WO2011069459A1 - Vecteur d'expression spécifique à une graine et ses procédés de construction et applications - Google Patents

Vecteur d'expression spécifique à une graine et ses procédés de construction et applications Download PDF

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WO2011069459A1
WO2011069459A1 PCT/CN2010/079620 CN2010079620W WO2011069459A1 WO 2011069459 A1 WO2011069459 A1 WO 2011069459A1 CN 2010079620 W CN2010079620 W CN 2010079620W WO 2011069459 A1 WO2011069459 A1 WO 2011069459A1
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Prior art keywords
apolipoprotein
mutant
milan
gene
oil
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PCT/CN2010/079620
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English (en)
Chinese (zh)
Inventor
安胜军
柴锡庆
王崑声
邵铁梅
焦展
温昕
李雪
刘培
卢海刚
陈云雨
胡良元
许海民
于成钢
Original Assignee
An Shengjun
Chai Xiqing
Wang Kunsheng
Shao Tiemei
Jiao Zhan
Wen Xin
Li Xue
Liu Pei
Lu Haigang
Chen Yunyu
Hu Liangyuan
Xu Haimin
Yu Chenggang
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Application filed by An Shengjun, Chai Xiqing, Wang Kunsheng, Shao Tiemei, Jiao Zhan, Wen Xin, Li Xue, Liu Pei, Lu Haigang, Chen Yunyu, Hu Liangyuan, Xu Haimin, Yu Chenggang filed Critical An Shengjun
Priority to US13/515,209 priority Critical patent/US9745362B2/en
Publication of WO2011069459A1 publication Critical patent/WO2011069459A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon

Definitions

  • the present invention relates to a seed-specific expression vector, a construction method and application thereof, and particularly to a seed-specific expression vector, a construction method thereof, and the use thereof A method for preparing apolipoprotein AI Milan mutants in oil sunflower.
  • BACKGROUND OF THE INVENTION Cardiovascular disease is the leading cause of human death worldwide, and it is estimated that by 2015, approximately 20 million people will die of cardiovascular disease. Many cardiovascular diseases (such as myocardial infarction and stroke) are the main complication of atherosclerosis. The pathogenesis of atherosclerosis is not fully understood.
  • Abnormal blood lipid metabolism is one of the major risk factors for the disease, including high levels of low density lipoproteins (LDL) and low levels of high density lipoprotein. (High density lipoproteins, HDL) are the two most important risk factors.
  • the traditional treatment strategy is to reduce the content of total cholesterol (TC) and low density lipoproteins cholesterol (LDL-C) in plasma.
  • Statins are currently the preferred lipid-lowering drugs, but these drugs cannot Clearing plaque that has deposited on the arterial wall does not achieve the ultimate goal of treating atherosclerosis.
  • Scholars from all over the world are turning their attention to another risk factor, a low level of high-density lipoprotein.
  • Apolipoprotein A-I apolipopratein A-I, apoA-I
  • HDL targeting treatment apolipopratein A-I, apoA-I
  • Apolipoprotein A-I is synthesized in the liver and small intestine, and its original translation product is a pre-pro protein form (prepraapo A-I) containing 267 amino acid residues.
  • the preproprotein is processed by signal peptidase to excise the 18-peptide pre-fragment into the original apolipoprotein form (proapo AI).
  • proapo AI signal peptidase
  • the original 6-peptide fragment is excised under the action of extracellular specific invertase. (Arg-His-Phe-Trp-Gln-Gln), transformed into plasma mature apolipoprotein AI.
  • the mechanism of action of apolipoprotein A-I is mainly to promote cholesterol exudation, anti-oxidation and anti-platelet aggregation.
  • Apolipoprotein A-IM is a natural mutant of apolipoprotein AI (Argl73--Cys), compared with apolipoprotein AI, arginine 173 Loss leads to a decrease in the ct helix content and an increase in the ability to bind to lipids.
  • the apolipoprotein AI Milan mutant is prone to form a dimer (A-IM/A-IM), which promotes the reverse transport of cholesterol more efficiently than apolipoprotein AI, thereby accelerating cholesterol excretion more efficiently.
  • the apolipoprotein AI Milan mutant can better attenuate the oxidation of low-density lipoprotein than apolipoprotein AI.
  • the apolipoprotein AI Milan mutant is the only proven pharmaceutical protein in the world that removes thrombus that has been deposited on the arterial wall. Has a very broad application prospects.
  • apolipoprotein AI Milan mutant has unprecedented speed and amplitude of atherosclerotic changes, and has few side effects. It has a broad application prospect and has become the focus and competition of the world pharmaceutical research and development field. Focus. Pfizer, the world's largest pharmaceutical company, predicts that any drug that reverses coronary plaques could achieve $1 billion in sales, so the development of apolipoprotein AI Milan mutants will inevitably bring huge economic benefits. And social benefits, but also enhance the world's influence in the field of cardiovascular disease and atherosclerotic disease drug development in China.
