CN103820403A - Delta4 fatty acid desaturase from siganus canaliculatus and application of Delta4 fatty acid desaturase - Google Patents
Delta4 fatty acid desaturase from siganus canaliculatus and application of Delta4 fatty acid desaturase Download PDFInfo
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Abstract
The invention discloses Delta4 fatty acid desaturase from siganus canaliculatus and the application of the Delta4 fatty acid desaturase. A protein is (1) a protein shown as (1) SEQ ID No.2, or (2) an identically functional protein obtained by the substitution and/or deletion and/or addition of one or more amino acid residues in an amino acid sequence shown as SEQ ID No.2. The Delta4 fatty acid desaturase can be used for promoting the production of important long-chain polyunsaturated fatty acid such as high-level docosahexaenoic acid in mammals, and can also be used for converting DPA (docosapentaenoic acid) produced by various methods into DHA in mammalian cells or the mammals.
Description
Technical field
The present invention relates to a kind of Δ 4 fatty acid desaturases and application thereof that derives from basket fish.
Background technology
Lipid acid is divided into saturated fatty acid, monounsaturated fatty acids and polyunsaturated fatty acid (PUFAs).Wherein, PUFAs has more unique function and significance to human body, is the lipid acid that a class is widely studied and receives much concern.PUFAs comprises two types of ω-3 and ω-6PUFAs, and each type comprises again multiple carbon chain lengths differences, unsaturated link(age) quantity member not etc.The PUFAs of carbon chain lengths >=20 is called as long chain polyunsaturated fatty acids (LCPUFAs), most important LCPUFAs comprises DHA(22:6n-3), EPA(20:5n-3) and ARA(20:4n-6, be arachidonic acid), they are three kinds of unsaturated fatty acidss that biological in vivo bioactivity is the strongest, have been proved and have promoted brain development and function maintains and at important role aspect the various diseases such as prevention and Cardiovarscular, inflammation, cancer.With respect to some other biology, the ability of the synthetic PUFAs of the mankind and other Mammalss self is very low, reason is as follows: first, Mammals can not start synthetic PUFAs from acetyl-CoA, only can be with food-based LA(18:2n-6, be linolic acid) and ALA(18:3n-3, i.e. alpha-linolenic acid) be substrate, utilize fatty acid desaturase and the active synthetic corresponding PUFAs of extending enzyme of self; Secondly, in Mammals, these fatty acid desaturases and extending enzyme activity are very limited, therefore can only finally synthesize limited sub-fraction PUFAs; Again, in Mammals, also lack fatty acid desaturase that some other biologicals have and extending enzyme active and make the especially synthetic very difficulty of DHA of some important long chain polyunsaturated fatty acidss.Therefore, human body mainly relies on from food source and obtains more PUFAs demand.But occurring in nature ω-6PUFAs is abundant, ω-3PUFAs is rare, thus cause in human body, take in and the ω-6PUFAs of accumulation too much and ω-3PUFAs is very few.And in Mammals, there is not the enzymic activity that makes ω-6PUFAs change ω-3PUFAs into, the ratio serious unbalance of ω-6PUFAs and ω-3PUFAs in old friend's body.Although ω-6PUFAs is also indispensable to human body, its excessive absorption is detrimental to health on the contrary.
