CN103820403B - A kind of Δ 4 fatty acid desaturase deriving from basket fish and application thereof - Google Patents
A kind of Δ 4 fatty acid desaturase deriving from basket fish and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of Δ 4 fatty acid desaturase deriving from basket fish and application thereof.A kind of albumen disclosed by the invention, shown in following (1) or (2): the albumen shown in (1) SEQ ID No.2；(2) by the aminoacid sequence shown in SEQ ID No.2 through the identical protein of the replacement of one or several amino acid residue and/or disappearance and/or interpolation and function.A kind of Δ 4 fatty acid desaturase disclosed by the invention can promote to produce in mammal the important long-chain polyunsaturated fatty acids such as high-caliber DHA, also the DPA utilizing various method to produce in mammalian cell or animal can be converted into DHA simultaneously.
Description
Technical field
The present invention relates to a kind of Δ 4 fatty acid desaturase deriving from basket fish and application thereof.
Background technology
Fatty acid is divided into satisfied fatty acid, monounsaturated fatty acid and polyunsaturated fatty acid (PUFAs).Wherein, PUFAs
Human body is had unique function and significance, is the class fatty acid that is widely studied and receives much concern.PUFAs includes
ω-3 and ω-6PUFAs two types, each type includes again that multiple carbon chain lengths is different, unsaturated bond quantity one-tenth not etc.
Member.The PUFAs of carbon chain lengths >=20 is referred to as long-chain polyunsaturated fatty acid (LCPUFAs), and most important LCPUFAs includes
DHA(22:6n-3), EPA(20:5n-3) and ARA(20:4n-6, i.e. arachidonic acid), they be biological in vivo bioactivity
Three kinds of strong unsaturated fatty acids, it is verified that promoting brain development and function to maintain and at prevention and treatment cardiovascular
The multiple disease aspect important roles such as disease, inflammation, cancer.Dynamic relative to some other biology, the mankind and other sucklings
The ability of thing self synthesis PUFAs is the lowest, and reason is as follows: first, and mammal can not start to synthesize PUFAs from S-acetyl-coenzyme-A,
It is only capable of with food-based LA(18:2n-6, i.e. linoleic acid) and ALA(18:3n-3, i.e. alpha-linolenic acid) it is substrate, utilize
The fatty acid desaturase of self and prolongation enzymatic activity synthesize corresponding PUFAs；Secondly, in mammal, these fatty acids go to satisfy
It is the most limited with enzyme and prolongation enzymatic activity, therefore can only finally synthesize limited sub-fraction PUFAs；Again, in mammal
Also lack fatty acid desaturase that some other biologicals have and extend enzymatic activity and make some many insatiable hungers of important long-chain
Synthesis with fatty acid especially DHA is extremely difficult.Therefore, more PUFAs demand is relied primarily on from food source by human body
Obtain.But ω-6PUFAs is abundant and ω-3PUFAs is rare in nature, thus cause the ω that takes in human body and accumulate-
6PUFAs is too much and ω-3PUFAs is very few.And mammal does not exist the enzyme making ω-6PUFAs be changed into ω-3PUFAs and lives
Property, the ratio serious unbalance of ω-6PUFAs and ω-3PUFAs in old friend's body.Although ω-6PUFAs is the most indispensable to human body,
But the absorption of its excess is detrimental to health on the contrary.
The picked-up that the life of modern and eating pattern result in fatty acid is the most uneven.This out-of-balance mass now with
Under several levels.First level is the imbalance between satisfied fatty acid and unsaturated fatty acid, i.e. majority of case servant
Body has taken in too much satisfied fatty acid (essentially from animal oil), and unsaturated fatty acids subacidity；Second level is to take the photograph
In the unsaturated fatty acid entered, ω-6 is unsaturated fatty acid and ω-3 is the imbalance between unsaturated fatty acid, i.e. ω-6 is
Unsaturated fatty acids hyper acid and ω-3 is that hypoacidity for unsaturated fatty acids；3rd level is in the unsaturated fatty acid taken in, short
Chain unsaturated fatty acid (18C) and the imbalance of long-chain unsaturated fatty acid (20C～22C), i.e. short chain unsaturated fatty acid is many,
Long-chain unsaturated fatty acid is few.Great majority conventional vegetable oil such as soybean oil, Oleum Arachidis hypogaeae semen, Oleum Helianthi etc. all contain high level
ω-6 i.e. LA(18:2n-6 of unsaturated fatty acid of short chain, i.e. linoleic acid), Semen Lini oil, Oleum Perillae etc., although containing Gao Shui
The omega-3 unsaturated fatty acid of flat short chain i.e. ALA(18:3n-3, i.e. alpha-linolenic acid), but they are not conventional in daily life
Oil.Bathypelagic fish oil is the main provider of long-chain polyunsaturated fatty acid EPA and DHA, but expensive, resource scarcity, edible
Crowd is limited, so it does not has changes human adipose's acid unbalanced state of absorption on a large scale.As can be seen here, fatty acid
Imbalance be just embodied in DHA(22:6n-3), EPA(20:5n-3) and ARA(20:4n-6) these three the most bioactive
The shortage of long-chain polyunsaturated fatty acid.That is, if it is possible to obtain abundant DHA(22:6n-3), EPA(20:5n-3)
And ARA(20:4n-6) resource for human consumption just can change current human adipose's acid picked-up unbalanced present situation.
