CN106349348A - Preparation method of GyV7 ring virus VP3 protein - Google Patents
Preparation method of GyV7 ring virus VP3 protein Download PDFInfo
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Abstract
The invention provides a preparation method of GyV7 ring virus VP3 protein. In the method, based on a recombinase Exnase TM II, a linearized pGEX-6p-1 carrier and a PCR product of GyV7 virus VP3 gene segment are directly treated by a recombinant cloning technology without an enzyme-cut link up reaction and then transformed into escherichia coli to realize expression of the VP3 protein, wherein the nucleotide sequences of the upstream and downstream primers applied to the PCR amplification of GyV7 virus VP3 gene are shown by SEQ ID No.3 and SEQ ID No.4 respectively. The method is easy to operate and quick and efficient; the obtained protein expressed and purified by the GyV7 virus VP3 can directly supply VP3 protein as a GyV7 diagnosis antigen; the VP3 protein serves as an immunogen to obtain an anti-VP3 polyclonal antibody; an effective diagnosis reagent is provided for developing GyV7 epidemiological investigation; and the VP3 protein is of great significance to further study of the biological functions of VP3.
Description
Technical field
The present invention relates to a kind of preparation method of gyv7 annular virus vp3 albumen.
Background technology
Chicken Anemia Virus (CAV) (chicken infectious anemia virus, cav) is considered as annulus always
Annular Tobamovirus (gyrovirus) unique member in Viraceae (circoviridae).Until 2011, rijsewijk etc.
(rijsewijk fa,dos santos hf,teixeira tf,cibulski sp,varela ap,dezen d,et
al.discovery of a genome of a distant relative of chicken anemia virus
reveals a new member of the genus gyrovirus.archives of virology.2011jun；156(6):
1097-100), New Ring-like Type virus sequence is detected from the blood serum sample of morbidity chicken, i.e. second member in annular Tobamovirus,
It is named as agv2.The same year, (sauvage v, cheval j, foulongne v, the gouilh ma, pariente such as sauvage
k,manuguerra jc,et al.identification of the first human gyrovirus,a virus
related to chicken anemia virus.journal of virology.2011aug；85 (15): 7948-50) exist
The first people source with agv2 very high homology annular virus hgyv sequence is detected in the skin cotton test agent of Healthy People.From 2012
Year, other New Ring-like Type viruses include gyv3, and gyv4, gyv5, gyv6, gyv7, gyv8 and gyv9 are found to identify successively, and have
There is potential public health meaning.However, there is no the serological method of detection gyv7 antigen and its antibody at present.Therefore, right
Gyv7 virus early expression albumen vp3 gene is cloned in vitro, builds vp3 expression vector, realizes its expression, will be deep
Carry out gyv7 proteantigen and its antibody test, serosurvey, specify infection duplication feelings in chicken group and crowd for the gyv7
Condition provides effective diagnostic reagent；And it is significant for probing into gyv7vp3 biological function.Structure in traditional expression vector
In, generally require design alternative restriction enzyme digestion sites, by the method carrier construction of enzyme action, connection, realize external source
The expression of gene.But sometimes due to can not find suitable restriction enzyme site, it is loaded down with trivial details to often lead to cloning procedure, inefficiency.
Content of the invention
The purpose of the present invention is to be to provide a kind of simple to operate, efficiency high, quick gyv7 annular virus vp3 albumen table
Reach the preparation method of carrier, and realize the expression of vp3 albumen.The principle of the present invention and most crucial key technology are: scientifically
Design amplifies pgex-6p-1 linearized vector and the primer of gyv7 virus vp3 genetic fragment, using the recombinase of commercialization
Without enzyme action coupled reaction, directly Quick Casting clones vp3 to exnasetm ii in vitro, converts escherichia coli, through iptg induction
Express, realize the vp3 albumen of gyv7 and the amalgamation and expression of gst, and obtain the vp3 fusion protein of purification.
To achieve these goals, technical scheme is specific as follows:
The invention provides a kind of primer expanding pgex-6p-1 linearized vector for pcr, its nucleotides sequence is classified as:
Forward primer 1:5 '-taatgacggtgaaaacctctgacacatgc-3 '；(seq id no.1)
Downstream primer 1:5 '-catgggcccctggaacagaacttccagat-3 '.(seq id no.2)
Amplification pgex-6p-1 linearized vector forward primer is located at pgex-6p-1 plasmid 1022-1047 position；And at 5 ' ends
With extra taa base；Amplification pgex-6p-1 linearized vector downstream primer is located at pgex-6p-1 plasmid 916-941 position；And
5, ' end carries extra cat base.
