CN107164408A - Express the dual-gene carrier for expression of eukaryon of PEDV M+N - Google Patents
Express the dual-gene carrier for expression of eukaryon of PEDV M+N Download PDFInfo
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- CN107164408A CN107164408A CN201710581141.0A CN201710581141A CN107164408A CN 107164408 A CN107164408 A CN 107164408A CN 201710581141 A CN201710581141 A CN 201710581141A CN 107164408 A CN107164408 A CN 107164408A
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Abstract
The invention belongs to biology field, and in particular to carrier for expression of eukaryon dual-gene a kind of expression PEDV M+N.By PEDV M and N genetic fragments, using Blunt M2 expression vectors, by seamless clone technology, the dual-gene carrier for expression of eukaryon of expression PEDV M+N, and the expression dual-gene fusion proteins of M+N in engineering cell are constructed.The present invention is that directly the M genetic fragments for purifying PEDV are connected in Blunt M2 mammal carrier for expression of eukaryon, require no connection in monoclonal carrier PMD19 T, then M+Blunt M2 plasmids pass through homologous recombination technique, seamless spliced N genetic fragments, so as to obtain the dual-gene fusion expression vectors of M+N, its plasmid is identified by round pcr, and the expression that succeeded in cell is transfected again after sequencing is accurate.Therefore, other eukaryotic expression technologies are compared in the preparation of the carrier for expression of eukaryon, with the features such as operating procedure is simple, the reaction time is short, are adapted to dual-gene expressing fusion protein.
Description
Technical field
The invention belongs to biology field, and in particular to eukaryotic expression dual-gene a kind of expression PEDV M+N is carried
Body.
Background technology
Pig epidemic diarrhea (PED) causes swinery vomiting, diarrhoea, dehydration and death by Porcine epidemic diarrhea virus (PEDV)
The digestive tract disease viral disease being characterized.The disease in 1971 first Britain report, China confirmed in 1984 the disease
In the presence of.Since second half 2010, because traditional PEDV is lacked on gene, inserted and made a variation, the disease is caused
Pathogenic enhancing to swinery, causes substantial amounts of swinery morbidity death, suffers heavy losses.Current pig-raising countries most of in the world
There is the report for the PEDV that morphs, it is especially serious with the morbidity of the country such as China, South Korea, Japan, Thailand and U.S., to pig industry
Huge economic loss is caused, and has turned into one of important epidemic disease that the global pig industry of influence develops in a healthy way.PEDV gene
Group leader mainly contains 4 structural proteins, respectively S, M, N and E protein about in 28000bp or so.At present, seen and passed through
PEDV individual gene such as N, S and M gene section piece, gives expression to single-gene albumen, but have no and pass through by prokaryotic expression method
PEDV M and N genetic fragments, using Blunt-M2 mammal carrier for expression of eukaryon, by seamless spliced technology, construct table
Up to PEDV M+N dual-gene carrier for expression of eukaryon.Therefore, dual-gene a kind of expression PEDV M+N carrier for expression of eukaryon
The foundation of method, to carrying out the diagnostic techniques of the disease or other cause of diseases and the research of recombinant vaccine from now on, with important meaning
Justice and application prospect.
The content of the invention
It is an object of the present invention to provide a kind of preparation side of carrier for expression of eukaryon dual-gene expression PEDV M+N
Method, and the carrier prepared by the preparation method.
It is a further object to provide the recombined engineering cell for having transfected carrier for expression of eukaryon.
It is also another object of the present invention to provide the preparation method for the fusion protein for having used carrier for expression of eukaryon.
What the present invention was realized in：
Present invention firstly provides a kind of preparation method of carrier for expression of eukaryon dual-gene expression PEDV M+N, including
Following steps：
(1) according to PEDV M, N gene and the fragment sequence of pEASY-Blunt-M2 expression vector genes, design and synthesis
Specific primer；
(2) using PEDV cDNA as template, M and N genetic fragments are amplified respectively by round pcr, and carries out reclaiming pure
Change, the M genetic fragments of purifying are connected on pEASY-Blunt-M2 expression vectors, M+Blunt-M2 plasmids are constructed；
(3) using M+Blunt-M2 plasmids as template, the genetic fragment of plasmid, recovery purifying M+Blunt-M2 gene pieces are expanded
Section；
(4) pEASY-Uni Seamless Cloning and Assembly kit are used, will by seamless clone technology
M+Blunt-M2 genes carry out restructuring with N genetic fragments and are connected, and connection product are converted again to Trans 1-T1 competent cells
In, it is coated on after the LB culture mediums of amicillin resistance are cultivated, extracts M+Blunt-M2+N plasmids and carry out sequencing identification,
Obtain the dual-gene carrier for expression of eukaryon of expression PEDV M+N.
