CN109535229A

CN109535229A - 2 type Rep albumen of recombinant porcine circovirus, ELISA detection kit and the preparation method and application thereof

Info

Publication number: CN109535229A
Application number: CN201811394114.3A
Authority: CN
Inventors: 荣俊; 陈清清; 李国攀; 徐保娟; 匡红艳; 郝尧光
Original assignee: Yangtze University; Qingdao Yebio Bioengineering Co Ltd
Current assignee: Yangtze University; Qingdao Yebio Bioengineering Co Ltd
Priority date: 2018-11-21
Filing date: 2018-11-21
Publication date: 2019-03-29
Anticipated expiration: 2038-11-21
Also published as: CN109535229B

Abstract

The present invention provides recombinant porcine circovirus 2 type (PCV2) Rep albumen, ELISA detection kit and the preparation method and application thereof.The preparation of recombination PCV2 Rep albumen includes the transformation of building PCV2 Rep protein gene cloning carrier and gene；Building recombination PCV2 Rep protein expression plasmid carrier, engineering bacteria；Recombinate the inducing expression and purifying of PCV2 Rep albumen；Porcine circovirus 2 type Rep protein antibodies ELISA detection kit is prepared with recombination PCV2 Rep albumen again, it can detect PCV2 wild virus infection antibody and PCV2 inactivated virus vaccine or totivirus attenuated vaccine immunity antibody in Swine serum, and it is negative to PCV2 Cap protein genetic engineering subunit vaccine immune swine Serum Antibody Detection；In conjunction with the application of PCV2 Cap protein antibody ELISA detection kit, subunit vaccine immune swine and wild virus infection pig can be distinguished, it is virus purified convenient for the PCV2 on pig farm.

Description

2 type Rep albumen of recombinant porcine circovirus, ELISA detection kit and preparation method thereof With application

Technical field

The present invention relates to 2 type Rep albumen of biopharmaceutical technology more particularly to recombinant porcine circovirus, ELISA to examine Test agent box and the preparation method and application thereof.

Background technique

Pig circular ring virus (Porcine Circovirus, PCV) belongs to circovirus section, Circovirus, and virus is without capsule Film is single-stranded negative crural ring shape DNA virus, is one of the smallest animal virus.Up to the present, it has been found that pig circular ring virus is deposited In 3 genotype: 1 type of pig circular ring virus (PCV1), porcine circovirus 2 type (PCV2) and 3 type of pig circular ring virus (PCV3).Its Middle PCV2 and PCV3 has pathogenic, and PCV1 no pathogenicity.Virion diameter about 17nm is in 20 face body symmetrical structures.Gene Group size is about 1.76kb, contains 2 main Open reading frames (ORF), wherein ORFl gene product and rdrp virus Related (involved in virus replication, rep), ORF2 gene product are to constitute viral capsid proteins (Capsid Protein, referred to as: cap) ingredient.

Canadian scientist John Harding resolves milk piglet multisystem exhaustion syndrome in report in 1991 (postweaning multisystemic wasting syndrome, PMWS) confirms pig gyrate virus II type by research (Porcine circovirus 2, PCV2) is the main pathogen of the syndrome.Cardinal symptom shows as gradual thin, breathing Difficult rapid, anaemia, diarrhea, jaundice, interstitial pneumonia, lymphnoditis and ephritis etc..In recent years it has also been found that pigskin inflammation and ephritis Syndrome (porcine dermatitis and nephropathy syndrome, PDNS), porcine respiratory syndrome (porcine respiratory disease complex, PRDC), the congenital chatter of A2 type (congenital tremor, CT), pig is Hypertrophic and necrotizing pneumonia (Porcine Proliferative and necrotizing Pneumonia, PNP), the diseases such as breeding difficulty (Reproductive failure) all have with PCV2 closely related.

PCV2 infection can cause a kind of clinical syndrome, main to encroach on 5-12 week old weanling pig, and the harm of PCV2 is The immune function for infecting pig can be made to be damaged, cause Abwehrkraft des Koepers to decline, often gone out in the form of subclinical infection It is existing, it is easily ignored.Since PCV2 infection makes immune system be damaged, secondary or concurrent other infectious diseases are easy, are caused Bigger harm.This disease is in world pop, finds to have caused to China's pig breeding industry there are since this disease for the first time from China in 2000 Sizable economic loss.But cytopathy is not generated when cultivating in vitro due to PCV2, virus multiplication ability is poor, using tradition Method to prepare the viral vaccine extremely difficult.

Preparing porcine circovirus 2 type (PCV2) vaccine with the method for genetic engineering is the important channel for controlling the disease.In weight The Cap protein gene of PCV2 is mostly important alternative gene in group vaccine research, because Cap albumen is that the virus is main Structural proteins contain type specific antigen determinant.There are research data report, the neutralizing monoclonal antibody and Duo Ke of PCV2 Grand pig antiserum can mutually identify with cap albumen and occur neutralization reaction.In recombinant vaccine, recombination engineering is sub- single Position vaccine is vaccine the safest, and its immune effect also determines the most, the energy as long as the amount of antigen protein is sufficiently large Generate corresponding specific antibody.Has the gene work of the PCV2 cap protein production of baculovirus expression on domestic market Journey subunit vaccine, and the genetic engineering subunit vaccine produced with the PCV2 Cap protein of Bacillus coli expression.

Vaccine immunity Swine serum antibody test is a kind of evaluation method of important immune effect, and there are many inspections both at home and abroad Survey the ELISA detection method of Swine serum PCV2 antibody.These ELISA detection methods are all the PCV2 Cap proteins with recombinant expression As indirect ELISA envelope antigen.Natural PCV2 virus infection and PCV2 genetic engineering subunit vaccine (Subunit Vaccine, also known as biosynthesis subunit vaccine or recombinant subunit vaccine refer to protective antigen gene in protokaryon or true It is expressed in nucleus, and vaccine is made with gene product-protein or polypeptide.) the detection knot that antibody positive can all occur is immunized Fruit.It cannot distinguish between vaccine immunity pig and natural infection pig.It is unfavorable for the pig for having infected porcine circovirus 2 type in farm It removes or eliminates, cause the virus in farm's long-term propagation, the virus purified target of pig farm PCV2 cannot be reached.

Summary of the invention

It is an object of the invention to overcome the defect of the prior art, provide 2 type Rep albumen of recombinant porcine circovirus, ELISA detection kit and the preparation method and application thereof.The method comprises the steps of firstly, preparing obtaining 2 type Rep albumen of recombinant porcine circovirus, Recombinating PCV2 Rep molecular weight of albumen is about 39kD, and the purity of protein after purification by an affinity chromatography is 95% or more；So Indirect ELISA detection is carried out to sample serum using PCV2 Rep recombinant protein as envelope antigen afterwards, it is natural that this method detects PCV2 It infects pig or Swine serum is immunized in PCV2 inactivated virus vaccine (or attenuated live vaccines), it may appear that positive testing result；And it examines It surveys the immune Swine serum of PCV2 cap protein subunit vaccine and negative findings then occurs.Therefore, with PCV2 Rep albumen of the invention Antibody ELISA detection kit and preparation method thereof, in conjunction with the indirect ELISA detection method of PCV2 Cap protein envelope antigen, The natural infection antibody (or whole virus vaccine immune swine antibody) and subunit vaccine immune antiboidy that PCV2 can be distinguished are pig The purification of pig circular ring virus provides foundation in.

The present invention is implemented as follows:

An object of the present invention is to provide a kind of 2 type Rep albumen of recombinant porcine circovirus, the Recombinant Swine circle The amino acid sequence of 2 type Rep albumen of circovirus virus is as shown in SEQ ID NO.1.

