CN106565829A - Preparation method of GyV9 circovirus VP3 protein - Google Patents
Preparation method of GyV9 circovirus VP3 protein Download PDFInfo
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Abstract
The invention provides a preparation method of a GyV9 circovirus VP3 protein. The preparation method comprises the steps of directly performing a recombinant cloning technology on a PCR product of a linear pGEX-6p-1 vector and a GyV9 virus VP3 gene segment based on a recombinase ExnaseTM II, wherein the PCR product is not subjected to an enzyme digestion connection reaction; and changing the PCR product into escherichia coli to realize VP3 protein expression, wherein when the GyV9 virus VP3 gene segment is amplified through PCR, the nucleotide sequence of the used primer is as follows: the upstream primer 1 is 5'-GTTCCAGGGGCCCATGGATGCGACGGGCAC-3', and the downstream primer 2 is 5'-GTTTTCACCGTCATTACAATCTTGCGCCTT-3'. The preparation method simplifies the cloning process, can build a prokaryotic expression vector of a GyV9 virus VP3 gene rapidly, and can realize rapid cloning expression of the VP3 gene. The GyV9 virus VP3 expressed and purified protein obtained by the invention can directly provide a VP3 protein to serve as a GyV9 diagnostic antigen, can serve as an immunizing antigen to obtain an anti-VP3 polyclonal antibody, can provide an effective diagnostic reagents for developing GyV9 epidemiological investigation and is of great significance in further investigation of VP3 biological functions.
Description
Technical field
The present invention relates to a kind of preparation method of GyV9 annulars virus VP 3 albumen.
Background technology
CAV (Chicken Infectious Anemia Virus, CAV) is considered as always annulus
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 in annular Tobamovirus
Member, is named as AGV2.The same year, Sauvage etc. (Sauvage V, Cheval J, Foulongne V, Gouilh MA,
Pariente K,Manuguerra JC,et al.Identification of the first human gyrovirus,a
virus related to chicken anemia virus.Journal of virology.2011Aug；85(15):
The first people source annular virus HGyV sequences with AGV2 very high homologies 7948-50) are detected in the skin cotton test agent of Healthy People
Row.From 2012, other New Ring-like Type viruses included GyV3, and GyV4, GyV5, GyV6, GyV7, GyV8 and GyV9 are had found successively
Identification, and with potential public health meaning.The serological method of detection GyV9 antigens and its antibody is so there is no at present.Cause
This, clones in vitro to GyV9 virus early expression VP 3 Genes, builds VP3 expression vectors, realizes its expression, will
To carry out GyV9 proteantigens and its antibody test, serosurvey in a deep going way, infection of the GyV9 in chicken group and crowd is specified
Duplication situation provides effective diagnostic reagent；And it is significant to probe into GyV9VP3 biological functions.In traditional expression vector
Structure in, generally require design alternative restriction enzyme digestion sites, it is real by digestion, the method carrier construction of connection
The expression of existing foreign gene.But sometimes due to can not find suitable restriction enzyme site, often lead to that cloning procedure is loaded down with trivial details, efficiency is low
Under.
The content of the invention
It is to provide a kind of simple to operate, efficiency high, quick GyV9 annular virus VP 3 albumen tables that the purpose of the present invention is
Up to the preparation method of carrier, and realize the expression of VP3 albumen.The principle and most crucial key technology of the present invention is scientifically to set
Meter amplifies the primer of GyV9 virus VP 3 gene fragments, and the recombinase ExnaseTM II using commercialization are anti-without digestion connection
Should, direct Quick Casting clone VP3 in vitro converts Escherichia coli, Jing IPTG abduction deliverings, realize the VP3 albumen of GyV9 with
The amalgamation and expression of GST, and obtain the VP3 fusion proteins of purifying.
To achieve these goals, technical scheme is specific as follows：
The invention provides a kind of PCR expands GyV9 virus VP 3 gene primers, its nucleotides sequence is classified as：
Upstream primer 1：5’-GTTCCAGGGGCCCATGGATGCGACGGGCAC-3’；
Downstream primer 1：5’-GTTTTCACCGTCATTACAATCTTGCGCCTT-3’.
Present invention also offers a kind of GyV9 annulars virus VP 3 protein preparation method, the method is based on recombinase
ExnaseTM II are connected linearizing pGEX-6p-1 carriers instead without digestion with GyV9 virus VP 3 gene fragment PCR products
Should, direct recombinant clone technology；And Escherichia coli are transformed into, realize VP3 protein expressions；Wherein, the described GyV9 of PCR amplifications is sick
The nucleotide sequence of the primer is as noted above during malicious VP3 genetic fragments.
