CN109777890A - A kind of PCV2, CSFV IgG antibody double fluorescent microballoon immunological detection method - Google Patents
A kind of PCV2, CSFV IgG antibody double fluorescent microballoon immunological detection method Download PDFInfo
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- CN109777890A CN109777890A CN201910064482.XA CN201910064482A CN109777890A CN 109777890 A CN109777890 A CN 109777890A CN 201910064482 A CN201910064482 A CN 201910064482A CN 109777890 A CN109777890 A CN 109777890A
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Abstract
The invention discloses a kind of PCV2, CSFV IgG antibody double fluorescent microballoon immunological detection methods.2 type pig circular ring virus Cap genetic fragments and E 2 gene of Classical Swine Fever segment, respectively as shown in NO:1~2 SEQ ID, the recombinant protein of coding is coupled with carboxylic fluorescent microballoon respectively as antigen and establishes dual FMIA detection method nucleotide sequence.This detection method speed is fast, high specificity, susceptibility are high；E2 antibody and Cap antibody detection method it is reproducible, with other Prevention of Common Occurrence Porcine Disease viral disease positive serum no cross reactions, high sensitivity, high specificity；Compared with commercial ELISA kit, sensitivity is higher.This method can be used for the rapid differential diagnosis of 2 type virus infection pig of swine fever virus and annulus and the detection of protection antibody simultaneously; experiment basis is established further to establish virus diseases of pigs antibody multiple detection method, the foundation for the novel serodiagnosis of other animal epidemic antibody provides thinking.
Description
Technical field
The present invention relates to immunologys and technical field of virus detection, more particularly, to a kind of PCV2, CSFV IgG antibody
Double fluorescent microballoon immunological detection method.
Background technique
China is the most country of pig raising, and pig raising has very important status in aquaculture, as China's economy is sent out
Exhibition, the scale of aquaculture will also be gradually expanded, and be intended to scale, intensive culture.Following problem also slowly emerges
Out, especially pig infectious disease is outstanding day by day to the harm of aquaculture.
Swine fever (Classical swine fever, CSF) is by swine fever virus (Classical swine fever
Virus, CSFV) caused by pig a kind of acute or chronic, hot and highly contagious disease.Swine fever is that China is most important
One of swine disease, this disease is included in legal A class infectious disease by World Organization for Animal Health (OIE), although there is many countries in the world
Swine fever, but China's complex geographical environment are eliminated, popular form is changeable, so annual country is all specified to be immunized.Swine fever
Serious economic loss is caused to pig breeding industry every year.The disease is with anxious, lasting high fever of falling ill, the general hair property dot bleeding of whole body, morbidity
Rate and death rate height are characterized, and pig (domestic pig and wild boar) is unique susceptible host.Swine fever is in worldwide distribution, due to its harm
Degree is high, causes huge loss to pig breeding industry in recent decades, and many countries successively take the measure for eliminating swine fever.
2 type pig circular ring virus (PCV2), a kind of small, nonencapsulated single-stranded DNA viruses are considered as postweaning multisystem
The main reason for exhaustion syndrome (PMWS).The clinical symptoms of PMWS include progressive weight loss, respiratory disease, jaundice
Increase with the death rate.Porcine circovirus 2 type to the very harmful of pig, cause pig it is a series of it is relevant face disease of diagnosing a disease, wherein wrapping
PMWS, dermatitis nephrotic syndrome (PDNS), sow breeding difficulty etc. are included, causes huge harm to pig breeding industry.
Pig virus infectious disease is mainly characterized by: spread speed is fast, popular wide, and harm is big.Swine fever, 2 type of pig circular ring virus 2
Both diseases have as a kind of acute viral infectious disease and propagate fast, popular wide, explosive feature, and typically exhibit
For mixed infection, breeding difficulty, immunosupress are caused, huge economic loss is brought to pig breeding industry.Therefore establish it is a kind of quickly,
Efficient detection method is of crucial importance.
Flexmap 3D detection system can detect 96 samples in 20min, detection single sample that can be qualitative or quantitative
Kind 500 kinds of molecules to be checked, have many advantages, such as flux height, high sensitivity, it is specific it is good be known as be the genome times afterwards comprehensively core
Chip technology.Using the multi objective Synchronization Analysis (FlexibleMulti-Analyte Profiling, xMAP) of Luminex
Technology, also referred to as liquid phase protein chip technology are used to be referred to as fluorescent microsphere immunological detection method when antibody test
(Fluorescent-microbead immunoassay, FMIA).By the development of more than ten years, liquid phase protein chip technology is
Through the new tool for becoming research proteomics and genomics.FMIA is that a kind of diagnosis for having Research Prospects and monitoring are immune
Analysis method is learned, compared with traditional ELISA immunization, the sample size needed is smaller, and time and economic cost are lower, and it
Overcome requirement limitation of the ELISA to single analyte
But so far without a kind of fluorescent microsphere immunology detection for detecting swine fever, 2 type both diseases of pig circular ring virus 2 simultaneously
Method.
Summary of the invention
It is dual glimmering the purpose of the invention is to overcome the deficiencies of the prior art and provide a kind of PCV2, CSFV IgG antibody
Light microballoon immunological detection method establishes experiment basis further to establish virus diseases of pigs antibody multiple detection method, is other
The foundation of the novel serodiagnosis of animal epidemic antibody provides thinking and theoretical basis.
To achieve the goals above, the present invention is achieved by the following technical programs:
The present invention utilizes prokaryotic expression carrier pMAL- by the amplified fragments of optimization PCV2 Cap and CSFV raq gene
C5X carries out solubility expression to CSFV E 2 protein, 2 type pig circular ring virus Cap proteins, using the recombinant protein of preparation as antigen
With microballoon be coupled, using Swine serum as measuring samples, using CSFV-E2, PCV2-Cap recombinant protein as antigen respectively with 60, No. 40
The coupling of pearl microballoon establishes individual event fluorescent microsphere immunological detection method (individual event FMIA) inspection of CSFV, PCV2, IgG antibody respectively
Survey method, and dual FMIA detection method is established on this basis.
Experiment early stage in PCV2 Cap and CSFV raq gene amplification procedure, carries out for several main epitopes
The PCR amplification of its gene, and expression plasmid is constructed, it finds and not all genetic fragment can give expression to soluble albumen,
After screening, the amino acid residue of PCV2 Cap and CSFV E2 are expressed in escherichia expression system, is given expression to solvable
Property albumen, as antigen carry out subsequent research after purified.At present in the PCV2 Cap and CSFV of expression in escherichia coli
Raq gene exists in the form of inclusion body mostly, however the purification step of inclusion body is complicated, it is also higher to require protein sample.
