CN106086067A - The recombinant plasmid of expression J subgroup avian leucosis virus gp85, p27, p10 gene of ubiquitin mediation and construction method thereof and application - Google Patents
The recombinant plasmid of expression J subgroup avian leucosis virus gp85, p27, p10 gene of ubiquitin mediation and construction method thereof and application Download PDFInfo
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Abstract
The invention discloses the recombinant plasmid of expression J subgroup avian leucosis virus gp85, p27, p10 gene of a kind of ubiquitin mediation, by J subgroup avian leucosis virus gp85, p27, p10 gene, chicken ubiquitin Ub gene and eukaryotic expression vector pVAX1 composition.Inventor also sets up corresponding construction method, and this method can be inserted in pVAX1 carrier after connecting J subgroup avian leucosis virus gp85, p27, p10 gene with chicken ubiquitin gene simultaneously.Experiment in vitro proves, by Transfected Recombinant Plasmid DF 1 cell, recombinant plasmid is successfully expressed.In chicken immune test, the antibody of immunity chicken turns sun rate and the antibody positive rate of offspring chick is improved.The present invention, by building the J subgroup avian leucosis virus polygenes coexpression DNA bacterin of ubiquitin mediation, is that the development of J subgroup avian leucosis virus new generation vaccine is laid a good foundation, and the purification for J subgroup avian leucosis virus provides a kind of effective supplementary means.
Description
Technical field
The invention belongs to J subgroup avian leucosis technical field, particularly relate to the white blood of expression J subgroup fowl of a kind of ubiquitin mediation
The recombinant plasmid of sick virus gp85, p27, p10 gene and construction method thereof and application.
Background technology
J subgroup avian leucosis virus (Avian leukosis virus subgroup J, ALV-J) be 1988 first
The new subgroup of a kind of avian leukosis virus of isolated from white meat-type chickens.ALV-J, as ALV-A, ALV-B, can cause machine
Body generation immunosupress, causes vaccine immunity failure, has a strong impact on again the production performance of bird, cause egg production and Egg Quality
Decline, have a strong impact on the sound development of aviculture.Compared to ALV-A, ALV-B, ALV-J horizontal transmission speed faster, harm more
Greatly, this disease has spread to many countries at present, and provisions fowl industrial belt carrys out heavy economic losses.As white plumage Breeder hens introduces ALV-J
Incoming China, and the isolated in the broiler chicken of upper level ground first in 1999, different regions, the chicken of different lines have separation afterwards
To the report of ALV-J, current ALV-J has spread in Local Chicken Breeds, the development of serious threat China aviculture.
Although there is no effective vaccine at present, but the supplementary means that development of new vaccine purifies as ALV is still by people
Concern.DNA vaccination have can in body continuous expression produce the cell of higher level and humoral immunity, safety, steady
The advantages such as fixed, preparation is easy, become the focus of research.
Gp85 gene expression virus surface proteins, determines that subgroup is specific, is that cell entry host cell replicates, induction is swollen
The necessary condition that knurl occurs.The surface protein that gp85 expresses causes a disease at ALV-J, has important function in tumorigenesis；P27 gene expression
Nucleocapsid protein, this albumen is main group specific antigen, high conservative in all subgroups；P10 gene expression albumen master
Participate in virus getting up early to infect and later stage assembling, have no p10 gene for building the report of gene vaccine at present.
Ubiquitin (Ubiquitin, Ub) is formed by 76 amino acid, high conservative, is prevalent in eukaryotic
Micromolecule polypeptide.Ubiquitin and mark modification (ubiquitination, ubiquitination or to protein
Ubiquityrmtion) the selective protein degradation process that intracellular ATP relies on is participated in, sending out of Ubiquitin-Proteasome Pathway
Existing, cause the extensive concern of researcher.In recent years, having scholar both at home and abroad utilizes ubiquitin to improve the immune effect of DNA vaccination,
Result of study shows that ubiquitin fusion plasmid DNA can induce stronger cell immune response.Strengthen bird with regard to ubiquitin at present sick
Former immune response research report is very few, and the research in particular for viral antigen have not been reported.