Modern's life and diet pattern have caused the picked-up of lipid acid seriously uneven.The present following level of this out-of-balance mass.First level is the imbalance between saturated fatty acid and unsaturated fatty acids, and in most cases human body has been taken in too much saturated fatty acid (mainly from animal grease), and unsaturated fatty acids subacidity; Second level is that in the unsaturated fatty acids of taking in, ω-6 are that unsaturated fatty acids and ω-3 are the imbalance between unsaturated fatty acids, and ω-6 are that hyper acid and ω-3 are that hypoacidity for unsaturated fatty acids for unsaturated fatty acids; The 3rd level is in the unsaturated fatty acids of taking in, the imbalance of short chain unsaturated fatty acids (18C) and long-chain unsaturated fatty acid (20C～22C), and short chain unsaturated fatty acids is many, and long-chain unsaturated fatty acid is few.The conventional vegetables oil of great majority as ω-6 unsaturated fatty acids that soybean oil, peanut oil, sunflower seeds wet goods all contain the short chain of high level be LA(18:2n-6, be linolic acid), linseed oil, perilla seed wet goods, although the omega-3 unsaturated fatty acid that contains high-caliber short chain is ALA(18:3n-3, be alpha-linolenic acid), but they are not common oil in daily life.Fish oil is the main provider of long chain polyunsaturated fatty acids EPA and DHA, but expensive, resource scarcity, and edible crowd is limited, takes in unbalanced state so it changes mankind's lipid acid in not having on a large scale.As can be seen here, the imbalance of lipid acid is just in time embodied in DHA(22:6n-3), EPA(20:5n-3) and the ARA(20:4n-6) shortage of these the three kinds bioactive long chain polyunsaturated fatty acidss of tool.That is to say, if can obtain abundant DHA(22:6n-3), EPA(20:5n-3) and ARA(20:4n-6) resource just can change the unbalanced present situation of current mankind's fatty acid uptake for mankind picked-up.
How to obtain abundant DHA(22:6n-3), EPA(20:5n-3) and ARA(20:4n-6) resource be the problem that within the scope of the world today, people are concerned about.Face resource exhaustion and pollute under day by day serious present situation at fish oil, utilize the EPA(20:5n-3 such as some marine algaes and Mycophyta), DHA(22:6n-3) initial production person obtain these long chain polyunsaturated fatty acidss and become good selection, exploitation at present also obtains very large success.But these products use and have certain limitation usually used as foodstuff additive, it is preserved needs the strict not good life-time service that is difficult to of measure, its taste.It is contemplated that, if terricole can provide DHA(22:6n-3 as domestic animals such as pig, cattle and sheeps as bathypelagic fish), EPA(20:5n-3) and the unsaturated fatty acids such as ARA(20:4n-6), the rich of its resource will produce revolutionary variation so, and in the situation that not affecting and change people's living habit, just can obtain these important lipid acid, this will produce far-reaching influence to human health.But as previously mentioned, Mammals is synthesized DHA(22:6n-3), EPA(20:5n-3) and ARA(20:4n-6) be extremely limited, improve its content, just must set about from corresponding polyunsaturated fatty acid synthetic enzyme.Relevant Research foundation shows, it is possible in Mammals, producing these long chain polyunsaturated fatty acidss by transgenic animal technology.1997, James P etc. from nematode C.elegans screening and cloning first animal Δ 15 delta 8 desaturase genes, called after fat-1, finds that in follow-up research fat-1 gene can become ω-3PUFAs by 16～20 carbon ω-6PUFAs desaturations.Fat-1 gene is proceeded to mammalian cell by Zhao B.Kang etc., and ω-6/ ω-3PUFAs drops to 1:1 than from 15:1, and ω-3PUFAs content that Mammals is inhaled improves significantly.In 2004, fat-1 gene is proceeded to mouse again afterwards, the ω-3PUFAs such as ALA, EPA, DPA are significantly increased.2006, the transgenic pig of the fat-1 of the preparation such as Lai, also showed and the similar situation of transgenic mice.But, in these transgenic animal bodies in the ω-3PUFAs of synthesized take ω-3PUFAs of 18C, 20C as main, and the DHA(22:6n-3 of even more important 22C) total amount is still little.In fact, some follow-up researchs show that the Δ 15 desaturase activity of fat-1 can not increase the DHA(22:6n-3 in these animal bodies) content.Therefore, fat-1 gene is for the practice that improves Mammals long chain polyunsaturated fatty acids content, and critical shortcoming can not increase DHA(22:6n-3 exactly) content.
In fact, DHA(22:6n-3) synthetic very complicated in Mammals, corresponding to one so-called " Sprecher path ", EPA(20:5n-3) extend and generate an intermediate product DPA(22:5n-3) after, further extend to again 24:5n-3, then generated 24:6n-3 by △ 6 desaturase effects rapidly, further produce DHA(22:6n-3 through betaoxidation afterwards), the mechanism of this complexity may be that Mammals is difficult to synthetic high-level DHA(22:6n-3) reason.Relevant polyunsaturated fatty acid biosynthetic pathway as shown in Figure 1.