  • apolipoprotein AI Milan mutant the clinical requirement of apolipoprotein AI is 5-6g per course, and the high therapeutic dose of apolipoprotein AI and the high prevalence of atherosclerosis predict a strong The market demand, which also provides an opportunity for the development of the apolipoprotein AI Milan mutant.
  • Esperion Company produces apolipoprotein A-I Milan mutants for experimental reagents, but it uses a biosynthetic method. The production cost is high and the yield is small, which is not suitable for large-scale production. Recombinant expression of proteins in bacterial systems is generally attractive, but the yield of this method is low, and E.
  • Plant bioreactor also known as Molecular medicine, refers to the use of plant biological systems for the mass production of exogenous proteins of great commercial and commercial value, especially for medical and diagnostic purposes. protein.
  • mammalian antibodies were successfully expressed for the first time in transgenic plants, and their heavy and light chains were expressed and correctly assembled in transgenic tobacco, demonstrating the feasibility of plants as bioreactors for the first time. Since then, research on genetically modified plants has gradually emerged.
  • SemBioSys Genetics a biotechnology company that develops a combination of protein drugs for metabolic and cardiovascular diseases in Canada, has applied for patents for transgenic safflower and Arabidopsis production of apolipoprotein AI and apolipoprotein AI milan mutant methods in China ( CN1906296A), the chimeric nucleic acid construct is introduced into Arabidopsis or safflower, and after seeding, apolipoprotein AI and apolipoprotein AI Milan mutant are expressed in the seed.
  • Arabidopsis thaliana is an annual or biennial herb whose genome is the smallest of the currently known plant genomes, and because of its highly homozygous gene, the physicochemical factors are used to treat mutations with high mutation rates and easy access to various metabolic functions. It has become a good material for genetic research and has been hailed by scientists as "the fruit fly in plants.” However, Arabidopsis is currently widely used in experimental pathways and there is no large-scale widespread planting. Safflower is an annual herb, the seed can be oiled, is an important oil crop, distributed in temperate zones, China is more prolific in the northwest, especially Xinjiang and Cambodia are the most important, followed by North China and Northeast China. However, the yield of safflower is relatively low (120-150 kg), and the oil content of achenes is not very high (34 ⁇ 55 %), resulting in a small amount of final product protein and high cost.
  • the applicant of the present invention has devoted a lot of creative labor through long-term research, and stably and efficiently produces apolipoprotein AI and apolipoprotein by constructing a specific expression vector and selecting oil sunflower as a bioreactor.
  • the AI Milan mutant thus completing the present invention.
  • the present invention relates to a method for producing apolipoprotein A-I or apolipoprotein A-I Milan mutant by recombinant DNA technology, and more particularly to a method for producing apolipoprotein A-I or apolipoprotein A-I Milan mutant using oil sunflower as a host.
  • the present invention relates to the use of a fusion protein gene of peanut oil body protein and apolipoprotein AI or apolipoprotein AI milan mutant in oil sunflower oil to be produced in large quantities for the treatment of atherosclerosis.
  • the important drugs for cardiovascular disease, apolipoprotein AI and apolipoprotein AI milan mutant are preferably prepared for apolipoprotein AI Milan mutant.
  • the present invention provides a seed-specific expression vector comprising a lipoprotein AI Milan mutant and a peanut oil body protein fusion gene or a fusion gene containing apolipoprotein AI and peanut oil body protein, preferably containing apolipoprotein AI A mutant gene of Milan mutant and peanut oil body protein, wherein the promoter of the vector is a promoter of the rapeseed oil protein gene.
  • the above vector is used to prepare apolipoprotein A-I or apolipoprotein A-I Milan mutant in oil sunflower, preferably apolipoprotein A-I Milan mutant.
  • the present invention also provides a method for constructing the above high-efficiency seed-specific expression vector, comprising the following steps:
  • the specific steps include:
  • rape variety can be disclosed or used in the prior art.
  • the rapeseed variety may be 14 varieties of green oil, mutual abundance 101, cold-tolerant high oil king, early oil 100 days, and Qinyou 2, etc., preferably 14 varieties of green oil.
  • the above rapeseed oil body protein promoter can be cloned between suitable sites of pUC19, preferably between the H ⁇ dl l BBamHI sites of pUC19.
  • the peanut variety may be a peanut variety that has been disclosed or used in the prior art, for example, it may be Huanhua No. 4, Yuyou No. 7, Baisha, Luhua 11, Haihua and Fenghua No. 1, etc., preferably ⁇ 4 ⁇ .
