CN116478277B - Improved lactoferrin gene, recombinant expression vector and application thereof - Google Patents
Improved lactoferrin gene, recombinant expression vector and application thereof Download PDFInfo
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- 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
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- 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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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Abstract
The invention belongs to the technical field of genetic engineering, and relates to an improved lactoferrin gene, the nucleotide sequence of which is shown as SEQ ID NO. 1. The invention also relates to a recombinant expression vector for expressing the lactoferrin and a construction method thereof, wherein the recombinant expression vector sequentially comprises the following elements from 5 'to 3': the promoter p35s from the cauliflower mosaic virus, the lactoferrin nucleotide sequence shown as SEQ ID NO. 1 and the terminator Nos of nopaline synthase are suitable for the expression vector used in industrial production. In addition, the invention also provides application of the lactoferrin gene and the recombinant expression vector in the production of lactoferrin. The invention uses genetic engineering technology to recombinant express lactoferrin, and provides a new direction for producing lactoferrin in future.
Description
Technical Field
The invention relates to the field of genetic engineering and transgenic plants, in particular to an improved lactoferrin gene, a recombinant expression vector for expressing the lactoferrin gene, a construction method of the recombinant expression vector and application of the recombinant expression vector in the production of lactoferrin.
Background
Lactoferrin is a protein widely distributed in tissues with secretory functions and its secretions, found in milk earliest in 1939, and isolated from milk and human milk in 1960. In human body, colostrum content is highest, up to 8mg/ml, 1.5-4mg/ml in milk, 2mg/ml in tears, and lower in other secretions (such as saliva, joint fluid, blood plasma), generally lower than 0.01mg/ml. It is believed to be associated with iron transport and storage, belonging to the Lactotransferrin (Lactotransferrin) family. Lactoferrin sequences are highly conserved among human, bovine, murine, porcine, etc., glycoproteins of about 690 amino acid residues and molecular weights of about 76-81kDa. In three-dimensional structure, lactoferrin comprises two N-and C-leaflets (lobes), each capable of binding a single fe3+ ion in the deep gap between the two domains [1].
In the digestive tract, lactoferrin is hydrolyzed into a small peptide with physiological functions, namely lactoferrin active polypeptide (Lfcin B), which is a polypeptide containing 25 amino acids, has strong alkalinity (pH is more than 8.15), and has the characteristics of heat resistance, difficult degradation in the digestive tract, immune activity regulation, broad-spectrum bacteriostasis and the like, and two reverse beta-sheet structures of hydrophilic and lipophilic are formed in aqueous solution. This is also the basis for the activity of oral lactoferrin. To date, research, development and application of lactoferrin have become important points of scientific research, and the existing research shows that lactoferrin has activities of resisting bacteria, resisting viruses, resisting inflammation, resisting cancer, regulating immunity, promoting bone formation, regulating intestinal flora and the like, and is widely applied in the directions of disease prevention, auxiliary treatment and the like.
At present, the preparation of the lactoferrin mainly depends on the extraction from milk, for example, a flying crane group applies an advanced chromatographic ultrafiltration technology, thereby realizing the extraction of the lactoferrin and building a first lactoferrin automatic production line in industry. However, the extraction of lactoferrin from milk has the risk of contamination by animal pathogens, and in addition, the natural content of lactoferrin is insufficient, and only 1g of protein can be extracted from about 14kg of milk, which limits the mass production of lactoferrin from milk, and also makes the price of lactoferrin as high as 7000 to 8000 yuan/kg.
For the mass production of lactoferrin, there are currently successful cases of expressing lactoferrin in escherichia coli, pichia pastoris, mice, cows, potatoes and rice, whereas proteins expressed by prokaryotes are usually not biologically active due to the lack of post-translational modifications. The expression of these proteins in yeast or animal cell culture is not only costly but also requires high conditions.
The plant fruits or seeds have stronger protein synthesis function, and if the transgenic plants can express the lactoferrin, the product is close to natural, i.e. has higher bioactivity, high expression quantity, low cost and simple and easy implementation. However, few in the prior art use plants, particularly soybeans, to produce lactoferrin in large quantities.
Disclosure of Invention
Aiming at the defects of the prior art, the invention utilizes a constitutive promoter and a tissue specific promoter to drive the expression of the lactoferrin gene so as to obtain better expression effect. To this end, the invention provides an improved lactoferrin gene, recombinant expression vector, method for preparing same, and use in the production of lactoferrin.
In a first aspect the invention provides an improved lactoferrin gene.
The nucleotide sequence of the lactoferrin gene (abbreviated as chlF) is shown as SEQ ID NO. 1, and the amino acid sequence of the lactoferrin encoded by the lactoferrin gene (abbreviated as hlF) is shown as SEQ ID NO. 2.