The second object of the present invention is to provide the preparation method of 2 type Rep albumen of recombinant porcine circovirus, including it is as follows Step:

Step 1, building 2 type of recombinant porcine circovirus (PCV2) Rep protein gene cloning carrier: design nest-type PRC primer PCV2 Rep protein gene is built into after carrying out DNA extraction and nested PCR amplification to PCV2 wild virus infection swine disease material collected Cloning vector；

Step 2, the transformation of 2 type of recombinant porcine circovirus (PCV2) Rep protein gene: expand for the resulting nest-type PRC of step 1 Increase production the optimization that object design primer realizes PCV2 Rep protein gene expression codon；And pass through the side of Over lapping PCR Method increases the sequence of coding His at 5 '-ends of PCV2 Rep protein gene expression sequence, and closes I digestion of Nco in former sequence Site；

Step 3, building 2 type of recombinant porcine circovirus (PCV2) Rep protein expression plasmid carrier；The pig circular ring virus of building The nucleotide sequence in the expression area of 2 type Rep protein expressing plasmids is as shown in SEQ ID NO.14.

Step 4, building 2 type of recombinant porcine circovirus (PCV2) Rep protein expression engineering bacteria；

The inducing expression and purifying of step 5,2 type of recombinant porcine circovirus (PCV2) Rep albumen.

Specifically, nest-type PRC primer is as follows in the step 1:

PCV2 rep nest-type PRC housing upstream primer PF: nucleotide sequence as shown in SEQ ID NO.2,

PCV2 rep nest-type PRC housing downstream primer PR: nucleotide sequence as shown in SEQ ID NO.3,

PCV2 rep nest-type PRC inner sleeve upstream primer PF: nucleotide sequence as shown in SEQ ID NO.4,

PCV2 rep nest-type PRC inner sleeve downstream primer PR: nucleotide sequence is as shown in SEQ ID NO.5；

Specifically, primer used in codon optimization is as follows in the step 2:

Codon optimization upstream primer sequence: nucleotide sequence is as shown in SEQ ID NO.6；

Codon optimization downstream primer sequence: nucleotide sequence is as shown in SEQ ID NO.7.

Specifically, Over lapping PCR includes Over lapping PCR A segment and Over in the step 2 Lapping PCR B segment,

The amplification template of Over lapping PCR A segment is load constructed by the amplified production after codon optimization The pET28a expression plasmid of PCV2 rep gene, the amplimer of the A segment are as follows:

The amplification upstream primer AP1 of A segment: nucleotide sequence is as shown in SEQ ID NO.8；

The amplification downstream primer AP2 of A segment: nucleotide sequence is as shown in SEQ ID NO.9；

The amplification template of Over lapping PCR B segment is load constructed by the amplified production after codon optimization The pET28a expression plasmid of PCV2 rep gene, the amplimer of the B segment are as follows:

The amplification upstream primer AP1 of B segment: nucleotide sequence is as shown in SEQ ID NO.10；

The amplification downstream primer AP2 of B segment: nucleotide sequence is as shown in SEQ ID NO.11；

The template of the amplification of Over lapping PCR AB segment is A fragment amplification product and B fragment amplification product, AB Fragment amplification primer are as follows:

AB fragment amplification upstream primer AP1: nucleotide sequence is as shown in SEQ ID NO.12；

AB fragment amplification downstream primer BP2: nucleotide sequence is as shown in SEQ ID NO.13.

In conclusion porcine circovirus 2 type Rep protein gene transformation the result is that:

(1) arginic Escherichia coli rare codon is encoded by the 8th of PCV2 Rep albumen protogene and the 16th, CGT is become from AGA and AGG, improves PCV2 Rep expressing quantity；

(2) the 207th His codon of former PCV2 Rep albumen is become into CAC from CAT, to close former sequence CCATGG (Nco I) restriction enzyme site；

(3) nucleotide sequence of 8 coding His is added to before the His tag at former 5 '-end of pET28a plasmid, it may be assumed that CAC CAT CAC CAC CAC CAC CAC CAT, with the affinity of Enhanced expressing albumen and Ni column；

(4) the whole codon TGA for eliminating former 3 '-end of PCV2 Rep protein gene makes to express one group 6 of PROTEIN C end increase × His Tag further enhances the affinity of expression albumen and Ni column.

Specifically, the purification condition in the step 5 after the protein induced expression of PCV2 Rep is: sample-loading buffer: 8M urine Element+0.05M pH7.4PBS；Washing buffer: 100mM imidazoles+sample-loading buffer；Elution buffer: 300mM imidazoles+loading is slow Fliud flushing.

The third object of the present invention is to provide a kind of porcine circovirus 2 type Rep protein expression vector, the expression is carried Body surface reaches the nucleotide sequence in area as shown in SEQ ID NO.14.

The fourth object of the present invention is to provide a kind of porcine circovirus 2 type Rep protein expression engineering bacteria, the engineering bacteria Include the porcine circovirus 2 type Rep protein expression vector.

The fifth object of the present invention is to provide a kind of ELISA detection reagent of porcine circovirus 2 type Rep protein antibodies Box, the ELISA detection kit include:

(A) with the 2 coated ELISA ELISA Plate of type Rep albumen of recombinant porcine circovirus；

(B) standard female serum: the Swine serum of PCV2 negative antibody；

(C) standard positive serum: PCV2 negative antibody pig acquires after being inoculated with SH plants of porcine circovirus 2 type inactivated vaccine Serum；

(D) ELIAS secondary antibody of horseradish peroxidase-labeled: anti-pig IgG or Anti-cavy IgG；

(E) sample diluting liquid, coating buffer, confining liquid, ELISA ELISA Plate cleaning solution, antibody diluent, developing solution and Terminate liquid.

The sixth object of the present invention is to provide the detection method of porcine circovirus 2 type Rep protein antibodies, the inspection Survey method the following steps are included:

(1) preparation detection ELISA ELISA Plate: buffer is coated with by the 2 type Rep albumen of recombinant porcine circovirus It is added in ELISA Plate and adsorbs after dilution, the dry coating washing lotion of sky adds coating to be closed with confining liquid；

(2) serum to be checked, negative serum, positive serum are loaded into ELISA ELISA Plate hole respectively as primary antibody and are incubated It educates；

(3) incubation of ELIAS secondary antibody: the ELIAS secondary antibody of horseradish peroxidase-labeled, anti-pig IgG or Anti-cavy IgG add Enter the washing of ELISA ELISA Plate cleaning solution, drying；

(4) developing solution is added, room temperature is protected from light incubation, and terminate liquid is added and terminates reaction；In microplate reader under 450nm wavelength Measure OD value.

The seventh object of the present invention is that providing the ELISA detection kit is distinguishing subunit vaccine immune swine The application in pig or whole virus vaccine immune swine is infected with totivirus.

The invention has the advantages that:

1, the present invention provides the ELISA detection kit and detection method of porcine circovirus 2 type Rep protein antibodies, first Porcine circovirus 2 type Rep albumen is prepared, is then envelope antigen between the progress of sample serum to recombinate PCV2 Rep albumen ELISA detection is connect, this method detects PCV2 natural infection pig or PCV2 inactivated virus vaccine (or attenuated live vaccines) immune swine Serum, it may appear that positive testing result；And detect PCV2 cap protein subunit vaccine and Swine serum is immunized, then there is negative knot Fruit.Therefore, with PCV2 Rep protein antibodies ELISA detection kit and preparation method thereof of the invention, in conjunction with PCV2 Cap egg The indirect ELISA detection method of white envelope antigen, so that it may distinguish natural infection antibody (or the whole virus vaccine immune swine of PCV2 Antibody) with subunit vaccine immune antiboidy, the purification for pig circular ring virus in pig farm provides foundation.