The PEDV preferably makes a variation Porcine epidemic diarrhea virus.
Wherein step (1) described specific primer sequence is as follows：
a、M
M-F:ATGTCTAACGGTTTTATTCC
M-R:GACTAAATGAAGCACTTTCT
b、M2+M
M2+M-F:AAGGGCGATCCGGAACAA
M-R:GACTAAATGAAGCACTTTCT
c、N
N-F:AGAAAGTGCTTCATTTAGTCGCTTCTGTCAGCTTTCAGGATCG
N-R:TTGTTCCGGATCGCCCTTATTTCCTGTATCGAAGATCTCGTTG。
Wherein step (2) is when building M+Blunt-M2 plasmids, pEASY-Blunt-M2 expression vectors and M genetic fragments
Molar concentration rate is 1:7, the reaction time is carried out by junction fragment size, and fragment length is in 0.1-1kb, and the reaction time is in 5-10
Minute, fragment length is in 1-2kb the reaction times at 10-15 minutes, and fragment length is in 2-3kb the reaction times at 15-20 points
Clock, more than 3kb the reaction times at 20-30 minutes.
Preferably, DMT enzymes are added in the recovery product that step (3) is obtained, template of degrading.
Secondly, the invention provides eukaryotic expression load dual-gene the expression PEDV prepared by the preparation method M+N
Body.
In addition, present invention also offers the eukaryotic expression vector transfection dual-gene M+N using described expression PEDV
Recombined engineering cell.
Preferably, the engineering cell is 293T cells.
Finally, present invention also offers a kind of preparation method of the expression PEDV dual-gene fusion proteins of M+N, methods described
Including：The described recombined engineering cell of culture, and extract fusion protein.
The invention has the advantages that：The present invention is that the M genetic fragments for purifying PEDV directly are connected into Blunt-M2 to feed
In newborn animal carrier for expression of eukaryon, it is not necessary to be connected in monoclonal carrier PMD19-T, then M+Blunt-M2 plasmids pass through same
Source recombinant technique, seamless spliced N genetic fragments, so as to obtain the dual-gene fusion expression vectors of M+N, its plasmid passes through round pcr
Identified, the expression that succeeded in cell is transfected again after sequencing is accurate.Therefore, the preparation of the carrier for expression of eukaryon compares it
His eukaryotic expression technology, with the features such as operating procedure is simple, the reaction time is short, is adapted to dual-gene expressing fusion protein.
Brief description of the drawings
The present invention is further illustrated in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is amplification M fragment electrophoretic figures, arrow logo for purpose fragment 678bp.
Fig. 2 is M2-M positive clone identification results, and (M fragments add Component Vectors sequence to band 834bp for the purpose of arrow logo
Row).
Fig. 3 is that M+M2 and N fragments distinguish PCR amplifications, wherein 1 represents the band 1326bp of N mesh, 2 represent M+M2 purposes
Band 6104bp.
Fig. 4 is N fragment PCR amplifications, wherein 1 represents the band 1326bp of N mesh.
Fig. 5 is the seamless spliced bacterium colony PCR qualification results of M+M2 and N fragments, wherein 9,10 represent M+N purpose fragments
2004bp。
Fig. 6 is cell growth state figure after 293T transfected plasmids 48h；Wherein A:293T without transfection, B:The unloaded matter of transfection
The 293T, C of grain (M2):Transfection builds the 293T of plasmid (M+M2+N).
Fig. 7 is the dual-gene fusion destination protein WB figures of M+N, and arrow is the destination protein of expression, molecular weight 75kD or so；Its
In 1：Transfect the testing result of unloaded 293T total protein of cell, 2：Transfection builds the 293T total protein of cell detection knot of plasmid
Really, 3：Transfect the 293T total protein of cell testing results of empty plasmid, M:Western Marker(25-90kDa).
Embodiment
Embodiment 1
Need to overcome following technical barrier before the technology for implementing the present invention：
(1) design of primers, it is former with reference to homologous recombination according to expressed gene and the gene order of Blunt-M2 carriers
Reason, design and and synthetic primer.
(2) when purpose fragment is with Blunt-M2 carrier reaction formings, preferably holds carrier and the molar concentration rate of fragment is
1:7, the reaction time is held by junction fragment size, such as fragment length is in 0.1-1kb, and the reaction time is at 5-10 points
Clock, 1-2kb the reaction times were at 10-15 minutes, and 2-3kb the reaction times existed at 15-20 minutes more than 3kb the reaction times
20-30 minutes.
