CN106047926A - Transgenic pig with transgenic delta 12 and delta 15 fatty acid desaturase genes as well as preparation method and applications of transgenic pig - Google Patents
Transgenic pig with transgenic delta 12 and delta 15 fatty acid desaturase genes as well as preparation method and applications of transgenic pig Download PDFInfo
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Abstract
The invention discloses a transgenic pig with transgenic delta 12 and delta 15 fatty acid desaturase genes as well as a preparation method and applications of the transgenic pig. Delta 12 and delta 15 fatty acid desaturase genes fat 2 and fat 1 genes are inserted into second introns of DCTN 5 genes of the No.10 chromosome of the pig in the fusion expression manner and through the transgenic method, and thus the transgenic pig with the transgenic fat1-fat 2 fused gene is obtained; a large number of n-3 and n-6 PUFAs are expressed in the tissues of the transgenic pig, and combined with the health care effect of PUFAs, the transgenic pig can be used for producing the tissues, such as the pork with the health care effect, thus being applied to the prevention and treatment for human diseases. The immune system and metabolism of the pig are very similar to those of the human beings, the PUFAs secreted by the pig can not generate the immunological rejection reaction when the human diseases are treated, therefore, the transgenic pig is safer and more efficient compared with the other similar transgenic animals, and thus the PUFAs can be easily taken in by people; in addition, comparing with the PUFAs exogenous additive medicines, the taking in through the transgenic pork causes little toxic and side effects, and therefore, the transgenic pork can become the new-generation exogenous PUFAs substitute.
Description
Technical field
The present invention relates to animal of being obtained by transgenic method and its preparation method and application, turn Δ particularly to one
12 and Δ 15 fatty acid desaturase gene pig and its preparation method and application.
Background technology
Δ 12 and Δ 15 fatty acid desaturase gene (fat2 and fat1) are polyunsaturated fatty acid in mammal body
Two kinds of key enzymes of (Polyunsaturated fatty acids, PUFAs) route of synthesis, control n-6 and n-3PUFAs respectively
Synthesis rate.Δ 15 fatty acid desaturase gene (fat1) is initially found in 1997 first by Spychalla, grinds subsequently
Study carefully this gene of display and can significantly improve n-3PUFAs content.And Δ 12 fatty acid desaturase gene (fat2) is demonstrate,proved the most under study for action
In fact n-6PUFAs is obviously improved.The growth promoter of people is played important physiological function by PUFAs.Controlled respectively by fat1 and fat2
Synthesis n-3 and n-6PUFAs neurocyte, stem cell differentiation with developmentally play an important role, DHA therein be considered and
The growth of human nervous system is closely bound up.In addition, both PUFAs for cardiovascular disease, skeletal diseases, cancer and
Inflammation has improvement and therapeutical effect, is accepted extensively by people and is applied in multiple therapeutic scheme.
For lacking Δ 12 and Δ 15 fatty acid desaturase gene (fat2 and fat1) in mammal body, causing cannot
Self normally synthesizes n-3 and n-6PUFAs.At present livestock is both needed to external source in feeding process and adds C18:3n-3 and C18:
2n-6 is to meet the original substrate accumulation of two kinds of PUFAs route of synthesis, it is simple to DHA direction composition.Due in mammal body
Lacking enough DHA and restrict its growth promoter, the multiple product rich in DHA in succession emerges and obtains health care medicine lot number.People
Affirmative and demand for DHA health benefit are considerably beyond its yield.
There is much research to attempt by transgenic animal productions such as pig, cattle, sheep at present and turned Δ 12 or Δ 15 fatty acid
Delta 8 desaturase genes (fat2 and fat1), but these transgenic animal can only solve Δ 12 or Δ 15 fatty acid desaturase gene
(fat2 and fat1) one of them, in other words, one of them route of synthesis of n-3 or n-6PUFAs can only be promoted, it is impossible to carry simultaneously
High n-3 and n-6PUFAs content, cures the symptoms, not the disease.For this present situation, the preparation of existing team turns Δ 12 and Δ 15 fat simultaneously
The Brachydanio rerio of acid delta 8 desaturase genes (fat1 and fat2 gene), can improve n-3 and n-6PUFAs content really simultaneously.But it becomes
Really shortcoming is: 1) transgenic zebrafish cannot be applied at once.Brachydanio rerio is that model organism is for studying from application existence one
Set a distance；2) to husbandry sector without directive function.There is bigger difference with mammal in Fish in PUFAs synthesis, Fish become
Fruit cannot be applied in husbandry sector, also need to make a search in mammal level again；3) its dual-gene taking is total to 2A sequence
Expression-form is attached expressing so that the expression of two genes exists larger difference, and especially 3 ' end gene expression amounts are relatively
Low.
