porcine circovirus and method of diagnosing porcine circovirus using the chip |
| GB0712160D0 (en) * | 2007-06-22 | 2007-08-01 | Univ Ghent | Methods and compositions in the treatment of procine circoviral infection |
| CN101629956B (en) * | 2009-07-09 | 2010-12-01 | 湖南农业大学 | ELISA kit for detecting porcine circovirus antibody II |
| CN101799471A (en) * | 2009-12-23 | 2010-08-11 | 湖南农业大学 | Indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) method established based on classical swine fever virus NS3 |
| CN103033614A (en) * | 2012-12-19 | 2013-04-10 | 北京伟嘉人生物技术有限公司 | ELISA kit for porcine circovirus type 2 antibody detection |
| KR20140118626A (en) * | 2013-03-29 | 2014-10-08 | 고려대학교 산학협력단 | PCV2 ORF3-specific primer/probe sets and the method for quantitative analysis of PCV2 DNA in the porcine blood using the same |
| CN104725490A (en) * | 2015-03-23 | 2015-06-24 | 湖北省农业科学院畜牧兽医研究所 | Porcine circovirus 2 type ELISA antibody detection kit |
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