2, the preparation method of porcine circovirus 2 type Rep albumen provided by the invention,

In step 1,2 sets of unique primers are designed, the PCV2 virus of Hubei province prevalence is cloned in by the method for nest-type PRC The Rep protein gene of strain；

In step 2, by designing one section of long primer, by the 8th of PCV2 Rep albumen protogene and the 16th coding essence The Escherichia coli rare codon of propylhomoserin, all becomes CGT from AGA and AGG, to improve PCV2 Rep expressing quantity；PCV2 Rep protein gene increase His sequence alterations purpose, be increase expression albumen and purifying affinity column (Ni column) it is affine Power, this transformation are completed by 2 genetic manipulation experiments.Final porcine circovirus 2 type Rep protein gene transformation the result is that:

(4) the whole codon TGA for eliminating former 3 '-end of PCV2 Rep protein gene increases by one group 6 at expression PROTEIN C end × His Tag further enhances the affinity of expression albumen and Ni column.

The recombination PCV2 Rep albumen for passing through expression and purity in the step 5, after SDS-PAGE electrophoresis, Carry out Peak area analysis with image J software, calculate purity of protein, after an affinity chromatography purity of protein can reach 95% with On.

Detailed description of the invention

Fig. 1 is recombination PCV2 Rep protein expression engineering bacteria E.coli BL21/ provided in an embodiment of the present invention PET28aPCV2 Rep M2 inducing expression electrophoresis detection figure；Wherein,

Swimming lane 1, the full bacterium of E.coli BL21/pET28a PCV2 Rep M2 sample 1；

Swimming lane 2,1 supernatant of E.coli BL21/pET28a PCV2 Rep M2 sample；

Swimming lane 3, the full bacterium of E.coli BL21/pET28a PCV2 Rep M2 sample 2；

Swimming lane 4,2 supernatant of E.coli BL21/pET28a PCV2 Rep M2 sample；

The full bacterium sample of swimming lane 5, E.coli BL21/pET28a；

Swimming lane 6, Blue Plus Protein Marker (14-100kDa), are respectively as follows: 14,25,30 from bottom to top, 40,50,70,100kDa；

Fig. 2 is that Fig. 2 provided in an embodiment of the present invention recombinates PCV2 Rep protein purification products electrophoretogram；Wherein,

Swimming lane 1,1 inclusion body of E.coli BL21/pET28a PCV2 Rep M2 sample cleaned with PBS after plus 8M urea+ 0.05M pH 7.4PBS redissolves；

Swimming lane 2,2 inclusion body of E.coli BL21/pET28a PCV2 Rep M2 sample cleaned with PBS after plus 8M urea+ 0.05M pH 7.4PBS redissolves；

Swimming lane 3, Blue Plus Protein Marker (14-100kDa), are respectively as follows: 14,25,30 from bottom to top, 40,50,70,100kDa；

Swimming lane 4, E.coli BL21/pET28a PCV2 Rep M2 sample No. 5 collecting pipes of 1 eluent；

Swimming lane 5, E.coli BL21/pET28a PCV2 Rep M2 sample No. 6 collecting pipes of 1 eluent；

Swimming lane 6, E.coli BL21/pET28a PCV2 Rep M2 sample No. 7 collecting pipes of 1 eluent；

Swimming lane 7, E.coli BL21/pET28a PCV2 Rep M2 sample No. 8 collecting pipes of 1 eluent.

Specific embodiment

The clone of 1 porcine circovirus 2 type of embodiment (PCV2) Rep protein gene

One, the extraction of pathological material of disease DNA

The inguinal lymph nodes sample 0.5g for taking sick dead pig after porcine circovirus type 2 infection, adds sterile saline 4.5ml is placed in glass homogenizer and homogenate is made.472 μ l lymph node homogenates are taken, 25 μ l 10%SDS and 2.5 μ l eggs are added White enzyme K (25mg/ml) is placed in 50 DEG C of water-baths and acts on 2 hours, 500 μ l saturated phenols are added, is vortexed 30 seconds, sets 12000rpm centrifugation 10 minutes.Supernatant is moved into an other centrifuge tube, add the phenol of 500 μ l: chloroform: isoamyl alcohol (25:24:1) mixes postposition 12000rpm is centrifuged 10 minutes.Supernatant is taken, 3mol/L sodium acetate (pH value 5.3) is added by 1/10 volume, 2.5 times of volumes are added Dehydrated alcohol is set in -20 DEG C of refrigerators 30 minutes.With 15000rpm centrifugation 10 minutes, 70% ethyl alcohol 1ml washing is added after abandoning supernatant Precipitating abandons supernatant, vacuum drying DNA precipitating with 15000rpm centrifugation 10 minutes.It is dissolved using 30 μ l distilled waters of preceding addition, set- It is saved backup in 20 DEG C of refrigerators.

Two, nested PCR amplification PCV2 Rep protein gene

1, design nest-type PRC primer is as shown in table 1, and the part of underscore is restriction endonuclease site, is passed through The method of nest-type PRC is cloned in the Rep protein gene of the PCV2 Strain of Hubei province prevalence.

Table 1

2, first round amplification is carried out with PCV2 rep housing upstream primer PF, PCV2 rep housing downstream primer PR, expanded Increasing system is as shown in table 2, thermal cycler parameters: 94 DEG C, 5min → (94 DEG C, 30S, → 62 DEG C, 45S, → 72 DEG C, 60S) × 30 →72℃,10min.Amplified production is further expanded for subsequent inner sleeve primer.

Table 2

3, using above-mentioned housing amplified production as template, with PCV2 rep nest-type PRC inner sleeve upstream primer PF, PCV2 Rep nest-type PRC inner sleeve downstream primer PR carries out inner sleeve amplification as primer pair, and amplification system is as shown in table 3, temperature cycles ginseng Number: 94 DEG C, 5min → (94 DEG C, 30S, → 62 DEG C, 45S, → 72 DEG C, 60S) × 35 → 72 DEG C, 10min.

Table 3

4, agarose gel electrophoresis recycles amplified band, is connected on pTOPO-TA plasmid vector.Bacillus coli DH 5 alpha is converted, Transformed bacteria name are as follows: E.coli DH5 α/pTOPO-PCV2 Rep serves the progress of marine growth Engineering Co., Ltd after PCR is identified Sequencing.Sequencing result PCV2 Rep wild-type protein gene order is as shown in SEQ ID NO.15.

5, it is compared by the Sequencing and Characterization of clone gene, finds the gene and many in GenBank publication The homology of PCV2 sequence reaches 100%, wherein representative sequence publication source place has: Henan Province, Sichuan Province, Budapest (Hungary), Jiangxi Province, Fujian Province, Shandong Province, Kangwon National University (South Korea), Hebei Province, Seoul National University (South Korea), Gansu Province, Montevideo (Uruguay), Jiangsu Province.Show this section Sequence is very conservative, has wide applicability, is suitable as the sequence tested and analyzed.