The invention provides a kind of primer expanding gyv7 virus vp3 gene for pcr, its nucleotides sequence is classified as:
Forward primer 2:5 '-gttccaggggcccatggcacacctatacac-3 '；(seq id no.3)
Downstream primer 2:5 '-gttttcaccgtcattacagtcttgcggcgt-3 '.(seq id no.4)
Amplification gyv7 virus vp3 upstream region of gene primer includes vp3 gene initiation codon atg and 14 bases thereafter, and 5
' end is with 16 and pgex-6p-1 linearized vector downstream primer reverse complemental base；Amplification gyv7 virus vp3 downstream of gene
Primer includes vp3 gene end password taa and its front 14 bases, and ' end is carried with pgex-6p-1 linearisation with 16 5
Body forward primer reverse complemental base.
Present invention also offers a kind of gyv7 annular virus vp3 protein preparation method, the method is based on recombinase
Linearizing pgex-6p-1 carrier is connected instead without enzyme action by exnasetm ii with gyv7 virus vp3 genetic fragment pcr product
Should, direct recombinant clone technology；And it is transformed into escherichia coli, realize vp3 protein expression；Wherein, for pcr amplification gyv7 virus
The primer of vp3 gene is divided into upstream and downstream primer, the nucleotide sequence of described forward primer as shown in seq id no.3, described under
The nucleotide sequence of trip primer is as shown in seq id no.4.
It specifically includes following steps:
1) pcr amplification pgex-6p-1 linearized vector and gyv7 virus vp3 genetic fragment: with pgex-6p-1 plasmid with
And gyv7 virus vp3 gene be template, using above-mentioned primer, pcr amplify respectively linearized vector containing pgex-6p-1 and
Gyv7 virus vp3 genetic fragment (accompanying drawing 1, step 1)；
2) gyv7 virus vp3 fragment quick clone is entered pgex-6p-1 carrier: carry out using recombinase exnasetm ii
Quick Casting clones (accompanying drawing 1, step 2)；
3) positive colony is transformed into escherichia coli, realizes vp3 protein expression (accompanying drawing 1, step 3).
Present invention also offers above-mentioned gyv7 annular virus vp3 protein preparation method, and above-mentioned primer is in preparation
Gyv7 diagnostic antigen, anti-vp3 polyclonal antibody and the application preparing gyv7 epidemiology efficient diagnosis reagent aspect.
Technical scheme has reached following beneficial effect:
1) in the present invention, external using the recombinase exnasetm ii not relying on restriction enzyme site and restricted enzyme
Recombinant technique clone's gyv7 virus vp3 gene, simplifies cloning procedure, realizes the expression of vp3 gene quick clone；
2) primer of this invention design and the Strategies For The Cloning based on recombinase exnasetm ii, can rapid build gyv7 disease
The prokaryotic expression carrier of malicious vp3 gene.
3) present invention obtains gyv7 virus vp3 expression and the albumen of purification, can directly provide vp3 albumen to examine as gyv7
Disconnected antigen；Obtain anti-vp3 polyclonal antibody as immunogen；There is provided effective diagnostic reagent for carrying out gyv7 Epidemiological study；
And it is significant for probing into vp3 biological function further.
4) present invention will obtain gyv7 annular virus vp3 protein expression vector, realize table in escherichia coli for the vp3 gene
Reach, and obtain the vp3 fusion protein of purification.The vp3 fusion protein of this expression vector establishment and purification will be for setting up detection
Gyv7 antigen and the serological diagnostic method of antibody, probing into vp3 biological function provides effective immunological reagent, fills up domestic
Outer blank.Therefore, the present invention has certain using value.
Brief description
Fig. 1 is a kind of gyv7 annular virus vp3 protein preparation method strategy.Step 1: with the gyv7 virus of synthetic
Vp3 gene is template, and using primer in table 1, pcr amplifies linearized vector containing pgex-6p-1 and gyv7 virus vp3 respectively
Genetic fragment.Step 2: carry out Quick Casting clone using recombinase exnasetm ii.Step 3: positive colony is transformed into large intestine
Bacillus, realizes vp3 protein expression.
Fig. 2 is pcr amplification gyv7 virus vp3 fragment.Swimming lane 1, gyv7 virus vp3 fragment pcr product；Swimming lane m, dna marker.