How to obtain abundant DHA(22:6n-3), EPA(20:5n-3) and ARA(20:4n-6) resource be world today's model
Enclose an interior people problem of interest.Face resource exhaustion in bathypelagic fish oil and pollute under the most serious present situation, utilizing
EPA(20:5n-3 such as some marine algae and Mycophytas etc.), DHA(22:6n-3) initial production person obtain the many insatiable hungers of these long-chains
Becoming well selection with fatty acid, current exploitation also obtains the biggest success.But, these products add usually used as food
Agent uses certain limitation, and it preserves needs strict measure, its taste is the best is difficult to life-time service.It is contemplated that such as
Really terraria can provide DHA(22:6n-3 such as domestic animals such as pig, cattle and sheeps as bathypelagic fish), EPA(20:5n-3) and
Etc. ARA(20:4n-6) if unsaturated fatty acid, then the rich of its resource will produce revolutionary change, and not
Just can obtain these important fatty acids in the case of impact and change people's living habit, this is deep by producing to human health
Remote impact.But, as previously mentioned, mammal synthesis DHA(22:6n-3), EPA(20:5n-3) and ARA(20:
It is 4n-6) extremely limited, its content will be improved, be necessary for setting about from corresponding polyunsaturated fatty acid synzyme.It is correlated with
Research foundation is it has been shown that produce these long-chain polyunsaturated fatty acids by Transgenic animal technology and be in mammal
Possible.1997, James P etc. is first animal Δ 15 desaturase base of screening and cloning from nematicide C.elegans
Cause, named fat-1, follow-up research find fat-1 gene 16～20 carbon ω-6PUFAs desaturations can be become ω-
3PUFAs.Fat-1 gene is proceeded to mammalian cell by Zhao B.Kang etc., and ω-6/ ω-3PUFAs drops to than from 15:1
1:1, the ω-3PUFAs content making mammal inhale significantly increases.In 2004, fat-1 gene is proceeded to mice the most again,
ω-the 3PUFAs such as ALA, EPA, DPA are significantly increased.2006, the transgenic pig of fat-1 prepared by Lai etc., also show
Go out the situation similar with transgenic mice.But, in ω-3PUFAs synthesized in these transgenic animal bodies with 18C,
ω-the 3PUFAs of 20C is main, and the DHA(22:6n-3 of even more important 22C) total amount is the most little.It is true that follow-up one
A little researchs show that the Δ 15 desaturase activity of fat-1 can not increase the DHA(22:6n-3 in these animal bodies) content.Cause
This, fat-1 gene is in the practice improving mammal long-chain polyunsaturated fatty acid content, and critical shortcoming is exactly not
DHA(22:6n-3 can be increased) content.
It is true that DHA(22:6n-3) synthesis in mammal is the most complicated, so-called through one accordingly
" Sprecher path ", EPA(20:5n-3) extend one intermediate product DPA(22:5n-3 of generation) after, further extend to
24:5n-3, is generated 24:6n-3 by △ 6 desaturase effect the most rapidly, produces DHA through betaoxidation the most further
(22:6n-3), the mechanism of this complexity is probably mammal and is difficult to synthesize high level DHA(22:6n-3) reason.Relevant many
Unsaturated fatty acid biosynthesis pathway is as shown in Figure 1.
Summary of the invention
It is an object of the invention to provide a kind of Δ 4 fatty acid desaturase deriving from basket fish and application thereof.