In consideration of it, in husbandry sector and human health research, be highly desirable to Δ 12 and Δ 15 fatty acid desaturation
Enzyme gene (fat2 and fat1) carries out clone amalgamation and expression, and preparation turns fat1-fat2 fusion gene pig so that this transgenic pig
Carnis Sus domestica in express Δ 12 and Δ 15 fatty acid desaturase simultaneously.
Summary of the invention
It is an object of the invention to provide one and turn Δ 12 and the pig of Δ 15 fatty acid desaturase gene (fat2 and fat1),
By transgenic method, Δ 12 and Δ 15 fatty acid desaturase gene fat2 and fat1 gene are inserted with amalgamation and expression form
In No. 10 chromosome DCTN5 gene intron 2 of pig, it is thus achieved that turn fat1-fat2 fusion gene pig, each group of this transgenic pig
Equal great expression n-3 and n-6PUFAs in knitting, can utilize this transgenic pig to produce have guarantor in conjunction with the health-care effect of PUFAs
The Carnis Sus domestica etc. of strong effect, is applied in treatment and the preventing and treating of human diseases.Due to pig immune system and metabolism and the mankind ten
Seemingly, the PUFAs of its secretion will not produce immunological rejection to split-phase when human disease treatment, compares other similar transgenic
Animal is more safe and efficient, it is simple to human foods is taken in.And add medicine, this transgenic pig relative to PUFAs external source
The picked-up toxic and side effects of meat is less, and becomes a new generation's external source PUFAs substitute.
According to an aspect of the invention, it is provided one turns Δ 12 and Δ 15 fatty acid desaturase gene pig, this Δ
12 and Δ 15 fatty acid desaturase gene pig genome in comprise Δ 12 and the fusion gene of Δ 15 fatty acid desaturase
Sequence, the fusion gene sequence of described Δ 12 and Δ 15 fatty acid desaturase is as shown in SEQ ID NO:1, and this transgenic pig
Carnis Sus domestica rich in n-3 and n-6PUFAs.Thus, the fat1-fat2 fusion gene sequence as shown in SEQ ID NO:1 is proceeded to
In gene pig so that this transgenic pig produces the Carnis Sus domestica rich in n-3 and n-6PUFAs, outside people can be by edible Carnis Sus domestica mode
Source obtains n-3 and n-6PUFAs, thus improves the present situation that mankind itself lacks PUFAs.In conjunction with the health-care effect of PUFAs, preventing and treating
Human diseases.Additionally, people can reduce the moment by form absorption external source PUFAs of food Carnis Sus domestica supplements PUFAs health product
Present situation, reduce health product absorbtivity, reduce human body toxic and side effects, meet to greatest extent people for growth promoter institute required
The consumption of PUFAs.
In some embodiments, the fusion gene sequence of this Δ 12 and Δ 15 fatty acid desaturase is by Δ 15 fatty acid
Delta 8 desaturase genes (fat1 gene) and Δ 12 fatty acid desaturase gene (fat2 gene) are directly connected to form fat1-fat2
Fusion gene.Thus, fat2 gene and fat1 gene share a promoter, and the two neighbouring fat1-fat2 that connects into merges base
Cause, after this fusion gene is incorporated into the chromosome of transgenic pig, is started by promoter CAG and transcribes, then translate into a fusion
Expressing protein, but this albumen has Δ 12 fatty acid desaturase and the effect of Δ 15 fatty acid desaturase, and two kinds simultaneously
Enzyme is close to equivalent and each plays effect and function thereof.Thus, it is possible to integrate a fat1-fat2 by transgenic technology to merge table
Reach gene to the chromosome of transgenic pig, and this amalgamation and expression gene plays Δ 12 fatty acid desaturase and Δ 15 fat simultaneously
The effect of fat acid desaturase, improves this transgenic pig and the health care of meat thereof and edibility.
In some embodiments, this turns fat1-fat2 fusion gene sequence and is incorporated into No. 10 dyeing of transgenic pig
On body.