Summary of the invention
The object of this invention is to provide a kind of Δ 4 fatty acid desaturases and application thereof that derives from basket fish.
A kind of albumen provided by the invention, shown in following (1) or (2):
(1) albumen shown in SEQ ID No.2;
(2) replacement and/or disappearance and/or interpolation and the identical protein of function through one or several amino-acid residue by the aminoacid sequence shown in SEQ ID No.2.
The encoding gene of above-mentioned albumen also belongs to protection scope of the present invention.
In above-mentioned encoding gene, described encoding gene be as lower at least one:
1) in SEQ ID No.1 from 5 ' end the DNA molecular shown in the 10th to the 1347th Nucleotide;
2) under stringent condition 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 more than 90% identity and the DNA molecular of code for said proteins.
The recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contain above-mentioned arbitrary described encoding gene also belong to protection scope of the present invention.
A kind of protein composition also belongs to protection scope of the present invention, is made up of the albumen shown in the albumen shown in SEQ ID No.2 and SEQ ID No.4.
A kind of protein composition also belongs to protection scope of the present invention, is made up of 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.
A kind of improving DHA(22:6n-3 in mammalian cell) method of synthesis capability also belongs to protection scope of the present invention, comprises the steps: the encoding gene of described albumen to import to and set out in cell, obtains transgenic cell; Compared with the cell that sets out, the DHA(22:6n-3 of transgenic cell) synthesis capability raising.
In aforesaid method, described encoding gene imports by recombinant expression vector, and described recombinant expression vector is that the multiple clone site that described encoding gene is inserted to the carrier pcDNA3.1 (-) that sets out obtains.
Above-mentioned albumen promotes DHA(22:6n-3 in preparation) application in synthetic product also belongs to protection scope of the present invention;
Or,
Above-mentioned arbitrary described protein composition promotes DHA(22:6n-3 in preparation) application in synthetic product also belongs to protection scope of the present invention.
Above-mentioned albumen also belongs to protection scope of the present invention in the application of preparing in the product that promotes DPA (22:5n-3) to change into DHA (22:6n-3);
Or,
Above-mentioned arbitrary described protein composition also belongs to protection scope of the present invention in the application of preparing in the product that promotes DPA (22:5n-3) to change into DHA (22:6n-3);
Or,
Above-mentioned albumen also belongs to protection scope of the present invention in the application of preparing in the product that promotes △ 15 fatty acid desaturases DPA (22:5n-3) to be changed into DHA (22:6n-3);
Or,
The application that above-mentioned albumen promotes △ 15 fatty acid desaturases LA (18:2n-6) and/or ARA (20:4n-6) to be changed in the product of DHA (22:6n-3) in preparation also belongs to protection scope of the present invention;
The application that the protein composition being made up of the albumen shown in the albumen shown in SEQ ID No.2 and SEQ ID No.4 changes into LA (18:2n-6) and/or ARA (20:4n-6) in preparation in the product of DHA (22:6n-3) also belongs to protection scope of the present invention;
The aminoacid sequence of described △ 15 fatty acid desaturases is as shown in SEQ ID No.4;
Or,
Above-mentioned albumen also belongs to protection scope of the present invention in the application of preparing in the product that promotes Δ 6-fatty acid desaturase and Δ 5-fatty acid desaturase DPA (22:5n-3) to be changed into DHA (22:6n-3);
The application that above-mentioned albumen promotes Δ 6-fatty acid desaturase and Δ 5-fatty acid desaturase LA (18:2n-6) and/or ALA (18:3n-3) to be changed in the product of DHA (22:6n-3) in preparation also belongs to protection scope of the present invention;
The application that the protein composition being made up of 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 changes into LA (18:2n-6) and/or ALA (18:3n-3) in preparation in the product of DHA (22:6n-3) also belongs to 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 desaturases provided by the invention, by coordinating or work in coordination with the effect of other fatty acid desaturases (Δ 15 or Δ 6/ Δ 5), can promote the mammalian cell of gene transfection and transgenic animal that the initial lipid acid substrate adding is transformed into high-caliber DHA(22:6n-3 as LA (18:2n-6) or ALA (18:3n-3)) etc. important long chain polyunsaturated fatty acids, also the DPA that utilizes the whole bag of tricks to produce in mammalian cell or animal can be converted into DHA simultaneously.