A kind of albumen that the present invention provides, shown in following (1) or (2):
(1) albumen shown in SEQ ID No.2；
(2) aminoacid sequence shown in SEQ ID No.2 through the replacement of one or several amino acid residue and/or is lacked
Lose and/or add and the identical protein of function.
The encoding gene of above-mentioned albumen falls within protection scope of the present invention.
In above-mentioned encoding gene, described encoding gene be following at least one:
1) in SEQ ID No.1 from 5 ' ends the DNA molecular shown in the 10th to the 1347th nucleotide；
2) under strict conditions with 1) DNA molecule hybridize that limits and the DNA molecular of code for said proteins；
3) with 1) or 2) DNA molecular that limits has the homogeneity of more than 90% and the DNA molecular of code for said proteins.
Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing any of the above-described described encoding gene fall within
Protection scope of the present invention.
A kind of protein composition falls within protection scope of the present invention, by the albumen shown in SEQ ID No.2 and SEQ ID
Albumen composition shown in No.4.
A kind of protein composition falls within protection scope of the present invention, by the albumen shown in SEQ ID No.2, SEQ ID
Albumen shown in No.6 and the composition of the albumen shown in SEQ ID No.8.
A kind of improving DHA(22:6n-3 in mammalian cell) method of synthesis capability falls within the protection model of the present invention
Enclose, comprise the steps: to import to set out in cell by the encoding gene of described albumen, obtain transgenic cell；Thin with setting out
Born of the same parents compare, the DHA(22:6n-3 of transgenic cell) synthesis capability raising.
In said method, described encoding gene is imported by recombinant expression carrier, described recombinant expression carrier be by
Described encoding gene insert the carrier pcDNA3.1 of setting out (-) multiple clone site obtain.
Above-mentioned albumen promotes DHA(22:6n-3 in preparation) application in the product that synthesizes falls within the protection model of the present invention
Enclose；
Or,
Any of the above-described described protein composition promotes DHA(22:6n-3 in preparation) application in the product that synthesizes also belongs to
In protection scope of the present invention.
In preparation, above-mentioned albumen promotes that the application that DPA (22:5n-3) changes in the product of DHA (22:6n-3) falls within
Protection scope of the present invention；
Or,
Any of the above-described described protein composition changes into the product of DHA (22:6n-3) in preparation promotion DPA (22:5n-3)
Application in product falls within protection scope of the present invention；
Or,
In preparation, above-mentioned albumen promotes that DPA (22:5n-3) is changed into DHA (22:6n-3) by △ 15 fatty acid desaturase
Product in application fall within protection scope of the present invention；
Or,
In preparation, above-mentioned albumen promotes that LA (18:2n-6) and/or ARA (20:4n-6) is turned by △ 15 fatty acid desaturase
Application in the product of chemical conversion DHA (22:6n-3) falls within protection scope of the present invention；
The protein composition being made up of the albumen shown in SEQ ID No.2 and the albumen shown in SEQ ID No.4 is in preparation
The application changed into by LA (18:2n-6) and/or ARA (20:4n-6) in the product of DHA (22:6n-3) falls within the present invention's
Protection domain；
The aminoacid sequence of described △ 15 fatty acid desaturase is as shown in SEQ ID No.4；
Or,
In preparation, above-mentioned albumen promotes that Δ 6-fatty acid desaturase and Δ 5-fatty acid desaturase are by DPA (22:5n-
3) application changed in the product of DHA (22:6n-3) falls within protection scope of the present invention；
In preparation, above-mentioned albumen promotes that Δ 6-fatty acid desaturase and Δ 5-fatty acid desaturase are by LA (18:2n-6)
And/or the application that ALA (18:3n-3) changes in the product of DHA (22:6n-3) falls within protection scope of the present invention；
By the albumen shown in the albumen shown in SEQ ID No.2, SEQ ID No.6 and the albumen shown in SEQ ID No.8
LA (18:2n-6) and/or ALA (18:3n-3) is changed into the product of DHA (22:6n-3) by the protein composition of composition in preparation
In application fall within protection scope of the present invention；
The aminoacid sequence of described Δ 6-fatty acid desaturase is as shown in SEQ ID No.6；
The aminoacid sequence of described Δ 5-fatty acid desaturase is as shown in SEQ ID No.8.
