CN103074347B - Humanized mfat1 gene for mammals to express and application of same - Google Patents
Humanized mfat1 gene for mammals to express and application of same Download PDFInfo
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- CN103074347B CN103074347B CN201310025915.3A CN201310025915A CN103074347B CN 103074347 B CN103074347 B CN 103074347B CN 201310025915 A CN201310025915 A CN 201310025915A CN 103074347 B CN103074347 B CN 103074347B
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Abstract
The invention discloses a humanized mfat1 gene for mammals to express. The complete sequence of the humanized mfat1 gene is shown as SEQ ID No.1. The invention also discloses application of the humanized mfat1 gene to improvement on the quality of a transgenic mammal. The humanized mfat1 gene disclosed by the invention can be efficiently expressed in the mammals, thereby effectively improving the quality of transgenic animals.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of humanized mfat1 gene.
Background technology
Omega-3 polyunsaturated fatty acid (omega-3polyunsaturated fatty acids, n-3PUFAs; Claim again n-3, ω-3), people's bulk-growth and healthy necessary material, belong to 18-24 carbon fatty acid family, have 3 above two keys in its carbochain, according to first pair of bond length, from methyl end carbon atom difference, being divided into n-3 system, n-6 system, n-7 system, n-9 is (to be n numbering system, also be ω numbering system), n-3PUFAs refers to from fatty acid carbon chain methyl end and counts, and the polyunsaturated fatty acid of first double bond position on the 3rd carbon atom, belongs to linolenic acids.Because cells of mamma animals does not have the enzyme that can increase after two 9 atoms of key to the, therefore, n-3PUFAs is essential amino acid, is the main component of cell membrane phospholipid, can not synthesize in vivo, must from food, provide.
N-3PUFAs mainly comprises alpha-linolenic acid (α-linolenic acid, ALA, 18:3n-3), timnodonic acid (eicosapentaenoic acid, EPA, 20:5n-3), docosahexenoic acid (docosahexaenoic acid, DHA, 22:6n-3).ALA is many from vegetables oil, and as linseed oil, walnut kernel and other seed oils, EPA and DHA are mainly from fish oil, these compositions, owing to can not synthesizing in body, can only absorb, therefore from food, be called as " essential " lipid acid (essential fatty acid, EFA).Wherein alpha-linolenic acid, as the precursor of n-3 polyunsaturated fatty acid, reacts with prolongation through desaturation in vivo, generates the n-3 polyunsaturated fatty acids such as EPA or DHA, the n-3 polyunsaturated fatty acid desaturase transforming to n-3PUFAs without catalysis n-6PUFAs in human body.N-3PUFAs mainly obtains from fish oil at present, but refining technique complexity and productive rate are low.
Derive from the n-3 polyunsaturated fatty acid dehydrogenase gene (fat-1) of C. Elegans Automatic Screening (Caenorhabditis elegans), its coded product is n-3 polyunsaturated fatty acid desaturase, can n-6PUFAs be preferential substrate, make its dehydrogenation be converted into n-3PUFAs.At present, the research of n-3 polyunsaturated fatty acid (n-3PUFA) main achievement aspect cardiovascular.It has reduction blood fat; change hemorheology; anticoagulant and thrombosis; effect (the Kobayashi M such as formation that suppress atherosclerotic plaque; Sasaki S; Hamada GS.et al.Serum n-3fatty acids, fish consumption and cancer mortality in six Japanese populations in Japan and Brazil.J Cancer Res.1999; 90 (9): 914-21.Saadatian-Elahi M; Toniolo P, Ferrari P.et al.Serum fatty acids and risk of breast cancer in a nested case-control study of the New York University Women's Health Study.Cancer Epidemiol Biomarkers Prev.2002; 11 (11): 1353-60)
Along with deepening continuously of research; it is found that n-3PUFA also has anti-tumor function; and more and more pay attention to its antitumor action (Barber MD; Ross JA; Preston T, et a1.Fish oil-enriched nutritional supplement attenuates progression of the acute-phase response in weight-losing patients with advanced pancreatic cancer.J Nutr.1999; 129 (6): 1120-5).Fatty acid content in blood of human body is measured and be studies show that, n-3PUFAs level and prostate cancer sickness rate relation (the Pratt VC that is inversely proportional in blood, Watanabe S, Bruera E, et al.Plasma and neutrophil fatty acid composition in advanced cancer patients and response to fish oil supplementation.Br J Cancer.2002,87 (12): 1370-1378).Result of study is found, n-3PUFAs can be by number of ways inhibition tumor cell propagation and inducing apoptosis of tumour cell (Stoll BA.N-3fatty acids and lipid peroxidation in breast cancer inhibition.Br J Nutr.2002, 87 (3): 193-198.Palakurthi SS, Fluckiger R, Aktas H, et al.Inhibition of translation initiation mediates the anticancer effect of the n-3polyunsaturated fatty acid eicosapentaenoic acid.Cancer Res.2000, 60 (11): 2919-2925).Based on these data, we transform fat1 gene, obtain mfat1 gene so that this gene can be in Mammals high efficient expression.We express humanized mfat1 gene at expection in Mammals, can effectively improve the quality of various transgenic animal.