  • apolipoprotein AI or apolipoprotein A-iman mutant gene design and synthesize apolipoprotein AI or apolipoprotein A-iman mutant gene according to plant-preferred codons.
  • the optimized gene is the apolipoprotein AI Milan mutant gene. Codons with a frequency less than 10% are considered to be excluded by rare codons, and the remaining codons are optimized according to the frequency of use of the oil sunflower codon.
  • the molecular weight of the optimized gene is 451.4, and the consistency of the sequence before and after optimization is greater than 60%. More than 65%, most preferably 72%, the molecular weight of the optimized gene is preferably 451.3.
  • the lipoprotein AI milan mutant gene or the apolipoprotein AI gene is constructed into a fusion gene, and the peanut oil body protein gene and the apolipoprotein AI milan mutant gene are preferably constructed into a fusion gene O/ e /3 ⁇ 4p -/ M ,
  • the fusion gene was ligated into pUCN to obtain the recombinant plasmid pUCNOA, and the preferred site was ligated between the BomHI and Sacl cleavage sites of pUCN, and the recombinant plasmid pUCNOA was ligated, preferably H dl ll and Sacl were digested with recombinant plasmid pUCNOA.
  • the 2202 bp exogenous fragment was recovered, and the exogenous fragment was ligated into the plant expression vector PBI 121 (the plant binary expression vector commonly used in plant transgenic engineering) to obtain pBINOA, ie, rapeseed oil body.
  • PBI 121 the plant binary expression vector commonly used in plant transgenic engineering
  • the present invention also provides a method for preparing apolipoprotein AI Milan mutant or apolipoprotein AI using the above seed-specific plant expression vector, comprising the following steps:
  • Apolipoprotein A-I Milan mutant or apolipoprotein A-I was isolated and purified from the above seeds.
  • the recipient plant is preferably oil sunflower, and it is preferred to isolate and purify the apolipoprotein A-I Milan mutant.
  • the specific steps include:
  • a seed-specific plant expression vector carrying the apolipoprotein AI Milan mutant gene or the apolipoprotein AI gene is introduced into the oil sunflower restorer explant.
  • the method for introducing a seed-specific plant expression vector into an oil sunflower restorer line may be Conventional introduction methods in the field include, but are not limited to, gene gun method, pollen tube passage method, ovary injection method, and Agrobacterium-mediated method, and Agrobacterium-mediated method is preferred.
  • a seed-specific plant expression vector carrying the apolipoprotein AI Milan mutant gene or the apolipoprotein AI gene is introduced into Agrobacterium, and Agrobacterium tumefaciens-mediated transformation of oil sunflower restorer explants.
  • the explants include four forms of sterile shoot tips, cotyledons, cotyledonary nodes, and live plants with one cotyledon removed, wherein preferably one shoot of the cotyledon is removed;
  • the regenerated plants obtained after transgenic are screened by resistance to obtain resistant seedlings. After the roots of the resistant seedlings are rooted, they are transplanted into the greenhouse for cultivation until the seeds are harvested. Among them, the resistant shoots were transplanted into the greenhouse for cultivation of vermiculite and nutrient soil mixture. PCR detection and Southern blotting were carried out at the seedling stage, and the oil protein and apolipoprotein AI Milan mutant fusion protein were obtained after harvesting the grain. Blotting detection;
  • a canola oil body protein gene promoter is selected. Experimental studies have shown that this promoter can greatly improve the expression efficiency of the apolipoprotein A-I milan mutant gene.
  • the Kozak sequence expression control element can be designed around the start of the oil body protein gene, which can further improve the gene expression efficiency.
  • fusion expression of oil body protein and apolipoprotein A-I Milan mutant or apolipoprotein A-I was also selected.
  • the target protein is specifically expressed in the oil body in the form of a fusion protein in the transgenic plant.
  • the oil is lipophilic and hydrophobic, and the seed of the transgenic plant is pulverized, liquid extracted, centrifuged, and the upper oil phase is recovered.
  • the fusion protein can be separated from other components in the cell to remove more than 90% of the seed protein.
  • a thrombin recognition site is designed between the oil body protein and the apolipoprotein AI Milan mutant or apolipoprotein AI to release the apolipoprotein AI Milan mutant from the oil body, simplifying The purification process of the expressed product improves the purification efficiency.
  • a preferred oil body protein is peanut oil body protein.
  • the fusion expression of peanut oil body protein and apolipoprotein A-I Milan mutant or apolipoprotein A-I has high expression efficiency and good effect.
  • the apolipoprotein AI milan mutant gene or the apolipoprotein AI gene in order to increase the expression efficiency of the apolipoprotein AI milan mutant gene or the apolipoprotein AI gene, according to the apolipoprotein AI milan mutant gene sequence or the apolipoprotein AI gene.