A kind of Δ 4 fatty acid desaturase that the present invention provides, by coordinating or other fatty acid desaturase (Δs collaborative
15 or Δ 6/ Δ 5) effect, mammalian cell that gene transfects and the initial fat that transgenic animal will be added can be promoted
Fat acid substrate such as LA (18:2n-6) or ALA (18:3n-3) transform into high-caliber DHA(22:6n-3) etc. important long-chain many
Unsaturated fatty acid, also can be converted into DHA by the DPA utilizing various method to produce in mammalian cell or animal simultaneously.
Accompanying drawing explanation
Fig. 1 is polyunsaturated fatty acid biosynthesis pathway.
Fig. 2 is pcDNA3.1-sScD4 carrier schematic diagram.
Fig. 3 is to turn the expression identification of genes of interest in sScD4 gene cell.
Fig. 4 is GC-MS testing result.
Fig. 5 is the qualification of transgenic cell in embodiment 2.
Fig. 6 is the synergism analysis of Δ 4 fatty acid desaturase and △ 15 fatty acid desaturase.
Fig. 7 is the qualification of transgenic cell in embodiment 3.
Fig. 8 is the synergism analysis of Δ 4 fatty acid desaturase and Δ 6/ Δ 5 fatty acid desaturase.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Chinese hamster ovary (CHO) cell is purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's preclinical medicine cell
The heart.
PEGFP-N1 is purchased from Clontech company.
PcDNA3.1 (-) purchased from Invitrogen company.
The construction method of pcDNA3.1-EGFP is as follows:
One, with pEGFP-N1 as template, with primer fG-s:5 '-TCTAGATCGCCACCATGGTGAGCAAGG-3 ' and fG-
A:5 '-CTCGAGCTTGTACAGCTCGTCCATGCCGAG-3 ' carries out PCR amplification, obtains pcr amplification product, and this product is
EGFP gene coding region (725bp)；
Two, the pcr amplification product that Xba I and Xho I double digestion step one obtain, obtains genetic fragment；Xba I and Xho
I double digestion pcDNA3.1(-), obtain carrier large fragment；Genetic fragment is connected with carrier large fragment, obtains recombiant plasmid, will
Its named pcDNA3.1-EGFP.
The construction method of pcDNA3.1-F2F1 is at document " Wang Kunfu, Zhu Guiming, Zhang Li etc..The many bases of fatty acid synthetase system
Structure because of expression vector.Aged in China magazine, 2013,33(17), 4178-4180 " disclosed in mistake, the public can be from Jiamusi
University obtains.
Embodiment 1, the synthesis of Δ 4 fatty acid desaturase gene (sScD4 gene) and functional verification thereof
One, the synthesis of Δ 4 fatty acid desaturase gene (sScD4 gene)
Δ 4 fatty acid desaturase gene to the Lan Ziyu (Siganus canaliculatus) submitted in GenBank
The cDNA sequence (encoder block total length is 1338bp) of (No. GenBank: GU594278) carries out codon optimized, is allowed to meet suckling
Animal gene codon usage bias.Use the secondary structure of its mRNA of software prediction after optimization, verify codon optimized whether
Rationally.
In the nucleotide sequence such as SEQ ID No.1 of the Δ 4 fatty acid desaturase gene (sScD4 gene) after optimization certainly
5 ' ends rise shown in the 10th to the 1347th nucleotide, and the aminoacid sequence of its coding is as shown in SEQ ID No.2.
Sequence after optimizing plus EcoRI and HindIII restriction enzyme site, then carries out the artificial of full-length gene at two ends
Synthesis, full length sequence is as shown in SEQ ID No.1.
Two, the DNA molecular shown in EcoRI and HindIII double digestion SEQ ID No.1, obtains genetic fragment；EcoRI and
HindIII double digestion eukaryotic expression vector pcDNA3.1 (-), obtain carrier large fragment；By genetic fragment with carrier large fragment even
Connect, obtain recombinant expression plasmid, by its named pcDNA3.1-sScD4.PcDNA3.1-sScD4 send order-checking, and result is correct.
The schematic diagram of pcDNA3.1-sScD4 carrier is as shown in Figure 2.
Three, the functional verification of Δ 4 fatty acid desaturase gene (sScD4 gene)
(1) cultivation of Chinese hamster ovary celI
Culture medium forms: DMEM high glucose medium, 10% hyclone, 1% mycillin (100 ×), 1% nonessential ammonia
Base acid, 1g/100ml Sodium Pyruvate, 0.22 μM of membrane filtration is degerming, and % all represents volumn concentration.
