CN109207441A - 3 type Cap protein of recombinant baculovirus expression pig circular ring virus and its construction method and primer - Google Patents
3 type Cap protein of recombinant baculovirus expression pig circular ring virus and its construction method and primer Download PDFInfo
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Abstract
The invention belongs to vaccine manufacturing technology fields.The invention discloses a kind of 3 type Cap proteins of recombinant baculovirus expression pig circular ring virus.The invention also discloses the primers for constructing 3 type Cap protein of recombinant baculovirus expression pig circular ring virus.The present invention discloses the construction method for 3 type Cap protein of recombinant baculovirus expression pig circular ring virus again, comprising the following steps: 1, pSK-sPCV3 of the building comprising PCV3 full-length genome；2, the building of transfer vector；3, the building of shuttle plasmid；4, the acquisition of recombinant baculovirus；Positive shuttle plasmid Bacmid-Cap is transfected into Sf9 cell, obtains the recombinant baculovirus rBac-Cap of expression 3 type Cap gene of pig circular ring virus.The Cap protein that the present invention expresses has good biological activity, and immune mouse is found to have good immunogenicity.
Description
Technical field
The present invention relates to the construction methods of the recombinant baculovirus of expression 3 type of pig circular ring virus (PCV3) Cap gene, are used for
Manufacture vaccine.
Background technique
Porcine circovirus desease (PCVD) is one of the important diseases for currently endangering pig breeding industry, and wherein PCV2 is to lead to such disease
The major virulent factor of disease.2016, the scholars such as Phan reported novel pig circular ring virus PCV3, which can cause pigskin scorching
With the diseases such as nephrotic syndrome (PDNS), pig breeding dysfunction respiratory disease and digestive system inflammation [Phan TG,
Giannitti F,Rossow S,et al.Detection of a novel circovirus PCV3 in pigs with
cardiac and multi-systemic inflammation[J].Virology Journal,2016,13(1):184.]。
It is found by PCV3 full-length genome phylogenetic analysis, PCV3 is in different from the virus of two genotype of other PCV1 and PCV2
Evolutionary branching, and there is also evolution differences between each separation strains of PCV3, are novel pig circular ring virus.From PCV3 be reported with
Come, domestic and international multiple areas detect the virus, and harm is just gradually taken seriously.At the beginning of 2017, what opening etc. is in China
PCV3 is detected for the first time.Domestic PCV3 strain is broadly divided into tri- kinds of genotype of 3a, 3b and 3c at present, wherein 3a and U.S. PCV3 ginseng
It examines strain and is in identical evolutionary branching [Fu X, Fang B, Ma J, et al.Insights into the epidemic
characteristics and evolutionary history of the novel porcine circovirus type
3 in southern China[J].Transboundary and Emerging Diseases,2018,65(2):e296-
e303.].The serum of domestic to Poland 14 commercializations the pig farm sow, piglet and bred pigs such as Stadejek carries out PCV3 detection,
Confirm PCV3 infection equally exist in European Region [Stadejek T,niak A,ek D,et al.First
detection of porcine circovirus type 3 on commercial pig farms in Poland[J]
.Transboundary and Emerging Diseases,2017,64(5):1350-1353.].It is control PCV3 into one
Step is popular, needs the commercialized vaccine that research and development are directed to the virus.
PCV3 is no togavirus as PCV1, PCV2, and virion is also in icosahedral structure of virus, the list with 2kb
Chain closed hoop minus-strand dna genome includes three most important open reading frame.ORF1 coding participates in viral genome duplication
GAP-associated protein GAP, and ORF2 encodes the Cap nucleocapsid structures albumen of leading immunogenicity, is to develop PCV3 subunit genetic engineering
Ideal target gene [Nawagitgul P, Morozov I, Bolin S R, the et al.Open reading frame 2 of vaccine
of porcine circovirus type 2 encodes a major capsid protein[J].Journal of
General Virology,2000,81(Pt 9):2281-2287.].PCV3 ORF3 encodes the function being made of 231 amino acid
It can agnoprotein matter.The Cap protein amino acid identity of PCV3 and PCV2 is lower, and only 30%, in addition, the two Cap protein
Epitope implies that the antigen cross protectiveness between PCV3 and PCV2 strain may be very low also without any similitude, existing
PCV2 vaccine can not effectively prevent PCV3 infection, and [profound ocean, Wang Dongliang, Wang Naidong wait the detection of 3 type of pig circular ring virus and its Cap
Structure sequence and antigenic forecast analysis [J] journal of animal science and veterinary medicine, 2017,48 (6): 1076-1084.].
