CN109679995A - The construction method of ox PLIN2 interaction albumen bimolecular fluorescence complementary carrier - Google Patents
The construction method of ox PLIN2 interaction albumen bimolecular fluorescence complementary carrier Download PDFInfo
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Abstract
The present invention relates to a kind of construction methods of ox PLIN2 interaction albumen bimolecular fluorescence complementary carrier, with the area the CDs full length amino acid sequence of ox PLIN2, pass through primer of the design PLIN2 on bimolecular fluorescence complementary VN-173 carrier, it is recombinated with pBiFC-VN173 carrier to construct destination protein expression vector, restriction enzyme site selects EcoRI and XbaI enzyme cutting, reaction temperature is 37 DEG C, and increase a base A behind the site EcoRI, frameshift mutation is prevented, ox PLIN2 interaction albumen bimolecular fluorescence complementary carrier pBiFC-VN173-PLIN2 is finally obtained.It, can the more efficient presence expressed and avoid false negative result compared with existing research interactions between protein method.Meanwhile certain verification experimental verification means also are provided further to study and constructing interactions between protein network and improve the integrality of interactions between protein theory.
Description
Technical field
The invention belongs to molecular biology, cell engineering field, and it is white mutually to be related to a kind of lipocyte differentiation associated protein
Make the screening of albumen, specifically, being a kind of construction method of ox PLIN2 interaction albumen bimolecular fluorescence complementary carrier.
Background technique
Fat drips (lipid droplets) are the storage places for the neutral fats being wrapped in by imitated vesicle structure, are widely present in machine
In all organs of body, it is related with many cell functions, such as energy metabolism balance, the self-protection to Fatty toxicity, signal transduction
Deng.The structure of fat drips periphery film is mainly single layer phospholipid molecule layer, has special albumen anchoring structure, neutral fats is (predominantly
ATGL, cholesteryl ester) constitute hydrophobic core.Perilipin (perilipin), the lipocyte differentiation associated protein being currently known
White (adipophilin), tail interaction protein (TIP47) three form PAT family and are anchored on fat drips surface, with fat drips knot
Structure is formed and is metabolized related.
PAT protein family is widely present in different plant species, in addition to occurring in fat cell, also in other types cell
Occur in (such as myocyte, epithelial cell etc.) fat drips cumulative process.Studies have shown that perilipin there are 5 seed types, ordered respectively
Entitled PLIN1~PLIN5.PLIN2 is originally found in the cell of fat and Steroidgenesis, is a kind of Adipocyte Factor,
Mainly in the early expression of Adipocyte Differentiation, play an important role to the formation for promoting intracellular fat drips, it is also referred to as fatty
Break up GAP-associated protein GAP (adipocytes differentiation-related protein, ADRP), it can be by hydrophobic
Effect connect with neutral fat drips core and combines fat drips, aggregation phosphatide, makes to form monolayer around each fat drips.In addition,
In the egg mother cell of preovulatory phase mouse, PLIN2 expression quantity will appear up-regulation trend, this, which may participate in energetic supersession with PLIN2, has
It closes.
Prokesch etc. in mouse galactophore epithelial cell the study found that turn in Adipocyte Differentiation test, in epidermal growth factor
5 (ELF5) of son stimulate PLIN2 expression quantity in lower galactophore epithelial cell to increase, and the marker protein of fat cell can be detected, because
This, PLIN2 also can be used as one and study the marker protein whether other cells successfully turn rouge differentiation.In vitro in test, grind
Study carefully first albumen for proposing occur when PLIN2 is Adipocyte Differentiation, can be used as preadipocyte differentiation is fat cell
Marker gene in the process.Therefore, PLIN2 has important physiological significance to Adipocyte Differentiation process.
Summary of the invention
The object of the present invention is to provide a kind of construction method of ox PLIN2 interaction albumen bimolecular fluorescence complementary carrier,
The obtained carrier finds the interaction protein classes functioned with it, for study certain particular biological phenomenons provide theory according to
According to.