Chinese hamster ovary (CHO) cell is with 5 × 105/ ml is inoculated in culture bottle, in 37 DEG C, 5%CO2And saturated humidity
Incubator in cultivate.
(2) liposome method transient transfection Chinese hamster ovary celI
1, the preparation before transfection: plasmid to be transfected is pcDNA3.1-sScD4(experimental group), with glimmering containing enhanced green
The recombiant plasmid pcDNA3.1-EGFP of photoprotein is that comparison carries out transfecting (matched group), monitors transfection efficiency.Day before transfection will
Cultivate Chinese hamster ovary celI be passaged to 60mm culture dish, add the fatty acid substrate DPA(22:5n-3 of 10 μm ol/L), when cell length extremely
90%-95% transfects.
2, transfection: add 500 μ l DMEM basal mediums in 1.5ml EP pipe, add 8 μ g pcDNA3.1-sScD4
Or mix gently after pcDNA3.1-EGFP, it is designated as A liquid；500 μ l DMEM basal mediums are added in another 1.5ml EP pipe, then
Add 20 μ l transfection reagent LipofectamineTMMix homogeneously after 2000, is designated as B liquid, incubated at room 5min.A and B is added to
Mixing in one pipe, room temperature stands 20min.The 60mm culture dish of the Chinese hamster ovary celI that then this mixed liquor is added to step 1 mixes gently
Even, 37 DEG C, 5%CO2After cultivating 4-6h, inhale and abandon the liquid in hole, change fresh culture (the same DPA containing 10 μm ol/L) and cultivate.
After transfection, next day starts available fluorescence microscope transfected condition, collects the cell of experimental group and the matched group obtained after 48h
For subsequent detection and experiment.
(3) RT-PCR carries out the qualification of transgenic cell
1, the extraction of total serum IgE: the cell TRizol reagent taking the experimental group after transfection 48h and matched group extracts total serum IgE,
And measure RNA concentration, finally the RNA of extraction is placed in-80 DEG C of ultra cold storage freezers and saves backup.
2, the reverse transcription of mRNA: be cDNA by the mRNA reverse transcription of experimental group and matched group, reverse transcription reaction condition is 42
℃30min；95℃5min；5℃5min；4 DEG C of preservations.After reverse transcription terminates, cDNA is placed in-20 DEG C and saves backup.
3, the expression of PCR testing goal gene sScD4, and with β-actin as reference gene
1. the detection primer of sScD4 gene:
D4-s:5 '-TCGGGCATCTGAGCGTGTTC-3 ' Tm:64.1 DEG C
D4-a:5 '-ATACTCCACGGGCTGGGTCTCC-3 ' Tm:66.2 DEG C
Purpose product length is 203bp.
2. β-actin reference gene detection primer:
Bact-s:5 '-CTGAGAGGGAAATCGTGCGTGAC-3 ' Tm:65.7 DEG C
Bact-a:5 '-TGCCACAGGATTCCATACCCAAG-3 ' Tm:65.7 DEG C
Purpose product length is 210bp.
With the cDNA of experimental group of step 2 preparation as template, carry out PCR amplification with D4-s and D4-a for primer, obtain PCR
Amplified production 1.
With the cDNA of matched group of step 2 preparation as template, carry out PCR amplification with Bact-s and Bact-a for primer,
To pcr amplification product 2.
Taking pcr amplification product 1 and 2 and carry out 1% agarose gel electrophoresis, result is as shown in Figure 3.
In Fig. 3,1 is pcr amplification product 2；2 is pcr amplification product 1.
Fig. 3 shows, transcribing of genes of interest sScD4 detected, and transfect in the Chinese hamster ovary celI of pcDNA3.1-sScD4 transfection
The Chinese hamster ovary celI of the recombiant plasmid pcDNA3.1-EGFP containing enhanced green fluorescence protein cannot detect genes of interest sScD4
Transcribe, it was demonstrated that experimental group turns sScD4 gene cell and successfully constructs.