In some embodiments, this turns fat1-fat2 fusion gene sequence and is incorporated into No. 10 dyeing of transgenic pig
On body in DCTN5 gene intron 2.DCTN5 gene is the known open gene (GenBank on No. 10 chromosome of pig
Accession number:NC_010452.3), and on No. 10 chromosome, DCTN5 gene region is that gene expression enlivens
District.Thus, it is incorporated on No. 10 chromosome of transgenic pig in DCTN5 gene intron 2 when fat1-fat2 fusion gene
Time, this transgenic pig can correctly express PUFAs in fat1-fat2 fusion gene, and the muscular tissue (Carnis Sus domestica) of this transgenic pig
Content is the highest, produces the Carnis Sus domestica rich in n-3 and n-6PUFAs, improves the present situation lacking PUFAs in Carnis Sus domestica, and people eat this pig
The effect preventing and treating multiple disease can be played after meat.
According to another aspect of the present invention, it is provided that a kind of system turning Δ 12 and Δ 15 fatty acid desaturase gene pig
Preparation Method, this transgenic pig is by fat1-fat2 amalgamation and expression gene integration to pig genome by transgenic technology, pig
Great expression in muscular tissue (Carnis Sus domestica), produces abundant n-3 and n-6PUFAs.In conjunction with the health-care effect of PUFAs, people are at food
The present situation that mankind itself lacks PUFAs can be improved during with this transgenic Carnis Sus domestica, prevent and treat human diseases.PUFAs can be reduced simultaneously
The demands status of health product, minimizing health product, to human body poison pair probability, meet people to greatest extent and are musted for growth promoter
Need the consumption of PUFAs.The transgenic pig obtained by the method, its Carnis Sus domestica is rich in n-3 and n-6PUFAs, and yield is high, will not be right
The mankind produce immunological rejection.Comparing other similar transgenic animal to seem more safely and efficient, the method includes as follows
Step:
(1) building transgenic fusion expression vector pCAG-fat1-fat2-EGFP, this fusion expression vector comprises fat1-
Fat2 amalgamation and expression gene order, CAG promoter sequence, EGFP sequence；
(2) the transgenic fusion expression vector transfection porcine fetus fibroblasts that will build, after drug screening, obtains
Positive pig cell；
(3) the positive pig cell that screening obtains is obtained clone embryos, by clone embryos after somatic cell nuclear transfer technique
Migrate to the intrauterine of receptor sow, cultivate primary transgenic pig；
(4) the primary transgenic pig of birth is detected, filter out and can stablize heredity Δ 12 and Δ 15 fat in offspring
Fat acid delta 8 desaturase genes, and rich in the transgenic pig of PUFAs in Carnis Sus domestica.
In some embodiments, the gene order of transgenic fusion expression vector pCAG-fat1-fat2-EGFP such as SEQ
Shown in ID NO:2.
In some embodiments, this transgenic fusion expression vector pCAG-fat1-fat2-EGFP is incorporated into described
On No. 10 chromosome of transgenic pig in DCTN5 gene intron 2.
In some embodiments, porcine fetus fibroblasts is boar fetal fibroblast, primary turn produced
Gene pig is transgenic boar.Transfect genes of interest using boar fetal fibroblast as target cell, and filter out positive thin
Born of the same parents, carry out body-cell neucleus transplanting using this positive cell as the donor cell of nuclear transplantation and obtain clone embryos, moved by clone embryos
Plant the intrauterine to receptor sow, cultivate primary transgenic boar.Thus, carry out using this primary transgenic boar as male parent
Breeding, can obtain in a large number can stable heredity Δ 12 and the offspring pig of Δ 15 fatty acid desaturase gene.When primary transgenic
When pig is boar, owing to not affected by period of pregnancy etc., than sow, there is higher reproductive capacity.