SEQ ID NO. 1 (chlF nucleotide sequence)
atgaagctcgtgtttctagtgctgctcttcttaggggcactggggctctgtttagccggaaggagacgcaggtcagtacagtggtgtgctgtctcacaacctgaggctacaaaatgcttccaatggcaaagaaacatgagaaaggtgagaggaccgcccgtatcgtgcataaagcgtgattcaccaatccaatgtattcaggctattgctgaaaacagagcagatgccgtaactcttgacggtggttttatatatgaagcaggcctcgctccatacaagttacgtcctgttgctgctgaggtttatggaaccgagagacagccaaggacacattattatgctgttgcagtagtgaagaagggcgggtctttccagttgaatgaattgcagggattaaaatcttgtcacaccggtttgcgaagaaccgccggatggaatgttccaattggcacgctacgacccttcttaaattggactgggcctcctgagcctattgaggcggccgtggctaggttcttttcagccagttgtgttcctggagctgacaaaggccagtttccaaacctgtgtaggttatgtgctgggactggtgaaaacaaatgtgcattctcttctcaagaaccatatttttcatacagcggtgctttcaagtgcctaagggatggtgcaggtgatgtggcgtttattagagaatctactgtttttgaagacctctccgacgaagctgaacgggacgagtatgagttgctttgtccagataacacaaggaaaccagttgataaatttaaagattgtcaccttgctagggtgccttcccacgccgtcgttgcacggagtgttaatggcaaggaggatgcaatttggaatttattaagacaagcacaagagaagtttggaaaggataaatcccctaagtttcagctcttcggtagccccagtggacagaaagacctactgttcaaagactccgcaatcggattcagccgtgtgcctccacggattgattcagggttgtacttgggttcagggtattttactgctatacaaaatttgaggaagtctgaagaagaggtggccgccagacgtgcaagagttgtttggtgcgccgtgggagagcaggagcttcgaaagtgcaatcagtggagtggattgagcgaaggaagtgtcacttgctccagtgcttctacgacagaagattgcatagctctagttctcaaaggagaagcggatgctatgtctctggatgggggctatgtttacactgcaggaaaatgtggacttgtgccggttttggcagaaaattataagtctcagcaatcgagtgatcctgaccctaactgcgttgatcgccctgtcgagggttacctggcagtagccgtggttcgccggtccgacacctcacttacatggaacagtgttaaaggtaaaaagtcatgtcatactgctgtagatagaacagctggttggaatattccaatgggactcctttttaatcaaacaggtagctgcaagtttgatgaatatttctctcagtcgtgcgctccaggttctgatccacgtagcaatctctgtgcgctttgtatcggagatgagcaaggggaaaataagtgtgtccccaactccaatgaaaggtactatggctacacaggcgcctttaggtgtttggcagagaatgctggggatgttgcgtttgtcaaagacgttacagttcttcaaaacactgatggaaacaacaatgaggcatgggcaaaagatttgaagttggctgattttgctttgctctgcttggacgggaagagaaaacccgtgacagaagcacgttcatgccatctagcaatggctccgaatcatgcagtagtatctaggatggataaagttgagcgtcttaagcaagtgcttcttcatcaacaagctaagttcggcagaaacggatctgactgccctgataagttttgtctctttcaaagtgaaaccaagaatcttttgttcaatgataatactgaatgtttagcgcgcttgcatggcaaaaccacctacgagaaatatcttggaccacaatatgtggcaggtatcacaaacttaaagaaatgctccacttcaccactgcttgaggcttgtgagtttcttagaaaaaaggatgaactttgataa
SEQ ID NO. 2 (hlF amino acid sequence)
MKLVFLVLLFLGALGLCLAGRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGPPVSCIKRDSPIQCIQAIAENRADAVTLDGGFIYEAGLAPYKLRPVAAEVYGTERQPRTHYYAVAVVKKGGSFQLNELQGLKSCHTGLRRTAGWNVPIGTLRPFLNWTGPPEPIEAAVARFFSASCVPGADKGQFPNLCRLCAGTGENKCAFSSQEPYFSYSGAFKCLRDGAGDVAFIRESTVFEDLSDEAERDEYELLCPDNTRKPVDKFKDCHLARVPSHAVVARSVNGKEDAIWNLLRQAQEKFGKDKSPKFQLFGSPSGQKDLLFKDSAIGFSRVPPRIDSGLYLGSGYFTAIQNLRKSEEEVAARRARVVWCAVGEQELRKCNQWSGLSEGSVTCSSASTTEDCIALVLKGEADAMSLDGGYVYTAGKCGLVPVLAENYKSQQSSDPDPNCVDRPVEGYLAVAVVRRSDTSLTWNSVKGKKSCHTAVDRTAGWNIPMGLLFNQTGSCKFDEYFSQSCAPGSDPRSNLCALCIGDEQGENKCVPNSNERYYGYTGAFRCLAENAGDVAFVKDVTVLQNTDGNNNEAWAKDLKLADFALLCLDGKRKPVTEARSCHLAMAPNHAVVSRMDKVERLKQVLLHQQAKFGRNGSDCPDKFCLFQSETKNLLFNDNTECLARLHGKTTYEKYLGPQYVAGITNLKKCSTSPLLEACEFLRKKDEL**
Specifically, primers adopted for constructing the lactoferrin gene are respectively shown in SEQ ID NO:9 and the nucleotide sequence shown in SEQ ID NO. 10.