Accompanying drawing explanation
Fig. 1 is polyunsaturated fatty acid biosynthetic pathway.
Fig. 2 is pcDNA3.1-sScD4 carrier schematic diagram.
Fig. 3 is the expression identification that turns goal gene in sScD4 gene cell.
Fig. 4 is GC-MS detected result.
Fig. 5 is the evaluation of transgenic cell in embodiment 2.
Fig. 6 is the synergy analysis of Δ 4 fatty acid desaturases and △ 15 fatty acid desaturases.
Fig. 7 is the evaluation of transgenic cell in embodiment 3.
Fig. 8 is the synergy analysis of Δ 4 fatty acid desaturases and Δ 6/ Δ 5 fatty acid desaturases.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
Chinese hamster ovary (CHO) cell is purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's preclinical medicine cell centre.
PEGFP-N1 is purchased from Clontech company.
PcDNA3.1 (-) is purchased from Invitrogen company.
The construction process of pcDNA3.1-EGFP is as follows:
One, take pEGFP-N1 as template, carry out pcr amplification with primer fG-s:5 '-TCTAGATCGCCACCATGGTGAGCAAGG-3 ' and fG-a:5 '-CTCGAGCTTGTACAGCTCGTCCATGCCGAG-3 ', obtain pcr amplification product, this product is EGFP gene coding region (725bp);
Two, the pcr amplification product that Xba I and Xho I double digestion step 1 obtain, obtains gene fragment; Xba I and Xho I double digestion pcDNA3.1(-), obtain carrier large fragment; Gene fragment is connected with carrier large fragment, obtains recombinant plasmid, by its called after pcDNA3.1-EGFP.
The construction process of pcDNA3.1-F2F1 is at document " Wang Kunfu, Zhu Guiming, Zhang Li etc.Fatty acid synthetase is the structure of polygene expression vector.Aged in China learn magazine, 2013,33(17), 4178-4180 " in disclosed, the public can obtain from Jiamusi University.
Synthetic and the functional verification of embodiment 1, Δ 4 fatty acid desaturase genes (sScD4 gene)
One, Δ 4 fatty acid desaturase genes (sScD4 gene) is synthetic
The cDNA sequence (encoder block total length is 1338bp) of the Δ 4 fatty acid desaturase genes (No. GenBank: GU594278) to the Lan Ziyu submitting in GenBank (Siganus canaliculatus) is carried out codon optimized, makes it to meet mammalian genes codon usage bias.After optimizing, use the secondary structure of its mRNA of software prediction, whether authentication password is optimized reasonable.
The nucleotide sequence of the Δ 4 fatty acid desaturase genes (sScD4 gene) after optimization as in SEQ ID No.1 from 5 ' end as shown in the 10th to the 1347th Nucleotide, the aminoacid sequence of its coding is as shown in SEQ ID No.2.
Sequence after optimizing is added to EcoRI and HindIII restriction enzyme site at two ends, then carry out the synthetic of full-length gene, 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 gene fragment; EcoRI and HindIII double digestion carrier for expression of eukaryon pcDNA3.1 (-), obtain carrier large fragment; Gene fragment is connected with carrier large fragment, obtains recombinant expression plasmid, by its called after pcDNA3.1-sScD4.Send order-checking by pcDNA3.1-sScD4, result is correct.The schematic diagram of pcDNA3.1-sScD4 carrier as shown in Figure 2.
Three, the functional verification of Δ 4 fatty acid desaturase genes (sScD4 gene)
(1) cultivation of Chinese hamster ovary celI
Substratum composition: DMEM high glucose medium, 10% foetal calf serum, 1% mycillin (100 ×), 1% non-essential amino acid, 1g/100ml Sodium.alpha.-ketopropionate, 0.22 μ M membrane filtration degerming, % all represents volumn concentration.
Chinese hamster ovary (CHO) cell is with 5 × 10
5/ ml is inoculated in culturing bottle, in 37 ℃, 5%CO
2and cultivate in the incubator of saturated humidity.