It specifically includes following steps：
1) PCR amplification pGEX-6p-1 linearized vectors and GyV9 virus VP 3 gene fragments：With pGEX-6p-1 plasmids with
And GyV9 virus VP 3 genes are template, design primer, PCR amplifies respectively linearized vector containing pGEX-6p-1 and GyV9 is sick
Malicious VP3 genetic fragments；Wherein, the nucleotide sequence of the primer is as above during PCR amplifications described GyV9 virus VP 3 gene fragments
Shown in stating；Preferably, the nucleotide sequence of PCR amplifications pGEX-6p-1 linearized vector primers is as follows：
Upstream primer 2：5’-TAATGACGGTGAAAACCTCTGACACATGC-3’；
Downstream primer 2：5’-CATGGGCCCCTGGAACAGAACTTCCAGAT-3’.
2) GyV9 virus VP 3 fragment quick clones are entered into pGEX-6p-1 carriers：Carried out using recombinase ExnaseTM II
Quick Casting is cloned；
3) positive colony is transformed into Escherichia coli, realizes VP3 protein expressions.
Present invention also offers above-mentioned GyV9 annulars virus VP 3 protein preparation method, and above-mentioned primer is in preparation
GyV9 diagnostic antigens, anti-VP3 polyclonal antibodies and prepare application in terms of GyV9 epidemiology efficient diagnosis reagents.
Technical scheme has reached following beneficial effect：
The primer and the Strategies For The Cloning based on recombinase ExnaseTM II of the invention design, simplifies cloning procedure, can be quick
The prokaryotic expression carrier of GyV9 virus VP 3 genes is built, realizes that VP3 genes quick clone is expressed.Using the method for the present invention, will
GyV9 annular virus VP 3 protein expression vectors are obtained, expression of the VP3 genes in Escherichia coli is realized, and obtains the VP3 of purifying
Fusion protein.The expression of GyV9 virus VP 3s and the albumen of purifying that the present invention is obtained, can directly provide VP3 albumen and examine as GyV9
Disconnected antigen；Anti- VP3 polyclonal antibodies are obtained as immunogene；Effective diagnostic reagent is provided to carry out GyV9 epidemiology surveys；
And it is significant further to probe into VP3 biological functions.The VP3 fusion proteins of this expression vector establishment and purifying will
To set up the serological diagnostic method of detection GyV9 antigens and antibody, probe into VP3 biological functions and effective immunology examination is provided
Agent, fills up domestic and international blank.Therefore, the present invention has certain using value.
Description of the drawings
Fig. 1 is a kind of GyV9 annulars virus VP 3 protein preparation method strategy.Step 1：It is viral with artificial synthesized GyV9
VP3 genes are template, and using primer in table 1, PCR amplifies respectively linearized vector containing pGEX-6p-1 and GyV9 virus VP 3s
Genetic fragment.Step 2：Quick Casting clone is carried out using recombinase ExnaseTM II.Step 3：Positive colony is transformed into large intestine
Bacillus, realizes VP3 protein expressions.
Fig. 2 is PCR amplification GyV9 virus VP 3 fragments.Swimming lane 1, GyV9 virus VP 3 fragment PCR products；Swimming lane M, DNA
Marker。
Fig. 3 is PCR amplification linearized vector pGEX-6p-1.Swimming lane 1, the PCR primer of linearized vector pGEX-6p-1；Swimming
Road M, DNA Marker.
Fig. 4 is the expression that SDS-PAGE analyzes GyV9 virus VP 3 genes.Swimming lane 1, VP3 ultrasonic degradation supernatants；Swimming lane 2,
VP3 ultrasonic degradations are precipitated；Swimming lane 4, the VP3 albumen of purifying；Swimming lane 5, GST ultrasonic degradation supernatants；Swimming lane 6, GST ultrasonic degradations sink
Form sediment；Swimming lane M, albumen Marker.
Fig. 5 is that Western blot identify anti-GyV9VP3 protein polyclone antibodies.1, transfect EGFP-VP3 expression plasmids
293T cell lysates；2, it is the 293T cell lysates of transfection control EGFP expression plasmids.
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 GyV9 virus VP 3 gene fragment primers：
Table 1:Amplification pGEX-6p-1 linearized vectors and the design of GyV9 virus VP 3s fragment primer:
Amplification pGEX-6p-1 linearized vectors upstream primer is located at pGEX-6p-1 plasmid 1022-1047 positions；And at 5 ' ends
With extra TAA bases；Amplification pGEX-6p-1 linearized vectors downstream primer is located at pGEX-6p-1 plasmid 916-941 positions；And
Extra CAT bases are carried at 5 ' ends.Amplification GyV9 virus VP 3 genes upstream primer is including VP3 genes codon ATG and thereafter
14 bases, and at 5 ' ends with 16 and pGEX-6p-1 linearized vector downstream primer reverse complemental bases；GyV9 is sick for amplification
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 upstream primer reverse complemental bases.Concrete primer sequence sees attached list 1, by LifeTechnologies Shanghai
Thermo Fischer Scient Inc. synthesizes.