Present invention employs mature prokaryotic protein expression system, expression vector has selected the pMAL-c5X of easy expression soluble protein
Carrier has MBP label on carrier, can increase the solubility of recombinant protein, while 6 His are added behind target gene
Label makes recombinant protein be more easier to purify.By continuing to optimize inductive condition in 16 DEG C, the condition of the final concentration of 0.3mM of IPTG
After 6~8h of lower inducing expression, successful expression goes out to have merged the pMAL-c5x-CSFV-E2 and pMAL- of His label and MBP label
C5x-PCV2-Cap recombinant protein.Discovery low temperature low speed extends induction time in an experiment, slows down protein expression speed, makes it more
It is intended to supernatant expression.
Therefore claimed the following contents:
2 type pig circular ring virus Cap genetic fragments are detecting 2 type pig circular ring virus or preparation 2 type pig circular ring virus detection examination
Application in agent box, nucleotide sequence is as shown in SEQ ID NO:1.
Application of the E 2 gene of Classical Swine Fever segment in detection swine fever virus or preparation swine fever virus detection kit, core
Nucleotide sequence is as shown in SEQ ID NO:2.
2 type pig circular ring virus Cap recombinant proteins are detecting 2 type pig circular ring virus or preparation 2 type pig circular ring virus detection examination
Application in agent box, amino acid sequence is as shown in SEQ ID NO:3.
Application of the swine fever virus E2 recombinant protein in detection swine fever virus or preparation swine fever virus detection kit, ammonia
Base acid sequence is as shown in SEQ ID NO:4.
A kind of carrier of recombination contains nucleotide sequence 2 type pig circular ring virus Cap gene piece as shown in SEQ ID NO:1
Section.
A kind of carrier of recombination contains nucleotide sequence E 2 gene of Classical Swine Fever segment as shown in SEQ ID NO:2.
Preferably, the carrier is pMAL-c5X carrier.
A kind of bacterial strain of recombination, contains the above recombinant vector.
Preferably, the bacterial strain is BL21 (DE3).
One group for detect the carboxylic fluorescent microballoon group of 2 type pig circular ring virus and swine fever virus, including is coupled and has amino
Acid sequence the carboxylic fluorescent microballoon of 2 type pig circular ring virus Cap recombinant proteins and coupling as shown in SEQ ID NO:3 are had the right
The carboxylic fluorescent microballoon of amino acid sequence swine fever virus E2 recombinant protein as shown in SEQ ID NO:4；
Wherein, 2 type pig circular ring virus Cap recombinant proteins and swine fever virus E2 recombinant protein are by by its coding DNA sequence
Column are connected into pMAL-c5X carrier, and are transferred to prokaryotic expression bacterium inducing expression,
Inductive condition is 12~37 DEG C, 6~8h of inducing expression under conditions of the final concentration of 0.3~1.0mM of IPTG.
Preferably, inductive condition be 16 DEG C, 6~8h of inducing expression under conditions of the final concentration of 0.3mM of IPTG.
Preferably, after inducing expression, it is crushed supernatant, with GE company Ni2+Affinity column is purified.
Preferably, when purifying, 2 type pig circular ring virus Cap recombinant proteins are washed twice through the imidazole solution 1~2 of 20~500mM
It is miscellaneous.
It is highly preferred that 2 type pig circular ring virus Cap recombinant proteins are washed miscellaneous twice through the imidazole solution 2 of 300mM when purifying, wash
The destination protein purity taken off is higher, and foreign protein removes substantially.
Preferably, when purifying, swine fever virus E2 recombinant protein is washed miscellaneous twice through the imidazole solution 1~2 of 20~500mM.
It is highly preferred that swine fever virus E2 recombinant protein is washed miscellaneous twice through the imidazole solution 2 of 200mM when purifying, elute
Destination protein purity it is higher, foreign protein removes substantially.
Preferably, what coupling had 2 type pig circular ring virus Cap recombinant proteins is No. 40 microballoons.
Preferably, what coupling had swine fever virus E2 recombinant protein is No. 60 microballoons.
The carboxylic fluorescent microballoon group answering in preparation 2 type pig circular ring virus of detection and the kit of swine fever virus
With.
A kind of kit detecting 2 type pig circular ring virus and swine fever virus, including the carboxylic fluorescent microballoon group.
Preferably, also contain streptomysin-phycoerythrin, the secondary antibody of biotin labeling.
Preferably, also contain PBS-TBN, PBST, sheath fluid.
Preferably, the secondary antibody is rabbit-anti pig IgG antibody.
Preferably, secondary antibody dilution is 1:1000~5000, streptomysin-phycoerythrin concentration is 5~10 μ g/ml and blood
Clear incubation time is 45~60min and secondary antibody incubation time is 45~60min.
It is highly preferred that when secondary antibody dilution is 1:1000, streptomysin-phycoerythrin concentration is 10 μ g/ml and serum is incubated for
Between be 60min and secondary antibody incubation time is 45min.
Preferably, test serum dilution is 1:50~100.
It is highly preferred that test serum dilution is 1:50.
A kind of kit detecting 2 type pig circular ring virus and swine fever virus, including coupling have amino acid sequence such as SEQ ID
No. 40 carboxylic fluorescent microballoons of 2 type pig circular ring virus Cap recombinant proteins shown in NO:3, coupling have the right amino acid sequence such as
No. 60 carboxylic fluorescent microballoons, the streptomysin-phycoerythrin of swine fever virus E2 recombinant protein shown in SEQ ID NO:4, biology
Secondary antibody (rabbit-anti pig IgG antibody), PBS-TBN, PBST and the sheath fluid that element marks；
Wherein, 2 type pig circular ring virus Cap recombinant proteins and swine fever virus E2 recombinant protein are by by its coding DNA sequence
Column are connected into pMAL-c5X carrier, and are transferred to prokaryotic expression bacterium inducing expression, and inductive condition is 16 DEG C, IPTG is final concentration of
6~8h of inducing expression under conditions of 0.3mM；After inducing expression, it is crushed supernatant, with GE company Ni2+Affinity column is purified,
Swine fever virus E2 recombinant protein is washed miscellaneous twice through the imidazole solution 2 of 200mM, and 2 type pig circular ring virus Cap recombinant proteins are through 300mM
Imidazole solution 2 wash twice it is miscellaneous.