(2) liposome method transient transfection Chinese hamster ovary celI
1, the preparation before transfection: the plasmid for the treatment of transfection is pcDNA3.1-sScD4(experimental group), carry out transfection (control group) take the recombinant plasmid pcDNA3.1-EGFP containing enhanced green fluorescence protein as contrast, monitoring transfection efficiency.Transfection is passaged to 60mm culture dish by the Chinese hamster ovary celI of cultivation the day before yesterday, adds the lipid acid substrate DPA(22:5n-3 of 10 μ mol/L), when growing to 90%-95%, cell carries out transfection.
2, transfection: add 500 μ l DMEM basic mediums in 1.5ml EP pipe, then mix gently after adding 8 μ g pcDNA3.1-sScD4 or pcDNA3.1-EGFP, be designated as A liquid; In another 1.5ml EP pipe, add 500 μ l DMEM basic mediums, then add 20 μ l transfection reagent Lipofectamine
tMafter 2000, mix, be designated as B liquid, incubated at room 5min.A and B are added in a pipe and are mixed, and room temperature leaves standstill 20min.Then the 60mm culture dish that this mixed solution is added to the Chinese hamster ovary celI of step 1 mixes gently, and 37 ℃, 5%CO
2cultivate after 4-6h, inhale the liquid of abandoning in hole, change fresh culture (containing equally the DPA of 10 μ mol/L) and cultivate.After transfection, start available fluorescence microscope transfection situation next day, and the experimental group that after 48h, collection obtains and the cell of control group are for subsequent detection and experiment.
(3) RT-PCR carries out the evaluation of transgenic cell
1, the extraction of total RNA: get experimental group after transfection 48h and the cell TRizol reagent of control group and extract total RNA, and measure RNA concentration, finally the RNA of extraction is placed in to-80 ℃ of Ultralow Temperature Freezers and saves backup.
2, the reverse transcription of mRNA: be cDNA by the mRNA reverse transcription of experimental group and control group, reverse transcription reaction condition is 42 ℃ of 30min; 95 ℃ of 5min; 5 ℃ of 5min; 4 ℃ of preservations.After reverse transcription finishes, cDNA is placed in to-20 ℃ and saves backup.
3, the expression of PCR testing goal gene sScD4, and take β-actin as reference gene
1. the detection primer of sScD4 gene:
D4-s:5’-TCGGGCATCTGAGCGTGTTC-3’ Tm：64.1℃
D4-a:5’-ATACTCCACGGGCTGGGTCTCC-3’ Tm：66.2℃
Object product length is 203bp.
2. β-actin reference gene detects primer:
Bact-s：5’-CTGAGAGGGAAATCGTGCGTGAC-3’ Tm：65.7℃
Bact-a：5’-TGCCACAGGATTCCATACCCAAG-3’ Tm：65.7℃
Object product length is 210bp.
The cDNA of the experimental group of preparing take step 2 is template, take D4-s and D4-a as primer carries out pcr amplification, obtains pcr amplification product 1.
The cDNA of the control group of preparing take step 2 is template, take Bact-s and Bact-a as primer carries out pcr amplification, obtains pcr amplification product 2.
Get pcr amplification product 1 and 2 and carry out 1% agarose gel electrophoresis, result 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 goal gene sScD4 in the Chinese hamster ovary celI of pcDNA3.1-sScD4 transfection, detected, and transcribing of goal gene sScD4 cannot be detected in the Chinese hamster ovary celI of transfection containing the recombinant plasmid pcDNA3.1-EGFP of enhanced green fluorescence protein, prove that experimental group turns sScD4 gene cell and successfully constructs.
(4) gas chromatography-mass spectrography analytical technology (GC-MS) detects the composition variation of lipid acid
1, the extraction of total fatty acids: the cell in the culture dish of the experimental group after transfection 48h and control group is digested to 3min with 37 ℃, 0.4ml trypsinase respectively, stop the effect of Trypsin containing blood serum medium with 1ml, with rinsed with deionized water once, 1000rpm, centrifugal 5min, adding 1ml volumn concentration is 2.5%H
2sO
4methanol solution, mix gently, 80 ℃ of water-bath 90min, to be cooled to room temperature, add NaCl solution and the 1ml normal hexane of 1.5ml0.9g/100ml, concussion, 2000rpm, centrifugal 5min, is extracted into lipid acid in organic phase (being normal hexane), the organic extractant phase thing of drawing experimental group or control group dries up after concentrated and detects and analyze for GC-MS through nitrogen, or saves backup in-80 ℃.