Content of the invention
The technical problem to be solved in the present invention be to provide a kind of ubiquitin mediation expression J subgroup avian leucosis virus gp85,
The recombinant plasmid of p27, p10 gene and construction method thereof and application, be that the development of J subgroup avian leucosis virus new generation vaccine is established
Basis.
For solving above-mentioned technical problem, the present invention by the following technical solutions:
The recombinant plasmid of expression J subgroup avian leucosis virus gp85, p27, p10 gene of ubiquitin mediation is white by J subgroup fowl
Blood virus gp85, p27, p10 gene, chicken ubiquitin Ub gene and eukaryotic expression vector pVAX1 composition.
The recombinant plasmid of expression J subgroup avian leucosis virus gp85, p27, p10 gene of above-mentioned ubiquitin mediation, be
pVAX1-Ub-gp85-p27-p10。
The recombinant plasmid of expression J subgroup avian leucosis virus gp85, p27, p10 gene of above-mentioned ubiquitin mediation has sequence
The base sequence of table SEQ.ID.No.13.
J subgroup avian leucosis virus is from Chinese pathogenic strain GX13HG03.
J subgroup avian leucosis virus gp85, p27, p10 gene is respectively provided with sequence table SEQ .ID.No.1 extremely
The base sequence of SEQ.ID.No.3, chicken ubiquitin Ub gene has sequence table SEQ .ID.No.12 base sequence.
The structure side of the recombinant plasmid of expression J subgroup avian leucosis virus gp85, p27, p10 gene of above-mentioned ubiquitin mediation
Method, uses PCR method amplification to obtain J subgroup avian leucosis virus gp85, p27, p10 gene and chicken ubiquitin gene, and amplification is used
Primers F/R-gp85, F/R-p27, F/R-p10, F/R-Ub is respectively provided with sequence table SEQ .ID.No.4's to SEQ.ID.No.11
Base sequence；The side that J subgroup avian leucosis virus gp85, p27, p10 gene is connected by restriction enzyme site with chicken ubiquitin gene
Method connects performing PCR amplification of going forward side by side, and PCR primer orients after Kpn I and Xho I double digestion insertion same with Kpn I and Xho I
In the eukaryotic expression vector pVAX1 that double digestion is processed, obtain J subgroup avian leucosis virus gp85, p27, p10 base of ubiquitin mediation
Eukaryon expression plasmid pVAX1-Ub-gp85-p27-p10 because of series connection.
The recombinant plasmid of expression J subgroup avian leucosis virus gp85, p27, p10 gene of above-mentioned ubiquitin mediation is pre-in preparation
Application in terms of anti-avian leukosis DNA vaccination.
The problem existing for current J subgroup avian leucosis prevention and control, inventor selects gp85, p27, p10 after considering
Gene as the target gene of nucleic acid vaccine, design and construct the expression J subgroup avian leucosis virus gp85 of ubiquitin mediation, p27,
The recombinant plasmid of p10 gene, by J subgroup avian leucosis virus gp85, p27, p10 gene, chicken ubiquitin Ub gene and eukaryotic expression
Carrier pVAX1 forms.Inventor also sets up corresponding construction method, this method can simultaneously by J subgroup avian leucosis virus gp85,
P27, p10 gene is inserted in pVAX1 carrier after connecting with chicken ubiquitin gene.Experiment in vitro proves, by Transfected Recombinant Plasmid DF-
1 cell, utilizes indirect immunofluorescence (IFA) method to determine that the recombinant plasmid building successfully is expressed.In chicken immune test,
The antibody of immunity chicken turns sun rate and the antibody positive rate of offspring chick is improved.The present invention is sub-by the J building ubiquitin mediation
Group's avian leukosis virus polygenes (gp85, p27, p10) coexpression DNA bacterin, is J subgroup avian leucosis virus new generation vaccine
Development is laid a good foundation, and the purification for J subgroup avian leucosis virus provides a kind of effective supplementary means.
Brief description
Fig. 1 is that pVAX1-Ub-gp85-p27-p10 is double cuts qualification result figure, in figure: M is DL15000DNA Maker；1 is
PVAX1-Ub-gp85-p27-p10 plasmid is through Kpn I+Xho I double digestion result.
Fig. 2 is immunofluorescence method testing result figure, in figure: A is that pVAX1-Ub-gp85-p27-p10 is in DF-1 cell
Transient expression result (positive), B is pVAX1 empty carrier transfection results (negative).