In order to realize that above-mentioned task, the present invention take following technical solution:
A kind of construction method of ox PLIN2 interaction albumen bimolecular fluorescence complementary carrier, which is characterized in that this method is with ox
The area the CDs full length amino acid sequence of PLIN2, by designing primer of the PLIN2 on bimolecular fluorescence complementary VN-173 carrier, with
PBiFC-VN173 carrier recombinates to construct destination protein expression vector, and restriction enzyme site selects EcoRI and XbaI enzyme cutting, reaction temperature
Degree is 37 DEG C, and increases a base A behind the site EcoRI, prevents frameshift mutation, and it is double to finally obtain ox PLIN2 interaction albumen
Molecular fluorescence complementing vector pBiFC-VN173-PLIN2.
According to the present invention, the area the CDs full length amino acid sequence of the ox PLIN2 are as follows:
atggcatctgttgcagttgaaccacaactgagtgtggtgaccagggtggccaacctccccttggtgag
ctccacgtatgatctggtctcctcggcttacatcagtagaaaggatcagtatccttacttgaagtctctgtgtgag
atggcagagaagggcatgaagaccatcacttctgtggctgtgaccagcgcgctgccaatcatccaaaagttagagc
cacaaattgctgttgccaatacctatgcgtgtaaggggctagacaggattgaggagaagctgcctattctgaatca
gccaacaaaccaggttgtggccaatgccaaaggggctatgactggggcaaaagatgctgtgacgactactgtgacc
ggggccaaggattctgtggccagcacaatcacgggggtggtggacaggaccaagggagctgtgactggtagtgtgg
agaagaccaagtccgtggtcagtggcagcattaacacagtcctacgaagtcgggtgatgcagctgatgagcagtgg
agtagaaaatgcactcaccaaatcagagctgctggtagaccagtacctccctcttaccaaggatgaactagaaaaa
gaagccaaaaaagtggaaggattcgatatggttcagaagccgagttactatgttagactgggatctctgtccacca
agctgcgctcacgggcctaccagcaggccctttgtagggttgaagaagctaagcgaaaaggccaggagaccatttc
tcagctccattccgccttcaacctgagtgaacttgccaggaagaatgtgcataatgccaaccagaaaattcaggat
gctcaggataagctctatctgtcctggctggagtggaagagaagcatcggctacgatgatacagatgaatcccact
gtgctgagcacattgagtcacgtactcttgctattgcccggaacctgactcagcagctccagaccatgtgccacac
cctcctgtccaacatccaggggttaccacagaacattcaagatcgggccaaccacttgggggtgatggctggtgac
atctactctgtgtttcgcaatgctgcctcctttaaagaagtgtctgatggcctcctcgcttccagcaaggggcagc
tgcagaaaatgaaggagtctttagatgatgtgatggattatcttgttaacaacacacccctcaactggctggtagg
tcccttttatccccaggtgaccgagtctgagagtgctcaggccccaggtacaaccaggaggcctggcaggtggagt
agaaaacaccctaaacccgtccctgtcagcaatgcagagggcagccagccagatgacagctcctcttga；
The primer sequence are as follows:
VN173-Plin2-F:
caagcttgcggccgcgaattcAGCATCTGTTGCAGTTGAACCA
VN173-Plin2-R:
ggtggcgatggatcttctagaAGAGGAGCTGTCATCTGGCTGG
Show the carrier ox PLIN2 interaction albumen bimolecular fluorescence complementary of above method building according to the experiment of applicant
Carrier pBiFC-VN173-PLIN2 into ox Primary adipocyte and can carry out follow-up study with Successful transfection.
The construction method of ox PLIN2 interaction albumen bimolecular fluorescence complementary carrier of the invention, it is mutual with existing research albumen
It is compared as method, it can the more efficient presence expressed and avoid false negative result.Meanwhile also further to study and construct albumen
Interaction network and the integrality for improving interactions between protein theory provide certain verification experimental verification means.