(4) the composition change of gas chromatography-mass spectrography analytical technology (GC-MS) detection fatty acid
1, the extraction of total fatty acids: the cell in the culture dish of the experimental group after transfection 48h and matched group is used respectively
0.4ml trypsin 37 DEG C digestion 3min, terminates the effect of Trypsin, rinses one with deionized water with 1ml containing blood serum medium
Secondary, 1000rpm, centrifugal 5min, adding 1ml volumn concentration is 2.5%H2SO4Methanol solution, mix gently, 80 DEG C of water-baths
90min, is cooled to room temperature, adds NaCl solution and the 1ml normal hexane of 1.5ml0.9g/100ml, concussion, 2000rpm, is centrifuged
5min, is extracted into fatty acid in organic facies (i.e. normal hexane), draws the organic extractant phase thing of experimental group or matched group through nitrogen
Analyze for GC-MS detection after drying up concentration, or save backup in-80 DEG C.
2, GC-MS detection: using instrument during detection is HP-5890/HP-5971 Gc-ms instrument, and experiment condition is pressed
The detection of more solito unsaturated fatty acid is analyzed requirement and is carried out, particular reference " Kang ZB, Ge Y, Chen Z, et
al.Adenoviral gene transfer of Caenorhabditis elegans n-3fatty acid
desaturase optimizes fatty acid composition in mammalian cells.Proc Natl Acad
Sci USA,2002,98(7):4050–4054”。
As shown in Figure 4, in Fig. 4, A is matched group GC-MS testing result to result；B is experimental group GC-MS testing result.
Fig. 4 shows, after adding DPA (22:5n-3) substrate, experimental group is notable compared with the synthetic quantity of matched group DHA (22:6n-3)
Increase, difference are extremely notable, illustrate that sScD4 gene expression △ 4 fatty acid desaturase is during synthesis DHA (22:6n-3)
Play effect, DPA (22:5n-3) has been directly translated into as DHA (22:6n-3).
Embodiment 2, in mammalian cell △ 4 fatty acid desaturase of sScD4 gene code and △ 15 fatty acid
The synergism of desaturase
One, according to research report, the fat-1 gene (No. GenBank: NM_ of nematicide C.elegans is derived from
001028389) expression product have △ 15 fatty acid desaturase activity, fat-1 gene is carried out codon optimized design,
The sequence of synthetic is as shown in SEQ ID No.3.
In the coding gene sequence such as SEQ ID No.3 of this △ 15 fatty acid desaturase from 5 ' ends the 13rd to
Shown in 1221 nucleotide, the aminoacid sequence of △ 15 fatty acid desaturase is as shown in SEQ ID No.4.
Recombinant expression plasmid pcDNA3.1-sD15 is obtained according to the method for embodiment 1 step 2.
Two, according to the cell transfecting method of step 3 in example 1, respectively by expression plasmid pcDNA3.1-EGFP,
PcDNA3.1-sD15, pcDNA3.1-sD15+pcDNA3.1-sScD4 transfection is plated on the Chinese hamster ovary celI of 60mm plate, Mei Gezhuan
Plasmid used by dye group is two kinds of each 4 μ g of plasmid in 8 μ g(pcDNA3.1-sD15+pcDNA3.1-sScD4), and cultivate with cell
Substrate linoleic acid LA (18:2n-6) and 10 μm ol/L arachidonic acid ARA (20:4n-6) of adding 10 μm ol/L in base are cultivated
After 48h, collecting cell, a part is used for extracting total serum IgE, carries out RT-PCR qualification and the transcriptional level of detection gene expression, side
Method is with (three) of step 3 in embodiment 1, and result is as shown in Figure 5；Another part is used for extracting fatty acid in cell, carries out GC-
MS analyzes fatty acid composition, and method is with (four) of step 3 in embodiment 1, and result is as shown in Figure 6.
In Fig. 5,1 represents pcDNA3.1-EGFP transfection group (matched group)；2 represent pcDNA3.1-sD15 transfection group；3 represent
PcDNA3.1-sD15+pcDNA3.1-sScD4 transfection group.
Fig. 5 shows, genes of interest sD15, sD15 and sScD4, it is anticipated that achieve expression in corresponding cell, do not contain
The pcDNA3.1-EGFP transfection group (matched group) of destination gene expression plasmid then can't detect the transcription product of genes of interest.
In Fig. 6, EGFP represents pcDNA3.1-EGFP transfection group (matched group)；SD15 represents pcDNA3.1-sD15 transfection
Group；SD15+sScD4 represents pcDNA3.1-sD15+pcDNA3.1-sScD4 transfection group.