Vaccine is still the main means of current prevention and control PCVD, therefore, can be become naturally for the vaccine of novel PCV3 infection
The hot spot of whole world research.Different from prokaryotic expression system, yeast and mammalian cell eukaryotic expression system, baculoviral table
Possess as one of eukaryotic expression system that external source insertion gene content is big, protein expression level is high, easy to operate and turn over up to system
Translate post-processing modification many advantages, such as and [Lin SY, Chen GY, Hu YC.Recent the patents on that is used widely
the baculovirus systems[J].Recent Patents on Biotechnology,2011,5(1):1-11.]。
Currently, the production about PCV2 subunit vaccine is based primarily upon Insect Cell/Baculovrius Expresion System expression system, such as Intervet, company is ground
Circumvent subunit vaccine [Baech NM, the Meng XJ.Efficacy and future prospects of of system
commercially available and experimental vaccines against porcine circovirus
type2(PCV2).Virus Res,2012(164):33-42.].Report at present about PCV3 vaccine is less, passes through building weight
Group baculoviral carries out the expression of PCV3 Cap protein, can lay the foundation for exploitation PCV3 subunit vaccine.
Summary of the invention
The present invention devises the primer of amplification PCV3 overall length by the screening to PCV3 strain；For PCV3 overall length Cap base
Because devising special primer, it is based on Bac-to-Bac system, the recombination for obtaining expression 3 type Cap protein of pig circular ring virus is rod-shaped
Virus.This method can be convenient the recombinant baculovirus for efficiently constructing expression PCV3 Cap protein, and can determine table
The Cap protein reached has good biological activity, and immune mouse is found to have good immunogenicity.
The present invention obtains the recombinant baculovirus that first purpose is to provide expression 3 type Cap protein of pig circular ring virus, recombinates bar
Shape virus rBac-Cap is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 10th, 2018
It is recommended that classification naming are as follows: expression 3 type Cap protein of pig circular ring virus recombinant baculovirus, deposit number CGMCC
No.15692。
The present invention obtains second purpose and is to provide for constructing 3 type Cap protein of recombinant baculovirus expression pig circular ring virus
Primer, referring to the PCV3 sequence announced on GenBank and the independent design of Cap full length gene sequence, the primer is shown in
Table 1:
Primer needed for table 1 and restriction enzyme site
The restriction enzyme site of primer Xho I-3F isCTCGAG；
The restriction enzyme site of primer EcoR I-3R isGAATTC；
The restriction enzyme site of primer Cap-F isGGATCC；
The restriction enzyme site of primer Cap-R isAAGCTT。
The present invention obtains the building side that third purpose is to provide 3 type Cap protein of recombinant baculovirus expression pig circular ring virus
Method, the screening of PCV3 strain；PCV3 overall length is expanded, to save PCV3 genome；Express PCV3 type Cap full length gene primer
Design, to expand PCV3 Cap full length gene；Transfer vector pFastBacTMThe building of HT A-Cap；Shuttle plasmid Bacmid-
The acquisition of Cap；The acquisition of recombinant baculovirus rBac-Cap；The expression identification of PCV3Cap albumen；Electronic Speculum observes virus-like
Grain；Immune mouse test.Finally obtain can be used for industrialized production expression 3 type Cap gene of pig circular ring virus recombination it is rod-shaped
Viral rBac-Cap；Specifically includes the following steps:
1, building includes the pSK-sPCV3 of PCV3 full-length genome:
Using PCV3 DNA as template, amplified with primer Xho I-3F described in claim 1 and primer EcoR I-3R
PCV3 full-length genome；The pBluescript SK+ carrier saved after the recycling of PCV3 full-length genome glue with this laboratory is used simultaneously
Xho I and EcoR I double digestion will be attached by the molar ratio of 3:1 after two kinds of digestion products purification and recoveries, finally be weighed
Group plasmid pSK-sPCV3；Identify that recombinant plasmid, digestion products are solidifying with 0.7% agarose by Xho I and EcoR I double digestion
Glue carries out electrophoresis, obtains the band that size is respectively 2958bp and 2000bp；
2, the building of transfer vector:
Using pSK-sPCV3 recombinant plasmid as template, expanded with primer Cap-F described in claim 1 and primer Cap-R