  • the codons and GC content of the sequence and the oily sunflower are optimized for the codons of the apolipoprotein AI Milan mutant gene or the apolipoprotein AI gene and the whole gene synthesis.
  • a preferred plant bioreactor is oil sunflower.
  • oil sunflower has a long history of planting in China and has the irreplaceable advantages of other crops.
  • Oil sunflower has high yield, as a drought-tolerant crop, it can be in saline-alkali land, arid areas or even sand. Planting in a harsh environment, such as desert, is suitable for large-scale extension of planting. It will not only compete with food for "land”, but also help to improve the utilization rate of China's mountainous land, arid and barren land, and ease the pressure on the country's cultivated land.
  • the selection of oil sunflower as a bioreactor, the large-scale production of apolipoprotein AI Milan mutant is a way to produce pharmaceutical protein suitable for China's national conditions.
  • the most advantageous is the use of oil sunflower as a bioreactor, which has a significant increase in productivity compared to safflower, which has now been used as a plant for the apolipoprotein AI Milan mutant or apolipoprotein AI production. Increased effect.
  • the production of human apolipoprotein AI milan mutant by this method has the following advantages:
  • a foreign protein expressed by a plant similar to a mammalian expressed protein, capable of proper folding, which is particularly important for the production of pharmaceutical proteins that must have in vivo activity.
  • the apolipoprotein A-I Milan mutant produced by the plant bioreactor is safer because it avoids the contamination of endotoxin and animal pathogens in E. coli.
  • the expression level of apolipoprotein A-I Milan mutant or apolipoprotein A-I is greatly improved, and the total protein content of the seed can be 1.5%.
  • Transformation of plants by Agrobacterium-mediated transformation not only reduces costs, improves transformation efficiency, but also improves the genetic stability of transgenic plants.
  • the present invention utilizes transgenic technology to develop a highly efficient plant bioreactor, and the apolipoprotein A-I milan mutant or apolipoprotein A-I produced is a special drug for treating cardiovascular diseases and atherosclerotic diseases.
  • LDL low density lipoproteins, low density lipoprotein
  • HDL high density lipoproteins, high density lipoprotein
  • TC total cholesterol, total cholesterol in plasma
  • LDL-C low density lipoproteins cholesterol, low density lipoprotein cholesterol
  • apoA-I apolipoproteinA-I Apo Moon Protein A-I
  • apoA-IM apolipoprotein A-I Milano apolipoprotein A-I milan mutant
  • A-IM/A-IM Apolipoprotein AI Milan mutant dimer pUC 19: Common E. coli cloning vector, purchased from MBI
  • PBI121 Plant expression vector commonly used in plant transgenic engineering
  • pUCN pUC19 vector carrying the rapeseed oil protein gene promoter (NOP), the insertion sites are H dlll and BamHI
  • Oleosin/apoA-IM fusion gene of peanut oil body protein and apolipoprotein A-I milan mutant
  • pUCNOA pUC 19 vector carrying the Brassica napus protein gene promoter (NOP), peanut oil body protein and apolipoprotein A-I mic mutant fusion gene, the insertion sites are ndm and ⁇ cl
  • pBINOA PBI121 vector carrying the Brassica napus protein gene promoter (NOP), peanut oil body protein and apolipoprotein A-I Milan mutant fusion gene, the insertion sites are H dlll and ⁇ cl
  • FIG. 1 Schematic diagram of the seed-specific plant expression vector pBINOA
  • FIG. 2 Schematic diagram of the construction process of seed-specific plant expression vector pBINOA;
  • Figure 3 Enzyme digestion and PCR detection of pUCN vector;
  • Figure 5 Identification of pUCNOA vector by restriction enzyme digestion
  • Figure 6 Identification of seed-specific plant expression vector pBINOA
  • Example 1 Seed-specific plant expression vector
  • the rapeseed oil body protein gene promoter (NOP) is first amplified by a PCR method, and the promoter is inserted between the H dm and Bamffl cleavage sites of pUC 19 to obtain pUCN.
  • the apolipoprotein AI milan mutant gene was designed and synthesized based on the apolipoprotein AI Milan mutant gene sequence and the oil sunflower preferred codon, and the synthetic gene was inserted into the 3' end of the peanut oil body protein gene 01.
  • the peanut oil body protein and the apolipoprotein AI milan mutant fusion gene were obtained, and a thrombin cleavage site was added between the peanut oil body protein gene and the apolipoprotein AI milan mutant gene. Furthermore, the fusion gene was inserted into the BamH MflcI cleavage site of pUCN to obtain ⁇ , «(1 ⁇ ⁇ 5 ⁇ double-cut pUCNOA, agarose gel recovered 2202 bp exogenous fragment, and the exogenous fragment was inserted Between the H ⁇ ffl MflcI restriction sites of the plant binary expression vector pBI121, the plant expression vector pBINOA provided by the present invention was obtained, and the expression cassette of pBINOA was O/e driven by the rapeseed oil protein gene promoter (NOP).