The a part of LA (18:2n-6) added is converted into ALA (18:3n-3) (this part by △ 15 fatty acid desaturase
ALA (18:3n-3) becomes EPA (20:5n-3) by ω-3 approach after two step metabolism), another part LA (18:2n-6) is by cell
The interior desaturase being had itself and prolongation enzymatic activity are transformed as ARA (20:4n-6) by two steps.This part ARA
(20:4n-6) it is partially converted into EPA (20:5n-3) by △ 15 fatty acid desaturase together with the ARA (20:4n-6) added.
Still some ARA (20:4n-6) is not converted to as EPA (20:5n-3) then by the intracellular prolongation enzyme being had itself
Active Transforming for ADA (22:4n-6).ADA (22:4n-6) now can be converted into DPA (22:5n-by △ 15 fatty acid desaturase
3), some DPA (22:5n-3) is produced through intracellular prolongation enzyme effect by EPA (20:5n-3).Pass through
The amount that DPA (22:5n-3) is transformed into DHA (22:6n-3) by Sprecher path is not notable, but transfects at sD15+sScD4
The DPA (22:5n-3) that then can be will build up on further by △ 4 fatty acid desaturase in group is converted into DHA (22:6n-
3), the increase of its quantity is the most significant.The metabolic conversion process of above-mentioned polyunsaturated fatty acid can be found in Fig. 1.
Fig. 6 shows, compared with matched group, in two groups of transfection group of sD15, sD15+sScD4 ω-6 be PUFAs such as LA (18:
2n-6), ARA (20:4n-6) and ADA (22:4n-6) all significantly reduce, and ω-3 is ALA in PUFAs (18:3n-3) and EPA
(20:5n-3) all dramatically increase.But, DPA (22:5n-3) and DHA (22:6n-3) situation is entirely different: compared with matched group,
In sD15 transfection group, DPA (22:5n-3) dramatically increases, and in sD15+sScD4 transfection group, DPA (22:5n-3) content is basic with right
Maintain an equal level according to group；In sD15 transfection group, DHA (22:6n-3) content is basic and matched group maintains an equal level, and DHA in sD15+sScD4 transfection group
(22:6n-3) content then dramatically increases (its content accounts for nearly the 10% of polyunsaturated fatty acid total content).
Result shows, △ 4 fatty acid desaturase of sScD4 gene code can collaborative with △ 15 fatty acid desaturase be made
With, in mammalian cell, utilize △ 4 fatty acid desaturase of sScD4 gene code can promote that △ 15 fatty acid goes
Saturated enzymatic activity, is converted into the DHA (22:6n-3) of higher level by the linoleic acid of interpolation and arachidonic acid, and only adds △
15 fatty acid desaturases are compared, and content improves about 1 times.
Embodiment 3, in mammalian cell △ 4 fatty acid desaturase of sScD4 gene code and △ 6/ △ 5 fat
The synergism of acid desaturase
PcDNA3.1-F2F1 be containing derive from mice Δ 6-fatty acid desaturase gene fads2 (No. GenBank:
And the double gene expression vector of Δ 5-fatty acid desaturase gene fads1 (No. GenBank: BC063053) BC057189).
The coding gene sequence of Δ 6-fatty acid desaturase as shown in SEQ ID No.5, the aminoacid sequence of this albumen
As shown in SEQ ID No.6.
The coding gene sequence of Δ 5-fatty acid desaturase as shown in SEQ ID No.7, the aminoacid sequence of this albumen
As shown in SEQ ID No.8.
According to the cell transfecting method of step 3 in example 1, respectively by expression plasmid pcDNA3.1-EGFP, pcDNA3.1-
F2F1, pcDNA3.1-F2F1+pcDNA3.1-sScD4 transfection is plated on the Chinese hamster ovary celI of 60mm plate, matter used by each transfection group
Grain is two kinds of each 4 μ g of plasmid in 8 μ g(pcDNA3.1-F2F1+pcDNA3.1-sScD4), and in cell culture medium, add 10
After fatty acid substrate linoleic acid LA (18:2n-6) of μm ol/L and 10 μm ol/L alpha-linolenic acid ALA (18:3n-3) cultivate 48h, receive
Collection cell, a part is used for extracting total serum IgE, carries out RT-PCR qualification and the transcriptional level of detection transgene expression, and method is with real
Executing (three) of step 3 in example 1, result is as shown in Figure 7；Another part is used for extracting fatty acid in cell, carries out GC-MS analysis
Fatty acid forms, and method is with (four) of step 3 in embodiment 1, and result is as shown in Figure 8.