The transformation and expression of embodiment 2PCV2 Rep protein gene

One, the transformation of PCV2 Rep protein gene

1, the applicant is by Plasmid DNA pTOPO-PCV2 Rep restriction endonuclease constructed by embodiment 1 It is inserted into expression plasmid pET 28a after I/Hind of BamH, III double digestion, constructs expression plasmid carrier.Name are as follows: pET28a PCV2 Rep.E. coli bl21 (DE3) is converted with connection product, screens transformant.It is built into PCV2 Rep protein expression engineering bacteria. Name are as follows: E.coli BL21/pET28a PCV2 Rep.Then inducing expression experiment is carried out, inducer is alpha-lactose, is worked dense Degree are as follows: 0.03mol/L.Induced product is detected with SDS-PAGE electrophoresis.There is apparent Rep protein expression as the result is shown, but It is that expression quantity is lower, the about 10%-15% of total protein.Expressing albumen is mainly inclusion body, insoluble.And use cOmplet Protein purification process under His-Tag purification Resin (Coche product) Denaturing, cannot hang up Ni column, nothing Method carries out affinity protein purification purifying.For this purpose, we have carried out following transformation to protogene.

2, the codon optimization of PCV2 Rep protein gene

(1) by designing new primer (codon optimization primer pair is as shown in table 4 below), by protogene the 8th and the 16th Encoding arginic Escherichia coli rare codon all becomes CGT from AGA and AGG, to improve PCV2 Rep expressing quantity.

Table 4

(2) amplification thermal cycler parameters used: 94 DEG C, 5min → (94 DEG C, 30S, → 62 DEG C, 45S, → 72 DEG C, 60S)×35→72℃,10min.Amplification system is as shown in table 5:

Table 5

(3) agarose gel electrophoresis recycles amplified band, is connected on pTOPO-TA plasmid vector.Convert bacillus coli DH 5 α, transformed bacteria name are as follows: E.coli DH5 α/pTOPO-PCV2 Rep M1.The restricted core of Plasmid DNA is extracted from transformed bacteria It is inserted into expression plasmid pET 28a after I/Hind of sour restriction endonuclease BamH, III double digestion, constructs expression plasmid carrier.Name are as follows: pET28a PCV2 Rep M1.E. coli bl21 (DE3) is converted with connection product, screens transformant.It is built into PCV2 Rep Protein expression engineering bacteria.Name are as follows: E.coli BL21/pET28a PCV2 Rep M1.

(4) inducing expression experiment is then carried out, inducer is alpha-lactose, working concentration are as follows: 0.03mol/L.Induced product It is detected with SDS-PAGE electrophoresis.There is apparent Rep protein expression as the result is shown, but expression quantity is higher, about total protein 25%-35%.Expressing albumen is mainly inclusion body, insoluble.And with cOmplet His-Tag purification Protein purification process under Resin (Coche product) Denaturing, can not hang up Ni column, can not carry out affinity protein purification Purifying.For this purpose, we increase to protogene the transformation of His sequence.

3, PCV2 Rep protein gene increases the transformation of His sequence

(1) sequencing result of embodiment 1 is shown, there is a Nco I digestion at the 618bp of PCV2 Rep protein gene Point (NcoI:C/CATGG) closes the restriction enzyme site with the method for Over lapping PCR.Simultaneously synthesizing one has 14 His The upstream primer of coded sequence has the Rep protein expression sequence of strong affinity to construct one with Ni column.Overlapping The primer of PCR design is as shown in table 6 below.

Table 6

(2) amplification of A segment

In table 6, A fragment amplification upstream primer sequence (AP1): the sequence of underscore is 8 groups of His codons of addition, His coded sequence for increasing pET28a expresses length, reinforces the affinity of expression albumen and Ni column.Using such as the following table 7 institute The amplification system shown is expanded, thermal cycler parameters: 94 DEG C, 5min → (94 DEG C, 30S, → 62 DEG C, 45S, → 72 DEG C, 60S)×20→72℃,10min.It is spare to retain amplified production.

Table 7

(3) B fragment amplification

B fragment amplification downstream primer sequence (BP2): removing the terminator codon TGA of former sequence in the design of primers, In case borrowing 6 × His sequence and terminator codon at carrier 3 '-end when constructing expression plasmid.

The primer amplification system such as table 8, thermal cycler parameters: 94 DEG C, 5min → (94 DEG C, 30S, → 62 DEG C, 45S, → 72℃,60S)×20→72℃,10min.It is spare to retain amplified production.

Table 8

(4) AB fragment amplification

Amplification system such as table 9, thermal cycler parameters: 94 DEG C, 5min → (94 DEG C, 30S, → 62 DEG C, 45S, → 72 DEG C, 60S)×35→72℃,10min。

Table 9

(5) with building expression plasmid carries in insertion pET 28a plasmid after I/Hind of Nco, III double digestion after amplified production recycling Body.It converts e. coli bl21 (DE3), transformed bacteria name are as follows: E.coli BL21/pET 28a-PCV2 Rep M2.By sequence Column measurement, expresses the nucleotide sequence in area as shown in SEQ ID NO.14, and the amino acid sequence such as SEQ ID NO.1 institute derived Show.

The genetic modification of this example the result is that: 1) by the 8th of PCV2 Rep albumen protogene and the 16th coding essence The Escherichia coli rare codon of propylhomoserin, all becomes CGT from AGA and AGG.To improve PCV2 Rep expressing quantity；It 2) will be former 207th His codon of PCV2 Rep albumen becomes CAC from CAT, to close CCATGG (Nco I) the digestion position of former sequence Point；3) the nucleotide sequence CAC CAT CAC of 8 coding His is added to before the His tag at former 5 '-end of pET28a plasmid CAC CAC CAC CAC CAT, with the ability of the affine Ni column of Enhanced expressing albumen；4) former PCV2 Rep protein gene is eliminated The whole codon TGA at 3 '-ends makes to express one group of 6 × His Tag of PROTEIN C end increase, further enhances the expression affine Ni column of albumen Ability.

The expression plasmid of this example building is named as pET 28aPCV2 Rep M2.

Two, the expression and purifying of PCV2 Rep protein gene are recombinated

1, with the recombination PCV2 Rep protein expression engineering bacteria E.coli BL21/pET28a PCV2 Rep M2 being built into Carry out inducing expression experiment.Inducer is alpha-lactose, working concentration are as follows: 0.03mol/L.Induced product 12%SDS-PAGE electricity Swimming is detected.Have apparent Rep protein expression as the result is shown and expression quantity be higher, according to Image J software analysis as a result, Destination protein content after 2 identical engineering bacterias are expressed in parallel is respectively the 37.5% and 39.4% of bacterial protein.Expression Albumen is mainly inclusion body, insoluble (recombination PCV2 Rep protein expression engineering bacteria E.coli BL21/pET28a PCV2 Rep M2 inducing expression electrophoresis detection figure is as shown in Figure 1).And with cOmplet His-Tag purification Resin (Coche Product) protein purification process under Denaturing, Ni column can be connected, affinity protein purification purifying can be carried out.

2, after cleaning E.coli BL21/pET28a PCV2 Rep M2 inclusion bodies of protein with enough PBS, with 8M urea Denaturation buffer dissolution precipitating.Centrifuging and taking supernatant, filtering are stand-by.2.5ml is loaded onto after the filtering of 1.5ml albuminate supernatant COmplet His-Tagpurification Resin (Coche product) chromatographic column.

Sample-loading buffer: 8M urea+0.05M pH 7.4PBS.