Its application method are as follows: after two kinds of microballoons are diluted to 50/μ of working concentration L with PBS-TBN, each 50 μ of two kinds of microballoons
The hole L/ (PCV2-Cap CSFV-E2 antigen is coupled each 2500 of microballoon) adds on Microplates96 orifice plate, is incubated at room temperature
After 30min, sample to be tested (monoclonal antibody or serum) 50 hole μ L/ is added, oscillation, which is protected from light, at room temperature is incubated for 1h, it is washed 2 times with PBST,
200 holes μ L/；100 hole μ L/ of rabbit-anti pig (1:1000) IgG antibody of biotin labeling is added, room temperature concussion (500r/min) is protected from light
It is incubated for 45min, is washed 2 times with PBST, 200 holes μ L/；Diluted 100 μ of streptomysin-phycoerythrin (SA-PE) of 10 μ g/ml is added
The hole L/, room temperature concussion (500r/min), which is protected from light, is incubated for 45min, is washed 2 times with PBST, 200 holes μ L/；125 hole μ L/ of sheath fluid is added,
Microballoon is resuspended, reading Analysis is carried out using Flex 3D liquid-phase chip detection system, obtains the corresponding MFI in each hole.
Compared with prior art, the invention has the following beneficial effects:
The present invention establishes high swine fever virus (CSFV) the E2 albumen and 2 of a kind of fast detection speed, high specificity, susceptibility
The double fluorescent microballoon immunological detection method of type circovirus (PCV2) Cap protein IgG antibody.E2 antibody of the invention and
Reproducible, the two and other Prevention of Common Occurrence Porcine Disease viral disease positive serum no cross reactions of Cap antibody detection method, high sensitivity,
High specificity.Compared with PCV2 and CSFV commercial ELISA kit, this method is better than commercial ELISA assay in sensitivity
Box.The foundation of this method can be used for the rapid differential diagnosis of 2 type virus infection pig of swine fever virus and annulus simultaneously and protectiveness resists
The detection of body has established experiment basis further to establish virus diseases of pigs antibody multiple detection method, anti-for other animal epidemics
The foundation of the novel serodiagnosis of body provides thinking.
Detailed description of the invention
Fig. 1 is the PCR amplification result of PCV2-Cap gene；M:DL-2000 standard molecular weight；1~2: the PCR amplification of sample
Product.
Fig. 2 is the PCR amplification result of CSFV-E2 gene；M:DL-2000 standard molecular weight；1~2: the PCR amplification of sample
Product.
Fig. 3 is recombinant plasmid pMAL-c5X-PCV2-Cap digestion qualification figure；M:DL-10,000 standard molecular weight；1:
PMAL-c5X-PCV2-Cap recombinant plasmid EcoRV+BamHI double digestion；2:pMAL-c5X-PCV2-Cap clone bacterium.
Fig. 4 is recombinant plasmid pMAL-c5x-CSFV-E2 digestion qualification figure；M:DL5000 standard molecular weight；1:pMAL-
C5x-CSFV-E2 recombinant plasmid EcoRV+BamHI double digestion；2:pMAL-c5x-CSFV-E2 clone bacterium.
Fig. 5 is pMAL-c5x-PCV2-Cap recombinant protein expression result chart；M: Protein Marker；1:pMAL-c5x
Before empty carrier induction；After the induction of 2:pMAL-c5x empty carrier；Before 3:pMAL-c5x-PCV2-Cap induction；4:pMAL-c5x-
After PCV2-Cap induction.
Fig. 6 is pMAL-c5x-CSFV-E2 recombinant protein expression result chart；M: Protein Marker；1:pMAL-c5x is empty
Before carrier induction；After the induction of 2 and 3:pMAL-c5x empty carrier；Before 4~6:pMAL-c5x-CSFV-E2 induction；7pMAL-c5x-
After CSFV-E2 induction.
Fig. 7 is pMAL-c5x-CSFV-E2 recombinant protein soluble analysis result figure；M: Protein Marker；1:
Before the induction of pMAL-c5x empty carrier；After the induction of 2pMAL-c5x empty carrier；Before 3:pMAL-c5x-PCV2-Cap induction；4:pMAL-
C5x-PCV2-Cap recombinant protein is crushed supernatant；The broken precipitating of 5:pMAL-c5x-PCV2-Cap recombinant protein.
Fig. 8 is pMAL-c5x-PCV2-Cap recombinant protein soluble analysis result figure；M: Protein Marker；1:
PMAL-c5x-CSFV-E2 recombinant protein is crushed supernatant；The broken precipitating of 2:pMAL-c5x-CSFV-E2 recombinant protein.
Fig. 9 is pMAL-c5x-CSFV-E2 recombinant protein Western-blot proof diagram；Note: figure is His label
Western-blot verifying；The albumen Marker of M:170kDa pre-dyed；Before 1:pMAL-c5x-CSFV-E2 induction；2:pMAL-
After c5x-CSFV-E2 induction；3:pMAL-c5x-CSFV-E2 recombinant protein is crushed supernatant；4:pMAL-c5x-CSFV-E2 recombinates egg
White broken precipitating.
Figure 10 is pMAL-c5x-CSFV-E2 recombinant protein Western-blot proof diagram；Note: figure is MBP label
Western-blot verifying；The albumen Marker of M:170kDa pre-dyed；1:pMAL-c5x-CSFV-E2 recombinant protein is crushed supernatant；
The broken precipitating of 2:pMAL-c5x-CSFV-E2 recombinant protein.
Figure 11 is pMAL-c5x-PCV2-Cap recombinant protein Western-blot proof diagram；Note: figure A is His label
Western-blot verifying；B is schemed for MBP label Western-blot verifying；The albumen Marker of M:170kDa pre-dyed；1:
PMAL-c5x-PCV2-Cap recombinant protein is crushed supernatant.
Figure 12 is the purifying SDS-PAGE analysis chart of pMAL-c5x-PCV2-Cap albumen；M:Marker；1:pMAL-c5x-
PCV2-Cap recombinant protein is crushed supernatant ultrafiltration；2:20mM imidazoles is washed miscellaneous；3:80mM imidazoles is washed miscellaneous；4:100mM imidazoles is washed miscellaneous；5:
200mM imidazoles is washed miscellaneous；6:300mM imidazoles is washed miscellaneous；7:400mM imidazoles is washed miscellaneous；The elution of 8:500mM imidazoles.
Figure 13 is the purifying SDS-PAGE analysis chart of pMAL-c5x-CSFV-E2 albumen；M:Marker；1: efflux；2:
20mM imidazoles is washed miscellaneous；3:60mM imidazoles is washed miscellaneous；4:80mM imidazoles is washed miscellaneous；5:100mM imidazoles is washed miscellaneous；6:200mM imidazoles is washed miscellaneous；7:
300mM imidazoles is washed miscellaneous；8.400mM imidazoles is washed miscellaneous.