PCV3 Cap overall length, the transfer vector pFastBac that PCR product is saved with this laboratory after purificationTMHT A carries out BamH jointly
The double digestion of I and Hind III；Carry out conventional connection reaction after purification again；Axygen small amount plasmid DNA extraction kit mentions
Take recombinant plasmid pFastBacTMHT A-Cap is utilized respectively the identification of III single endonuclease digestion of restriction enzyme BamH I and Hind, with
And the double digestion identification of BamH I-Hind III；Digestion products are subjected to electrophoresis with 0.7% Ago-Gel, BamH I and
Hind III distinguishes single endonuclease digestion pFast BacTMHT A-Cap plasmid then obtains the single band of 5501bp；BamH I-Hind III pair
Digestion recombinant plasmid pFastBacTMHT A-Cap then obtains two bands of 4856bp and 645bp；
3, the building of shuttle plasmid:
III double digestion of BamH I-Hind is identified to obtain the correct recombinant vector of two bands of 4856bp and 645bp
pFastBacTMHT A-Cap conversion enters DH10 α Bac competent cell；Blue hickie screens swivel base successfully white single colonie, ginseng
It examines Invirtogen company Bac-to-Bac baculovirus expression system specification and carries out corresponding PCR identification, after identification is correct, take out
Mention recombinant shuttle plasmid Bacmid-Cap；With the amplifiable about 3075bp band out of primer pUC/M13；With primer pUC/M13 F+
CapR amplifies about 2500bp purpose band；1000bp purpose band is amplified with primer Cap F+pUC/M13R；With primer Cap
F/R amplifies 645bp purpose band respectively；
4, the acquisition of recombinant baculovirus:
Positive shuttle plasmid Bacmid-Cap is transfected into Sf9 cell, obtains the weight of expression 3 type Cap gene of pig circular ring virus
Group baculoviral rBac-Cap.
Compared with prior art, the invention has the following advantages:
The present invention obtains PCV3 full-length genome first, then carries out conventional connection reaction and realizes the mono- structure copied of PCV3
It builds.PCV3 overall length Cap gene is obtained simultaneously, it was demonstrated that the gene can be expressed using insect baculovirus expression system, be obtained
High titre recombinant baculovirus, the recombinant baculovirus titre after amplification is up to 108.3TCID50/ mL or more is small with His-tag
Mouse monoclonal antibody and the anti-PCV3 positive serum of mouse are that primary antibody carries out Western Blot identification, and discovery expression albumen has good exempt from
Epidemic disease reactivity, immune mouse test discovery can stimulate mouse to generate the specific antibody for being directed to PCV3, finally obtain completely suitable
The recombinant baculovirus rBac-Cap of expression 3 type Cap gene of pig circular ring virus for the factorial production.It can be used as genetic engineering
Subunit vaccine uses, while having established material base for the function and immune mechanism of further investigation PCV3 virus related gene.
Detailed description of the invention
Fig. 1 is the electrophoretogram that recombinant plasmid pSK-sPCV3 double digestion is copied comprising PCV3 full-length genome list.
Fig. 2 is transfer vector pFastBacTMHT A-Cap restriction enzyme digestion and electrophoresis figure.
The PCR that Fig. 3 is shuttle vector Bacmid-Cap identifies electrophoretogram.
Fig. 4 is the PCR identification electrophoretogram for extracting recombinant baculovirus rBac-Cap.
Fig. 5 is the total serum IgE electrophoretogram for extracting recombinant baculovirus rBac-Cap.
The electrophoretogram that PCR is identified after Fig. 6 is RT-PCR.
Fig. 7-1 is the IFA figure for being inoculated with the Sf9 cell of recombinant baculovirus rBac-Cap.
Fig. 7-2 is the IFA comparative diagram for being inoculated with the Sf9 cell of wild-type baculovirus.
Fig. 7-3 is the IFA comparative diagram of health Sf9 cell.
Fig. 8 is the SDS-PAGE qualification figure for expressing 3 type Cap protein of pig circular ring virus.
Fig. 9 is to express the Western Blot qualification figure of 3 type Cap protein of pig circular ring virus (with His-tag mouse monoclonal work
For primary antibody).
Figure 10 is to express the Western Blot qualification figure of 3 type Cap protein of pig circular ring virus (with the anti-PCV3 positive blood of mouse
It is used as primary antibody clearly).
Figure 11 PCV3 Cap VLPs observes transmission electron microscope picture.
Figure 12 test mice antibody level figure.