Washing buffer: sample-loading buffer+100mM imidazoles

Elution buffer: sample-loading buffer+300mM imidazoles

3, No. 5-8 each 500 μ L of eluent of pipe is taken to be collected by centrifugation overnight plus 4 DEG C of ammonium sulfate of 500 μ L saturated liquid respectively heavy It forms sediment.Then albumen precipitation is redissolved with the PBS liquid of 100 μ L urea containing 8M respectively, with 12%SDS-PAGE electrophoresis detection.As a result see Attached drawing 2.It is analyzed with result of the Image J software to attached drawing 2, No. 5, No. 6, No. 7, the purpose in No. 8 elution collecting pipes Purity of protein is respectively as follows: 95.1%, 96.6%, 95.4% and 97.4%.

The ELISA detection kit and application method of the antibody of 3 porcine circovirus 2 type Rep albumen of embodiment

1, coated elisa plate

(1) coating buffer: 9.5 carbonic acid buffer of 0.05M pH

(2) coating washing lotion: 0.01M pH7.2 kaliumphosphate buffer

K₂HPO₄·3H₂O 1.56g

KH₂PO₄ 0.38g

PH to 7.2 is adjusted, 1000ml is settled to.

(3) method for coating: the PCV2 Rep recombinant protein purified in Example 2 is diluted to protein content with coating buffer For 25 μ g/ml, every hole adds 100 μ L in 96 hole elisa Plates, sets 2-8 DEG C and adsorbs 24 hours.Sky goes coating buffer, coating washing lotion board-washing 3 times.

(4) when determining antigen protein peridium concentration, the recombination PCV2 Rep albumen of purifying is first diluted to egg with coating buffer Bai Hanliang is that 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 25 μ g/ml, 30 μ g/ml, 40 μ g/ml add to the micropore of 96 hole elisa Plates In, every hole adds 100 μ L, sets 2-8 DEG C and adsorbs 24 hours.Therefrom select the peridium concentration of an optimization, i.e. 25 μ g/ml.

2, it closes:

(1) coating confining liquid:

(2) " locked in " operation: the dry coating washing lotion of sky adds 150 hole μ L/ of coating confining liquid, sets 2-8 DEG C overnight.Removal closing It is spontaneously dried 24 hours after liquid.

3, the incubation (primary antibody incubation) of yin and yang attribute serum

(1) prepared by serum dilution: picking E.coli BL21 single colonie in LB solid medium tablets, switching is in containing LB In the test tube of fluid nutrient medium, 37 DEG C are cultivated 12 hours, do seed bacterium solution.The seed bacterium solution is inoculated in 5% ratio In 250ml LB liquid medium, 37 DEG C are cultivated 7 hours.It is placed in centrifuge tube, 8000 revs/min are centrifuged 10 minutes, collect thallus. Thallus is resuspended in PBS liquid in the ratio of 1:10 (weight/volume), ultrasonic wave is carried out and breaks bacterium.Be placed under 12000 revs/min from The heart 10 minutes, precipitating is discarded, collects supernatant.Contain total protein is diluted to PBS liquid after supernatant progress determining the protein quantity Amount is 1.2mg/ml.

(2) prepared by standard female serum: the sow litter of PCV2 negative antibody carries out artificial feeding after removing afterbirth To 4-5 week old, takes a blood sample and separate serum.Will separation serum make 1:800 times dilution after it is spare.

(3) prepared by standard positive serum: the sow litter of PCV2 negative antibody carries out artificial feeding after removing afterbirth To 3 week old.It is inoculated with (SH plants) of porcine circovirus 2 type inactivated vaccine, every pig musculi colli injects 2ml.One exempts from 3 weeks latter, use Same vaccine same dose carries out two and exempts from.Two exempt from after 2 weeks, attack that malicious (viral level is with FJ plants of porcine circovirus 2 type 10^6.25TCID50/ml), every pig collunarium 1ml, musculi colli inject 2ml.It takes a blood sample within 27 days after attacking poison, separates serum.Use sample Dilution is made spare after 1:800 dilution.

(4) cleaning solution: (10 times of concentration)

(5) operate: serum (Swine serum or substitution experimental animal guinea pig serum) to be checked makees serum dilution with PBS liquid, presses 1:400 times of dilution proportion is added in coating plate hole, 100 holes μ L/.Directly draw standard positive serum or standard female serum It is added in coating plate hole, 100 holes μ L/.ELISA Plate is set in 37 DEG C, 30min.

It is used cleaning solution board-washing 5 times after empty net serum dilution.

4, the incubation of ELIAS secondary antibody

The ELIAS secondary antibody (anti-pig IgG, or Anti-cavy IgG) of horseradish peroxidase-labeled is diluted with serum dilution To working concentration, 100 holes μ L/ are added into ELISA Plate hole, set 37 DEG C, 30min.

5, it develops the color:

Substrate solution A50 μ L, 50 μ L of substrate solution B, jog mixing, 37 DEG C of reaction 10min are added.

(1) substrate solution A

(2) substrate solution B

6, it terminates:

Terminate liquid: 2mol/L H₂SO₄。

After colour developing plus 100 hole μ L/ of terminate liquid.

7, read plate:

Under 450nm wavelength, ELISA Plate absorbance value is read.

It should be noted that 2 type Rep albumen of recombinant porcine circovirus provided by the invention is applied to preparation ELISA reagent Box can also be used for preparing the various forms of productions such as immune colloidal gold detection test paper strip, immunofluorescence, immunoturbidimetry, chemiluminescence Product.

The application of the ELISA detection kit of 4 porcine circovirus 2 type Rep protein antibodies of embodiment

One, the application in experiment pig Serum Antibody Detection

1, experiment pig source: porcine circovirus 2 type antibody feminine gender sow institute galactopoiesis pig carries out artificial feeding after removing afterbirth To 4 week old.It takes 20 to survive piggy, takes a blood sample in 4 week old, and separate serum.Will separation serum make 1:800 times dilution after it is spare. WithPCV2 antibody assay kit is confirmed as PCV2 negative antibody serum.

2, experiment pig is grouped: experiment pig is divided into 4 groups.First group, 5 pigs are negative control；Second group, 5 pig inoculations PCV2 Fujian Strain (YEBIO Bioengineering Co., Ltd of Qingdao's separation saves) live virus, viral generation was the 5th generation, and virus titer is 10^6.51TCID50/ml, every collunarium 1ml/ pigs, intramuscular injection 2ml；Third group, 5 pig muscle inoculation Qingdao Yi Bangsheng Porcine circovirus 2 type gene engineered subunit (Cap protein) vaccine of object Engineering Co., Ltd, 2ml/ pigs；4th group, 5 Pig inoculation Cimmeria company PCV2 inactivated virus vaccine (MERIALInactivated oil Adjuvant vaccine), 2ml/ pigs.

3, experimental method: taking a blood sample with 28 days after immune before immune, separate serum, in the method detection serum of embodiment 3 Anti- PCV2 Rep protein antibodies.

4, experimental result: the judgment basis of result is: the OD of sample₄₅₀It is worth (S)/standard female OD₄₅₀It is worth (N) less than 1.6 For feminine gender；It is suspicious that S/N, which is greater than or equal to 1.6 but is less than 2.1,；S/N is greater than or equal to 2.1 for the positive.Testing result is as follows:

10 experiment pig PCV2 Rep protein antibodies testing result of table

When PCV2 Rep protein antibodies indirect ELISA test experience Swine serum antibody, inoculation PCV2 poison living infects pig and exempts from Swine serum antibody after epidemic disease injection MERIAL PCV2 inactivated virus vaccine is the positive；Immune Qingdao Yi Bang bio-engineering corporation The Swine serum antibody of PCV2 Cap protein subunit vaccine is feminine gender.