According to the third aspect of the present invention, it is provided that a kind of turn Δ 12 and Δ 15 fatty acid desaturase gene pig should
With, this application comprises rich in Δ 12 and the preparation method of the Carnis Sus domestica of Δ 15 fatty acid desaturase, turns from there through setting up to utilize
Gene pig produces the method rich in n-3 and n-6PUFAs Carnis Sus domestica, in conjunction with the health-care effect of PUFAs, can be widely applied to the mankind
In the treatment of disease and preventing and treating, safer, more efficient, comprise the steps of:
1) Δ 12 and Δ 15 fatty acid desaturase gene pig are turned according to said method preparation；
2) using step 1) in obtain turn Δ 12 and Δ 15 fatty acid desaturase gene pig is bred as male parent, obtain
Heredity must be stablized and turn Δ 12 and Δ 15 fatty acid desaturase gene, and rich in the transgenic pig of PUFAs in Carnis Sus domestica；
3) by step 2) in turn Δ 12 and Δ 15 fatty acid desaturase gene pig is butchered, obtain rich in PUFAs
Carnis Sus domestica.
In some embodiments, the Carnis Sus domestica rich in PUFAs is applied in human health care as pork product, for mankind's disease
Disease plays preventive and therapeutic effect.
Accompanying drawing explanation
Fig. 1 is for turning fat1-fat2 fusion gene carrier structural representation.
Fig. 2 is for turning fat1-fat2 fusion expression vector PCR testing result: in figure, 1,2 expand for EGFP gene PCR；3,4
Expand for fat1-fat2 fusion gene PCR；" M " is Marker labelling.
Fig. 3 is for positive transgenic cell fluorescence testing result after drug screening: " Bright field " is indicated in white light
Lower compared with control cells；" Blue Light field " indicates cell under blue light illumination and expresses green fluorescence.
Fig. 4 is the fluorescence results figure of primary transgenic pig: " Normal light " be denoted as under white light transgenic pig and
Tissue control；" Blue light " indicates transgenic pig and expresses green fluorescence under blue light illumination.
Fig. 5 is primary transgenic pig PCR detection electrophoresis result figure: " H in figure2O " represent blank；" Neg. " represents non-
Transgenic pig；" Pos. " represents positive vector comparison；" M " represents Marker labelling；“Transgenic pigs(935800-
935802) " represent that each primary transgenic pig individuality is numbered；" Fat1fat2gene " is the fat1-fat2 fusion gene proceeded to；
" EGFP gene " is green fluorescence protein gene；" GADPH gene " is pig reference gene.
Fig. 6 is the Southern blot result figure of primary transgenic pig: in figure, " M " represents Marker labelling；“0.5c-
2c " represent 3 positive copy gradients；" Transgenic pigs (935800-935802) " represents each primary transgenic pig
Body is numbered.
Fig. 7 is the mRNA relative expression quantity testing result of fat1-fat2 gene in primary transgenic pig muscular tissue: " WT "
Represent non-transgenic pig；" 935800-935802 " represents that each primary transgenic pig individuality is numbered；In " β-actin " represents pig
Ginseng gene.
Fig. 8 is fat1-fat2 gene mRNA relative expression quantity difference between each transgenic pig individuality different tissues:
" 935800-935802 " represents that each primary transgenic pig individuality is numbered；Figure A is muscular tissue；Figure B is skin histology；Figure C is
Fatty tissue.
Fig. 9 is the comparison that fat1-fat2 fusion gene sequence is incorporated on No. 10 chromosome of No. 935801 transgenic pigs
Interpretation of result figure.
Detailed description of the invention
The present invention is further detailed explanation below in conjunction with the accompanying drawings.
Pst-fat1 plasmid origin is in Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health；PcDNA3.1 carrier is purchased from
Excellent precious biological；PEGFP-N1 empty carrier is purchased from excellent precious biological；Fat2 gene order derives from GenBank accession
Number:NM070159；Fat1 gene order derives from GenBank accession number:178291.
1, fat1-fat2 fusion gene carrier and PCR augmentation detection thereof are built.
Utilizing round pcr to amplify fat1 gene from pst-fat1 plasmid, fat2 gene is announced according to GenBank
Sequence direct labor synthesis.Utilize overlap extension pcr, be connected as between fat1 and the fat2 gene after amplification
Fat1-fat2 fusion gene (as shown in SEQ ID NO:1), shares a promoter after the neighbouring connection of fat1 and fat2 gene
CAG, transcription and translation is a fusion protein, and this fusion protein can play the function of fat1 gene and fat2 gene simultaneously
Function.From pcDNA3.1 carrier, design primer amplification CMV, NEO, bGH-polyA sequence simultaneously.Utilize enzyme action interconnection technique,
Fat1-fat2 fusion gene and a series of synthetic gene are connected to pEGFP-N1 carrier, and this carrier contains EGFP gene,
Transgenic animal and cell screening can be done.Finally build fat1-fat2 integrative gene expression vector: pCAG-fat1-
Fat2-EGFP (as shown in SEQ ID NO:2).This fusion expression vector total length 15300bp.CMV promoter starts EGFP gene
Express green fluorescence；CAG promoter starts genes of interest fat1-fat2 amalgamation and expression.Accompanying drawing 1 is shown in by its structure collection of illustrative plates.