Fig. 3 is pcr amplification linearized vector pgex-6p-1.Swimming lane 1, the pcr product of linearized vector pgex-6p-1；Swimming
Road m, dna marker.
Fig. 4 is the expression that sds-page analyzes gyv7 virus vp3 gene.Swimming lane m, albumen marker；Swimming lane 1,2 generation respectively
The supernatant of the ultrasonic degradation pgex-6p-1 sample that table induces through iptg, precipitation；Swimming lane 3,4,5 represents respectively through iptg induction
Ultrasonic degradation sample vp3 supernatant, precipitation and after purification vp3 albumen.
Fig. 5 is that western blot identifies anti-gyv7vp3 protein polyclone antibody.1, transfection egfp-vp3 expression plasmid
293t cell lysate；2, it is the 293t cell lysate of transfection control egfp expression plasmid.
Specific embodiment
In order to illustrate technical scheme and technical purpose, below in conjunction with the accompanying drawings and specific embodiment is to the present invention
It is described further.
Embodiment 1:
1) design amplification linearized vector containing pgex-6p-1 and gyv7 virus vp3 genetic fragment primer:
Amplification pgex-6p-1 linearized vector forward primer is located at pgex-6p-1 plasmid 1022-1047 position；And at 5 ' ends
With extra taa base；Amplification pgex-6p-1 linearized vector downstream primer is located at pgex-6p-1 plasmid 916-941 position；And
Carry extra cat base at 5 ' ends.Amplification gyv7 virus vp3 upstream region of gene primer includes vp3 gene initiation codon atg and thereafter
14 bases, and at 5 ' ends with 16 and pgex-6p-1 linearized vector downstream primer reverse complemental base；Amplification gyv7 disease
Malicious vp3 downstream of gene primer includes vp3 gene end password taa and its front 14 bases, and carries 16 and pgex- at 5 ' ends
6p-1 linearized vector forward primer reverse complemental base.Concrete primer sequence is shown in Table 1, by life technologies Shanghai
Thermo Fischer Scient Inc. synthesizes.
Table 1: amplification pgex-6p-1 linearized vector and gyv7 virus vp3 fragment primer design:
2) pgex-6p-1 linearized vector and gyv7 virus vp3 genetic fragment pcr amplification:
With the gyv7vp3 gene of pgex-6p-1 plasmid and synthesis as masterplate, described in table 1, primer carries out pcr expansion for primer
Increase.As the step 1 in Fig. 1.Pcr amplification reaction system is: 40 μ l water, 5 10 times of μ l buffer, 1 μ l 10mm dntp, 1 μ l
10 μm of ol forward primer, 1 10 μm of μ l ol downstream primer, the pgex-6p-1 plasmid of 1 μ l 10ng/ μ l or pcgyv7-vp3 plasmid,
The phanta super-fidelity dna polymerase of 1 μ l commercialization.Pcr amplified reaction loop parameter is: 5 points of 94 DEG C of degeneration
Clock, subsequently carries out 30 circulations (94 DEG C of degeneration 30 seconds, anneal 30 seconds for 58 DEG C, and 72 DEG C extend 3 minutes), last 72 DEG C extend 10 points
Clock.After pcr terminates, pcr product carries out electrophoresis in 1% agarose gel.As shown in Fig. 2 wherein swimming lane m is dna
Marker, wherein swimming lane 1 are gyv7 virus vp3 fragment pcr product.As shown in figure 3, wherein swimming lane m is dna marker, swimming lane
1 is the pcr product of linearized vector pgex-6p-1.
3) gyv7 virus vp3 fragment quick clone enters pgex-6p-1 carrier:
The expression linearized vector pgex-6p-1 and gyv7 virus vp3 fragment pcr product of above purification is recombinated in commercialization
Carry out recombinant clone in the presence of enzyme exnasetm ii.As the step 2 in Fig. 1.Concrete recombining reaction system is as follows: the gyv7 of purification
Viral vp3 fragment products 50-100ng, pgex-6p-1 expression the linearized vector 50ng, the exnasetm ii of 2 μ l commercializations of purification
Enzyme, the buffer of 45 times of μ l, other benefit adds water to 20 μ l.Reactant, after 37 DEG C of effects 30 minutes, puts 5 minutes on ice.Subsequently will
20 μ l reactants are transformed into conventional competence antibacterial, apply lb plate.Next day picking bacterial clone carries out plasmid preparation, positive clone identification.