2, GC-MS detects: when detection, use instrument for HP-5890/HP-5971 Gc-ms instrument, experiment condition is analyzed and is required to carry out according to the detection of conventional unsaturated fatty acids, concrete 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 control group GC-MS detected result to result; B is experimental group GC-MS detected result.
Fig. 4 shows, add after DPA (22:5n-3) substrate, experimental group compared with the resultant quantity of control group DHA (22:6n-3) significantly increase, difference is extremely remarkable, illustrate that sScD4 genetic expression △ 4 fatty acid desaturases have brought into play effect in the process of synthetic DHA (22:6n-3), DPA (22:5n-3) is directly transformed into DHA (22:6n-3).
One, root according to research reports, the expression product that derives from the fat-1 gene (No. GenBank: NM_001028389) of nematode C.elegans has △ 15 fatty acid desaturase activity, fat-1 gene is carried out to the sequence of codon optimized design, synthetic as shown in SEQ ID No.3.
The coding gene sequence of these △ 15 fatty acid desaturases as in SEQ ID No.3 from 5 ' end as shown in the 13rd to the 1221st Nucleotide, the aminoacid sequence of △ 15 fatty acid desaturases is as shown in SEQ ID No.4.
Method according to embodiment 1 step 2 obtains recombinant expression plasmid pcDNA3.1-sD15.
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, each transfection group plasmid used is in 8 μ g(pcDNA3.1-sD15+pcDNA3.1-sScD4 two kinds of each 4 μ of plasmid g), and with cell culture medium in add the substrate linolic acid LA (18:2n-6) of 10 μ mol/L and 10 μ mol/L arachidonic acid ARA (20:4n-6) cultivate after 48h, collecting cell, a part is used for extracting total RNA, carry out the transcriptional level of RT-PCR Identification and detection genetic expression, method is with (three) of step 3 in embodiment 1, result as shown in Figure 5, another part is used for extracting cell lipid acid, carries out GC-MS and analyzes lipid acid composition, and method is with (four) of step 3 in embodiment 1, and result as shown in Figure 6.
In Fig. 5,1 represents pcDNA3.1-EGFP transfection group (control group); 2 represent pcDNA3.1-sD15 transfection group; 3 represent pcDNA3.1-sD15+pcDNA3.1-sScD4 transfection group.
Fig. 5 shows, goal gene sD15, sD15 and sScD4 have realized expression according to expection in corresponding cell, not can't detect the transcription product of goal gene containing the pcDNA3.1-EGFP transfection group (control group) of destination gene expression plasmid.
In Fig. 6, EGFP represents pcDNA3.1-EGFP transfection group (control 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) adding is converted into ALA (18:3n-3) (this part ALA (18:3n-3) becomes EPA (20:5n-3) after two step metabolism by ω-3 approach) by △ 15 fatty acid desaturases, and another part LA (18:2n-6) is transformed as ARA (20:4n-6) by two steps by the desaturase and the extending enzyme activity that itself are had in cell.This part ARA (20:4n-6) is EPA (20:5n-3) by △ 15 fatty acid desaturase Partial Conversions with the ARA (20:4n-6) adding together.Still some ARA (20:4n-6) is not converted to and is converted into ADA (22:4n-6) by itself extending enzyme activity of being had in cell for EPA (20:5n-3).△ 15 fatty acid desaturases now can be converted into ADA (22:4n-6) DPA (22:5n-3), and some DPA (22:5n-3) is produced through extending enzyme effect in cell by EPA (20:5n-3).The amount that DPA (22:5n-3) is transformed into DHA (22:6n-3) through Sprecher path is not remarkable, but in sD15+sScD4 transfection group, can further the DPA (22:5n-3) of accumulation be converted into DHA (22:6n-3) by △ 4 fatty acid desaturases, the increase of its quantity is very significant.The metabolic conversion process of above-mentioned polyunsaturated fatty acid can be referring to Fig. 1.