2) pGEX-6p-1 linearized vectors and GyV9 virus VP 3 genes fragment PCR are expanded：
With pGEX-6p-1 plasmids and the GyV9VP3 genes of synthesis as masterplate, primer described in table 1 enters performing PCR and expands for primer
Increase.Such as the step 1 in Fig. 1.Pcr amplification reaction system is：40 μ l water, 5 10 times of μ l buffer solutions, 1 μ l 10mM dNTP, 1 μ l
10 μm of ol upstream primers, 1 10 μm of μ l ol downstream primers, the pGEX-6p-1 plasmids or pcGyV9-VP3 plasmids of 1 μ l 10ng/ μ l,
The Phanta Super-Fidelity archaeal dna polymerases of 1 μ l commercializations.Pcr amplification reaction loop parameter is：94 DEG C of 5 points of denaturation
Clock, subsequently carries out 30 circulations (94 DEG C of denaturation 30 seconds, 58 DEG C are annealed 30 seconds, and 72 DEG C extend 3 minutes), and last 72 DEG C extend 10 points
Clock.After PCR terminates, PCR primer carries out electrophoresis in 1% Ago-Gel.As shown in Fig. 2 wherein swimming lane M is DNA
Marker, wherein swimming lane 1 are GyV9 virus VP 3 fragment PCR products.As shown in figure 3, wherein swimming lane M is DNA Marker, swimming lane
1 is the PCR primer of linearized vector pGEX-6p-1.
3) GyV9 virus VP 3s fragment quick clone enters pGEX-6p-1 carriers：
The expression linearized vector pGEX-6p-1 and GyV9 virus VP 3 fragment PCR products of purifying are in commercialization by more than
Recombinant clone is carried out in the presence of recombinase ExnaseTM II.Such as the step 2 in Fig. 1.Concrete recombining reaction system is as follows：It is pure
The GyV9 virus VP 3 fragment products 50-100ng of change, the pGEX-6p-1 expression linearized vector 50ng of purifying, 2 μ l commercializations
ExnaseTM II enzymes, the buffer solution of 45 times of μ l, other benefits add water to 20 μ l.After reactant is acted on 30 minutes in 37 DEG C, ice is put
Upper 5 minute.Subsequently 20 μ l reactants are transformed into into conventional competence bacterium, apply LB plates.Next day picking bacterial clone carries out plasmid
Prepare, positive clone identification.
Embodiment 2
1) the protein induced expression of GyV9 virus VP 3s:
The positive colony (being named as pGEX-VP3) containing GyV9 virus VP 3 genes for obtaining is transformed into after BL21 bacteriums,
Turn to be inoculated in LB fluid nutrient mediums of the 3ml containing Amp (100 μ g/mL), 37 DEG C of 250rpm shake overnight incubation.Next day presses 1:100 turns
LB culture mediums are inoculated in, 37 DEG C of 1500rpm shake 2.5h, with 37 DEG C of inductions of final concentration of 1mM IPTG.By bacterium solution after induction 5h
It is transferred in centrifuge tube, 6000rpm centrifugation 5min remove supernatant, washed once with PBS, 600 μ L sterilizing DDW suspended bacterials, 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.30 μ L bacteria lysis supernatants precipitation is taken respectively, adds 5 μ L6 × sds gel sample-loading buffer to mix
It is even, boil 5min；Expression for SDS-PAGE (5% concentration glue, 10% separation gel) electrophoretic analysis recombinant protein and its
Expression-form.In the diagram, VP3 albumen can be in ultrasonication Sample supernatants with soluble form presence.
2) purifying of GyV9 virus VP 3s albumen:
It is determined that on the basis of the solubility expression of VP3, ultrasonication Sample supernatants have been carried out into VP3 by GST purification columns
The purifying of albumen.Briefly step is as follows：The DDW that 5 volumes are added in purification column removes ethanol；With the combination of 10 volumes
Buffer (140mM Nacl, 2.7mM KCl, 10mM Na2HPO4,1.8mM KH2PO4) balances pillar；By protein sample 5-
10mL/ every time, repeated post twice, albumen is fully combined on post.In combination Buffer column scrubbers with 10 volumes
Foreign protein；Destination protein is eluted with the wash-out Buffer (50mM Tris-HCl, 10mM reductive glutathione) of 5 volumes, with
Add afterwards and fully dialysed with 1 × PBS solution in bag filter, after packing, in -70 DEG C of preservations；The albumen for taking after purification adds in proportion
Enter 5 × Loading Buffer, after boiling sample 5min, use SDS-PAGE electrophoretic analysis.As a result show that VP3 albumen is obtained good
Purifying is (as shown in swimming lane 4 in Fig. 4).