  • NOP rapeseed oil protein gene promoter
  • 3 ⁇ 4p -/ M fusion gene pBINOA structure shown in Figure 1, 1 : rapeseed oil protein gene promoter, 2: peanut oil body protein gene, 3: thrombin cleavage site, 4: apolipoprotein AI Milan mutation Somatic gene.
  • the sequence of the expression cassette was obtained by sequencing pBINOA as shown in SEQ ID NO: 15, and the length was 2202 bp.
  • the rapeseed oil protein gene promoter was cloned. Rape is an important oil crop with high oil content (42 ⁇ 45%), and the amount of 20kD oil body protein in rapeseed oil is 10 times that of 24kD oil body protein.
  • the forward primer pBINOA-1 was designed according to the promoter sequence of the rapeseed oil protein gene promoter (Genbank No. AF134411) : CCC AAG CTT TTC AAC
  • the product was recovered by agarose gel electrophoresis, and the obtained product was double-digested with H dm and fiiimHI, and ligated with Hdm and mffl double-digested pUC19, and the ligation product was compared with 20 ( ⁇ L DH5 ⁇ State cells (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), ice bath for 30 min, heat shock at 42 °C for 1.5 min, ice bath
  • PCR method to select transformants pBINOA-1 and BpBINOA- 2 is a primer, the PCR conditions are: 94 ° C lmin,
  • M DNA Molecular Weight Marker ⁇ DNA/EcoT14I
  • LI Hindm digests the pUCN plasmid product into a 3565 bp fragment
  • L2 H dm and Bflmffl double-cleave the pUCN plasmid product is 2662 bp.
  • L3 PCR detection of pUCN plasmid to obtain a 903 bp promoter.
  • the pUCN plasmid was sequenced, and the sequencing steps were as follows: (1) PCR was carried out by using pUC19 as a template and pUC19 universal sequencing primer to obtain a PCR product;
  • the base C is mutated to the base T, and a thrombin cleavage site is added to the 5' end of the gene.
  • the nucleotide sequence is CTGGTCCCAA GGGGTAGC as shown in SEQ ID NO: 6, and the amino acid sequence is SEQ ID NO.
  • LVPRGS shows LVPRGS
  • the artificially synthesized apolipoprotein AI Milan mutant gene is 750 bp
  • the molecular weight is 462.4 kDa
  • the sequence is shown in SEQ ID NO: 8.
  • the gene-encoded protein consists of 249 amino acid residues with a molecular weight of 28.585 kDa.
  • pBINOA -4 and pBINOA-5 are mutually complementary sequences.
  • pBINOA-3 CGC-GGA TCC AGC AAA GCC GCC ACC ATG GCT ACT GCT ACT GAT CG pBINOA-4: GCT ACC CCT TGG GAC CAG TGA TGA TGA CCT CTT AAC pBINOA-6: C GAG CTC TTA TTG TGT GTT AAG TTT CTT TG
  • PCR conditions were: 94 °C lmin, 50-55 °C Lmin, 68 °C lmin, 30 After cycling, the extension of lOmin at 68 °C; using pBINOA-5 / pBINOA-6 as a primer, the optimized apolipoprotein AI Milan mutant gene as a template to amplify the apolipoprotein AI Milan mutant gene, the PCR conditions are : 94°C lmin, 63-73 °C lmin, 68 °C lmin, 30 cycles, 68 °C extension lOmin; agarose gel electrophoresis and recovery of two PCR amplification products, the molar ratio of the two products 1 : 1 mixed as a template, using pBINOA-3
  • the PCR conditions were: 94 ° C lmin, 50-55 ° C lmin, 68 ° C 2 min, after 30 cycles, 68 ° C extension lOmin, agarose gel electrophoresis and recovery of the amplified product to obtain O / e / 3 ⁇ 4p - / M fusion gene.
  • the construction of the O/e/3 ⁇ 4p -/ M fusion gene is shown in Figure 4, M: DNA Molecular Weight Marker DL2000, LI : pBINOA-3/ pBINOA-4 is the primer, and peanut (variety safflower 4) genomic DNA is used as the template.
  • the mutant gene was 750 bp (containing a nucleotide sequence encoding a thrombin cleavage site), and L3 was an overlapped PCR with pBINOA-3/pBINOA-6 as a primer to obtain an O/e/ ⁇ 3 ⁇ 4wA-/ M fusion gene.