Embodiment 2:
The protein induced expression of gyv7 virus vp3 and its purification: the positive colony (life containing gyv7 virus vp3 gene will be obtained
Entitled pgex-vp3) conversion bl21 antibacterial, collect antibacterial after iptg induction (0.1mmol/ml), carry out ultrasonic (40 hertz) and break
Broken.After ultrasonication sample is centrifuged point supernatant and precipitation carry out sds-page (5% concentration glue, 10% separation gel) and
Western blot analysis (anti-for one with the gst monoclonal antibody in anti-Mus source, the igg of sheep anti mouse hrp labelling resists for two) identification expression.
It specifically comprises the following steps that
1) the gyv7 virus protein induced expression of vp3:
The positive colony (being named as pgex-vp3) obtaining containing gyv7 virus vp3 gene is transformed into after bl21 antibacterial,
Turn and be inoculated in the lb fluid medium that 3ml contains amp (100 μ g/ml), 37 DEG C of 250rpm shake overnight incubation.Next day is turned by 1:100
It is inoculated in lb culture medium, 37 DEG C of 1500rpm shake 2.5h, with final concentration of 37 DEG C of inductions of 1mm iptg.By bacterium solution after induction 5h
It is transferred in centrifuge tube, 6000rpm is centrifuged 5min, removes supernatant, be washed once with pbs, 600 μ l sterilizing ddw suspended bacterial, and
Ultrasonic treatment, 30hz, 10min are carried out on ice bath.10000rpm is centrifuged 5min, takes supernatant, precipitates with 300 μ l sterilizing ddw
Resuspended, -20 DEG C frozen standby.Take 30 μ l bacteria lysis supernatant precipitations respectively, add 5 μ l 6 × sds Gel Loading buffer
Mix, boil 5min；Expression for sds-page (5% concentration glue, 10% separation gel) electrophoretic analysiss recombiant protein and
Its expression-form.In the diagram, vp3 albumen can be existed with soluble form in ultrasonication Sample supernatants.
2) purification of gyv7 virus vp3 albumen:
On the basis of the solubility expression determining vp3, ultrasonication Sample supernatants are carried out vp3 albumen by gst purification column
Purification.Briefly step is as follows: adds the ddw of 5 volumes to remove ethanol in purification column；Combination buffer with 10 volumes
(140mm nacl, 2.7mm kcl, 10mm na2hpo4,1.8mm kh2po4) balances pillar；By protein sample 5-10ml/ every time, weight
Answered post twice, and so that albumen is fully combined on post.With the foreign protein in the combination buffer column scrubber of 10 volumes；With 5 volumes
Eluting buffer (50mm tris-hcl, 10mm reductive glutathione) eluting destination protein, be subsequently added in bag filter with 1 ×
Pbs solution is fully dialysed, and after subpackage, preserves at -70 DEG C；Take albumen to be after purification proportionally added into 5 × loading buffer, boil sample
After 5min, use sds-page electrophoretic analysiss.Result shows that vp3 albumen obtains good purification (as shown in swimming lane 5 in Fig. 4).
3) immunoreactivity of gyv7 virus vp3 albumen:
By the vp3 albumen peritoneal immunity 6 week old balb/c mice of purification, immunity three times, every minor tick 14 days, dosage is 50 μ
G//time.Mix with isopyknic complete Freund's adjuvant when head exempts to Water-In-Oil shape, again during immunity with isopyknic not exclusively not
Family name's adjuvant mixes, and immunity afterwards is not added with adjuvant.Three exempt from after gather mice serum, in 293t cell expression vp3 albumen conduct
Antigen carries out anti-vp3 specific antibody in western blot analysis serum.Result shows the anti-gyv7 virus vp3 albumen of acquisition
Mice multi-resistance can carry out specific reaction (Fig. 5) with the vp3 albumen of expression in 293t cell.This result shows present invention expression
Vp3 albumen has reactivity and immunogenicity well, will have a good application prospect in gyv7 serodiagnosiss.