Fig. 6 shows, compared with control group, in sD15, two groups of transfection group of sD15+sScD4, to be PUFAs all significantly reduce as LA (18:2n-6), ARA (20:4n-6) and ADA (22:4n-6) in ω-6, and to be ALA in PUFAs (18:3n-3) and EPA (20:5n-3) all significantly increase in ω-3.But, DPA (22:5n-3) is completely different with DHA (22:6n-3) situation: compared with control group, DPA in sD15 transfection group (22:5n-3) significantly increases, and in sD15+sScD4 transfection group, DPA (22:5n-3) content is basic fair with control group; DHA in sD15 transfection group (22:6n-3) content is basic to maintain an equal level with control group, significantly increase of DHA in sD15+sScD4 transfection group (22:6n-3) content (its content account for polyunsaturated fatty acid total content nearly 10%).
Result shows, △ 4 fatty acid desaturases of sScD4 genes encoding can act synergistically with △ 15 fatty acid desaturases, in mammalian cell, utilize △ 4 fatty acid desaturases of sScD4 genes encoding can promote △ 15 fatty acid desaturase activity, the linolic acid of interpolation and arachidonic acid are converted into the DHA (22:6n-3) of higher level, compared with only adding △ 15 fatty acid desaturases, content improves approximately 1 times.
PcDNA3.1-F2F1 contains to derive from the Δ 6-fatty acid desaturase gene fads2 (No. GenBank: BC057189) of mouse and the double gene expression vector of Δ 5-fatty acid desaturase gene fads1 (No. GenBank: BC063053).
The coding gene sequence of Δ 6-fatty acid desaturase is as shown in SEQ ID No.5, and the aminoacid sequence of this albumen is as shown in SEQ ID No.6.
The coding gene sequence of Δ 5-fatty acid desaturase is as shown in SEQ ID No.7, and the aminoacid sequence of this albumen is 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, each transfection group plasmid used is in 8 μ g(pcDNA3.1-F2F1+pcDNA3.1-sScD4 two kinds of each 4 μ of plasmid g), and the lipid acid substrate linolic acid LA (18:2n-6) and the 10 μ mol/L alpha-linolenic acid ALA (18:3n-3) that in cell culture medium, add 10 μ mol/L cultivate after 48h, collecting cell, a part is used for extracting total RNA, carry out the transcriptional level of RT-PCR Identification and detection transgene expression, method is with (three) of step 3 in embodiment 1, result as shown in Figure 7, another part is used for extracting cell lipid acid, carries out GC-MS and analyzes lipid acid composition, and method is with (four) of step 3 in embodiment 1, and result as shown in Figure 8.
In Fig. 7,1 represents pcDNA3.1-EGFP transfection group (control group); 2 represent pcDNA3.1-F2F1 transfection group; 3 represent pcDNA3.1-F2F1+pcDNA3.1-sScD4 transfection group.
Fig. 7 shows, the goal gene fads2 of transfection and fads1, fads2, fads1 and sScD4 have realized overexpression according to expection in corresponding cell, and its transcriptional level is significantly higher than control group; Not can't detect the transcription product of sScD4 gene containing the pcDNA3.1-EGFP transfection group (control group) of destination gene expression plasmid.
In Fig. 8, EGFP represents pcDNA3.1-EGFP transfection group (control 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 as described in example 2 above.
Fig. 8 shows, compared with control group, in pcDNA3.1-F2F1 transfection group, the increase of Δ 6/ Δ 5 fatty acid desaturase activity can impel the lipid acid substrate LA (18:2n-6) of interpolation to change significantly ARA (20:4n-6) into.The increase of Δ 6/ Δ 5 fatty acid desaturase activity makes the lipid acid substrate A LA (18:3n-3) adding change more EPA (20:5n-3) and DPA (22:5n-3) into, but the active content that does not improve significantly DHA (22:6n-3) of Δ 6/ Δ 5 fatty acid desaturases.Compared with pcDNA3.1-F2F1 transfection group, in pcDNA3.1-F2F1+pcDNA3.1-sScD4 transfection group, the activity of collaborative Δ 6/ Δ 5 fatty acid desaturases of △ 4 fatty acid desaturases of sScD4 genes encoding, DPA (22:5n-3) is directly transformed into higher levels of DHA (22:6n-3), the content of DHA (22:6n-3) has improved approximately 1.5 times compared with pcDNA3.1-F2F1 transfection group, accounts for polyunsaturated fatty acid total content nearly 30%.