Figure 14 is pMAL-c5x-PCV2-Cap recombinant protein antigen proof diagram；In A: the albumen of M:170kDa pre-dyed
Marker；1:PCV2 positive serum；In B: the albumen Marker of M:170kDa pre-dyed；1:PCV2 negative serum
Figure 15 is pMAL-c5x-CSFV-E2 recombinant protein antigen proof diagram；In A: the albumen of M:170kDa pre-dyed
Marker；1~2:CSFV positive serum；In B: the albumen Marker of M:170kDa pre-dyed；1:CSFV negative serum.
Figure 16 is that pMAL-c5x-PCV2-Cap schemes in conjunction with No. 40 microballoons.
Figure 17 is that pMAL-c5x-CSFV-E2 schemes in conjunction with No. 60 microballoons.
Figure 18 is the verification result figure of coupling and detection method feasibility.
Figure 19 is the testing result figure of PCV2-Cap monoclonal antibody dilution gradient.
Figure 20 is the testing result figure of CSFV-E2 monoclonal antibody dilution gradient.
Figure 21 is CSFV serum dilution gradient testing result figure.
Figure 22 is PCV2 serum dilution gradient testing result figure.
Figure 23 is PCV2 specificity analysis chart.
Figure 24 is CSFV specificity analysis chart.
Figure 25 is CSFV-E2IgG antibody detection method ROC curve figure.
Figure 26 is CSFV-E2IgG antibody detection method ROC curve analysis chart.
Figure 27 is PCV2-Cap IgG antibody detection method ROC curve figure.
Figure 28 is PCV2-Cap IgG antibody detection method ROC curve analysis chart.
Figure 29 is the evaluation figure that dual FMIA detection method detects PCV Cap IgG antibody effect.
Figure 30 is the evaluation figure that dual FMIA detection method detects CSFV E2IgG antibody effects.
Specific embodiment
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
1, bacterial strain and plasmid
It clones competent cell E.coli DH5 α and expression bacterium competence cell Rosetta (DE3) and BL21 is purchased from Beijing
Tiangeng biochemical technology Co., Ltd；Prokaryotic expression carrier pMAL-c5X is purchased from NEB company.Cloning vector pMD18-T Vector is
TaKaRa Products；PMD18-T-PCV-Cap and pMD18-T-CSFV-E2 plasmid is College of Veterinary Medicine, South China Agricultural University's infection
Sick teaching and research room saves.
2, serum sample
Swine fever virus (CSFV), 2 type circovirus (PCV-2), Pseudorabies virus (PRV), aftosa (FMDV), B-mode brain
Scorching virus (JEV), pig parvoviral (PPV) positive serum and PCV-2 and CSFV negative serum are by Agricultural University Of South China's veterinary science
Infectious disease teaching and research room, institute saves.CSFV128 parts, PCV-2128 parts pig farms from Guangdong Province of clinical Swine serum sample.
3, main agents
CSFV monoclonal antibody, PCV-2 monoclonal antibody；Goat-anti pig (HRP label) antibody is by Agricultural University Of South China animal doctor
Infectious disease teaching and research room, institute saves；IRDye 800CW Goat anti-Mouse IgG (H+L) is purchased from LI-COR Products；
Rabbit F (ab) Anti-Pig IgG H&L (Biotin), GoatAnti-Mouse IgG H&L (Biotin) are purchased from Abcam
Company；Anti-MBP Monoclonal Antibody (MBP antibody) is purchased from NEB company；Anti-His Antibody(His)
Antibody is purchased from Beijing Tiangeng biochemical technology Co., Ltd；PCV2 and CSFV ELISA kit purchased from South Korea Jin Nuo company and
IDEXX company.Carboxylic fluorescent encoding microsphere, correction, calibrating reagent, sheath fluid, 1.5mL magnetic frame are public purchased from U.S. Luminex
Department；Ethyl -3- (3- dimethylaminopropyl) carbodiimide hydrochloride (EDC), succinimide base sulfonic group biotin (Sulfo-
NHS-Biotin), 2- [N- morpholino] ethanesulfonic acid (MES), Sodium azide are purchased from Sigma Co., USA；Streptomysin-phycoerythrin
(SA-PE) it is purchased from Invitrogen company；96 orifice plate of Microplates is purchased from Greiner company.
4, key instrument
96 microwell plate concussion instruments: Jiangsu Haimen Kylin-Bell；Flex 3D liquid-phase chip detection system: Guangzhou Mi Libo
Co., Ltd；Board-washing machine: BIOTEK company；Odyssey two-color laser analysis system is U.S. LI-COR Products.
The amplification of 1 PCV-Cap, CSFV-E2 gene of embodiment
One, experimental method
Experiment early stage carries out it for several main epitopes in CV2Cap and CSFV raq gene amplification procedure
The PCR amplification of gene, and expression plasmid is constructed, it finds and not all genetic fragment can give expression to soluble albumen, warp
After crossing screening, the amino acid residue of PCV2 Cap and CSFV E2 are expressed in escherichia expression system, gives expression to solubility
Albumen, as antigen carry out subsequent research after purified.PCV-Cap, CSFV-E2 are designed according to homologous recombination cloning mechanisms
The primer of the target fragment of gene simultaneously carries out PCR amplification.
Design of primers general principle: two terminal homologous of linearized vector is introduced at 5 ' ends of the forward and reverse amplimer of Insert Fragment
Sequence, make amplification after the least significant end of Insert Fragment 5 ' and 3 ' be respectively provided with and linearize two end of cloning vector correspond to it is consistent together
Source sequence (15bp~20bp does not include restriction enzyme site).If final primer length is more than 40bp, recommend to select in primer synthesis
Purified with PAGE, clone's success rate can be improved.When calculating amplimer annealing temperature, gene-specific amplification sequence need to be only calculated
Tm value, the homologous sequence and restriction enzyme site of introducing should not participate in calculating.CSFV-E2 and PCV2-Cap gene order is set respectively
A pair of of specific primer is counted, (table 1) is synthesized by Invitrogen (Shanghai) Trading Co., Ltd..
1 design of primers of table:
Two, experimental result
The amplification of PCV-Cap, CSFV-E2 gene is detected by 1% agarose gel electrophoresis, is amplified respectively
Purpose band size about 612bp (nucleotide sequence is as shown in SEQ ID NO:1), 564bp (nucleotide sequence such as SEQ ID NO:
Shown in 2), it is consistent with expection, testing result is as depicted in figs. 1 and 2.