EGFP gene on plasmid and fat1-fat2 fusion gene carry out PCR amplification inspection respectively, and band is clearly single
One, result is correct, and amplification is shown in accompanying drawing 2.
2, positive transgenic cell fluorescence detection.
Utilize liposomal transfection teclmiques that fat1-fat2 integrative gene expression vector is imported boar fetal fibroblast,
Cell transient expression green fluorescence.After G418 screens 20 days, remaining cell is the Green fluorescent colonies group of stable transfection, positive
Cell fluorescence testing result is shown in accompanying drawing 3.
3, preparation and the qualification thereof of fat1-fat2 fusion gene pig are turned.
The cell clone group of green fluorescence is expressed as donorcells, by body-cell neucleus transplanting method, 3 after screening
Head sow goes up 868 pieces of clone embryos of co-transplantation.2 sows return feelings, and 1 farrowing sow gives birth to 3 young offspring boars that live, and these are 3 years old
No. ID of head transgenic pig is respectively 935800,935801,935802.Progeny transgenic pig carries out butchering survey 120 day age respectively
Determine content of polyunsaturated fatty acid.
By the genomic DNA of 3 transgenic pig ear tissues by Tissue DNA extraction kit (purchased from Omega
Company) test kit is stripped.Fat1-fat2 gene, EGFP gene and pig reference gene GADPH are respectively adopted three pairs of primers pair
It carries out corresponding PCR augmentation detection.3 transgenic pigs all can detect that fusion gene fat1-fat2, EGFP gene, internal reference
The existence of gene GADPH, band is single, and length is respectively 1942bp, 2118bp, 182bpPCR, and PCR testing result is shown in accompanying drawing 5.
Using portable fluorescent lamp, the transgenic pig green fluorescence detecting 3 day age is expressed.Seen from perusal, 3 turn base
Green fluorescent label is all had, for transgenic positive pig because of pig.Green fluorescence detection is again carried out when 120 day age of transgenic pig,
Transgenic pig green fluorescence is expressed stable.The green fluorescence of transgenic pig body surface is concentrated mainly on transgenic pig snout, hoof
Express.Finding after dissecting sampling, the shallower tissue of the color such as the heart of primary transgenic pig, tongue has obvious green fluorescence,
Transgenic pig fluoroscopic examination is shown in accompanying drawing 4.
3, exogenous gene integration mode in different transgenic pigs and expression compare.
Extract appropriate transgenic pig genomic DNA, the integration feelings of Southern blot detection fat1-fat2 fusion gene
Condition.Result shows that this approving and forwarding gene pig exists two kinds of Integration Modes, and wherein 935800 and 935802 have 3 hybridization positive band；
935801 hybridize positive band for wall scroll, thus it is speculated that 935800 and 935802 from same clone cell group, produce 3 and integrate position
Point, more than 2 copies in single integration site；And 935801 are derived from another cloning cluster, only one of which integration site, purpose base
Because being integrated into single allele, copy number is about 2 copies.The Southern blot result of transgenic pig is shown in accompanying drawing 6.
Detection transgenic pig different times different tissues mRNA expression find, 120 day age transgenic pig (935800-
935802) all having fat1-fat2 gene mRNA expression in, result is shown in accompanying drawing 7.In 120 day age, No. 935802 transgenic pigs are at flesh
In meat, fat and skin histology, expression is minimum.In muscular tissue, 935800 and No. 935801 transgenic pigs are No. 935802 turns
4.36 times and 3.94 times of gene pig mrna expression amount, result is shown in accompanying drawing 8A；In skin histology, the mRNA of 935800 and 935801
Expression is 2.65 times and 3.58 times of 935802, and result is shown in Fig. 8 B；Fatty tissue is similar to skin histology expression, and 935800
With 935801 mrna expression amount be 1.91 times and 2.30 times of 935802, result is shown in Fig. 8 C.