When (3) 2 genes carry out seamless spliced, first genetic fragment and the recombinant vector after M2 vector constructions need to enter
Recovery purifying recombinates purpose fragment after performing PCR, becomes linear carrier, and DMT enzyme degradative plasmids are added preferably in recovery product
Template, then with the 2nd purpose fragment according to homologous arm section progress homologous recombination.
Biological material source：PEASY-Blunt M2 expression vectors, pEASY-Uni Seamless Cloning and
Assembly kit are purchased from Beijing Quanshijin Biotechnology Co., Ltd.
First, according to disclosed in GenBank M the and N genes and Blunt-M2+M of Porcine epidemic diarrhea virus (PEDV)
Plasmid sequence, the related amplimer of design synthesis, sequence such as table 1 below.
The primer sequence of table 1
2nd, RT reacts：With PEDV, (PEDV lactation piggy occurs from the scale pig farm of Fujian in 2013 for the Strain
Small intestine, by small intestine with PBS liquid grind, draw supernatant, use commercial kit extract RNA) RNA for template, carry out instead
Transcription, synthesizes cDNA, its system such as table 2 below.
The reverse transcription system of table 2
Reaction condition：25℃10min；50℃30min；85℃5s.
3rd, the PCR amplifications of M fragments：Using cDNA as template, enter performing PCR amplification, amplification system such as table 3 below.
The PCR amplification system of the M fragments of table 3
Reaction condition is as follows：
PCR primer is detected by 1.5% agarose gel electrophoresis, purpose fragment, such as Fig. 1 is obtained.
4th, M genetic fragments are connected to M2 carriers
1st, linked system is as follows：2.0 μ l M fragments, 1.0 μ l Blunt-M2 carriers, use ddH2O polishings volume to 5.0 μ
L, 25 DEG C of connection 10min；
2nd, connection product is converted into Trans1-T1 competent cells, flicks mixing, ice bath 30min；
3rd, 42 DEG C of heat shock 30s, are immediately placed on 2min on ice；
4th, balance is added to the LB culture mediums of room temperature, 250rpm, 37 DEG C of culture 1h；
5th, 4000rpm centrifuges 1min, discards part supernatant, retains 100 μ l, and Amp+ resistant panels are applied to after flicking resuspension
On, 37 DEG C of overnight incubations；
6th, monoclonal bacterial strain is chosen respectively into 10 μ l sterilized waters, is vortexed and is mixed laggard performing PCR identification, obtains purpose fragment such as
Fig. 2.Reaction system such as table 4 below.
The PCR reaction systems of table 4
Reaction condition is as follows：
7th, take positive bacterium colony to send sequencing company to be sequenced, sequence alignment is carried out afterwards, take comparison purpose plasmid to enter as template
Performing PCR is expanded, and obtains linear carrier.
5th, the PCR amplifications of M+M2 fragments,
Using M+M2 plasmids as template, expanded, amplification system such as table 5 below.
The amplification system of table 5
Reaction system is as follows, and PCR primer is detected using 1.5% agarose gel electrophoresis, obtains purpose band such as Fig. 3.
6th, the PCR amplifications of N fragments
Using PEDV cDNA as template, expanded, amplification system such as table 6 below.
The amplification system of table 6
Reaction system is as follows, and PCR primer is detected using 1.5% agarose gel electrophoresis, obtains purpose fragment such as Fig. 4.
7th, purified fragments
The N fragments of glue reclaim purifying PCR amplifications and the M+M2 fragments of PCR amplifications, are carried out pure using purification kit respectively
Change.
8th, it is seamless spliced
1. recommend system to carry out recombining reaction with pEASY-Uni Seamless Cloning and Assembly Kit,
Linked system is as follows：2×Assembly Mix 5μl；The μ l of N fragments 2 are reclaimed in purifying；The μ l of M2+M fragments 3 are reclaimed in purifying；In PCR instrument
In 50 DEG C connection 15min；
2. connection product is totally converted in Trans 1-T1 competent cells；
3.42 DEG C of heat shock 30s, stand 2min on ice, add in 400 μ l LB culture mediums, 37 DEG C of 200rpm shake bacterium 1h；
4. take the bacterium solution 4000rpm that has shaken to abandon part supernatant after centrifuging 1min, take bacterial strain blow it is outstanding after be applied to resistance containing Amp+
LB flat boards on, the overnight incubation in 37 DEG C of incubators；
Flat board is taken out after 5.18h from incubator, the competence grown after random picking conversion extracts plasmid and enters performing PCR
Obtain purpose fragment such as Fig. 5；
6. positive colony shakes and is sequenced after bacterium, homology illustrates that splicing restructuring is accurate more than 99%.