The building of the recombinant expression carrier of embodiment 2 PCV-Cap, CSFV-E2
One, experimental method
By PCV2 Cap of acquisition, CSFV raq gene segment respectively with pMAL-c5X expression vector EcoRV and BamHI
Restriction enzyme connects after carrying out double digestion respectively.Obtained recombinant plasmid pMAL-c5x-PCV2-Cap, pMAL-c5x-
CSFV-E2 is converted respectively into E.coli DH5 α Escherichia coli, expands bacterium, PCR, electrophoresis；And through plasmid extraction kit
Plasmid Mini Kit extracts to obtain recombinant plasmid, using restriction enzyme EcoRV and BamHI to two recombinant plasmids into
Row double digestion, rear electrophoresis.Meanwhile the plasmid of extracting being sent to Invitrogen (Shanghai) Trading Co., Ltd. and is sequenced.
Two, experimental result
PCR product gel electrophoresis is shown, obtains the specific band of about 612bp, 564bp respectively, in the same size with expection；
Double enzyme digestion product gel electrophoresis shows that pMAL-c5x-PCV2-Cap digestion obtains two about bands of 5677bp and 612bp,
(Fig. 3) consistent with expected results；PMAL-c5x-CSFV-E2 digestion obtains two about bands of 5677bp and 564bp, and pre-
Phase result is consistent (Fig. 4).Mentioned sequencing result is consistent with the sequence of target gene, and two restriction enzyme sites are correct, illustrates weight
Group plasmid construction success.
Embodiment 3 recombinates PCV2 Cap, the expression of CSFV E2 albumen and soluble analysis
One, experimental method
Recombination bacterium solution will be obtained in recombinant plasmid transformed to BL21 (DE3), and the inducer isopropyl of final concentration of 0.3mM is added
Base thiogalactoside (IPTG), inducing expression 8h in 16 DEG C of shaking tables, can largely lure since supernatant expression quantity is relatively fewer
It leads expression and collects supernatant.The bacterium solution 10000rpm/min induced is centrifuged 10min, abandons supernatant, thallus is resuspended with PBS.It will weigh
The ultrasonication under condition of ice bath of outstanding thallus, 200W, work 3sec, interval 5sec, is crushed to bacterium solution and becomes clarification.Bacterium solution warp
12000rpm, 4 DEG C of centrifugation 20min collect supernatant and precipitating respectively, and precipitating is resuspended with PBS, carry out the solubility of recombinant protein
Analysis.
Take pMAL-c5X empty carrier as control simultaneously, to full bacterium solution induction front and back, supernatant, precipitating, unloaded induction front and back
Carry out SDS-PAGE electrophoretic analysis.
Two, experimental result
Coomassie brilliant blue staining as the result is shown pMAL-c5X empty carrier induction after 45KDa occur a thick protein band
It is identical as destination protein size, Cap, E2 destination protein size with it is expected be consistent (amino acid sequence respectively such as SEQ ID NO:3 and
Shown in SEQ ID NO:4), there is a purpose band (Fig. 5 and Fig. 6) at about 65KD, 64.5KD respectively, and destination protein is upper
There is expression in cleer and peaceful precipitating, illustrates that the albumen has soluble (Fig. 7 and Fig. 8).
Embodiment 4 recombinates the Western-blot verifying of PCV2 Cap, CSFV E2 albumen
One, experimental method
Soluble recombination PCV2 Cap, CSFV E2 albumen will be obtained, carries out recombinant C ap, E2 according to the method that document provides
Purifying.Purified product is transferred on nitrocellulose filter (NC) film after polyacrylamide gel electrophoresis (SDS-PAGE)；With
Anti-MBP monoclonal antibody, Anti-His monoclonal antibody are primary antibody, IRDye 800CW Goat anti-Mouse IgG (H+L) sheep anti mouse is anti-
Body is secondary antibody, carries out detected by Western blot verifying (Western-blot) analysis: 5% 37 DEG C of skimmed milk power closing 2h；1:10
Diluted PCV2 and CSFV positive and negative serum is as primary antibody, 4 DEG C of overnight incubations；The label goat-anti pig of 1:5000 dilution HRP is added
Antibody is incubated at room temperature 1h；Chromogenic assay is carried out using enhanced HRP-DAB substrate colour reagent box.
Two, experimental result
Specific reaction can occur for Cap, E2 albumen and respective monoclonal antibody as the result is shown, generate single band (Fig. 9, Figure 10,
Figure 11).Illustrate MBP label protein and His the label protein successful expression in recombinant plasmid.The destination protein of expression has antigen
Property, it can be used for the foundation of fluorescent microsphere immunological detection method.
The purifying of 5 PCV-Cap, CSFV-E2 recombinant protein of embodiment
One, experimental method
Since pMAL-c5x-PCV2-Cap, pMAL-c5x-CSFV-E2 recombinant protein supernatant expression quantity are few compared to precipitating,
So obtaining pMAL-c5x-CSFV-E2, pMAL-c5x-PCV2-Cap to a large amount of inducing expressions of the two recombinant plasmids recombinates egg
White broken supernatant amount increases, with GE company Ni2+Affinity column is purified, and is taken its efflux, is washed miscellaneous liquid, eluent progress
SDS-PAGE and Coomassie brilliant blue are analyzed.
PCV-Cap, CSFV-E2 recombinant protein respectively through 20mM, 80mM, 100mM, 200mM, 300Mm, 400mM, 500mM,
The imidazole solution of concentration each 2 washes miscellaneous twice
Two, experimental result
The results show that recombinant protein pMAL-c5x-PCV2-Cap through the imidazole solution of 300Mm concentration each 2 wash twice it is miscellaneous after,
The destination protein purity eluted is higher, and foreign protein removes (Figure 12) substantially；Recombinant protein pMAL-c5x-CSFV-E2 result
Display through the imidazole solution of 200mM, concentration each 2 wash twice it is miscellaneous after, the destination protein purity eluted is higher (Figure 13).
The coupling of 6 antigen of embodiment and magnetic carboxylic fluorescent encoding microsphere
One, experimental method
It is complete works of according to the xMAP technology of Luminex, it is coupled using two step amide reaction methods: by the way that NaH is added2PO4It is slow
Fliud flushing, Sulfo-NHS and the activated phosphorylated microballoon of EDC solution form CSFV-E2, PCV2-Cap antigen with 60, No. 40 microballoons
Covalent amido bond, then with PBS-TBN solution be resuspended conjugate complexes, be placed in 4 DEG C be kept in dark place it is spare.
Using PCV2 Cap, the positive and negative serum of CSFV E2, PBS as positive control, negative control, blank control
Sample carries out machine testing reading Analysis, and whether verifying microballoon coupled antigen succeeds.PCV2-Cap, CSFV-E2 albumen are used simultaneously
Monoclonal antibody with 1:10 gradient dilution, 3 repetitions of each concentration detect coupling efficiency, while determining the detection limit of antibody.
There is the microballoon of antigen to carry out upper machine testing to positive and negative serum, PBS with coupling.