Summary of the invention
Technical problem to be solved by this invention be to provide a kind of humanized can be in Mammals the mfat1 gene of high efficient expression.
Sequence of the present invention is as shown in SEQ ID No:1.
Above-mentionedly be applicable to the application in improving transgene mammal quality of humanization mfat1 gene that Mammals expresses.
Wherein, described Mammals is mouse.
Wherein, described raising transgene mammal quality, for humanization mfat1 gene completes the function that is converted into Omega-3 polyunsaturated fatty acid in mammalian body; In islet tissue, improve the level of islet cells excreting insulin; Improve the apoptosis capacity of islet cells opposing cytokine induction.
Beneficial effect: humanization mfat1 gene of the present invention can be for expressing in Mammals, complete in vivo the function that is converted into Omega-3 polyunsaturated fatty acid simultaneously, in islet tissue, can effectively improve the level of islet cells excreting insulin, in addition, can improve the apoptosis capacity of islet cells opposing cytokine induction.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the pST181 transgene carrier that builds in the embodiment of the present invention.
Fig. 2, after mfat1 transgenosis is distinguished separated islet tissue with wild-type control mice, uses the glucose of different concns and the glucagon-like-peptide-1 co-treatment of 10nmol/l 30 minutes, measures insulin secretion level.Result is presented under glucose and glucagon-like-peptide-1 stimulation, and the pancreas islet of mfat1 transgenic mice has higher levels of insulin secretion.
Fig. 3 is mfat1 transgenosis and the separated islet tissue of wild-type control mice difference, then the glucose of use 1.1mmol/l and 10mmol/l leucine and 10mmol/l glutaminate are processed 60 minutes, contrast is the dextrose treatment of only using 1.1mmol/l, measures insulin secretion level.Result is presented at glucose, and under leucine and glutaminate stimulate, the pancreas islet of mfat1 transgenic mice has higher levels of insulin secretion.
Fig. 4 is mfat1 transgenosis and the separated islet tissue of wild-type control mice difference, with 10 μ mol/l prostaglandin E2 (PGE2) pre-treatment, use respectively afterwards 50ng/ml Toxins, pertussis (PTX) to process, do not have Toxins, pertussis to process in contrast, then study the insulin secretion level that glucose stimulates.The islet tissue of result demonstration mfat1 transgenic mice is secreted more Regular Insulin.
Fig. 5 is the apoptosis test of cytokine induction.From the pancreas islet that mfat1 transgenic mice is separated with contrast wild-type mice, by cytokine, process 48 hours, then with confocal microscope, analyze.Result shows that mfat1 transgenic mice can obviously suppress the apoptosis of cytokine induction.
Fig. 6 is the quantitative statistical analysis of the apoptosis test of cytokine induction.Result shows that mfat1 transgenic mice can obviously suppress the apoptosis of cytokine induction.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described content of embodiment is only for the present invention is described, and should also can not limit the present invention described in detail in claims.
The structure of embodiment 1:pST181 plasmid.
First according to the cDNA sequence of nematode fat1 gene, according to Mammals Nucleotide frequency of utilization, be optimized, the cDNA sequence (being synthesized by Geneart) of having synthesized high efficient expression fat1 in Mammals, called after mfat1, this transgenic fragment that can extensively express fat1 in mouse includes the promotor of CMV associating avian beta-actin (CMV-chicken-β-actin promoter) gene of 1.7kb, muscle creatine kinase enhanser muscle creatine kinase (MCK) enhancer, cDNA (the Mammals mfat-1cDNA of mouse fat1 gene, its coding region has effective translation after optimizing in mammalian cell system) and bovine growth hormone gene (bGH) 3 ' end untranslated region, and with screening resistant gene-penbritin (ampicillin) gene (see figure 1) of screening use in resistant gene Liu Suanyan NEOMYCIN SULPHATE (Neomycin) gene of use and prokaryotic cell prokaryocyte in mammalian cell.The promotor of β-skeletal actin (CMV-chicken-β-actin promoter) gene that this energy popularity is expressed can guarantee the expression of downstream gene popularity.