Detailed description of the invention
1.1 strain
J subgroup avian leucosis virus strain GX13HG03 (preservation of inventor laboratory).
1.2 method
1.2.1 design of primers
According to J subgroup avian leucosis virus gp85, p27, p10 gene order delivered in GenBank and chicken ubiquitin base
It because of primers, and on different primers, Hind III, BamH I, EcoR I, Xho I and Kpn I enzyme have been separately added into
Cutting site, all restriction endonucleases are all bought in precious bioengineering (Dalian) Co., Ltd.Gp85, p27, p10 and ubiquitin gene amplification
Primer sequence is shown in Table 1.
Table 1 primer sequence
In table 1, italicized item is restriction enzyme site, and primer is synthesized by Beijing Hua Da genome company.
1.2.2 the amplification of gp85, p27, p10, ubiquitin gene
The amplification of GX13HG03 strain gp85, p27, p10 gene: with strain GX13HG03 proviral DNA as template, respectively
Amplification gp85, p27, p10 gene.PCR response procedures is 94 DEG C of denaturations 5 minutes, 94 DEG C of denaturation 30 seconds, anneals 30 seconds for 60 DEG C,
72 DEG C extend 30 seconds, totally 35 circulations, and last 72 DEG C extend 10 minutes.
The amplification of ubiquitin gene: (Beijing health is century, ultrapure RNA extraction agent according to RNA extraction agent box specification
Box) extract chicken muscle total serum IgE, with for template with reference to MLV reverse transcriptase (treasured bioengineering (Dalian) Co., Ltd,
TaKaRa MLV, 10000units) specification carries out reverse transcription, it is thus achieved that cDNA.PCR system configuration is reacted with reference to specification, PCR
Program is 94 DEG C of denaturations 5 minutes, 94 DEG C of denaturation 30 seconds, anneals 45 seconds for 62 DEG C, and 72 DEG C extend 30 seconds, totally 35 circulations, finally
72 DEG C extend 10 minutes.
1.2.3 genes of interest is identified
Be connected genes of interest gp85, p27, p10 and ubiquitin respectively with pUC-T cloning vector, linked system is shown in Table 2.
Table 2 gp85, p27, p10, ubiquitin, clone's linked system
Above-mentioned reactant is mixed gently after connecting 22 DEG C of connection 30min in instrument, converts DH5 α competent cell afterwards,
Concrete operations are as follows:
1. the DH5 α competent cell of 1 pipe 100 μ L, thawed on ice 10min is taken out at-80 DEG C of refrigerators；
2. the connection product of 10 μ L is all joined in the competent cell of 100 μ L, ice bath 30min after mixing gently；
3.42 DEG C of heat stress 90s, are immediately placed on 2min-5min on ice afterwards；
4. add 500 μ L LB fluid nutrient mediums, 37 DEG C of 225r/min shaken cultivation 1h-2h；
5. the X-gal of the Amp, the IPTG of 8 μ L 500mM and the 40 μ L 20mg/mL that take 10 μ L 100 μ g/mL is spread evenly across
On LB flat board, placing 30min for 37 DEG C makes it fully absorb；
6. take the bacterium solution bed board that 100 μ L cultivate, 37 DEG C of incubated overnight.
After incubated overnight, white colony on 3 LB flat boards of random picking contains the LB training of 100 μ g/mLAmp in 300 μ L
Supporting in base, 1h-2h are cultivated in 37 DEG C of 225r/min shakings, carry out the qualification of bacterium solution PCR, take the positive bacterium solution of 10 μ L and join 5mL and contain
There is incubated overnight in the LB culture medium of 100 μ g/mL Amp, use plasmid extraction kit extracting plasmid, plasmid is digested
Identify, after identifying correctly, send Beijing Hua Da genome company to check order positive bacterium solution.It is 906bp that sequencing result obtains size
(SEQ.ID.No.1), the mesh of 717bp (SEQ.ID.No.2), 186bp (SEQ.ID.No.3) and 228bp (SEQ.ID.No.12)
Fragment, be all consistent with theoretical value.Sequencing result shows, the external source genes of interest of insertion is correct, base mutation does not occurs.