In Fig. 7,1 represents pcDNA3.1-EGFP transfection group (matched group)；2 represent pcDNA3.1-F2F1 transfection group；3 represent
PcDNA3.1-F2F1+pcDNA3.1-sScD4 transfection group.
Fig. 7 shows, genes of interest fads2 and fads1 of transfection, fads2, fads1 and sScD4 are it is anticipated that accordingly
Achieving overexpression in cell, its transcriptional level is significantly higher than matched group；PcDNA3.1-without destination gene expression plasmid
EGFP transfection group (matched group) then can't detect the transcription product of sScD4 gene.
In Fig. 8, EGFP represents pcDNA3.1-EGFP transfection group (matched group)；F2F1 represents pcDNA3.1-F2F1 transfection
Group；F2F1+sScD4 represents pcDNA3.1-F2F1+pcDNA3.1-sScD4 transfection group.
In Fig. 8, the conversion process of each material is as described in example 2 above.
Fig. 8 shows, compared with matched group, in pcDNA3.1-F2F1 transfection group, and Δ 6/ Δ 5 fatty acid desaturase activity
Increase can promote add fatty acid substrate LA (18:2n-6) be changed into ARA (20:4n-6) significantly.Δ 6/ Δ 5 fat
Acid desaturase activity increase make the fatty acid substrate ALA (18:3n-3) of interpolation be changed into more EPA (20:5n-3) and
DPA (22:5n-3), but Δ 6/ Δ 5 fatty acid desaturase activity does not significantly increase the content of DHA (22:6n-3).With
PcDNA3.1-F2F1 transfection group is compared, in pcDNA3.1-F2F1+pcDNA3.1-sScD4 transfection group, and sScD4 gene code
△ 4 fatty acid desaturase work in coordination with the activity of Δ 6/ Δ 5 fatty acid desaturase, DPA (22:5n-3) is directly translated into into
The content relatively pcDNA3.1-F2F1 transfection group of higher levels of DHA (22:6n-3), DHA (22:6n-3) improves about 1.5 times,
Account for polyunsaturated fatty acid total content nearly 30%.
Result shows, in mammalian cell, △ 4 fatty acid desaturase of sScD4 gene code can work in coordination with Δ
The alpha-linolenic acid ALA (18:3n-3) that ω-3 is is converted into high level DHA (22:6n-by the effect of 6/ Δ 5 fatty acid desaturase
3)。
Claims (4)
1. the encoding gene of an albumen, it is characterised in that: described encoding gene be in SEQ ID No.1 from 5 ' ends the 10th
Position is to the DNA molecular shown in the 1347th nucleotide.
2. contain the recombinant vector of encoding gene described in claim 1, expression cassette or recombinant bacterium.
3. improve a method for DHA 22:6n-3 synthesis capability in mammalian cell, comprise the steps: SEQ ID
In No.1, from 5 ' ends, the DNA molecular shown in the 10th to the 1347th nucleotide imports to set out in cell, obtains turning base
Because of cell；Compared with the cell that sets out, the DHA 22:6n-3 synthesis capability of transgenic cell improves.
Method the most according to claim 3, it is characterised in that: DNA molecular is imported by recombinant expression carrier, described
Recombinant expression carrier be by DNA molecular insert set out carrier pcDNA3.1 (-) multiple clone site obtain.
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Non-Patent Citations (6)
| Title |
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| A more desirable balanced polyunsaturated fatty acid composition achieved by heterologous expression of Δ15/Δ4 desaturases in mammalian cells;Zhu G et al.;《Plos one》;20131231;第8卷(第12期);第2页第1栏第1段，第2页第2栏最后1段，第3页第1栏第1段，第3页第2栏第2-3段，第5页第1栏第1段，第5页第2栏第3段、第2页第1栏第3段、第2页第2栏第4段、图1、图1图注 * |
| Cloning and functional identification of delta5 fatty acid desaturase gene and its 5"-upstream region from marine fungus Thraustochytrium sp.FJN-10;Huang JZ et al.;《Mar Biotechnol》;20100401;第13卷(第1期);第12-21页 * |
| GenBank Accession：ADJ29913.1;Li Y et al.;《GenBank》;20101001;第1-2页 * |
| GenBank Accession：GU594278.1;Li Y et al.;《GenBank》;20101001;第1-2页 * |
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| 鱼类高度不饱和脂肪酸合成的影响因素及其机理;谢帝芝等;《中国水产科学》;20130331;第20卷(第2期);图1 * |
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