The complete sequence of the transgene carrier of the general expression mfat1 that the present invention builds is as shown in SEQ ID No:1, the promoter region that wherein 586-2285 base is gene, 317-533 base is MCK enhancer, 2286-3516 base is the coding region of mfat1 gene, 3572-3796 base is bGH3 ' end untranslated region, 4275-5778 base is Liu Suanyan NEOMYCIN SULPHATE (Neomycin) resistant gene, and 6972-7971 base is penbritin (Ampicillin) resistant gene.
Embodiment 2: utilize the method for procaryotic injection build transgenic mice and identify.
By the pST181 transgene carrier building, with Bgl II and Rsr II digestion with restriction enzyme, reclaim required segment, by the method for procaryotic injection, directly inject zygote, make exogenous origin gene integrator in DNA, then zygote is implanted in the 4-5 female mouse body of replace-conceive C57BL/6 in age in week.The genome that extracts mfat-1 transgenic mice afterbody, utilizes PCR primer to increase, and carries out genotype identification.
Forward primer is: 5 '-GGACCTTGGTGAAGAGCATCCG-3 ',
Reverse primer is: 5 '-GCGTCGCAGAAGCCAAAC-3 '.
The positive segment of fragment of the 438bp obtaining.
The phenotypic function analysis of embodiment 3:mfat-1 transgenic mice.
1. the determination of fatty acid of Mice Body inner tissue, mfat1 proceeds in Mice Body, can effectively Omega-6 polyunsaturated fatty acid in Mice Body be converted into Omega-3 polyunsaturated fatty acid.
Extract the lipid acid of mouse mouse tail and islet tissue, method by chromatography of gases is measured unsaturated fatty acid content in mouse mouse tail and islet tissue, the mouse tail of the transgenic mice that discovery embodiment 2 makes is compared compared with wild-type control mice with islet tissue, contain higher levels of Omega-3 polyunsaturated fatty acid, and the Omega-6 polyunsaturated fatty acid of lower level.As shown in table 1.Table 1 is in the embodiment of the present invention 3, the determination of fatty acid result of transgenic mice and contrast wild-type mice mouse tail and islet tissue, and mfat1 transgenic mice and wild-type control group mice are taken respectively tail tissue and 200 pancreas islet of 50mg.Presentation of results proceeds to mfat1 in Mice Body, can effectively the Omega-6 polyunsaturated fatty acid in Mice Body be converted into Omega-3 polyunsaturated fatty acid.
Table 1
* representative p<0.001 when mfat1 group is compared with corresponding wild-type control group has significant difference.
2. separating mouse islet tissue, processes separated mouse islets tissue with high concentration glucose and glucagon-like-peptide-1, and the islet cells excreting insulin level of transgenic mice separation is significantly increased.
Separating mouse pancreas islet by the following method: use Hanks ' the buffered saline solution (HBSS) that contains collagenase (type V) to carry out perfusion digestion by ductus pancreaticus.Digestion shreds later, be placed in HBSS, 37 ° of C, 15 minutes, then with the cell sieve of 500 μ m, filter, HBSS cleans, Ficoll-gradient gradient centrifugation purification pancreas islet, finally under inverted microscope, choose by hand separated pancreas islet, the pancreas islet of every 10 similar sizes is divided into one group, in Krebs-Ringer bicarbonate buffer (KRBB) (adding 10mmol/l HEPES and0.5%BSA), carry out pre-treatment, with 2.8mmol/l glucose, at 37 ° of C, process 1 hour, with KRBB, wash once, then with the KRBB of the fresh configuration of 1ml that contains low concentration glucose (2.8mmol/l) or high concentration glucose (22.2mmol/l), process 30 minutes.Control group is that separated pancreas islet is processed with the KRBB that contains high concentration glucose and 10nmol/l glucagon-like-peptide-1 glucagon-like peptide-1 (GLP-1), as a control group.After processing, collect supernatant in KRBB, by the method for ELISA, measure insulin content.After glucose stimulates, the islet tissue of transgenic mice contrasts and compares, and excreting insulin level is significantly increased.As shown in Figure 2.
3. the mouse islets tissue of separation, uses amino acid stimulation process, and the islet cells excreting insulin level of transgenic mice separation is significantly increased.
In addition, the pancreas islet of every 10 similar sizes is divided into one group, at KRBB and 1.1mmol/l glucose, at 37 ° of C, process 1 hour, then containing 1.1mmol/l glucose, in the KRBB of the fresh configuration of 0.5ml of 10mmol/l leucine and 10mmol/l glutaminate, process 1 hour, collect afterwards supernatant, pancreas islet is collected in KRBB, measure insulin content, as Fig. 3.Result demonstration, after amino acid stimulates, the islet tissue excreting insulin level of transgenic mice is significantly increased.