9th, big upgrading grain
Take the viable bacteria that correct M+M2+N is sequenced to be transferred after overactivation into LB ammonia benzyl resistance culture bases, be put into 37 DEG C of shaking table,
After 200rpm cultures 16h, collect bacterium solution and extract plasmid.
Tenth, transfect
(1) cell is inoculated with
293T cells are seeded in cell culture plate, 12-24h is cultivated, cell density during transfection is reached 70% remittance
It is right；
(2) dilution of plasmid and transfection reagent
Through removing endotoxin M+M2+N plasmids and M2 empty plasmids, plasmid is diluted in 50 μ l Opti-MEM culture mediums,
TransIn EL transfection reagents addition is 16 μ l, and transfection reagent is added into the above-mentioned Opti-MEM culture mediums containing dilution plasmid
In, soft mix is stored at room temperature 20min；
(3) transfect
Respectively will only plus culture medium, the EL transfection reagent mixtures of-TransIn containing empty plasmid and plasmid containing M+M2+N-
TransIn EL transfection reagent mixtures add cell, in changing culture medium after 37 DEG C of CO2 incubator cultures, 4h, cultivate to 48h
Afterwards, as shown in fig. 6, atrophy does not occur in cell comes off, illustrate that cell growth state is good, do not interfere with the expression of albumen, so
After extract total protein of cell.
11, the expression of WB verifying purposes albumen
Total protein of cell is extracted
Collect after the cell for building plasmid (M+M2+N) and empty plasmid (M2) transfectional cell and untransfected, extract thin respectively
Born of the same parents' total protein, measure protein concentration is 3.6mg/ml.
SDS-PAGE electrophoresis is carried out for 10% glue using resolving gel concentration, upper sample is 12ug, 200V constant pressure electrophoresis
45min。
Transferring film：Transferring film, 100V constant pressure transferring films 1h are carried out using pvdf membrane.
Closing：Use 5% skim milk (0.5%TBST dilutions), 37 DEG C of closing 2h.
Primary antibody is incubated：Using 0.5%TBST dilution primary antibodies (PEDV rabbit-antis hyper-immune serum), dilution ratio is 1:4000,4 DEG C
Night incubation.
Washing：Washed 4 times using TBST, each 10min.
Secondary antibody is incubated：Use 0.5%TBST dilution secondary antibodies (PEDV anti-rabbit HRP enzymes), dilution ratio 1:4000, room temperature is rocked
It is incubated 2h.
Colour developing：Washed 4 times using TBST, each 10min is detected using chemiluminescence imaging instrument.As a result 75KD is obtained
The purpose fragment of left and right such as Fig. 7, illustration purpose albumen is succeeded expression in 293T cells, and to PEDV rabbit-anti hyper-immune serums
With reaction, illustrate that expressing protein has immunogenicity.
Although the foregoing describing the embodiment of the present invention, those familiar with the art should manage
Solution, the specific embodiment described by us is merely exemplary, rather than for the restriction to the scope of the present invention, is familiar with this
The equivalent modification and change that the technical staff in field is made in the spirit according to the present invention, should all cover the present invention's
In scope of the claimed protection.
SEQUENCE LISTING
<110>Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<120>Express the dual-gene carrier for expression of eukaryon of PEDV M+N
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
atgtctaacg gttttattcc 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
gactaaatga agcactttct 20
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence
<400> 3
aagggcgatc cggaacaa 18
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
gactaaatga agcactttct 20
<210> 5
<211> 43
<212> DNA
<213>Artificial sequence
<400> 5
agaaagtgct tcatttagtc gcttctgtca gctttcagga tcg 43
<210> 6
<211> 43
<212> DNA
<213>Artificial sequence
<400> 6
ttgttccgga tcgcccttat ttcctgtatc gaagatctcg ttg 43
Claims (9)
1. a kind of preparation method of carrier for expression of eukaryon dual-gene expression PEDV M+N, it is characterised in that：Including following step
Suddenly：
(1) according to PEDV M, N gene and the fragment sequence of pEASY-Blunt-M2 expression vector genes, design is special with synthesis
Property primer；
(2) using PEDV cDNA as template, M and N genetic fragments are amplified respectively by round pcr, and carries out recovery purifying, will
The M genetic fragments of purifying are connected on pEASY-Blunt-M2 expression vectors, construct M+Blunt-M2 plasmids；
(3) using M+Blunt-M2 plasmids as template, the genetic fragment of plasmid, recovery purifying M+Blunt-M2 genetic fragments are expanded；
(4) pEASY-Uni Seamless Cloning and Assembly kit are used, by seamless clone technology by M+
Blunt-M2 genes carry out restructuring with N genetic fragments and are connected, and connection product are converted again into Trans 1-T1 competent cells,
It is coated on after the LB culture mediums of amicillin resistance are cultivated, extracts M+Blunt-M2+N plasmids and carry out sequencing identification, obtain
To the dual-gene carrier for expression of eukaryon of expression PEDV M+N.