Detailed description of the invention
Fig. 1 is the area the CDs augmentation detection figure of PLIN2；
Fig. 2 is progress bacterium solution PCR race glue figure after homologous recombination；
Fig. 3 is 18 hours detection luciferase expression situations after plasmid corotation.
The present invention is described in further detail with example with reference to the accompanying drawing.
Specific embodiment
Mentality of designing of the invention is, the recombination of ox PLIN2 interaction carrier and its is deposited with destination protein in screening with verifying
Other albumen spatially to interact, the interactions between protein Study of Support is by by encoding amino acid sequence and pBiFC-
VN173 (bimolecular fluorescence complementary carrier N-terminal) carrier recombinates to construct destination protein expression vector.The pBiFC-VN173- of building
PLIN2 has the area the CDs overall length of complete ox PLIN2, and agarose gel electrophoresis detection PLIN2 target sequence is reassembled into carrier
A function pacing sequence testing goal carrier of going forward side by side recombinates successfully.Ox PLIN2 interactions between protein carrier is reassembled as the egg of in vitro study from now on
White interaction protein type provides the vector modality for stablizing expression.It efficiently solves in conventional authentication interactions between protein model
The condition of the difficult expression of ox PLIN2 (fat drips memebrane protein) in the common double miscellaneous experiments of yeast, it is strong to transfect observation fluorescence using ordinary cells
The method of degree can verify that other albumen for generating interaction with ox PLIN2 albumen, while can also save other experimental proteins
The complex steps such as extraction purification interact between ox PLIN2 albumen interaction target protein and following protein to find from now on
Functional experiment provides new reliable research method.
Carrier that following embodiment is related to, bacterial strain, cell, reagent source are as follows:
(1) DH5 α is purchased from Tiangeng biochemical technology Co., Ltd；
(2) primer synthesis is completed in Takara company and Invitrogen company；
(3) bimolecular fluorescence complementary carrier is purchased from BD biotechnology company；
(4) plasmid extraction kit is purchased from OMEGA Reagent Company；
(5) seamless Cloning Kit is purchased from Vazyme Reagent Company；
(6) the conventional molecular biologicals laboratory operating procedures such as digestion, connection, recycling, conversion, recombination, PCR amplification are detailed in
" molecular cloning (third edition) ".
(7) fetal calf serum be purchased from Invitrogen company, DMEM culture medium, it is dual anti-be purchased from Hyclone company, transfection reagent
Purchased from Roche company.
The present embodiment provides a kind of construction method of ox PLIN2 interaction albumen bimolecular fluorescence complementary carrier, with ox PLIN2
The area CDs full length amino acid sequence, by homologous recombination strategy design PLIN2 on bimolecular fluorescence complementary VN-173 carrier
Primer is recombinated the area CDs of PLIN2 by primer and pBiFC-VN173 carrier using KOD enzyme to construct destination protein expression and carry
Body, restriction enzyme site select EcoRI and XbaI enzyme cutting, and reaction temperature is 37 DEG C, and increases a base A behind the site EcoRI, is prevented
Only frameshift mutation finally obtains ox PLIN2 interaction albumen bimolecular fluorescence complementary carrier pBiFC-VN173-PLIN2.