Owing to 935800 and 935801 transgenic pigs expression of fat1-fat2 gene mRNA in each tissue is suitable,
And in 935801 transgenic pigs fat1-fat2 gene only one of which copy number, in the heredity of offspring, more stable, therefore
Select this transgenic pig with for further study.
4, exogenous gene integrates the analysis of position in No. 935801 primary transgenic pig genomes.
In order to determine exogenous gene integration position in transgenic pig genome, test uses inverse PCR technique analysis
No. 935801 primary transgenic pigs integrate exogenous gene positions.Design primer, by checking order to Inverse PCR products and right
Sequencing result finds with pCAG-fat1-fat2-EGFP carrier and pig genome sequence comparison respectively, and this turns fat1-fat2 and merges
Gene order is incorporated on No. 10 chromosome of this transgenic pig in DCTN5 gene intron 2.DCTN5 gene is pig
Known open gene (GenBank accession number:NC_010452.3) on No. 10 chromosomes, and this gene place
Region is gene expression active region, and comparison result is shown in accompanying drawing 9.
5, transgenic pig muscular tissue PUFAs content analysis in No. 935801 transgenic pig familys.
Breed as male parent using No. 935801 primary transgenic pigs, and build No. 935801 transgenic pig familys, and
It is further analyzed, content of polyunsaturated fatty acid during transgenic pig is respectively organized in detection family.And dissect 120 day age 935801
Transgenic pig in number transgenic pig family, muscle of sampling respectively, fat, skin samples (about 10g), put into rapidly-80 DEG C of ice
Case preserves.Take petroleum ether method to extract polyunsaturated fatty acid in tissue sample, entrust China Guangzhou Analysis &. Test Center, depend on
Detecting according to organic mass spectrometry method general rule (JY/T 003-1996), result uses GC/MS area normalization method to calculate.
When 120 day age, from No. 935801 transgenic pig familys, select 3 transgenic pigs, and select non-turn base age 3 on the same day
Because the muscular tissue of pig detects, n-3PUFAs total amount relatively non-transgenic pig improves 41.84%.Wherein C18:3n-3, C20:
5n-3, C22:5n-3 and C22:6n-3 content is respectively increased 40.38%, 133.33%, 33.33% and 130.00%.Simultaneously
C22:4n-6 and C20:4n-6 content increases by 9.52% and 84.09%.N-6PUFAs total amount relatively non-transgenic pig is improved
33.36%.In transgenic pig, n-6/n-3PUFAs ratio is adjusted downward to 12.28 from accounting 13.06, declines and reaches 5.97%, and result is shown in
Following table:
Thus, turn fat1-fat2 fusion gene pig, by transgenic technology, fat1 and fat2 fusion gene is incorporated into pig
In genome, heredity that can be stable in the offspring of transgenic pig, and great expression in Swine muscle (Carnis Sus domestica), produce rich
Rich n-3 and n-6PUFAs.In conjunction with the health-care effect of PUFAs, people can improve mankind itself when this transgenic Carnis Sus domestica edible
Lack the present situation of PUFAs, prevent and treat human diseases.
Above-described is only some embodiments of the present invention.For the person of ordinary skill of the art, not
On the premise of departing from the invention design, it is also possible to making some deformation and improvement, these broadly fall into the protection domain of invention.
Claims (10)
1. one kind turns Δ 12 and Δ 15 fatty acid desaturase gene pig, it is characterised in that described turns Δ 12 and Δ 15 fat
The genome of acid delta 8 desaturase genes pig comprises Δ 12 and the fusion gene sequence of Δ 15 fatty acid desaturase, described Δ 12
With the fusion gene sequence of Δ 15 fatty acid desaturase as shown in SEQ ID NO:1.
The most according to claim 1 turn Δ 12 and Δ 15 fatty acid desaturase gene pig, it is characterised in that described Δ
12 and the fusion gene sequence of Δ 15 fatty acid desaturase by Δ 15 fatty acid desaturase gene order and Δ 12 fatty acid
Delta 8 desaturase genes sequence is directly connected to form fat1-fat2 fusion gene sequence.
The most according to claim 2 turn Δ 12 and Δ 15 fatty acid desaturase gene pig, it is characterised in that described Δ
12 and the fusion gene sequence of Δ 15 fatty acid desaturase be incorporated on No. 10 chromosome of described transgenic pig.