2. the preparation method of carrier for expression of eukaryon dual-gene expression PEDV according to claim 1 M+N, its feature exists
In：The PEDV is variation Porcine epidemic diarrhea virus.
3. the preparation method of carrier for expression of eukaryon dual-gene expression PEDV according to claim 1 M+N, its feature exists
In：Step (1) described specific primer sequence is as follows：
a、M
M-F:ATGTCTAACGGTTTTATTCC
M-R:GACTAAATGAAGCACTTTCT
b、M2+M
M2+M-F:AAGGGCGATCCGGAACAA
M-R:GACTAAATGAAGCACTTTCT
c、N
N-F:AGAAAGTGCTTCATTTAGTCGCTTCTGTCAGCTTTCAGGATCG
N-R:TTGTTCCGGATCGCCCTTATTTCCTGTATCGAAGATCTCGTTG。
4. the preparation method of carrier for expression of eukaryon dual-gene expression PEDV according to claim 1 M+N, its feature exists
In：Step (2) is when building M+Blunt-M2 plasmids, pEASY-Blunt-M2 expression vectors and the molar concentration of M genetic fragments
Than for 1:7, the reaction time is carried out by junction fragment size, and fragment length is in 0.1-1kb, and the reaction time was 5-10 minutes, piece
Segment length is in 1-2kb the reaction times at 10-15 minutes, and fragment length, at 15-20 minutes, is more than in 2-3kb the reaction times
3kb the reaction times were at 20-30 minutes.
5. the preparation method of carrier for expression of eukaryon dual-gene expression PEDV according to claim 1 M+N, its feature exists
In：DMT enzymes are added in the recovery product that step (3) is obtained, template of degrading.
6. carrier for expression of eukaryon dual-gene expression PEDV prepared by the preparation method as described in claim any one of 1-5 M+N.
7. the recombined engineering of the eukaryotic expression vector transfection dual-gene M+N of the expression PEDV described in utilization claim 6 a kind of
Cell.
8. recombined engineering cell according to claim 7, it is characterised in that：The engineering cell is 293T cells.
9. a kind of preparation method of the expression PEDV dual-gene fusion proteins of M+N, it is characterised in that：Methods described includes：Culture
Recombined engineering cell as claimed in claim 7 or 8, and extract fusion protein.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805106A (en) * | 2014-05-14 | 2015-07-29 | 北京市农林科学院 | Fusion gene containing transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) protective antigens as well as encoding protein and application thereof |
| CN106188307A (en) * | 2016-07-06 | 2016-12-07 | 长沙爱科博生物科技有限公司 | A kind of fusion protein, its preparation method and boar oral vaccine or a medicine |
-
2017
- 2017-07-17 CN CN201710581141.0A patent/CN107164408A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805106A (en) * | 2014-05-14 | 2015-07-29 | 北京市农林科学院 | Fusion gene containing transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) protective antigens as well as encoding protein and application thereof |
| CN106188307A (en) * | 2016-07-06 | 2016-12-07 | 长沙爱科博生物科技有限公司 | A kind of fusion protein, its preparation method and boar oral vaccine or a medicine |
Non-Patent Citations (5)
| Title |
|---|
| TRANSGENE: "pEASY-Blunt M2", 《PEASY-BLUNT M2 EXPRESSION KIT》 * |
| 任玉鹏: "携PEDV M、N基因的减毒沙门氏菌株构建及其免疫原性研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 吴万军等: "日本血吸虫外分泌蛋白、跨膜蛋白基因筛选与无缝克隆", 《湖北医药学院学报》 * |
| 安颖等: "重症肌无力患者肌细胞中LRP4胞内区与SNX17的相互作用", 《郑州大学学报（医学版）》 * |
| 程向朝等编著: "《动物基因工程》", 31 May 2008, 中国农业出版社 * |
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