1.2.4 construction of recombinant plasmid and qualification
Gp85, p27, p10 and ubiquitin gene are after 1.2.3 qualification, and according to the system that is digested through being digested, double digestion system is such as
Under:
The condition of endonuclease reaction: 37 DEG C 3 hours.
After being digested end, the Ago-Gel of 1% carries out electrophoresis and reclaims the fragment purifying after being digested.According to Kang Weishi
The operation of kit specification is reclaimed in discipline gel electrophoresis.
Restriction enzyme site is utilized to enter in pVAX-1 carrier by fragment clone by ubiquitin, gp85, p27, p10 order, reactant
It is as follows:
16 DEG C of connection instrument connect 20h, the Plastid transformation DH5 α competent cell obtaining, and coated plate choosing colony is taken out after identifying the positive
Plasmid utilizes restriction enzyme site to enter in pVAX-1 carrier by fragment clone according to ubiquitin, gp85, p27, p10 order, recombinant plasmid
It is pVAX1-Ub-gp85-p27-p10.
Double digestion method is used to be digested the recombinant plasmid pVAX1-Ub-gp85-p27-p10 obtaining, endonuclease reaction
Enter row agarose gel electrophoresis analysis after end to product, judge recombination whether insertion vector (result according to electrophoresis result
See Fig. 1).Result shows that genes of interest is properly inserted in carrier, and purpose clip size is consistent with expected.
By be digested identify the order-checking of correct recombinant plasmid sample presentation after identify further construction of recombinant plasmid whether correct and
Whether purpose fragment is properly inserted in expression vector.Result show purpose fragment (SEQ.ID.No.1, SEQ.ID.No.2,
SEQ.ID.No.3, SEQ.ID.No.12) it is properly inserted in expression vector and sequencing result is completely the same with expected.
1.2.6 vivoexpression and the product thereof of recombinant plasmid detects
1.2.6.1 the in-vitro transfection of recombinant plasmid
By pVAX1-Ub-gp85-p27-p10 Transfected Recombinant Plasmid DF-1 cell, method is according to transfection reagent
(Invitrogen company, Lipofectamine 2000,1.5ml) specification is carried out.
1.2.6.2 the gene expression after indirect immunofluorescence method detection Transfected Recombinant Plasmid
1. transfect latter 48 hours, rinse DF-1 cell 3 times with the PBS (pH value=7.4) of precooling, 5 minutes every time；
2. fixing 10 minutes with the paraformaldehyde of 4 DEG C of precoolings, PBS washs 3 times, natural air drying；
3. drip J subgroup avian leucosis virus monoclonal antibody JE9 (1: 250 dilution), be positioned over the wet box effect of 37 DEG C
60 minutes, PBS rinsing 3 times, 5 minutes every time；
4. in each hole, drip rabbit anti-mouse igg two anti-(1: 300 dilution) respectively, be positioned over the wet box lucifuge effect 45 points of 37 DEG C
Clock, PBS rinsing 3 times, 5 minutes every time；
5. the neutral glycerine of mounting configuration, is observed by inverted fluorescence microscope afterwards and takes pictures.
As in figure 2 it is shown, left figure is that the DF-1 after having transfected recombinant plasmid pVAX1-Ub-gp85-p27-p1048 hour is thin
Born of the same parents, occur in that specific fluorescence signal；Right figure is the DF-1 cell after having transfected empty carrier pVAX148 hour, spy does not occurs
The fluorescence signal of the opposite sex.
1.2.7 plant chicken immune test
Plasma viral is selected to separate detection avian leukosis p27 antigen, Virus monitory avian leukosis ALV-A/B, ALV-J antibody
It is all 180 negative age in days kind chickens, often organize 25, be divided into control group with immune group.182 age in days head exempt from the DNA of immune group kind chicken
Plasmid immunizations is injected through chicken, and the PBS of control group same volume carries out immunity.196 age in days secondary immunities.Immunization ways is same
On.