Fluorescent microsphere immunological detection method is complete works of referring to the xMAP technology of Luminex company, the specific steps are as follows:
The microballoon being coupled with vortex oscillator oscillation 1min, is diluted to 50/μ of working concentration L for microballoon with PBS-TBN
Afterwards, 50 holes μ L/ (i.e. 2500 microballoons) add on 96 orifice plate of Microplates, and sample to be tested (monoclonal antibody or serum) 50 is added
The hole μ L/ vibrates (500r/min) at room temperature and is protected from light incubation 1h, washed 2 times with the PBST of pH7.4,200 holes μ L/；Biotin is added
100 hole μ L/ of rabbit-anti pig (1:1000) IgG antibody of label, room temperature concussion (500r/min), which is protected from light, is incubated for 45min, is washed with PBST
It washs 2 times, 200 holes μ L/；Diluted 100 hole μ L/ of streptomysin-phycoerythrin (SA-PE) 10 μ g/ml is added, room temperature shakes (500r/
Min it) is protected from light and is incubated for 45min, washed 2 times with PBST, 200 holes μ L/；125 hole μ L/ of sheath fluid is added, microballoon is resuspended, using Flex
3D liquid-phase chip detection system carries out reading Analysis, obtains the corresponding MFI in each hole.
Two, experimental result
The magnetic bead number of this experiment purchase is 40, No. 60, and in conjunction with this laboratory Flex 3D chip detector, magnetic bead is three
The position tieed up in reference axis is not identical (Figure 16 and Figure 17), and display purchase magnetic bead is correct and available.
The result shows that the MFI (mean fluorescence intensities) of positive serum is greater than 10000, and it is greater than 5
The MFI of times negative control, the MFI of blank control illustrate the coupling method and detection method are feasible less than 100 (Figure 18).
The single FMIA detection method feasibility analysis of embodiment 7
One, experimental method
PCV2-Cap monoclonal antibody is subjected to 10 times of gradient dilutions from initial concentration 5mg/mL, CSFV-E2 monoclonal antibody is from initial concentration
32mg/mL carries out 10 times of gradient dilutions, is detected with the FMIA detection method of foundation, MFI value is fitted three using SPSS software
Equation of n th order n.
Two, experimental result
The result shows that the related coefficient (R2) of PCV2, CSFV, fluorescent microsphere immunological detection method be respectively 0.9932,
0.9901, illustrate that curvilinear correlation is good (Figure 19, Figure 20).
The orthogonal test of the reaction principal element of embodiment 8
One, experimental method
According to each step reaction condition, it is biotin labelled antibodies respectively that filtering out 4, which influences more significant factor on experiment,
Dilution, SA-PE depth, the time of magnetic bead and seroreaction, magnetic bead, serum and detection antibody response time.This experiment
Each factor is divided into three position grades, according to four factors, three position grades, design factor position grade table (table 2).
2 optimum seeking table of table:
Two, experimental result
As a result as shown in Table 3 and Table 4.
3 CSFVL of table9(34) orthogonal arrage:
4 table 4PCV2L of table9(34) orthogonal arrage
Show that PCV2-Cap optimal conditions is to be averaged measured by A1B2C3D3 respectively according to 4 factor, 3 grade orthogonal tests
Fluorescence intensity be it is highest, i.e., secondary antibody dilution is that 1:1000, SA-PE concentration are 10 μ g/ml and serum incubation time is
It is optimum reaction condition that 60min and secondary antibody incubation time, which are 60min,.CSFV-E2 optimal conditions is A1B1C2D2, i.e. secondary antibody is dilute
Degree of releasing is that 1:1000, SA-PE concentration are 5 μ g/ml, are 45min, are that 45min is with secondary antibody incubation time with serum incubation time
Optimum reaction condition.And secondary antibody dilution is all most important influence factor for PCV2-Cap, CSFV-E2.
The determination of the dilute best degree of releasing of 9 serum of embodiment
One, experimental method
PCV2 Cap, CSFV E2 positive and negative serum are subjected to 1:2 gradient dilution with PBS-1%BSA: 1:50,1:100,
1:200,1:400,1:800,1:1600,1:3200,1:6400,1:12800,1:25600,3 repetitions of each dilution, with true
Determine serum optimum diluting multiple.
Two, experimental result
Testing result: PCV2 serum optimum dilution degree is 1:50；CSFV serum optimum dilution degree is 1:100；It is positive at this time
Serum MFI value is greater than 10000, and is greater than 5 times (Figure 21,22) of negative serum MFI value.
10 individual event fluorescent microsphere immunological detection method of embodiment
One, the detection of PCV2 virus
The microballoon being coupled with vortex oscillator oscillation 1min, is diluted to 50/μ of working concentration L for microballoon with PBS-TBN
Afterwards, 50 holes μ L/ (i.e. 2500 microballoons) add on 96 orifice plate of Microplates, and sample to be tested (monoclonal antibody or serum) 50 is added
The hole μ L/ vibrates (500r/min) at room temperature and is protected from light incubation 1h, washed 2 times with the PBST of pH7.4,200 holes μ L/；Biotin is added
100 hole μ L/ of rabbit-anti pig (1:1000) IgG antibody of label, room temperature concussion (500r/min), which is protected from light, is incubated for 60min, is washed with PBST
It washs 2 times, 200 holes μ L/；Diluted 100 hole μ L/ of streptomysin-phycoerythrin (SA-PE) 10 μ g/ml is added, room temperature shakes (500r/
Min it) is protected from light and is incubated for 60min, washed 2 times with PBST, 200 holes μ L/；125 hole μ L/ of sheath fluid is added, microballoon is resuspended, using Flex
3D liquid-phase chip detection system carries out reading Analysis, obtains the corresponding MFI in each hole.
Two, the detection of CSFV virus
The microballoon being coupled with vortex oscillator oscillation 1min, is diluted to 50/μ of working concentration L for microballoon with PBS-TBN
Afterwards, 50 holes μ L/ (i.e. 2500 microballoons) add on 96 orifice plate of Microplates, and sample to be tested (monoclonal antibody or serum) 50 is added
The hole μ L/ vibrates (500r/min) at room temperature and is protected from light incubation 1h, washed 2 times with the PBST of pH7.4,200 holes μ L/；Biotin is added
100 hole μ L/ of rabbit-anti pig (1:1000) IgG antibody of label, room temperature concussion (500r/min), which is protected from light, is incubated for 45min, is washed with PBST
It washs 2 times, 200 holes μ L/；Diluted 100 hole μ L/ of streptomysin-phycoerythrin (SA-PE) 5 μ g/ml is added, room temperature shakes (500r/
Min it) is protected from light and is incubated for 45min, washed 2 times with PBST, 200 holes μ L/；125 hole μ L/ of sheath fluid is added, microballoon is resuspended, using Flex
3D liquid-phase chip detection system carries out reading Analysis, obtains the corresponding MFI in each hole.