  • the O/e/ ⁇ 3 ⁇ 4wA-/ M fusion gene was sequenced, and the sequencing result showed that the O/e 3 ⁇ 4p -/ M gene sequence was as shown in SEQ ID NO: 13, and was 1278 bp in length and molecular weight 787.9 kDa.
  • the predicted amino acid residue sequence is set forth in SEQ ID NO: 14, consisting of 425 amino acid residues and having a molecular weight of 46.994 kDa.
  • the O/e 3 ⁇ 4p -/ M fusion gene was double-digested with BflmH MflcI and ligated with the same double-digested pUCN, and the ligation product was combined with 20 ( ⁇ L DH5 a competent cells (purchased from Tiangen). Biochemical Technology (Beijing) Co., Ltd.) Mix, ice bath for 30 min, heat shock at 42 °C for 1.5 min, ice bath for 3 min, add 800 ⁇ LB medium for 37 min at 37 ° C, apply 100 ⁇ ⁇ / mL ampicillin The LB plate was cultured overnight at 37 ° C.
  • the transformants were screened by PCR, pBINOA-3 and pBINOA-6 were used as primers.
  • the PCR conditions were: 94 °C lmin, 60-73 °C lmin, 72 °C 1.5 min, 30 After a loop,
  • the positive transformant was named pUCNOA, the positive transformant was subjected to liquid shock culture, the plasmid was extracted by alkaline lysis, and the plasmid was subjected to H dm single digestion.
  • the product of the pUCNOA plasmid is a 3571 bp vector fragment and a 1278 bp exogenous fragment.
  • the pUCNOA plasmid was sequenced, and the sequencing result of SEQ ID NO: 15, the full length of 2202 bp, the molecular weight was 1357.5 kDa, including the rapeseed oil protein gene promoter and the 0 / « ⁇ ' «- ⁇ 4- fusion gene.
  • the results of the digestion (as shown in Figure 5) and the sequencing results (as in the sequence listing) SEQ ID NO: 15 indicate that the peanut oil body protein and the apolipoprotein AI milan mutant driven by the rapeseed oil body protein gene promoter have been obtained. Fusion gene expression cassette, and the expression cassette has been successfully Clone to vector pUC19.
  • Double-digested PBI121 was ligated, and the ligation product was mixed with 20 ( ⁇ L DH5 ⁇ competent cells (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), ice bath for 30 min, heat shock at 42 °C for 1.5 min, ice bath After 3 min, add 800 ⁇ LB medium for 45 min at 37 ° C, and apply LB plate containing 10 ( ⁇ g/mL kanamycin, culture overnight at 37 ° C.
  • PCR method to select transformants pBINOA-1 and pBINOA-6
  • the primers were: 94 ° C lmin, 60-73 ° C lmin 72 ° C 2 min, after 30 cycles, 72 ° C extension lOmin, the PCR product was subjected to agarose gel electrophoresis detection and screening results, the positive transformation
  • the sub-named pBINOA, the positive transformants were cultured in liquid shock, the plasmid was extracted by alkaline lysis, and the plasmid was identified by H ⁇ din single enzyme digestion and H mffl MflcI double enzyme digestion.
  • the results of agarose gel electrophoresis showed that the results are shown in Fig. 6.
  • M DNA Molecular Weight Marker ⁇ DNA/EcoT14I
  • LI HmdIII digested the pBINOA plasmid product was a 14205 bp fragment
  • L2 H mffl ⁇ flcI double-cleavage of the pBINOA plasmid product was a 12003 bp vector fragment and a 2202 bp fragment Source fragment (containing the rapeseed oil protein gene promoter and o/ Eo ⁇ i--/ M fusion gene.
  • the pBINOA plasmid was sequenced, and the sequencing result was SEQ ID NO: 15, and the full-length nucleotide sequence of the vector was as shown in SEQ ID NO: 16.
  • the entire expression cassette was 2202 bp in length and the molecular weight was 1357.5kDa, including the Brassica napus protein gene promoter and ⁇ / « ⁇ ' «- ⁇ -/ ⁇ fusion gene.
  • the rapeseed oil protein gene promoter is a seed-specific strong promoter that drives in the seeds of transgenic plants.
  • the apolipoprotein ⁇ - ⁇ Milan mutant is specifically expressed in the oil body together with the peanut oil body protein in the form of a fusion protein, and the peanut oil body protein carries the apolipoprotein ⁇ - ⁇ Milan mutant anchored on the oil body surface.
  • the transgenic plant seeds are pulverized, liquid extracted, centrifuged, and the upper oil phase is recovered to separate the fusion protein from other components in the cell, and more than 90% of the seed protein can be removed.