Ultimate principle, principal character and the advantages of the present invention of the present invention have been shown and described above.The technology of the industry
, it should be appreciated that the present invention is not restricted to the described embodiments, the simply explanation described in above-described embodiment and description is originally for personnel
The principle of invention, without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, the present invention
Claimed scope is by appending claims, description and its equivalent thereof.
sequence listing
<110>Yangzhou University
<120>a kind of gyv7 annular virus vp3 protein preparation method
<160> 4
<170> patentin version 3.5
<210> 1
<211> 29
<212> dna
<213>artificial primer sequence
<400> 1
taatgacggt gaaaacctct gacacatgc 29
<210> 2
<211> 29
<212> dna
<213>artificial primer sequence
<400> 2
catgggcccc tggaacagaa cttccagat 29
<210> 3
<211> 30
<212> dna
<213>artificial primer sequence
<400> 3
gttccagggg cccatggcac acctatacac 30
<210> 4
<211> 30
<212> dna
<213>artificial primer sequence
<400> 4
gttttcaccg tcattacagt cttgcggcgt 30
Claims (6)
1. a kind of gyv7 annular virus vp3 protein preparation method is it is characterised in that the method is based on recombinase exnasetm ii
By linearizing pgex-6p-1 carrier with gyv7 virus vp3 genetic fragment pcr product without enzyme action coupled reaction, directly recombinate
Clone technology；And it is transformed into escherichia coli, realize vp3 protein expression；Wherein, expand drawing of gyv7 virus vp3 gene for pcr
Thing is divided into upstream and downstream primer, the nucleotide sequence of described forward primer as shown in seq id no.3, the core of described downstream primer
Nucleotide sequence is as shown in seq id no.4.
2. as claimed in claim 1 a kind of gyv7 annular virus vp3 protein preparation method it is characterised in that specifically include as
Lower step:
1) pcr amplification pgex-6p-1 linearized vector and gyv7 virus vp3 genetic fragment: with pgex-6p-1 plasmid and
Gyv7 virus vp3 gene is template, designs primer, and pcr amplifies linearized vector containing pgex-6p-1 and gyv7 virus respectively
Vp3 genetic fragment；Wherein, the primer expanding gyv7 virus vp3 gene for pcr is divided into upstream and downstream primer, described forward primer
Nucleotide sequence as shown in seq id no.3, the nucleotide sequence of described downstream primer is as shown in seq id no.4；
2) gyv7 virus vp3 fragment quick clone is entered pgex-6p-1 carrier: carry out quickly using recombinase exnasetm ii
Recombinant clone；
3) positive colony is transformed into escherichia coli, realizes vp3 protein expression.
3. as claimed in claim 2 a kind of gyv7 annular virus vp3 protein preparation method it is characterised in that step 1) in institute
The primer expanding pgex-6p-1 linearized vector for pcr stated is divided into upstream and downstream primer, the nucleotide of described forward primer
, as shown in seq id no.1, the nucleotide sequence of described downstream primer is as shown in seq id no.2 for sequence.
4. a kind of expand the primer of pgex-6p-1 linearized vector it is characterised in that the nucleoside of described forward primer for pcr
, as shown in seq id no.1, the nucleotide sequence of described downstream primer is as shown in seq id no.2 for acid sequence.
5. a kind of expand the primer of gyv7 virus vp3 gene it is characterised in that the nucleotides sequence of described forward primer for pcr
, as shown in seq id no.3, the nucleotide sequence of described downstream primer is as shown in seq id no.4 for row.
6. a kind of gyv7 annular virus vp3 protein preparation method as described in any one in claim 1-3, and as right
The primer described in 4 or 5 is required to have in preparation gyv7 diagnostic antigen, anti-vp3 polyclonal antibody and preparation gyv7 epidemiology
The application of effect diagnostic reagent aspect.
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| CN106995486A (en) * | 2017-04-17 | 2017-08-01 | 扬州大学 | A kind of preparation method of the annular virus VP 3 soluble protein of GyV8 types |
| CN108929881A (en) * | 2018-08-06 | 2018-12-04 | 扬州大学 | A kind of expression vector and its construction method and protein expression of the avian leukosis virus gp37 albumen lacking transmembrane domains |
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| CN104195116A (en) * | 2014-08-13 | 2014-12-10 | 吉林大学 | Recombinant Newcastle disease virus for expressing goose parvovirus VP3 genes and construction method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106995486A (en) * | 2017-04-17 | 2017-08-01 | 扬州大学 | A kind of preparation method of the annular virus VP 3 soluble protein of GyV8 types |
| CN108929881A (en) * | 2018-08-06 | 2018-12-04 | 扬州大学 | A kind of expression vector and its construction method and protein expression of the avian leukosis virus gp37 albumen lacking transmembrane domains |
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