Specific embodiment
One, the building of the recombinant baculovirus rBac-Cap of 3 type Cap gene of pig circular ring virus is expressed:
Primer sequence designed for amplification is as follows:
Primer needed for table 1 and restriction enzyme site
Note: underscore part is each restriction enzyme site
1, building includes the pSK-sPCV3 of PCV3 full-length genome:
Using the PCV3 DNA of extraction as template, PCV3 full genome is amplified with primer Xho I-3F and primer EcoR I-3R
Group；PBluescript SK (+) carrier saved after the recycling of PCV3 full-length genome glue with laboratory is used into Xho I and EcoR simultaneously
I double digestion will be attached after two kinds of digestion products purification and recoveries by the molar ratio of 3:1, finally obtain recombinant plasmid pSK-
sPCV3.Identify that recombinant plasmid, digestion products carry out electricity with 0.7% Ago-Gel by Xho I and EcoR I double digestion
Swimming, obtains the band that size is respectively 3000bp and 2000bp or so, as shown in Figure 1.In Fig. 1, M:DL5000 DNA
Marker；1:Xho I enzyme and EcoR I enzyme double digestion pSK-sPCV3 plasmid obtain 2958bp and 2000bp or so segment.
2, the building of transfer vector:
Using pSK-sPCV3 recombinant plasmid as template, PCV3 Cap overall length is expanded with primer Cap-F and primer Cap-R, it will
The transfer vector pFastBac that PCR product is saved with laboratory after purificationTMHT A carries out the double of BamH I and Hind III jointly
Digestion；Carry out conventional connection reaction after purification again.Axygen small amount plasmid DNA extraction kit extracts recombinant plasmid in a small amount
pFastBacTMHT A-Cap is utilized respectively the identification of III single endonuclease digestion of restriction enzyme BamH I and Hind and BamH I-
The double digestion of Hind III is identified.Digestion products are subjected to electrophoresis with 0.7% Ago-Gel, BamH I and Hind III distinguishes
Single endonuclease digestion pFast BacTMThe single band of 5501bp then can be obtained in HT A-Cap plasmid.III double digestion of BamH I-Hind recombinates matter
Grain pFastBacTMTwo bands of 4856bp and 645bp then can be obtained, as shown in Figure 2 in HT A-Cap.In Fig. 2, M:DL5000
DNA Marker；1:BamH I enzyme digestion pFastBacTMHT A-Cap plasmid obtains 5501bp or so segment；2:Hind III enzyme
Digestion pFastBacTMHT A-Cap plasmid obtains 5501bp or so segment；3:BamH I enzyme and Hind III enzyme double digestion
pFastBacTMHT A-Cap plasmid obtains 4856bp and 6_45bp or so segment.
4, the building of shuttle plasmid:
It will identify correct recombinant vector pFastBacTMHT A-Cap conversion enters DH10 α Bac competent cell；Lan Bai
Spot screens swivel base successfully white single colonie, extracts recombinant shuttle plasmid Bacmid-Cap after PCR identification is correct.With primer pUC/
M13 can amplify 300bp or so band；3075bp purpose band can be amplified with primer pUC/M13F+CapR；Use primer
Cap F+pUC/M13R can amplify 2500bp or so purpose band；The left side 645bp can be amplified respectively with primer Cap F/R
Right purpose band.As shown in Figure 3.In Fig. 3, M:DL5000 DNA Marker；1:pUC/M13 expands wild type shuttle plasmid
The product of Bacmid about 300bp；The product of 2:pUC/M13 amplification recombinant shuttle plasmid Bacmid-Cap about 3000bp；3:M13F+
The product of CapR amplification recombinant shuttle plasmid Bacmid-Cap about 2500bp；4:CapF+M13R expands recombinant shuttle plasmid
The product of Bacmid-Cap about 1000bp；The product of 5:CapF+CapR amplification recombinant shuttle plasmid Bacmid-Cap about 645bp；
6:Negative control。
4, the acquisition of recombinant baculovirus:
Positive shuttle plasmid Bacmid-Cap is transfected into Sf9 cell, obtains the weight of expression 3 type Cap gene of pig circular ring virus
Group baculoviral rBac-Cap.
Identification: extracting the DNA of recombinant baculovirus rBac-Cap, is utilized respectively primer Cap F/R and carries out PCR identification.With
0.7% Ago-Gel carries out electrophoresis, as shown in Figure 4: M:DL5000 DNA Marker；1:Negative control；2:
Cap F/R expands rBac-Cap about 645bp or so target fragment.
As seen from Figure 4: having the Cap purpose band of 645bp or so size in electroresis appraisal PCR product, illustrate to obtain weight
Group baculoviral rBac-Cap.