Result above SPSS statistical analysis software 17.0 carries out one-way analysis of variance, and uses Student- Newman-Keuls carries out multiple comparative test.The result is as follows:

11 experiment pig PCV2 Rep protein antibodies testing result statistical analysis table of table

Note: the Student- of the One-Way ANOVA program of detection data 17.0 statistical analysis software of SPSS The Newman-Keuls method of inspection carries out Multiple range test analysis, with poor between the group with different alphabetical shoulders number (a, b, c) in column Different significant (P < 0.05).

11 statistical result of table shows:

OD of (1) 4 experimental group before immune₄₅₀Value and S/N value there are no significant difference；

(2) 28 days after vaccine immunity, Qingdao Yi Bang bio-engineering corporation PCV2 Cap protein subunit vaccine immune group with OD between negative control group₄₅₀There was no significant difference with S/N value for value；

(3) 28 days after vaccine immunity, PCV2 Fujian Strain live virus inoculation group is significantly higher than Qingdao Yi Bang bio-engineering corporation PCV2 Cap protein subunit vaccine immune group and negative control group；

(4) 28 days after vaccine immunity, the OD of MERIAL PCV2 totivirus inactivated vaccine immune group₄₅₀Value and S/N value, significantly Higher than PCV2 Fujian Strain live virus inoculation group, higher than Qingdao Yi Bang bio-engineering corporation PCV2 Cap protein subunit vaccine Immune group and negative control group.

5, pig or whole virus vaccine immune swine, Wo Menyong are infected for the ease of distinguishing subunit vaccine immune swine and totivirus The PCV2 Cap protein antibody ELISA detection kit of YEBIO Bioengineering Co., Ltd of Qingdao's production carries out parallel control inspection It surveys, as a result as follows:

As a result judgment basis is: the OD of sample₄₅₀It is worth (S)/standard female OD₄₅₀Being worth (N) less than 1.6 is feminine gender；S/N More than or equal to 1.6 but 2.1 are less than to be suspicious；S/N is greater than or equal to 2.1 for the positive.

12 experiment pig PCV2 Cap protein antibody test result of table

The results show that PCV2 cap protein antibodies indirect ELISA reagent kit test experience Swine serum Antibody Results are as follows: connect Swine serum antibody after PCV2 poison infection pig living and immune MERIAL PCV2 inactivated virus vaccine is the positive；Immune Qingdao is easy The Swine serum antibody of bio-engineering corporation, nation PCV2 cap protein subunit vaccine is also positive.

13 experiment pig PCV2 Cap protein antibody test result statistical analysis table of table

Note: the Student- of the One-Way ANOVA program of detection data 17.0 statistical analysis software of SPSS The Newman-Keuls method of inspection carries out Multiple range test analysis, between the group with different alphabetical shoulders number (a, b, c, d) in column Significant difference (P < 0.05).

13 statistical result showed of table:

(2) 4 experimental groups are 28 days after vaccine immunity, OD₄₅₀Value and S/N value have significant difference；

(3) it connects after poison 28 days, the OD of PCV2 Fujian Strain live virus inoculation group₄₅₀Value and S/N value are significantly higher than negative control Group；

(4) 28 days after vaccine immunity, Qingdao Yi Bang bio-engineering corporation PCV2 Cap protein subunit vaccine immune group OD₄₅₀Value and S/N value are significantly higher than MERIAL PCV2 totivirus inactivated vaccine immune group, and it is living that the two is also significantly greater than PCV2 Fujian Strain Virus inoculation group and negative control group；

It can be seen that between joint PCV2 Rep protein antibodies indirect ELISA testing kit and PCV2 cap protein antibodies ELISA detection kit is connect, subunit vaccine immune swine and totivirus infection pig or whole virus vaccine immune swine can be distinguished.

Two, the application in the antibody test of experimental model animal guinea pig serum

Because the ratio for the pig PCV2 antibody positive raised in its natural state is higher, the experiment pig of negative antibody is more difficult It looks for.And the blood-serum P CV2 antibody of cavy is all negative, background is uniform, is the alternative model animal of relatively good experiment pig.Use globefish The immunization experiment of mouse can further confirm that real reliability of the invention.

1, cavy source: Hartley cavy 30, it is purchased from Hubei Province's Animal Experimental Study center.

2, experimental guinea pig is grouped: 30 cavys are randomly divided into 3 groups.First group of 10 cavy is negative control；Second group 10 The PCV2 Cap protein genetic engineering subunit vaccine of YEBIO Bioengineering Co., Ltd of Qingdao is immunized in cavy；Third group 10 Cimmeria company PCV2 inactivated virus vaccine (MERIAL is immunized in cavyInactivated oil adjuvantvaccine)。

3, the judgment basis of result is: the OD of sample₄₅₀It is worth (S)/standard female OD₄₅₀Being worth (N) less than 1.6 is feminine gender；S/ It is suspicious that N, which is greater than or equal to 1.6 but is less than 2.1,；S/N is greater than or equal to 2.1 for the positive.

The testing result of 14 cavy PCV2 Rep protein antibodies indirect ELISA testing kit of table

4, cavy PCV2 Rep protein antibodies indirect ELISA testing result is shown, immune MERIAL PCV2 totivirus goes out Serum antibody after live vaccine is the positive；The globefish of immune Qingdao Yi Bang bio-engineering corporation PCV2 Cap protein subunit vaccine Mouse serum antibody is feminine gender.

5, statistical analysis

15 cavy PCV2 Rep protein antibodies testing result statistical analysis table of table

Note: the Student- of the One-Way ANOVA program of detection data 17.0 statistical analysis software of SPSS The Newman-Keuls method of inspection carries out Multiple range test analysis, with difference between the group with different alphabetical shoulders number (a, b) in column Significantly (P < 0.05).

15 statistical result showed of table:

(1) negative control group, MERIAL PCV2 totivirus inactivated vaccine immune group, Qingdao Yi Bang bio-engineering corporation PCV2 The cavy of Cap protein subunit vaccine immune group anti-PCV2 rep protein antibodies in preimmune serum are feminine gender, between group OD₄₅₀With S/N value there are no significant difference；

(2) 28 days after being immunized, the OD of MERIAPCV2 totivirus inactivated vaccine immune group₄₅₀It is negative right to be significantly higher than with S/N value According to group；

(3) 28 days after being immunized, the OD of Qingdao Yi Bang bio-engineering corporation PCV2 Cap protein subunit vaccine immune group₄₅₀ There was no significant difference between S/N value and negative control group.

6, same, in order to illustrate the present invention in distinguishing the immune animal of subunit vaccine and the immune animal of whole virus vaccine Effect, we compare experiment with PCV2 cap protein ELISA kit.As a result as follows:

The testing result of 16 cavy PCV2 Cap protein antibody indirect ELISA detection kit of table

It is shown with the PCV2 Cap protein antibody ELISA kit test result of YEBIO Bioengineering Co., Ltd of Qingdao: Serum antibody after immune MERIAL PCV2 inactivated virus vaccine is the positive；Immune Qingdao Yi Bang bio-engineering corporation PCV2 The Swine serum antibody of Cap protein subunit vaccine is also positive.

17 cavy PCV2 Cap protein antibody test result statistical analysis table of table

17 statistical result showed of table:

(1) negative control group, MERIAL PCV2 totivirus inactivated vaccine immune group, Qingdao Yi Bang bio-engineering corporation PCV2 The cavy of Cap protein subunit vaccine immune group anti-PCV2 cap protein antibodies in preimmune serum are feminine gender, between group OD₄₅₀With S/N value there are no significant difference；

(2) 28 days after being immunized, MERIAL PCV2 totivirus inactivated vaccine immune group and Qingdao Yi Bang bio-engineering corporation PCV2 Cap protein subunit vaccine immune group, anti-PCV2 cap protein antibodies are the positive, OD₄₅₀Feminine gender is all remarkably higher than with S/N value Control group；

(3) MERIAL PCV2 totivirus inactivated vaccine immune group and Qingdao Yi Bang bio-engineering corporation PCV2 Cap protein are sub- OD between subunit vaccine immune group₄₅₀There was no significant difference with S/N value.