The most according to claim 3 turn Δ 12 and Δ 15 fatty acid desaturase gene pig, it is characterised in that described Δ
12 and the fusion gene sequence of Δ 15 fatty acid desaturase be incorporated on No. 10 chromosome of described transgenic pig
In the intron 2 of DCTN5 gene.
5. the preparation method turning Δ 12 and Δ 15 fatty acid desaturase gene pig, it is characterised in that described preparation method
Comprise the steps of:
(1) building transgenic fusion expression vector pCAG-fat1-fat2-EGFP, this fusion expression vector comprises fat1-fat2
Amalgamation and expression gene order, CAG promoter sequence, EGFP sequence；
(2) the transgenic fusion expression vector transfection porcine fetus fibroblasts that will build, after drug screening, obtains the positive
Pig cell；
(3) the positive pig cell that screening obtains is obtained clone embryos as donor cell after somatic cell nuclear transfer technique, will
Clone embryo transplantation, to the intrauterine of receptor sow, cultivates primary transgenic pig；
(4) the primary transgenic pig of birth is detected, filter out and can stablize heredity Δ 12 and Δ 15 fatty acid in offspring
Rich in the transgenic pig of PUFAs in delta 8 desaturase genes, and Carnis Sus domestica.
The preparation method turning Δ 12 and Δ 15 fatty acid desaturase gene pig the most according to claim 5, its feature exists
In, the gene order of described transgenic fusion expression vector pCAG-fat1-fat2-EGFP is as shown in SEQ ID NO:2.
The preparation method turning Δ 12 and Δ 15 fatty acid desaturase gene pig the most according to claim 6, its feature exists
In, described transgenic fusion expression vector pCAG-fat1-fat2-EGFP is incorporated into No. 10 dye of described transgenic pig
In the intron 2 of the DCTN5 gene on colour solid.
The preparation method turning Δ 12 and Δ 15 fatty acid desaturase gene pig the most according to claim 5, its feature exists
In, described porcine fetus fibroblasts is boar fetal fibroblast, and described primary transgenic pig is transgenic boar.
9. turning Δ 12 and an application for Δ 15 fatty acid desaturase gene pig, described application comprises rich in PUFAs Carnis Sus domestica
Preparation method, it is characterised in that comprise the steps of:
1) Δ 12 and Δ 15 fatty acid desaturase gene pig, institute are turned according to the method preparation described in any one of claim 5-8
State and turn Δ 12 and Δ 15 fatty acid desaturase gene pig is according to turning Δ 12 and Δ 15 fat described in any one of claim 1-4
Fat acid delta 8 desaturase genes pig；
2) using step 1) described in turn Δ 12 and Δ 15 fatty acid desaturase gene pig is bred as male parent, it is thus achieved that stable
Heredity turns Δ 12 and Δ 15 fatty acid desaturase gene, and rich in the transgenic pig of PUFAs in Carnis Sus domestica；
3) by step 2) described in turn Δ 12 and Δ 15 fatty acid desaturase gene pig is butchered, obtain rich in PUFAs
Carnis Sus domestica.
The application turning Δ 12 and Δ 15 fatty acid desaturase gene pig the most according to claim 9, it is characterised in that
Described is applied in human health care as pork product rich in PUFAs Carnis Sus domestica, plays preventive and therapeutic effect for human diseases.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206675A (en) * | 2011-03-23 | 2011-10-05 | 中国农业科学院北京畜牧兽医研究所 | Novel vector for raising content of polyunsaturated fatty acids in animal body |
| CN104059881A (en) * | 2013-03-21 | 2014-09-24 | 中国农业大学 | Method for producing polyunsaturated fatty acid-containing transgenic animal |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206675A (en) * | 2011-03-23 | 2011-10-05 | 中国农业科学院北京畜牧兽医研究所 | Novel vector for raising content of polyunsaturated fatty acids in animal body |
| CN104059881A (en) * | 2013-03-21 | 2014-09-24 | 中国农业大学 | Method for producing polyunsaturated fatty acid-containing transgenic animal |
Non-Patent Citations (1)
| Title |
|---|
| SHAO-CHEN PANG，ET AL: "Double Transgenesis of Humanized fat1 and fat2 Genes Promotes Omega-3 Polyunsaturated Fatty Acids Synthesis in a Zebrafish Model", 《MAR BIOTECHNOL》 * |
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