The area the CDs full length amino acid sequence of the ox PLIN2 are as follows:
atggcatctgttgcagttgaaccacaactgagtgtggtgaccagggtggccaacctccccttggtgag
ctccacgtatgatctggtctcctcggcttacatcagtagaaaggatcagtatccttacttgaagtctctgtgtgag
atggcagagaagggcatgaagaccatcacttctgtggctgtgaccagcgcgctgccaatcatccaaaagttagagc
cacaaattgctgttgccaatacctatgcgtgtaaggggctagacaggattgaggagaagctgcctattctgaatca
gccaacaaaccaggttgtggccaatgccaaaggggctatgactggggcaaaagatgctgtgacgactactgtgacc
ggggccaaggattctgtggccagcacaatcacgggggtggtggacaggaccaagggagctgtgactggtagtgtgg
agaagaccaagtccgtggtcagtggcagcattaacacagtcctacgaagtcgggtgatgcagctgatgagcagtgg
agtagaaaatgcactcaccaaatcagagctgctggtagaccagtacctccctcttaccaaggatgaactagaaaaa
gaagccaaaaaagtggaaggattcgatatggttcagaagccgagttactatgttagactgggatctctgtccacca
agctgcgctcacgggcctaccagcaggccctttgtagggttgaagaagctaagcgaaaaggccaggagaccatttc
tcagctccattccgccttcaacctgagtgaacttgccaggaagaatgtgcataatgccaaccagaaaattcaggat
gctcaggataagctctatctgtcctggctggagtggaagagaagcatcggctacgatgatacagatgaatcccact
gtgctgagcacattgagtcacgtactcttgctattgcccggaacctgactcagcagctccagaccatgtgccacac
cctcctgtccaacatccaggggttaccacagaacattcaagatcgggccaaccacttgggggtgatggctggtgac
atctactctgtgtttcgcaatgctgcctcctttaaagaagtgtctgatggcctcctcgcttccagcaaggggcagc
tgcagaaaatgaaggagtctttagatgatgtgatggattatcttgttaacaacacacccctcaactggctggtagg
tcccttttatccccaggtgaccgagtctgagagtgctcaggccccaggtacaaccaggaggcctggcaggtggagt
agaaaacaccctaaacccgtccctgtcagcaatgcagagggcagccagccagatgacagctcctcttga。
The step of specific implementation, is:
1, ox PLIN2 gene order is inquired in ncbi database, design includes the primer including restriction enzyme site, passes through
Pcr clone ox PLIN2 gene C DS full length sequence；
2, ox PLIN2 gene is connected on BiFC-VN173 carrier by seamless clone, by being transformed into DH5 α large intestine
Bacillus competent cell, and select positive cell clone and carry out plasmid extraction, sequencing, obtain PLIN2-VN73 gene cloning load
Body；
3, that the PLIN2-VN73 recombinated is transfected into 293T with the general CGI-58-VC155 carrier previously built is thin
In born of the same parents, after 24 hours microscope detect luciferase expression situation, thus detect PLIN2 whether with CGI-58 occur interaction.
It is the specific embodiment that inventor provides below.
Embodiment 1: ox PLIN2 gene C DS sequence clone
This experiment uses BiFC expression vector system, the ox Plin2 gene order (Accession announced with GenBank
Number:NM_173980) it is template, designs and synthesizes primer, expands ox Mef2a gene C DS full length sequence.
The primer sequence is as follows:
Upstream primer (Forward primer):
5-caagcttgcggccgcgaattcAGCATCTGTTGCAGTTGAACCA-3；
Downstream primer (Reverse primer):
5-ggtggcgatggatcttctagaAGAGGAGCTGTCATCTGGCTGG-3；
Wherein, the protection base that " A " of upstream primer overstriking adds after being EcoRI, otherwise gene order meeting frameshift mutation, on
Swimming selected primer is EcoRI, and primer selected by downstream is XbaI.
This experiment carries out overall length amplification, amplification system with CDS area of the KOD-PLus High fidelity PCR enzyme to ox PLIN2 gene
And amplification condition is shown in Table 1, table 2, augmentation detection the result is shown in Figure 1.
Table 1:PCR amplification system
2:PCR response procedures
The building of embodiment 2:PLIN2-VN73
Under the conditions of 37 DEG C, digestion 2h is carried out to BiFC-VN73 zero load with EcoRI+XbaI enzyme, isometric PCR product is used
Homologous recombination is carried out with ClonExpress II One Step Cloning Kit with carrier after digestion, 37 DEG C of reaction 2h pass through
It is transformed into DH5 α competent escherichia coli cell, and selects positive cell clone and carries out plasmid extraction, sequencing etc., obtains PLIN2-
VN73 gene clone carrier., carry out bacterium solution PCR after homologous recombination and run glue figure as shown in Fig. 2, linked system see the table below 3.
Run glue as the result is shown: purpose band becomes clear single, and size is correct, and can carry out subsequent glue recovery test is homologous recombination
It prepares.