Recombinant baculovirus rBac-Cap is preserved in Chinese microorganism strain preservation conservator on May 10th, 2018
Can common micro-organisms center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), it is proposed that classification naming are as follows: expression pig circle
The recombinant baculovirus of 3 type Cap protein of circovirus virus, deposit number are CGMCC No.15692.
Two, the Identification of Biological Characteristics of the recombinant baculovirus of 3 type Cap gene of pig circular ring virus is expressed:
Material: recombinant baculovirus rBac-Cap, health Sf9 cell.
The anti-PCV3 positive serum in pig source (self-control serum), the anti-PCV3 positive serum of source of mouse (self-control serum), commercialization His-
Tag monoclonal antibody.
1, the cDNA detection of the recombinant baculovirus of 3 type Cap gene of pig circular ring virus is expressed
By F2The recombinant baculovirus rBac-Cap in generation is inoculated with health Sf9 cell respectively, and TRIzol cracking is extracted total after 72h
RNA, as shown in Figure 5.RT-PCR is carried out after removal genomic DNA, then passes through PCR testing goal genetic transcription situation, PCR method
Detect PCV3Cap gene specific band, as shown in Figure 6.
In Fig. 5, M:DL5000 DNA Marker；1:rBac-Cap total serum IgE.
In Fig. 6, M:200bp DNA Marker；645bp band after 1:rBac-Cap total serum IgE reverse transcription；2:Negative
control。
2, indirect immunofluorescence (IFA) detection of the recombinant baculovirus of 3 type Cap gene of pig circular ring virus is expressed:
Using the anti-PCV3 positive serum in pig source as primary antibody, the goat-anti pig IgG of FITC fluorescent marker is secondary antibody, in fluorescence microscopy
It arrives under the microscope, in the Sf9 cell of the recombinant baculovirus rBac-Cap through inoculation expression 3 type Cap gene of pig circular ring virus
There is apparent specific fluorescence (as shown in Fig. 7-1)；And it is inoculated with wild-type baculovirus (as shown in Fig. 7-2) and normal control
Do not occur then in cell fluorescence (as shown in Fig. 7-3), it is seen that the rod-shaped disease of recombination of infection expression 3 type Cap gene of pig circular ring virus
The cell of malicious rBac-Cap can express PCV3 Cap protein.
2, the SDS-PAGE detection of the recombinant baculovirus of 3 type Cap gene of pig circular ring virus is expressed
After F2 is inoculated with Sf9 cell 96h for recombinant baculovirus rBac-Cap, collect after protein sample boils for SDS-
PAGE electrophoretic analysis is observed after coomassie brilliant blue staining.It can be seen that there is purpose band (Fig. 8) in expected size position, it can
See the destination protein band that size is about 30kD.
3, the Western Blot identification of the recombinant baculovirus of 3 type Cap gene of pig circular ring virus is expressed
Western Blot is carried out with His-tag mouse monoclonal (Fig. 9) and anti-PCV3 mouse positive serum (Figure 10) respectively
The truncation Δ Cap protein of identification, the overall length Cap protein either expressed, or removal nuclear localization signal is in SDS-PAGE
Purpose band position is correct, and illustration purpose albumen is succeeded expression.
4, the TCID of the recombinant baculovirus of 3 type Cap gene of pig circular ring virus is expressed50Measurement
The Sf9 cell that will be grown fine dispenses 96 orifice plates, will express 3 type Cap base of pig circular ring virus with Grace culture medium
The recombinant baculovirus liquid of cause carries out 10 times of doubling dilutions (10-1~10-10Dilution).It is inoculated with after 96 orifice plates in 37 DEG C, 5%CO2Training
It supports and continues to cultivate 96h after adsorbing 1.5 hours in case.Culture solution is discarded, PBS cleaning pats dry the fixed 20min of rear cold methanol -20, abandons
Fixer is removed, PBS is washed 3 times, each 5min, patted dry on blotting paper.The addition diluted pig source PCV3 positive serum of PBS (1:
500) 50 hole μ L/, 37 DEG C of incubation 1h, PBS ibid washings, pats dry.It is protected from light the sheep that the diluted FITC label of 1:200 of 30 μ L is added
Anti- pig fluorescence secondary antibody (IgG), is protected from light and is incubated for 45min, and PBS is ibid washed, patted dry.Carry out indirect immunofluorescence (IFA) observation, disease
Malicious TCID50It is calculated by Reed-Muench Er Shi method.