The specific test of 11 detection method of embodiment
One, experimental method
The detection method that embodiment 10 is established examines CSFV, PCV-2, PRV, FMDV, JEV, pig PPV positive serum
It surveys, every group of 3 repetitions, to verify the specificity of the detection method.
Two, experimental result
As the result is shown: when the PCV2-Cap fluorescent microsphere immunological detection method that application is established, only PCV2 positive serum
Testing result is the positive, and other diseases positive serum testing result is negative (Figure 23).Using the CSFV-E2 fluorescent microsphere of foundation
When immunological detection method, only CSFV positive serum testing result is the positive, and other diseases positive serum testing result is yin
Property (Figure 24).
The repetitive test of 12 detection method of embodiment
One, experimental method
Repetitive test is carried out to the detection method that embodiment 10 is established.
It is repeated in batch: taking each 4 parts of positive serums of PCV2, CSFV, 1 part of negative serum, 10 repetitions of every part of serum pass through meter
Its coefficient of variation is calculated to evaluate the withinrun precision of this method.
It is repeated between batch: taking each 11 parts of positive serums of PCV2, CSFV, 1 part of negative serum, 3 repetitions of every part of serum, when different
Between do 3 times, by calculating the coefficient of variation to evaluate the betweenrun precision of this method.
Two, experimental result
The variation within batch coefficient (CV) of CSFV, PCV2 fluorescent microsphere immunological detection method is respectively 6.7-8.5%, 3.6-
9.0% (table 5,6), interassay coefficient of variation are respectively as follows: 2.6-12.6%, 2.2-9.8% (table 7,8).Its average variation within batch system
Number is respectively as follows: 7.6%, 6.4%, and average interassay coefficient of variation is respectively as follows: 6.9%, 6.7%.Meet Luminex companyRequire in technology complete works: criticizing interior CV is no more than 10%, and CV is not more than 20% between batch, illustrates being somebody's turn to do for this research foundation
Detection method has good repeatability.
It repeats to test in 5 CSFV liquid phase protein chip detection method of table batch:
It repeats to test in 6 PCV2 liquid phase protein chip detection method of table batch:
It repeats to test between 7 CSFV liquid phase protein chip detection method of table batch
It repeats to test between 8 PCV2 liquid phase protein chip detection method of table batch
12 fluorescent microsphere immunological detection method of embodiment is compared with ELISA
One, experimental method
PCV2128 parts, CSFV128 parts of clinical serums are taken, the FMIA detection method established using this research and IDEXX company
ELISA kit and Jin Nuo company, South Korea ELISA kit detect simultaneously, testing result MFI value using MedCalc software into
The analysis of row ROC curve, determines optimal critical value, specificity and susceptibility.And Chi-square Test is carried out to testing result and evaluates it
Coincidence rate.
Two, experimental result
As a result ROC analysis is carried out by MedCalc software, obtains ROC curve (Figure 25).It is glimmering using CSFV-E2IgG antibody
Light immune microsphere detection method and ELISA kit detect 128 parts of CSFV clinic pig anteserum samples simultaneously, the results show that CSFV-
The specificity of E2IgG antibody FMIA detection method is 96.77%, sensitivity 98.96%, and critical value is 5939 (Figure 26).
128 parts are detected simultaneously using PCV2-Cap IgG antibody fluorescence immunoassay microballoon detection method and ELISA kit
As a result PCV2 clinic pig anteserum sample carries out ROC analysis by MedCalc software, obtains ROC curve (Figure 27).The results show that
The sensitivity of PCV2-Cap IgG antibody fluorescence immunoassay microballoon detection method is 99.04%, and specificity is 91.3%, and critical value is
2882.5 (Figure 28).
Chi-square Test shows: the relevance of CSFV, PCV2 fluorescence immunoassay microballoon detection method and commercial kit ELISA
Significantly (P < 0.01), two methods can react same index.
A kind of foundation of the double check method of embodiment 13
A kind of kit detecting 2 type pig circular ring virus and swine fever virus, including coupling have amino acid sequence such as SEQ ID
No. 60 carboxylic fluorescent microballoons of 2 type pig circular ring virus Cap recombinant proteins shown in NO:3, coupling have the right amino acid sequence such as
No. 40 carboxylic fluorescent microballoons, the streptomysin-phycoerythrin of swine fever virus E2 recombinant protein shown in SEQ ID NO:4, biology
Secondary antibody (rabbit-anti pig IgG antibody), PBS-TBN, PBST and the sheath fluid that element marks；
Wherein, 2 type pig circular ring virus Cap recombinant proteins and swine fever virus E2 recombinant protein are by by its coding DNA sequence
Column are connected into pMAL-c5X carrier, and are transferred to prokaryotic expression bacterium inducing expression, and inductive condition is 16 DEG C, IPTG is final concentration of
6~8h of inducing expression under conditions of 0.3mM；After inducing expression, it is crushed supernatant, with GE company Ni2+Affinity column is purified,
Swine fever virus E2 recombinant protein is washed miscellaneous twice through the imidazole solution 2 of 200mM, and 2 type pig circular ring virus Cap recombinant proteins are through 300mM
Imidazole solution 2 wash twice it is miscellaneous.
It is complete works of according to the xMAP technology of Luminex, after two kinds of microballoons are diluted to 50/μ of working concentration L with PBS-TBN,
Each hole 50 μ L/ of two kinds of microballoons (PCV2-Cap CSFV-E2 antigen is coupled each 2500 of microballoon) adds to 96 orifice plate of Microplates
On, after being incubated at room temperature 30min, sample to be tested (monoclonal antibody or serum) 50 hole μ L/ is added, oscillation, which is protected from light, at room temperature is incubated for 1h, uses
PBST is washed 2 times, 200 holes μ L/；100 hole μ L/ of rabbit-anti pig (1:1000) IgG antibody of biotin labeling, room temperature concussion is added
(500r/min), which is protected from light, is incubated for 45min, is washed 2 times with PBST, 200 holes μ L/；The diluted streptomysin of 10 μ g/ml-algae red egg is added
100 hole μ L/ white (SA-PE), room temperature concussion (500r/min), which is protected from light, is incubated for 45min, is washed 2 times with PBST, 200 holes μ L/；It is added
Microballoon is resuspended in 125 hole μ L/ of sheath fluid, carries out reading Analysis using Flex 3D liquid-phase chip detection system, it is corresponding to obtain each hole
MFI.