The kind chicken of immunity pVAX1-Ub-gp85-p27-p10 recombinant plasmid second week (196 age in days) after first immunisation has 3
It is only ALV-J antibody positive, positive rate 12% (3/25).After secondary immunity, second week has 5 is ALV-J antibody positive, positive
Rate is 20% (5/25).After first immunisation, two weeks kind chicken offspring chick are without antibody positive, and after secondary immunity, second week has 1 to present
For ALV-J antibody positive.The all feminine genders of control group.This result proves the pVAX1-Ub-gp85-p27-p10 restructuring matter building
Grain have expressed corresponding albumen in kind of chicken body, and stimulates body to create corresponding antibodies.
Claims (7)
1. a recombinant plasmid for expression J subgroup avian leucosis virus gp85, p27, p10 gene of ubiquitin mediation, its feature exists
In by J subgroup avian leucosis virus gp85, p27, p10 gene, chicken ubiquitin Ub gene and eukaryotic expression vector pVAX1 composition.
2. the weight of expression J subgroup avian leucosis virus gp85, p27, p10 gene of ubiquitin according to claim 1 mediation
Group plasmid, it is characterised in that be pVAX1-Ub-gp85-p27-p10.
3. the weight of expression J subgroup avian leucosis virus gp85, p27, p10 gene of ubiquitin according to claim 1 mediation
Group plasmid, it is characterised in that there is the base sequence of sequence table SEQ .ID.No.13.
4. the weight of expression J subgroup avian leucosis virus gp85, p27, p10 gene of ubiquitin according to claim 1 mediation
Group plasmid, it is characterised in that: described J subgroup avian leucosis virus is from Chinese pathogenic strain GX13HG03.
5. the weight of expression J subgroup avian leucosis virus gp85, p27, p10 gene of ubiquitin according to claim 1 mediation
Group plasmid, it is characterised in that: described J subgroup avian leucosis virus gp85, p27, p10 gene is respectively provided with sequence table
The base sequence of SEQ.ID.No.1 to SEQ.ID.No.3, described chicken ubiquitin Ub gene has sequence table SEQ .ID.No.12 base
Sequence.
6. the recombinant plasmid of expression J subgroup avian leucosis virus gp85, p27, p10 gene of ubiquitin mediation described in claim 1
Construction method, it is characterised in that use PCR method amplification to obtain J subgroup avian leucosis virus gp85, p27, p10 gene and chicken
Ubiquitin gene, amplification the primer F/R-gp85, F/R-p27, F/R-p10, F/R-Ub are respectively provided with sequence table SEQ .ID.No.4
Base sequence to SEQ.ID.No.11；Chicken ubiquitin gene is passed through with J subgroup avian leucosis virus gp85, p27, p10 gene
The method that restriction enzyme site is connected connects performing PCR amplification of going forward side by side, and PCR primer orients after Kpn I and Xho I double digestion insertion same
In the eukaryotic expression vector pVAX1 with Kpn I and the process of Xho I double digestion for the sample, the J subgroup avian leucosis obtaining ubiquitin mediation is sick
The eukaryon expression plasmid pVAX1-Ub-gp85-p27-p10 of poison gp85, p27, p10 gene tandem.
7. the recombinant plasmid of expression J subgroup avian leucosis virus gp85, p27, p10 gene of ubiquitin mediation described in claim 1
Application in terms of preparation prevention avian leukosis DNA vaccination.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107523531A (en) * | 2017-07-04 | 2017-12-29 | 山东农业大学 | A kind of genetic engineering bacterium containing pMG36e pgsA gp85 recombinant plasmids |
| CN107586788A (en) * | 2017-07-04 | 2018-01-16 | 山东农业大学 | A kind of construction method of pMG36e pgsA gp85 recombinant plasmids |
| CN113341140A (en) * | 2021-06-02 | 2021-09-03 | 贵州大学 | Indirect ELISA (enzyme-linked immunosorbent assay) method for detecting avian leukosis P27 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107523531A (en) * | 2017-07-04 | 2017-12-29 | 山东农业大学 | A kind of genetic engineering bacterium containing pMG36e pgsA gp85 recombinant plasmids |
| CN107586788A (en) * | 2017-07-04 | 2018-01-16 | 山东农业大学 | A kind of construction method of pMG36e pgsA gp85 recombinant plasmids |
| CN113341140A (en) * | 2021-06-02 | 2021-09-03 | 贵州大学 | Indirect ELISA (enzyme-linked immunosorbent assay) method for detecting avian leukosis P27 |
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Application publication date: 20161109 |