It is detected, expresses the TCID of the recombinant baculovirus of 3 type Cap gene of pig circular ring virus50It is 108.3/ mL or more is full
Foot prepares the requirement of vaccine.
5, transmission electron microscope observing
By be inoculated with recombinant baculovirus Sf9 cell and culture multigelation 3 times, in 4 DEG C, 4000rpm is centrifuged 20min
Remove cell fragment.Virus liquid is added on sucrose cushions, 4 DEG C, after ultracentrifugation, appropriate PBS, which suspends, to be precipitated.A drop weight is added dropwise
For suspension on copper mesh, 2% phosphotungstic acid carries out negative staining.It draws extra dye liquor and dries, transmission electron microscope observing virus-like particle is formed
Situation, as shown in figure 11: the uniform virus-like particle of form is observed after phosphotungstic acid negative staining under transmission electron microscope, it is rounded, directly
Diameter about 20nm.
6, animal immune experiment
6 week old BALB/c mouses 20 are only randomly divided into 4 groups, wherein the rBac-Cap (10 of 1 injection inactivation of group6.5TCID50/
Only), the rBac-Cap (10 of 2 injection inactivation of group7.5TCID50/ only), group 3 injects the wild-type baculovirus of inactivation with group 4 respectively
The Sf9 cells and supernatant and PBS of infection, each group carry out first immunisation after emulsifying completely with the Freund's complete adjuvant of equivalent,
Secondary booster immunization is carried out with after incomplete Freund's adjuvant emulsification after two weeks.Sinus under socket of the eye weekly is immunized after preceding and first immunisation to adopt
Blood separates and saves serum.
By the Cap protein coated elisa plate of the PCV3 removal nuclear localization signal of prokaryotic expression, 100 holes μ L/, 4 DEG C are wrapped overnight
Quilt, PBST wash 3 times, 6min/ times, pat dry on blotting paper；200 μ L confining liquids, 37 DEG C of closing 3h are added in every hole；Board-washing 3 times again
Afterwards, the mice serum through the appropriate doubling dilution of PBS is added, 100 holes μ L/, are incubated for 1h, while with mouse PCV3 positive blood by 37 DEG C
Make positive control clearly；The sheep anti-mouse igg secondary antibody marked with the PBS containing 4%FBS by 1:8000 dilution HRP, 100 holes μ L/, 37 DEG C,
It is incubated for 1h；200 μ L TMB developing solutions, 37 DEG C of colour developing 15min are added in every hole；50 μ L terminate liquids are added in each hole to terminate instead
It answers, then measures absorbance at 450nm with microplate reader.
It is as shown in figure 12 that indirect ELISA detects PCV3 specific antibody level result: head, which exempts from rear second week, can be detected
PCV3 specific antibody generate, third week antibody level rise rapidly, 4th week antibody level continues to rise, high dose immune group
Potency can reach 1:4000 or so.
<110>Yangzhou University
<120>3 type Cap protein of recombinant baculovirus expression pig circular ring virus and its construction method and primer
<160>6
SEQ ID NO.1
<210>1
<211>34
<212>DNA
<213>artificial sequence
<400>1
TTTCTCGAGA TTGGCGAAGA TTCCTCTTCG GGTA 34
SEQ ID NO.2
<210>2
<211>33
<212>DNA
<213>artificial sequence
<400>2
CCGGAATTCG TAATCCCCCT CTTTCTTGCA ATA 33
SEQ ID NO.3
<210>3
<211>31
<212>DNA
<213>artificial sequence
<400>3
CGCGGATCCC ATGAGACACA GAGCTATATT C 31
SEQ ID NO.4
<210>4
<211>31
<212>DNA
<213>artificial sequence
<400>4
CCCAAGCTTT TAGAGAACGG ACTTGTAACG A 31
SEQ ID NO.5
<210>5
<211>17
<212>DNA
<213>artificial sequence
<400>5
GTTTTCCCAG TCACGAC 17
SEQ ID NO.6
<210>6
<211>17
<212>DNA
<213>artificial sequence
<400>6
CAGGAAACAG CTATGAC 17
Claims (7)
1. 3 type Cap protein of recombinant baculovirus expression pig circular ring virus, deposit number is CGMCC No.15692.