3) immunoreactivity of GyV9 virus VP 3s albumen:
By the week old BALB/C mice of VP3 albumen peritoneal immunity 6 of purifying, immunity three times, every minor tick 14 days, dosage is
50 μ g//times.Mix with isopyknic complete Freund's adjuvant when head exempts to Water-In-Oil shape, again during immunity with it is isopyknic not
Complete Freund's adjuvant mixes, and immunity afterwards is not added with adjuvant.Three exempt from after gather mice serum, in 293T cells express VP3
Albumen carries out anti-VP3 specific antibodies in western blot analysis serum as antigen.As a result show that the anti-GyV9 for obtaining is sick
How anti-the mouse of malicious VP3 albumen is can carry out specific reaction (Fig. 5) with the VP3 albumen expressed in 293T cells.This result
The VP3 albumen for showing present invention expression has reactivity and immunogenicity well, will be with good in GyV9 serodiagnosises
Good application prospect.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel it should be appreciated that the present invention is not restricted to the described embodiments, the simply explanation described in above-described embodiment and specification this
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, specification and its equivalent thereof.
SEQUENCE LISTING
<110>Yangzhou University
<120>A kind of GyV9 annulars virus VP 3 protein preparation method
<130> 20161101
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
<213>Artificial primer sequence
<400> 1
gttccagggg cccatggatg cgacgggcac 30
<210> 2
<211> 30
<212> DNA
<213>Artificial primer sequence
<400> 2
gttttcaccg tcattacaat cttgcgcctt 30
Claims (5)
1. a kind of PCR expands GyV9 virus VP 3 gene primers, it is characterised in that the nucleotides sequence of described primer is classified as：
Upstream primer 1：5’-GTTCCAGGGGCCCATGGATGCGACGGGCAC-3’；
Downstream primer 1：5’-GTTTTCACCGTCATTACAATCTTGCGCCTT-3’.
2. a kind of GyV9 annular virus VP 3 protein preparation method, it is characterised in that described method is based on recombinase ExnaseTM
II without digestion coupled reaction, directly weighs linearizing pGEX-6p-1 carriers and GyV9 virus VP 3 genes fragment PCR products
Group clone technology；And Escherichia coli are transformed into, realize VP3 protein expressions；Wherein, the described GyV9 virus VP 3 genes of PCR amplifications
The nucleotide sequence of the primer is as shown in claim 1 during fragment.
3. a kind of GyV9 annulars virus VP 3 protein preparation method as claimed in claim 2, it is characterised in that specifically include as
Lower step：
1) PCR amplification pGEX-6p-1 linearized vectors and GyV9 virus VP 3 gene fragments：With pGEX-6p-1 plasmids and
GyV9 virus VP 3 genes are template, design primer, and PCR amplifies respectively linearized vector containing pGEX-6p-1 and GyV9 is viral
VP3 genetic fragments；Wherein, the nucleotide sequence such as right of the primer when PCR expands described GyV9 virus VP 3 gene fragments
Require shown in 1；
2) GyV9 virus VP 3 fragment quick clones are entered into pGEX-6p-1 carriers：Carried out quickly using recombinase ExnaseTM II
Recombinant clone；
3) positive colony is transformed into Escherichia coli, realizes VP3 protein expressions.
4. a kind of GyV9 annulars virus VP 3 protein preparation method as claimed in claim 3, it is characterised in that step 1) in,
The nucleotide sequence of the primer used during PCR amplification pGEX-6p-1 linearized vectors is as follows：
Upstream primer 2：5’-TAATGACGGTGAAAACCTCTGACACATGC-3’；
Downstream primer 2：5’-CATGGGCCCCTGGAACAGAACTTCCAGAT-3’.
5. a kind of GyV9 annular virus VP 3 albumen described in primer as claimed in claim 1, and claim 2,3 or 4
Preparation method, is preparing GyV9 diagnostic antigens, anti-VP3 polyclonal antibodies and is preparing GyV9 epidemiology efficient diagnosis reagent side
The application in face.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105037503A (en) * | 2015-06-25 | 2015-11-11 | 扬州大学 | AGV2 (avian gyrovirus 2) type soluble VP3 (viral protein 3) and preparation method thereof |
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2016
- 2016-11-01 CN CN201610937115.2A patent/CN106565829A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105037503A (en) * | 2015-06-25 | 2015-11-11 | 扬州大学 | AGV2 (avian gyrovirus 2) type soluble VP3 (viral protein 3) and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| PHAN,T. G. ET AL.，: "Gyrovirus GyV9，partial genome", 《GENEBANK》 * |
| 布日额等: "鹅细小病毒VP1与VP3非重叠序列的克隆与原核表达", 《中国兽医杂志》 * |
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