The result shows that use in conjunction PCV2 Rep protein antibodies indirect ELISA testing kit and PCV2 Cap protein are anti- Body indirect ELISA testing kit, can distinguish PCV2 cap protein subunit vaccine immune guinea pig and whole virus vaccine is immune Cavy.

Three, the application in the antibody test of field trial Swine serum

From Shandong, Hebei, Fujian, the Infection of Porcine circovirus pig blood final proof of the not immune pig circular ring virus vaccine of Sichuan acquisition Product, at the same it is anti-with PCV2 Rep protein antibodies ELISA detection method of the invention and the PCV2 Cap protein of Qingdao Yi Bang company Body ELISA detection kit is detected.

As a result judgment basis is: the OD of sample₄₅₀It is worth (S)/standard female OD₄₅₀Being worth (N) less than 1.6 is feminine gender；S/N More than or equal to 1.6 but 2.1 are less than to be suspicious；S/N is greater than or equal to 2.1 for the positive.The result is as follows:

Infect Swine serum PCV2 rep protein antibodies and PCV2 cap protein antibodies testing result in 18 field of table

The above results show that the pig anteserum sample of pig circular ring virus wild virus infection, anti-with PCV2 Rep albumen of the invention The PCV2 Cap protein antibody ELISA detection kit of body ELISA detection method and Qingdao Yi Bang company is detected, as a result For the positive, the coincidence rate as a result judged is 100%.

The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.

Sequence table

<110>Changjiang University, YEBIO Bioengineering Co., Ltd of Qingdao

<120>porcine circovirus 2 type Rep albumen, ELISA detection kit and the preparation method and application thereof

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 369

<212> PRT

<213>pig circular ring virus (Porcine circovirus)