  • a thrombin recognition site was designed between the apolipoprotein ⁇ - ⁇ Milan mutants to release the apolipoprotein ⁇ - ⁇ Milan mutant from the oil body.
  • Agrobacterium-mediated genetic transformation of oil sunflower explants Four types of explants of shoot tips, cotyledons, cotyledonary nodes and seedlings with one cotyledon removed from the seedlings of the oil sunflower seeds germinated for 3 to 4 days, Soak in the above Agrobacterium liquid for 6 ⁇ 8min, and transfer to MS solid medium for co-cultivation for 3d (25 °C dark). A preferred mode of this is to remove the live plants of one cotyledon.
  • Receptor plant material is cultivated into a complete plant, and the seed is obtained.
  • the above transformed explants were transferred to MS medium containing cephalosporin 300 mg/L to continue the culture, and after about 7 days, they were transferred to MS resistance screening medium (containing cephalosporin 300 mg/L and Kana).
  • MS resistance screening medium containing cephalosporin 300 mg/L and Kana.
  • Selective culture of 70mg/L of mycin change the medium every 15 ⁇ 20d, obtain resistant buds after screening three times, transfer 2 ⁇ 3cm resistant buds to rooting medium MS2 (MS + IBAO. l mg/L+ Kan 70 mg/L+cef 300 mg/L), after the roots of the resistant seedlings are transplanted into the greenhouse, the mixture of vermiculite and nutrient soil is cultured until the seeds are harvested.
  • PCR was performed on the apolipoprotein AI Milan mutant gene at the seedling stage. After harvesting the grain, the peanut oil body protein and the apolipoprotein AI Milan mutant fusion protein were detected by Western blotting. PCR detection and PCR-Southem blotting detection of transgenic oil sunflower seedlings
  • the genomic DNA of the young leaves of resistant oil sunflower seedlings was extracted by SDS method as a template. PCR amplification was performed with two pairs of primers: npmF/npmR and pBINOA-5/pBINOA-6.
  • the primer sequences were: nptUF: ATGAAC TGC AGGACGAGG (SEQ ID NO: 17) npt!
  • IR GCG ATA CCG TAA AGC ACG (SEQ ID NO: 18)
  • the conditions for PCR amplification of wpllF/wpnR and pBINOA-5/pBINOA-6 are: 94 ° C lmin, 60 ° C lmin, 72 °C lmin, after 30 cycles, 72 °C extended lOmin, respectively amplified the expected 567 bp fragment (partial gene) and 750 bp ⁇ - / ⁇ gene fragment, the results are shown in Figure 7 and Figure 8. Show. In Fig.
  • M DNA Molecular Weight Marker DL2000
  • LI-L4 nF/ «pnR is a primer
  • a 567 bp fragment is amplified from the genomic DNA extracted from the kana-resistant oil sunflower, which is a positive plant.
  • L5 Non-resistant oil sunflower is used as a control.
  • M DNA Molecular Weight Marker DL2000
  • LI-L4 pBINOA-5/pBINOA-6 is a primer
  • a 750 bp fragment is amplified by using genomic DNA extracted from kana-resistant oil sunflower as a template.
  • Positive plants L5: Non-resistant oil sunflower control.
  • the genomic DNA of the true leaves of transgenic oil sunflower seedlings which were positive by -S/ M was extracted by SDS method, and the genomic DNA was amplified by PCR using pBINOA-1/pBINOA-6 as a primer.
  • the PCR reaction conditions were 94 ° C lmin, 60 ° C lmin, 72 ° C 2.5 min, 30 cycles, and 72 ° C extension for 10 min.
  • Hybridized and washed membranes were rinsed with rinse buffer, rinsed once; immersed in 100 mL of blocking solution for 30 min; soaked in 20 mL of antibody solution for 30 min; rinsed with 100 mL of wash buffer twice for 15 min each; in 20 mL assay buffer
  • the medium was equilibrated for 2-5 min; the DNA of the membrane was placed upside down into the hybridization bag, and 1 mL of CSPD was added; the membrane was wetted at 37 ° C for 10 min to allow the chemical fluorescence to fully react; and exposed to X-ray film at room temperature.
  • the results are shown in Fig. 9.
  • transgenic oil sunflower seeds were ground in 5V grinding buffer (50mM Tris-HCl H 7.5, 0.4 M sucrose, 0.5M NaCl), centrifuged 10 X g for 30 min, divided into three parts, the oil phase was taken, and resuspended in an equal volume. In the grinding buffer, mix, gently add 5V pre-cooled 50mM Tris-HCl pH 7.5 buffer, centrifuge 10 X g for 30min, and take the oil phase.