It is compared through sequencing result, ox PLIN2 is without mutant nucleotide sequence success and bimolecular fluorescence complementary carrier VN-173 carrier weight
Group can be used for the interaction development test of its subsequent albumen as a purpose.
Table 3:PLIN2-VN73 linked system
Embodiment 3:PLIN2-VN73 and CGI-58-VC155 corotation 293T cell line
It is 80% progress transfection assay by 293T cell line culture to cell density, after plasmid passes through transfection reagent cotransfection
(Fig. 3) is observed under fluorescence microscope after 18 hours, finds apparent yellow fluorescence occur in 293T cytoplasm at this time, then
Illustrate destination protein of the ox PLIN2 albumen as research, is that there are interactions with ox CGI-58 albumen.Picture scale is
100μm。
Nucleotides sequence list
<110>Xibei Univ. of Agricultural & Forest Science & Technology
<120>construction method of ox PLIN2 interaction albumen bimolecular fluorescence complementary carrier
<160>
<210>1
<211>1353
<212>area the CDs full length amino acid sequence of ox PLIN2
<213>DNA
<220>
<400>
atggcatctgttgcagttgaaccacaactgagtgtggtgaccagggtggccaacctccccttggtgagctcc
acgtatgatctggtctcctcggcttacatcagtagaaaggatcagtatccttacttgaagtctctgtgtgagatgg
cagagaagggcatgaagaccatcacttctgtggctgtgaccagcgcgctgccaatcatccaaaagttagagccaca
aattgctgttgccaatacctatgcgtgtaaggggctagacaggattgaggagaagctgcctattctgaatcagcca
acaaaccaggttgtggccaatgccaaaggggctatgactggggcaaaagatgctgtgacgactactgtgaccgggg
ccaaggattctgtggccagcacaatcacgggggtggtggacaggaccaagggagctgtgactggtagtgtggagaa
gaccaagtccgtggtcagtggcagcattaacacagtcctacgaagtcgggtgatgcagctgatgagcagtggagta
gaaaatgcactcaccaaatcagagctgctggtagaccagtacctccctcttaccaaggatgaactagaaaaagaag
ccaaaaaagtggaaggattcgatatggttcagaagccgagttactatgttagactgggatctctgtccaccaagct
gcgctcacgggcctaccagcaggccctttgtagggttgaagaagctaagcgaaaaggccaggagaccatttctcag
ctccattccgccttcaacctgagtgaacttgccaggaagaatgtgcataatgccaaccagaaaattcaggatgctc
aggataagctctatctgtcctggctggagtggaagagaagcatcggctacgatgatacagatgaatcccactgtgc
tgagcacattgagtcacgtactcttgctattgcccggaacctgactcagcagctccagaccatgtgccacaccctc
ctgtccaacatccaggggttaccacagaacattcaagatcgggccaaccacttgggggtgatggctggtgacatct
actctgtgtttcgcaatgctgcctcctttaaagaagtgtctgatggcctcctcgcttccagcaaggggcagctgca
gaaaatgaaggagtctttagatgatgtgatggattatcttgttaacaacacacccctcaactggctggtaggtccc
ttttatccccaggtgaccgagtctgagagtgctcaggccccaggtacaaccaggaggcctggcaggtggagtagaa
aacaccctaaacccgtccctgtcagcaatgcagagggcagccagccagatgacagctcctcttga
<210>2
<211>43
<212>primer sequence VN173-Plin2-F
<213>DNA
<220>
<400>
caagcttgcggccgcgaattcAGCATCTGTTGCAGTTGAACCA
<210>3
<211>43
<212>primer sequence VN173-Plin2-R
<213>DNA
<220>
<400>
ggtggcgatggatcttctagaAGAGGAGCTGTCATCTGGCTGG
Claims (2)
1. a kind of construction method of ox PLIN2 interaction albumen bimolecular fluorescence complementary carrier, which is characterized in that this method is with ox
The area the CDs full length amino acid sequence of PLIN2, by designing primer of the PLIN2 on bimolecular fluorescence complementary VN-173 carrier, with
PBiFC-VN173 carrier recombinates to construct destination protein expression vector, and restriction enzyme site selects EcoRI and XbaI enzyme cutting, reaction temperature
Degree is 37 DEG C, and increases a base A behind the site EcoRI, prevents frameshift mutation, and it is double to finally obtain ox PLIN2 interaction albumen
Molecular fluorescence complementing vector pBiFC-VN173-PLIN2.