The evaluation of 14 fluorescence immunoassay microballoon double check method of embodiment
One, experimental method
The individual event fluorescent microsphere immunological detection method and embodiment 13 established with embodiment 10 establish double fluorescent microballoon
Immunological detection method detects 96 parts of clinical Swine serum samples simultaneously, and testing result carries out linear regression analysis, corresponded to
Related coefficient (R2), examine the multiple FMIA detection method of foundation with the presence or absence of cross reaction, while verifying multiple FMIA inspection
The feasibility of survey method.
Two, experimental result
Testing result shows that PCV2, CSFV the double fluorescent microballoon immunological detection method established and substance fluorescent microsphere are exempted from
The related coefficient (R2) of the MFI value of epidemiology detection method is respectively 0.9849,0.9642 (Figure 29,30), when illustrating double check
Both PCV2-Cap-40, CSFV-E2-60 microballoons will not influence each other, no cross reaction.
Sequence table
<110>Agricultural University Of South China
<120>a kind of PCV2, CSFV IgG antibody double fluorescent microballoon immunological detection method
<160> 4
<170> SIPOSequenceListing 1.0
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gatatcatga acggaatctt caacaccaga ctgtcacgca cattcggata caccatcaag 60
cgcacaaccg tgaaaacacc atcttgggca gtggacatga tgcgcttcaa catcaacgac 120
ttcctgccac caggaggagg atcaaatccc agatcagtgc cattcgagta ctaccgcatc 180
agaaaggtga aggtggagtt ttggccttgc tcaccaatca cacagggaga tagaggagtg 240
ggatcatcag cagtcattct ggacgacaac ttcgtgacaa aggcaacagc actgacatac 300
gacccatacg tgaactactc ctccagacac accatcacac agcccttctc ataccactca 360
cgctacttca cacccaaacc agtgctggac tcaacaatcg actacttcca gcccaactcc 420
aagagaaacc agctctggct gagactgcag acaacaggaa acgtggatca cgtgggactg 480
ggaacagcat tcgagaactc catctacgac caggagtaca acatccgcgt gacaatgtac 540
gtgcagttca gagaattcaa cctgaaggac ccaccactga atccccatca tcatcatcat 600
cactaaggat cc 612
<210> 2
<211> 564
<212> DNA
<213>artificial sequence (Artificial Sequence)
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gatatccggc tagcctgcaa ggaagattac aggtacgcaa tatcgtcaac cgatgagata 60
gggctacttg gggccggagg tctcaccacc acctggaagg aatacaacca cgatttgcaa 120
ctgaatgacg ggaccgtcaa ggccagttgc gtggcaggtt cctttaaagt cacagcactt 180
aatgtggtca gtaggaggta tttggcgtca ttgcataaga aggctttacc cacttccgtg 240
acattcgagc tcctgttcga cgggaccaac ccatcaactg aggaaatggg agatgacttc 300
aggtccgggc tgtgcccgtt tgatacgagt cctgttgtta agggaaagta caatacgacc 360
ttgttgaacg gtagtgcttt ctatcttgtc tgcccaatag ggtggacggg tgtcatagag 420
tgcacagcag tgagcccaac aactctgagg acagaagtgg taaagacctt caggagagac 480
aagccctttc cgcacagaat ggattgtgtg accaccatag tggaaaatga agatttacat 540
catcatcatc atcactaagg atcc 564
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35 40 45
Asn Pro Arg Ser 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 Ser Ala Val Ile Leu Asp Asp Asn Phe Val Thr Lys Ala Thr
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 Ser Thr Ile Asp Tyr Phe Gln Pro Asn Ser 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 Glu Tyr Asn Ile Arg
165 170 175
Val Thr Met Tyr Val Gln Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro
180 185 190
Leu Asn Pro His His His His His His Gly Ser
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<211> 187
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35 40 45
Ser Cys Val Ala Gly Ser Phe Lys Val Thr Ala Leu Asn Val Val Ser
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Arg Arg Tyr Leu Ala Ser Leu His Lys Lys Ala Leu Pro Thr Ser Val
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Gly Asp Asp Phe Arg Ser Gly Leu Cys Pro Phe Asp Thr Ser Pro Val
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Val Lys Gly Lys Tyr Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe Tyr
115 120 125
Leu Val Cys Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val
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Ser Pro Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe Arg Arg Asp
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Claims (10)
1.2 type pig circular ring virus Cap genetic fragments are detecting 2 type pig circular ring virus detection reagents of 2 type pig circular ring virus or preparation
Application in box, which is characterized in that its nucleotide sequence is as shown in SEQ ID NO:1.
2. application of the E 2 gene of Classical Swine Fever segment in detection swine fever virus or preparation swine fever virus detection kit, feature
It is, nucleotide sequence is as shown in SEQ ID NO:2.
3.2 type pig circular ring virus Cap recombinant proteins are detecting 2 type pig circular ring virus detection reagents of 2 type pig circular ring virus or preparation
Application in box, which is characterized in that its amino acid sequence is as shown in SEQ ID NO:3.
4. application of the swine fever virus E2 recombinant protein in detection swine fever virus or preparation swine fever virus detection kit, feature
It is, amino acid sequence is as shown in SEQ ID NO:4.
5. one group for detecting the carboxylic fluorescent microballoon group of 2 type pig circular ring virus and swine fever virus, which is characterized in that including idol
It is associated with the carboxylic fluorescent microballoon and idol of amino acid sequence 2 type pig circular ring virus Cap recombinant proteins as shown in SEQ ID NO:3
It is associated with the carboxylic fluorescent microballoon of amino acid sequence swine fever virus E2 recombinant protein as shown in SEQ ID NO:4,
Wherein, 2 type pig circular ring virus Cap recombinant proteins and swine fever virus E2 recombinant protein are by connecting its DNA sequences encoding
PMAL-c5X carrier is accessed, and is transferred to prokaryotic expression bacterium inducing expression,
Inductive condition is 12~37 DEG C, 6~8h of inducing expression under conditions of the final concentration of 0.3~1.0mM of IPTG.
6. right want 5 described in carboxylic fluorescent microballoon group preparation detection 2 type pig circular ring virus and swine fever virus kit
In application.
7. a kind of kit for detecting 2 type pig circular ring virus and swine fever virus, which is characterized in that including described in claim 5
Carboxylic fluorescent microballoon group.
8. kit according to claim 7, which is characterized in that also contain streptomysin-phycoerythrin, biotin labeling
Secondary antibody.
9. kit according to claim 8, which is characterized in that secondary antibody dilution is 1:1000~10000, streptomysin-
Phycoerythrin concentration is 5~10 μ g/ml and serum incubation time is 45~60min and secondary antibody incubation time is 45~60min.
10. kit according to claim 7, which is characterized in that test serum dilution is 1:50~100.
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Application publication date: 20190521 |