  • 5V grinding buffer 50mM Tris-HCl H 7.5, 0.4 M sucrose, 0.5M NaCl
  • the above procedure was repeated twice to further remove the remaining water-soluble and insoluble components to obtain a pure oil body (the oil body components include: neutral lipid phospholipids, oil body proteins).
  • the oil body components include: neutral lipid phospholipids, oil body proteins.
  • 2V diethyl ether was added to the oil body, centrifuged, and the neutral lipid was left in the upper diethyl ether phase.
  • the phospholipid was in the lower aqueous phase, and the middle protein layer was taken, resuspended in 0.1 M sucrose buffer, and added with chloroform methanol ( 2 : 1 ) Mixture, extract twice, take the intermediate protein layer, extract with ether once, dissolve in sterile water.
  • the expression amount is 1.1% of the total protein of the seed, which exceeds the minimum commercialization requirement of recombinant protein expression in plants (1%). Therefore, the plant oil body expression system is used to realize the industrialization of the apolipoprotein AI Milan mutant. Feasibility and broad application prospects.
  • Step 1 Separate the oil body from the other ingredients in the seed
  • Seeds were ground in 5V grinding buffer (50mM Tris-HCl pH 7.5, 0.4M sucrose, 0.5 M NaCl), centrifuged (10xg) for 30min, divided into three parts: the bottom is insoluble precipitate (seed shell, fibrous material) , insoluble sugar, protein and other insoluble soils), the middle is the aqueous phase, contains soluble cellular components (storage proteins), and the uppermost layer is the oil body and the oil body protein bound to it.
  • 5V grinding buffer 50mM Tris-HCl pH 7.5, 0.4M sucrose, 0.5 M NaCl
  • centrifuged (10xg) for 30min, divided into three parts: the bottom is insoluble precipitate (seed shell, fibrous material) , insoluble sugar, protein and other insoluble soils), the middle is the aqueous phase, contains soluble cellular components (storage proteins), and the uppermost layer is the oil body and the oil body protein bound to it.
  • Step 2 Washing the oil body
  • Step 3 Enzymatic cleavage release of apolipoprotein A-I milan mutant protein
  • Step 4 Purification of apolipoprotein A-I milan mutant protein by HPLC
  • Example 4 Oil sunflower as a bioreactor and safflower as a bioreactor to prepare apolipoprotein AI Milan mutants of the same quality (280 mg) of the apolipoprotein AI Milan mutant gene of oil sunflower seeds And safflower seeds were isolated and purified according to Example 3 to obtain apolipoprotein AI Milan mutant protein, and the amount of the sample was one tenth of the total amount obtained, and Western blotting was performed. The results are shown in Fig.
  • M protein Molecular weight standard
  • L1 Apolipoprotein AI Milan mutant purified from the transgenic safflower, size 28.9kDa, consistent with the expected size, quantified 50ng
  • L2 Apolipoprotein AI Milan mutant purified from the transgenic oil sunflower The size is 28.9kDa, which is consistent with the expected size and is quantified as 80ng. From this, it was estimated that 2.85 g of apolipoprotein AI Milan mutant can be obtained from 1 kg of transgenic oil sunflower seeds, and 1.78 g of apolipoprotein AI Milan mutant can be obtained under the same conditions of 1 kg of transgenic safflower seeds. Moreover, the yield per mu of oil sunflower is about 250kg.
  • the yield per mu of safflower is about 200kg, whether it is from the yield of seed apolipoprotein AI milan mutant or the yield of apolipoprotein AI milan mutant per plant area. Oil sunflowers must take precedence over safflower.

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Abstract

La présente invention concerne un vecteur d'expression spécifique à une graine et ses procédés de construction et applications. La cassette d'expression de protéine de fusion constituée par le gène des oléosines de l'arachide - gène mutant de l'apolipoprotéine A-I Milano (A-IM) actionnée par le promoteur du gène des oléosines de Brassica campestris est insérée entre les sites d'enzymes de restriction Hind III et Sac I du vecteur d'expression binaire de la plante pBI121, et le vecteur d'expression de la plante pBINOA décrit dans l'invention est obtenu. La présente invention concerne également un procédé qui utilise le vecteur d'expression pour transformer de l'huile de tournesol pour construire un bioréacteur végétal pour la préparation du mutant apolipoprotéine A-IM. Le procédé peut non seulement améliorer le rendement du mutant apolipoprotéine A-IM, mais réduit également grandement les frais de production, et convient pour la production industrielle.
PCT/CN2010/079620 2009-12-09 2010-12-09 Vecteur d'expression spécifique à une graine et ses procédés de construction et applications WO2011069459A1 (fr)

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CN109706150B (zh) * 2019-01-24 2020-07-28 山东省花生研究所（山东省农业科学院花生工程技术研究中心） 一种花生种子特异表达启动子ahssp29及其应用

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