2. the primer for constructing 3 type Cap protein of recombinant baculovirus expression pig circular ring virus, the primer are shown in Table 1:
Primer needed for table 1 and restriction enzyme site
The restriction enzyme site of primer Xho I-3F isCTCGAG；
The restriction enzyme site of primer EcoR I-3R isGAATTC；
The restriction enzyme site of primer Cap-F isGGATCC；
The restriction enzyme site of primer Cap-R isAAGCTT。
3. the construction method of 3 type Cap protein of recombinant baculovirus expression pig circular ring virus described in claim 1, characterized in that
The construction method the following steps are included:
1, building includes the pSK-sPCV3 of PCV3 full-length genome:
Using PCV3 DNA as template, it is complete that PCV3 is amplified with primer Xho I-3F described in claim 1 and primer EcoR I-3R
Genome；Xho I and EcoR I double digestion will be used simultaneously with pBluescript SK+ carrier after the recycling of PCV3 full-length genome glue,
It will be attached after two kinds of digestion products purification and recoveries, finally obtain recombinant plasmid pSK-sPCV3；
2, the building of transfer vector:
Using pSK-sPCV3 recombinant plasmid as template, PCV3 is expanded with primer Cap-F described in claim 1 and primer Cap-R
Cap overall length, by PCR product after purification with transfer vector pFastBacTMHT A carries out double enzymes of BamH I and Hind III jointly
It cuts；It carries out conventional connection reaction after purification again, obtains recombinant plasmid pFastBacTMHT A-Cap；Extract recombinant plasmid
pFastBacTMHT A-Cap is utilized respectively the identification of III single endonuclease digestion of restriction enzyme BamH I and Hind and BamH I-
The double digestion of Hind III is identified；Digestion products are subjected to electrophoresis with 0.7% Ago-Gel, BamH I and Hind III distinguishes
Single endonuclease digestion pFast BacTMHT A-Cap plasmid then obtains the single band of 5501bp；III double digestion recombinant plasmid of BamH I-Hind
pFastBacTMHT A-Cap then obtains two bands of 4856bp and 645bp；
3, the building of shuttle plasmid:
III double digestion of BamH I-Hind is identified to obtain the correct recombinant vector of two bands of 4856bp and 645bp
pFastBacTMHT A-Cap conversion enters DH10 α Bac competent cell；Blue hickie screens swivel base successfully white single colonie,
After PCR identification is correct, recombinant shuttle plasmid Bacmid-Cap is extracted；2430bp is amplified plus insertion mesh with primer pUC/M13
Clip size band, i.e. 3075bp band；Recombination Bacmid DNA ideograph is identified according to PCR, thus it is speculated that calculates pUC/M13
Institute's amplified band size is applied in combination it is found that with primer pUC/M13F+CapR in upstream and downstream primer and target fragment upstream and downstream primer
Amplify the 2500bp purpose band of Bacmid-Cap recombinant shuttle plasmid；Similarly, it is amplified with primer Cap F+pUC/M13R
1000bp purpose band；645bp purpose band is amplified respectively with primer Cap F/R；
4, the acquisition of recombinant baculovirus:
It will identify correct recombinant shuttle plasmid Bacmid-Cap transfection Sf9 cell, and obtain expression 3 type Cap base of pig circular ring virus
The recombinant baculovirus rBac-Cap of cause.
4. the construction method of 3 type Cap protein of recombinant baculovirus expression pig circular ring virus according to claim 3, special
Sign is Xho I and EcoR I double digestion to be used simultaneously with pBluescript SK+ carrier after the recycling of PCV3 full-length genome glue, by two
It is attached after kind digestion products purification and recovery by the molar ratio of 3:1.
5. the construction method of 3 type Cap protein of recombinant baculovirus expression pig circular ring virus according to claim 3, special
Sign is, identifies recombinant plasmid pSK-sPCV3 by Xho I and EcoR I double digestion, digestion products with 0.7% Ago-Gel
Electrophoresis is carried out, the band that size is respectively 2958bp and 2000bp is obtained.
6. the construction method of 3 type Cap protein of recombinant baculovirus expression pig circular ring virus according to claim 3, special
Sign is to extract recombinant plasmid pFastBacTMHT A-Cap is carried out using Axygen small amount plasmid DNA extraction kit.