<400> 1

Met Gly Ser Ser His His His His His His His His His His His His

1 5 10 15

His His Ser Ser Gly Leu Val Pro Arg Gly Ser His Met Ala Ser Met

20 25 30

Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Met Pro Ser Lys Lys Ser

35 40 45

Gly Arg Ser Gly Pro Gln Pro His Lys Arg Trp Val Phe Thr Leu Asn

50 55 60

Asn Pro Ser Glu Asp Glu Arg Lys Lys Ile Arg Glu Leu Pro Ile Ser

65 70 75 80

Leu Phe Asp Tyr Phe Ile Val Gly Glu Glu Gly Asn Glu Glu Gly Arg

85 90 95

Thr Pro His Leu Gln Gly Phe Ala Asn Phe Val Lys Lys Gln Thr Phe

100 105 110

Asn Lys Val Lys Trp Tyr Phe Gly Ala Arg Cys His Ile Glu Lys Ala

115 120 125

Lys Gly Thr Asp Gln Gln Asn Lys Glu Tyr Cys Ser Lys Glu Gly Asn

130 135 140

Leu Leu Ile Glu Cys Gly Ala Pro Arg Ser Gln Gly Gln Arg Ser Asp

145 150 155 160

Leu Ser Thr Ala Val Ser Thr Leu Leu Glu Ser Gly Ser Leu Val Thr

165 170 175

Val Ala Glu Gln His Pro Val Thr Phe Val Arg Asn Phe Arg Gly Leu

180 185 190

Ala Glu Leu Leu Lys Val Ser Gly Lys Met Gln Lys Arg Asp Trp Lys

195 200 205

Thr Asn Val His Val Ile Val Gly Pro Pro Gly Cys Gly Lys Ser Lys

210 215 220

Trp Ala Ala Asn Phe Ala Asp Pro Glu Thr Thr Tyr Trp Lys Pro Pro

225 230 235 240

Arg Asn Lys Trp Trp Asp Gly Tyr His Gly Glu Glu Val Val Val Ile

245 250 255

Asp Asp Phe Tyr Gly Trp Leu Pro Trp Asp Asp Leu Leu Arg Leu Cys

260 265 270

Asp Arg Tyr Pro Leu Thr Val Glu Thr Lys Gly Gly Thr Val Pro Phe

275 280 285

Leu Ala Arg Ser Ile Leu Ile Thr Ser Asn Gln Thr Pro Leu Glu Trp

290 295 300

Tyr Ser Ser Thr Ala Val Pro Ala Val Glu Ala Leu Tyr Arg Arg Ile

305 310 315 320

Thr Ser Leu Val Phe Trp Lys Asn Ala Thr Glu Gln Ser Thr Glu Glu

325 330 335

Gly Gly Gln Phe Val Thr Leu Ser Pro Pro Cys Pro Glu Phe Pro Tyr

340 345 350

Glu Ile Asn Tyr Lys Leu Ala Ala Ala Leu Glu His His His His His

355 360 365

His

<210> 2

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

cagcggcagc acctcggcag cacc 24

<210> 3

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

acgaccagga ctacaatatc cgtataacc 29

<210> 4

<211> 36

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

cccggatcca tgcccagcaa gaagagtgga agaagc 36

<210> 5

<211> 41

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

cccaagcttt cagtaattta tttcatatgg aaattcaggg c 41

<210> 6

<211> 61

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

gggggatcca tgcccagcaa gaagagtgga cgtagcggac cccaaccaca taaacgttgg 60

g 61

<210> 7

<211> 41

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

gggaagcttt cagtaattta tttcatatgg aaattcaggg c 41

<210> 8

<211> 87

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

aaaaacatgg gcagcagcca ccatcaccac caccaccacc atcatcatca tcatcatcac 60

agcagcggcc tggtgccgcg cggcagc 87

<210> 9

<211> 74

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

caataacaac cacttcttca ccgtggtaac catcccacca cttgtttcta ggtggtttcc 60

agttaccacg gtga 74

<210> 10

<211> 60

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

caagtggtgg gatggttacc acggtgaaga agtggttgtt attgatgact tttatggctg 60

<210> 11

<211> 38

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

gggaagcttg taatttattt catatggaaa ttcagggc 38

<210> 12

<211> 87

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

aaaaccatgg gcagcagcca ccatcaccac caccaccacc atcatcatca tcatcatcac 60

agcagcggcc tggtgccgcg cggcagc 87

<210> 13

<211> 38

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

cccaagcttg taatttattt catatggaaa ttcagggc 38

<210> 14

<211> 1110

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

atgggcagca gccaccatca ccaccaccac caccatcatc atcatcatca tcacagcagc 60

ggcctggtgc cgcgcggcag ccatatggct agcatgactg gtggacagca aatgggtcgc 120

ggatccatgc ccagcaagaa gagtggaaga agcggacccc aaccacataa aaggtgggtg 180

ttcacgctga ataatccttc cgaagacgag cgcaagaaaa tacgggagct cccaatctcc 240

ctatttgatt attttattgt tggcgaggaa ggtaatgagg agggccgaac accccaccta 300

caggggttcg ctaattttgt gaagaagcaa acttttaata aagtgaagtg gtattttggt 360

gcccgctgcc acatcgagaa agcgaaagga acagatcagc agaataaaga atattgcagt 420

aaagaaggca acttactgat agaatgtgga gctcctagat ctcaaggaca acggagtgac 480

ctctctactg ctgtgagtac cttgttggag agcgggagtc tggtgaccgt tgcagagcag 540

caccctgtaa cgtttgtcag aaatttccgc gggctggctg aacttttgaa agtgagcggg 600

aaaatgcaga agcgtgattg gaagacgaat gtacacgtca ttgtggggcc acctgggtgt 660

ggcaaaagca aatgggctgc taattttgca gacccggaaa ccacatactg gaaaccacct 720

agaaacaagt ggtgggatgg ttaccacggt gaagaagtgg ttgttattga tgacttttat 780

ggctggctgc cgtgggatga tctactgaga ctctgtgatc gatatccttt gactgttgag 840

actaaaggtg gaactgtacc ttttttggcc cgcagtattc tgattaccag caatcagacc 900

ccgttggaat ggtactcctc aactgctgtc ccagctgtag aagctctcta tcggaggatt 960

acttccttgg tattttggaa gaatgctaca gaacaatcca cggaggaagg gggccagttc 1020

gtcacccttt cccccccatg ccctgaattt ccatatgaaa taaattacaa gcttgcggcc 1080

gcactcgagc accaccacca ccaccactga 1110

<210> 15

<211> 945

<212> DNA

<213>pig circular ring virus (Porcine circovirus)

<400> 15

atgcccagca agaagagtgg aagaagcgga ccccaaccac ataaaaggtg ggtgttcacg 60

ctgaataatc cttccgaaga cgagcgcaag aaaatacggg agctcccaat ctccctattt 120

gattatttta ttgttggcga ggaaggtaat gaggagggcc gaacacccca cctacagggg 180

ttcgctaatt ttgtgaagaa gcaaactttt aataaagtga agtggtattt tggtgcccgc 240

tgccacatcg agaaagcgaa aggaacagat cagcagaata aagaatattg cagtaaagaa 300

ggcaacttac tgatagaatg tggagctcct agatctcaag gacaacggag tgacctctct 360

actgctgtga gtaccttgtt ggagagcggg agtctggtga ccgttgcaga gcagcaccct 420

gtaacgtttg tcagaaattt ccgcgggctg gctgaacttt tgaaagtgag cgggaaaatg 480

cagaagcgtg attggaagac gaatgtacac gtcattgtgg ggccacctgg gtgtggcaaa 540

agcaaatggg ctgctaattt tgcagacccg gaaaccacat actggaaacc acctagaaac 600

aagtggtggg atggttacca tggtgaagaa gtggttgtta ttgatgactt ttatggctgg 660

ctgccgtggg atgatctact gagactctgt gatcgatatc ctttgactgt tgagactaaa 720

ggtggaactg tacctttttt ggcccgcagt attctgatta ccagcaatca gaccccgttg 780

gaatggtact cctcaactgc tgtcccagct gtagaagctc tctatcggag gattacttcc 840

ttggtatttt ggaagaatgc tacagaacaa tccacggagg aagggggcca gttcgtcacc 900

ctttcccccc catgccctga atttccatat gaaataaatt actga 945

Claims

1. a kind of 2 type Rep albumen of recombinant porcine circovirus, which is characterized in that the 2 type Rep albumen of recombinant porcine circovirus Amino acid sequence as shown in SEQ ID NO.1.

2. a kind of preparation method of 2 type Rep albumen of recombinant porcine circovirus, which comprises the steps of:

Step 1, building 2 type Rep protein gene cloning carrier of recombinant porcine circovirus: design nest-type PRC primer pair is collected PCV2 wild virus infection swine disease material carries out DNA and extracts and be built into PCV2 Rep protein gene cloning carrier after nested PCR amplification；

Step 2, the transformation of 2 type Rep protein gene of recombinant porcine circovirus: for the resulting nested PCR amplification product design of step 1 The optimization of primer realization PCV2 Rep protein gene expression codon；And pass through the method for Over lapping PCR, in PCV2 5 ' ends of Rep protein gene expression sequence increase the sequence of coding His, and close I restriction enzyme site of Nco in former sequence；

Step 3, building 2 type Rep protein expression plasmid carrier of recombinant porcine circovirus；

Step 4, building 2 type Rep protein expression engineering bacteria of recombinant porcine circovirus；

The inducing expression and purifying of step 5,2 type Rep albumen of recombinant porcine circovirus.

3. preparation method as claimed in claim 2, which is characterized in that nest-type PRC primer is as follows in the step 1:

Primer used in codon optimization is as follows in the step 2:

4. preparation method as claimed in claim 2, which is characterized in that Over lapping PCR includes in the step 2 Over lapping PCR A segment and Over lapping PCR B segment,

The amplification template of Over lapping PCR A segment be codon optimization after amplified production constructed by be loaded with The pET28a expression plasmid of PCV2 rep gene, the amplimer of the A segment are as follows:

The amplification template of Over lapping PCR B segment be codon optimization after amplified production constructed by be loaded with The pET28a expression plasmid of PCV2 rep gene, the amplimer of the B segment are as follows:

The template of the amplification of Over lapping PCR AB segment is A fragment amplification product and B fragment amplification product, AB segment Amplimer are as follows:

5. preparation method as claimed in claim 2, which is characterized in that in the step 5 after the protein induced expression of PCV2 Rep Purification condition be: sample-loading buffer: 8M urea+0.05M pH7.4PBS；Washing buffer: 100mM imidazoles+loading buffer Liquid；Elution buffer: 300mM imidazoles+sample-loading buffer.

6. a kind of porcine circovirus 2 type Rep protein expression vector, which is characterized in that the nucleosides in the expression area of the expression vector Acid sequence is as shown in SEQ ID NO.14.

7. a kind of porcine circovirus 2 type Rep protein expression engineering bacteria, which is characterized in that the engineering bacteria includes described in claim 6 Porcine circovirus 2 type Rep protein expression vector.

8. a kind of ELISA detection kit of the antibody of porcine circovirus 2 type Rep albumen, which is characterized in that the ELISA Detection kit includes:

(A) it is coated with the ELISA ELISA Plate of 2 type Rep albumen of recombinant porcine circovirus described in claim 1；

(B) standard female serum: the Swine serum of PCV2 negative antibody；

(C) standard positive serum: the pig of PCV2 negative antibody, with the serum after SH plants of porcine circovirus 2 type inactivated vaccine inoculations；

(E) sample diluting liquid, coating buffer, confining liquid, ELISA ELISA Plate cleaning solution, antibody diluent, developing solution and termination Liquid.

9. a kind of detection method of the antibody of porcine circovirus 2 type Rep albumen, which is characterized in that the detection method includes Following steps:

(1) preparation detection ELISA ELISA Plate: buffer is coated with by 2 type Rep egg of recombinant porcine circovirus described in claim 1 It is added in ELISA Plate and adsorbs after white dilution, the dry coating washing lotion of sky adds coating to be closed with confining liquid；

(2) respectively as primary antibody, sample-adding is incubated for into ELISA ELISA Plate hole for serum to be checked, negative serum, positive serum；

(3) incubation of ELIAS secondary antibody: the ELIAS secondary antibody of horseradish peroxidase-labeled, anti-pig IgG or Anti-cavy IgG are added ELISA ELISA Plate, cleaning solution washing, drying；

(4) developing solution is added, room temperature is protected from light incubation, and terminate liquid is added and terminates reaction；OD is measured under 450nm wavelength in microplate reader Value.

10. ELISA detection kit according to any one of claims 8 is in differentiation subunit vaccine immune swine and totivirus infection pig or entirely Application in virus vaccine pig.