Result shows, in mammalian cell, △ 4 fatty acid desaturases of sScD4 genes encoding can be worked in coordination with the alpha-linolenic acid ALA (18:3n-3) that the effect of Δ 6/ Δ 5 fatty acid desaturases by ω-3 is and are converted into high-level DHA (22:6n-3).
Claims (10)
1. an albumen, shown in following (1) or (2):
(1) albumen shown in SEQ ID No.2;
(2) replacement and/or disappearance and/or interpolation and the identical protein of function through one or several amino-acid residue by the aminoacid sequence shown in SEQ ID No.2.
2. the encoding gene of albumen described in claim 1.
3. encoding gene according to claim 2, is characterized in that: described encoding gene for as lower at least one:
1) in SEQ ID No.1 from 5 ' end the DNA molecular shown in the 10th to the 1347th Nucleotide;
2) under stringent condition with 1) protein DNA molecule described in the DNA molecule hybridize that limits and coding claim 1;
3) with 1) or 2) DNA molecular that limits has protein DNA molecule described in more than 90% identity and coding claim 1.
4. contain recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium of encoding gene described in claim 2 or 3.
5. a protein composition, is made up of the albumen shown in the albumen shown in SEQ ID No.2 and SEQ ID No.4.
6. a protein composition, is made up of 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.
7. improve DHA(22:6n-3 in mammalian cell) method of synthesis capability, comprise the steps: the encoding gene of albumen described in claim 1 to import to and set out in cell, obtain transgenic cell; Compared with the cell that sets out, the DHA(22:6n-3 of transgenic cell) synthesis capability raising.
8. method according to claim 7, is characterized in that: described encoding gene imports by recombinant expression vector, and described recombinant expression vector is that the multiple clone site that described encoding gene is inserted to the carrier pcDNA3.1 (-) that sets out obtains.
9. albumen claimed in claim 1 promotes DHA(22:6n-3 in preparation) application in synthetic product;
Or,
Protein composition described in claim 5 or 6 promotes DHA(22:6n-3 in preparation) application in synthetic product.
10. albumen claimed in claim 1 is in the application of preparing in the product that promotes DPA (22:5n-3) to change into DHA (22:6n-3);
Or,
Protein composition described in claim 5 or 6 is in the application of preparing in the product that promotes DPA (22:5n-3) to change into DHA (22:6n-3);
Or,
Albumen claimed in claim 1 is in the application of preparing in the product that promotes △ 15 fatty acid desaturases DPA (22:5n-3) to be changed into DHA (22:6n-3);
Or,
Albumen claimed in claim 1 promotes △ 15 fatty acid desaturases LA (18:2n-6) and/or ARA (20:4n-6) to be changed into the application in the product of DHA (22:6n-3) in preparation;
Protein composition claimed in claim 5 is in the application of preparing in the product that promotes LA (18:2n-6) and/or ARA (20:4n-6) to change into DHA (22:6n-3);
The aminoacid sequence of described △ 15 fatty acid desaturases is as shown in SEQ ID No.4;
Or,
Albumen claimed in claim 1 is in the application of preparing in the product that promotes Δ 6-fatty acid desaturase and Δ 5-fatty acid desaturase DPA (22:5n-3) to be changed into DHA (22:6n-3);
Or,
Albumen claimed in claim 1 promotes Δ 6-fatty acid desaturase and Δ 5-fatty acid desaturase LA (18:2n-6) and/or ALA (18:3n-3) to be changed into the application in the product of DHA (22:6n-3) in preparation;
Protein composition claimed in claim 6 is in the application of preparing in the product that promotes LA (18:2n-6) and/or ALA (18:3n-3) to change into DHA (22:6n-3);
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.
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| CN1884500A (en) * | 2006-06-15 | 2006-12-27 | 中国人民解放军军事医学科学院生物工程研究所 | Omega-3-aliphatic acid desaturase, and its encoding gene and use thereof |
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| CN111455064B (en) * | 2020-04-14 | 2022-06-28 | 汕头大学 | Application of miRNA-sc-miR-145 in fish LC-PUFA synthesis |
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