7. the construction method of 3 type Cap protein of recombinant baculovirus expression pig circular ring virus according to claim 3, special
Sign is that the identification identification of PCR described in step 4 is said with reference to Invirtogen company Bac-to-Bac baculovirus expression system
Bright book carries out.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110438156A (en) * | 2019-08-19 | 2019-11-12 | 军事科学院军事医学研究院军事兽医研究所 | Recombinate rod-shaped plasmid and its application in expression PCV3 Cap protein, vaccine |
| WO2020206452A1 (en) | 2019-04-04 | 2020-10-08 | Boehringer Ingelheim Animal Health USA Inc. | Porcine circovirus type 3 (pcv3) vaccines, and production and uses thereof |
| CN112326963A (en) * | 2020-11-09 | 2021-02-05 | 扬州大学 | Application of eukaryotic expression influenza A virus PB2cap protein |
| CN114014916A (en) * | 2019-01-18 | 2022-02-08 | 南京农业大学 | Porcine circovirus type 3Cap recombinant protein, coding gene thereof and application thereof in ELISA antibody detection |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991001332A1 (en) * | 1989-07-21 | 1991-02-07 | The Upjohn Company | Feline calicivirus capsid protein and nucleotide sequence |
| CN102839195A (en) * | 2012-07-18 | 2012-12-26 | 华南农业大学 | Method for expression of PCV 2 Cap protein by pFast Bac Dual baculovirus |
| WO2017066772A1 (en) * | 2015-10-16 | 2017-04-20 | Kansas State University Research Foundation | Porcine circovirus type 3 immunogenic compositions and methods of making and using the same |
| CN107854688A (en) * | 2017-11-06 | 2018-03-30 | 陕西诺威利华生物科技有限公司 | Porcine circovirus 2 type and the type bivalent inactivated vaccine of pig circular ring virus 3 and preparation method thereof |
| CN108359677A (en) * | 2018-02-01 | 2018-08-03 | 福建农林大学 | The method for improving porcine circovirus 2 type and 3 type Cap protein expressing in series efficiency |
-
2018
- 2018-08-12 CN CN201810912587.1A patent/CN109207441B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991001332A1 (en) * | 1989-07-21 | 1991-02-07 | The Upjohn Company | Feline calicivirus capsid protein and nucleotide sequence |
| CN102839195A (en) * | 2012-07-18 | 2012-12-26 | 华南农业大学 | Method for expression of PCV 2 Cap protein by pFast Bac Dual baculovirus |
| WO2017066772A1 (en) * | 2015-10-16 | 2017-04-20 | Kansas State University Research Foundation | Porcine circovirus type 3 immunogenic compositions and methods of making and using the same |
| CN107854688A (en) * | 2017-11-06 | 2018-03-30 | 陕西诺威利华生物科技有限公司 | Porcine circovirus 2 type and the type bivalent inactivated vaccine of pig circular ring virus 3 and preparation method thereof |
| CN108359677A (en) * | 2018-02-01 | 2018-08-03 | 福建农林大学 | The method for improving porcine circovirus 2 type and 3 type Cap protein expressing in series efficiency |
Non-Patent Citations (2)
| Title |
|---|
| 王旻: "《生物工程》", 31 August 2009 * |
| 赵爱春: "《家蚕转基因技术及应用》", 31 December 2017 * |
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| CN114014916A (en) * | 2019-01-18 | 2022-02-08 | 南京农业大学 | Porcine circovirus type 3Cap recombinant protein, coding gene thereof and application thereof in ELISA antibody detection |
| CN114014916B (en) * | 2019-01-18 | 2023-04-25 | 南京农业大学 | Porcine circovirus 3 type Cap recombinant protein, encoding gene thereof and application thereof in ELISA antibody detection |
| WO2020206452A1 (en) | 2019-04-04 | 2020-10-08 | Boehringer Ingelheim Animal Health USA Inc. | Porcine circovirus type 3 (pcv3) vaccines, and production and uses thereof |
| US11701419B2 (en) | 2019-04-04 | 2023-07-18 | Boehringer Ingelheim Animal Health USA Inc. | Porcine Circovirus Type 3 (PCV3) vaccines, and production and uses thereof |
| US11896659B2 (en) | 2019-04-04 | 2024-02-13 | Boehringer Ingelheim Animal Health USA Inc. | Porcine Circovirus Type 3 (PCV3) vaccines, and production and uses thereof |
| CN110438156A (en) * | 2019-08-19 | 2019-11-12 | 军事科学院军事医学研究院军事兽医研究所 | Recombinate rod-shaped plasmid and its application in expression PCV3 Cap protein, vaccine |
| CN112326963A (en) * | 2020-11-09 | 2021-02-05 | 扬州大学 | Application of eukaryotic expression influenza A virus PB2cap protein |
| CN112326963B (en) * | 2020-11-09 | 2024-02-06 | 扬州大学 | Application of eukaryotic expression type A influenza virus PB2cap protein |
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