4. the mouse islets tissue of separation, uses Toxins, pertussis and prostaglandin E2 stimulation process, and the islet cells excreting insulin level of transgenic mice separation is significantly increased.
The pancreas islet of every 6 similar sizes is divided into one group, with the glucose containing 2.8mmol/l, there is or do not have the KRBB of the Toxins, pertussis of 50ng/ml and the prostaglandin E2 (PGE2) of 10 μ mol/l to process 12 hours, before carrying out the glucose stimulation test of 22.2mmol/l.Collect nutrient solution, measure insulin concentration.As shown in Figure 4, the islet tissue of mfat1 transgenic mice is secreted more Regular Insulin.
5. the mouse islets tissue of separation, by different cytokine stimulation process, genetically modified islet cells can be resisted the necrocytosis of cytokine induction completely.
After pancreas islet vitro culture 48 hours, by following cytokine, process: 5ng/ml human interleukin-1 β (IL-1 β interleukin-), 100ng/ml γ-interferon (IFN-IFN-γ), and 10ng/ml tumor necrosis factor-α (TNF-cachectin) 48 hours.Every group of experiment in triplicate.(PI) propidium iodide of propidium iodide for pancreas islet and Hoescst nuclear dye redye, and use confocal microscopical analysis.As Fig. 5, shown in 6.Result shows that genetically modified islet cells can resist the necrocytosis of cytokine induction completely.
In the modern society mankind's food, the ratio of the corresponding Omega-3 polyunsaturated fatty acid of Omega-6 polyunsaturated fatty acid is up to 20:1, but not the 1:1 of desired proportions, relatively derive from the quasi-eicosane acid product of Omega-3 polyunsaturated fatty acid, the quasi-eicosane acid product of Omega-6 polyunsaturated fatty acid is more potential inflammatory factor, the unbalance corresponding Omega-3 polyunsaturated fatty acid of Omega-6 polyunsaturated fatty acid ratio highly promotes inflammatory reaction by being, many modern diseases are brought out in this unbalance ratio regular meeting, autoimmune disease for example, diabetes etc.
In cell, the rising of Omega-3 levels of polyunsaturated fatty acids has very favorable impact for the survival of islet cells under inflammatory stimulus, and what this also can be interpreted as can effectively reduce the generation of type 1 diabetes in the intake of young stage increase Omega-3 polyunsaturated fatty acid.
Claims (3)
1. be applicable to the humanization mfat1 gene that Mammals is expressed, its complete sequence is as shown in SEQ ID No:1.
2. be claimed in claim 1ly applicable to the application in improving transgene mammal quality of humanization mfat1 gene that Mammals expresses;
Wherein, described raising transgene mammal quality, for humanization mfat1 gene completes the function that Omega-6 is converted into Omega-3 polyunsaturated fatty acid in mammalian body; In islet tissue, improve the level of islet cells excreting insulin; Improve the apoptosis capacity of islet cells opposing cytokine induction.
3. application according to claim 2, is characterized in that, described Mammals is mouse.
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Non-Patent Citations (8)
| Title |
|---|
| Fat-1转基因小鼠模型的构建;刘晶;《中国优秀硕士学位论文全文数据库 基础科技辑》;20110915(第9期);第四章 * |
| n-3多不饱和脂肪酸对疾病的预防与治疗作用;陈银基 等;《中国油脂》;20081231;第31卷(第9期);第32-33页 * |
| Protein FAT-1[Caenorhabditis elegans];Sulston J.E.;《Genbank》;20120629;全文 * |
| Sulston J.E..Protein FAT-1[Caenorhabditis elegans].《Genbank》.2012, |
| 刘晶.Fat-1转基因小鼠模型的构建.《中国优秀硕士学位论文全文数据库 基础科技辑》.2011,(第9期),A006-67. |
| 华晓敏 等.Fat-1基因及其功能的研究进展.《青岛农业大学学报（自然科学版）》.2012,第1卷(第29期),41-47. * |
| 郑征.n-3多不饱和脂肪酸饮食对小鼠肥胖的影响及fat-1基因小鼠打靶载体的构建.《中国优秀硕士学位论文全文数据库 医药卫生科技辑(月刊)》.2011,(第3期),E055-24. * |
| 陈银基 等.n-3多不饱和脂肪酸对疾病的预防与治疗作用.《中国油脂》.2008,第31卷(第9期),31-33. |
Cited By (1)
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|---|---|---|---|---|
| CN104745627A (en) * | 2015-01-09 | 2015-07-01 | 青岛农业大学 | Method for synthesis, transfection and detection of anti-aging gene of goat |
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