2. the method as described in claim 1, which is characterized in that the area the CDs full length amino acid sequence of the ox PLIN2 are as follows:
atggcatctgttgcagttgaaccacaactgagtgtggtgaccagggtggccaacctccccttggtgagctcc
acgtatgatctggtctcctcggcttacatcagtagaaaggatcagtatccttacttgaagtctctgtgtgagatgg
cagagaagggcatgaagaccatcacttctgtggctgtgaccagcgcgctgccaatcatccaaaagttagagccaca
aattgctgttgccaatacctatgcgtgtaaggggctagacaggattgaggagaagctgcctattctgaatcagcca
acaaaccaggttgtggccaatgccaaaggggctatgactggggcaaaagatgctgtgacgactactgtgaccgggg
ccaaggattctgtggccagcacaatcacgggggtggtggacaggaccaagggagctgtgactggtagtgtggagaa
gaccaagtccgtggtcagtggcagcattaacacagtcctacgaagtcgggtgatgcagctgatgagcagtggagta
gaaaatgcactcaccaaatcagagctgctggtagaccagtacctccctcttaccaaggatgaactagaaaaagaag
ccaaaaaagtggaaggattcgatatggttcagaagccgagttactatgttagactgggatctctgtccaccaagct
gcgctcacgggcctaccagcaggccctttgtagggttgaagaagctaagcgaaaaggccaggagaccatttctcag
ctccattccgccttcaacctgagtgaacttgccaggaagaatgtgcataatgccaaccagaaaattcaggatgctc
aggataagctctatctgtcctggctggagtggaagagaagcatcggctacgatgatacagatgaatcccactgtgc
tgagcacattgagtcacgtactcttgctattgcccggaacctgactcagcagctccagaccatgtgccacaccctc
ctgtccaacatccaggggttaccacagaacattcaagatcgggccaaccacttgggggtgatggctggtgacatct
actctgtgtttcgcaatgctgcctcctttaaagaagtgtctgatggcctcctcgcttccagcaaggggcagctgca
gaaaatgaaggagtctttagatgatgtgatggattatcttgttaacaacacacccctcaactggctggtaggtccc
ttttatccccaggtgaccgagtctgagagtgctcaggccccaggtacaaccaggaggcctggcaggtggagtagaa
aacaccctaaacccgtccctgtcagcaatgcagagggcagccagccagatgacagctcctcttga；
The primer sequence are as follows:
VN173-Plin2-F:
CaagcttgcggccgcgaattcAGCATCTGTTGCAGTTGAACCA:
VN173-Plin2-R:
ggtggcgatggatcttctagaAGAGGAGCTGTCATCTGGCTGG。
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| CN105177041A (en) * | 2015-11-04 | 2015-12-23 | 中国热带农业科学院热带生物技术研究所 | Expression vector for bimolecular fluorescence complementation) research and application thereof |
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| CN105177041A (en) * | 2015-11-04 | 2015-12-23 | 中国热带农业科学院热带生物技术研究所 | Expression vector for bimolecular fluorescence complementation) research and application thereof |
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| Title |
|---|
| NIELSEN R.L等: "NM_173980.1", 《NCBI》 * |
| PEIWEI LI等: "The Expression Pattern of PLIN2 in Differentiated Adipocytes from Qinchuan Cattle Analysis of Its Protein Structure and Interaction with CGI-58", 《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》 * |
| Y. JOHN SHYU等: "Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions", 《BIOTECHNIQUES》 * |
| 胡濒月等: "PLIN2对脂类代谢的调控研究", 《中国医药科学》 * |
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