SEQ ID NO. 9 primer 1 (ChlF-F1): aaaggagaagcggatgctat
SEQ ID NO. 10 primer 2 (ChlF-R1): gcctcattgttgtttccatc
In a second aspect, the present invention provides a recombinant expression vector comprising, in order from 5 'to 3', the following elements: promoter p35s from cauliflower mosaic virus, lactoferrin nucleotide sequence as shown in SEQ ID NO. 1, terminator Nos of nopaline synthase.
Specifically, the nucleotide sequence of the promoter p35s from the cauliflower mosaic virus is shown as SEQ ID NO. 5;
SEQ ID NO. 5 (p 35s nucleotide sequence)
ccatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaagactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacacgcttgtctactccaaaaatatcaaagatacagtctcagaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcggattccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagaggaca
Specifically, the nucleotide sequence of the terminator Nos of nopaline synthase is shown as SEQ ID NO. 4:
SEQ ID NO. 4 (tNos nucleotide sequence)
gatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatc
In a third aspect, the present invention provides a method for constructing the recombinant expression vector according to the second aspect of the present invention.
The method comprises the following steps: (1) Connecting the lactoferrin nucleotide sequence of the first aspect of the invention to a cloning vector pEASY-T5 to obtain a recombinant cloning vector JK01-T; (2) Respectively enzyme-cutting a recombinant cloning vector JK01-T by using restriction endonucleases NcoI and EcoRI, inserting the cut lactoferrin nucleotide sequence fragment between NcoI and EcoRI sites of the recombinant cloning vector JK01-T, and constructing a recombinant expression vector JK-PT01; (3) Cutting recombinant cloning vector JK01CK-T by HindIII and NcoI, inserting the cut p35s promoter sequence into the recombinant expression vector JK-PT01, and replacing pGmA1aB1b nucleotide sequence on the recombinant expression vector JK-PT01 to obtain recombinant expression vector JK-PT01CK; the nucleotide sequence of pGmA1aB1b is shown as SEQ ID NO. 3.
Specifically, the pGmA1aB1b nucleotide sequence is shown as SEQ ID NO. 3.
SEQ ID NO. 3 (pGmA 1aB1b nucleotide sequence)
cttggtgatattgatgatgcattggactctgaaaaaggtaacgttcttcacattattaactgattggagtattatttgtcattttggccttgttcttatcgtaatattactgcattgtatctacaagttgcatagcaagtaaatctacaagttgcatagcaagcaataatttactttgtgcagctgttggagaactactctcacattttatctgaccaagtaccccggctctgcgagtcctaaatgtacaaaactcagttgcagggtccacattagaggttaccttgcaaatgtggatagtaccttccttcccttagcacgtgctggagtgagtttatcacgtagctaggtttgtcagattgggcttagagtgatgaacattcaccaatcaccagtttcttgattcaagttgcagagcatggagtgatgaagattgaaccacgcaagggtgactacaagttatatattctacactttttaaaatttagctcataattagttaacaatatatatagtgctatatatttcttctctcaataaacaatgtagtactataatggaataagaaacttgaaatatgtagaacaatatatagctccatcattaagcaagaaaagggttttttgattggacaaaatttaaatatagttcttaacatgctgtttgtcatgttctgttattagaattgaaatttatctcaagatttgtactaaaaaaaaatatgtagattaaattaaactccaattttaattggagaacaatacaaacaacacttaaaacctgtaattaatttttcttctttttaaaagtggttcaacaacacaagcttcaagttttaaaaggaaaaatgtcagccaaaaactttaaataaaatggtaacaaggaaattattcaaaaattacaaacctcgtcaaaataggaaagaaaaaaagtttagggatttagaaaaaacatcaatctagttccaccttattttatagagagaagaaactaatatataagaactaaaaaacagaagaatagaaaaaaaaagtattgacaggaaagaaaaagtagctgtatgcttataagtactttgaggatttgaattctctcttataaaacacaaacacaatttttagattttatttaaataatcatcaatccgattataattatttatatatttttctattttcaaagaagtaaatcatgagcttttccaactcaacatctattttttttctctcaacctttttcacatcttaagtagtctcaccctttatatatataacttatttcttaccttttacattatgtaacttttatcaccaaaaccaacaactttaaaattttattaaatagactccacaagtaacttgacactcttacattcatcgacattaacttttatctgttttataaatattattgtgatataatttaatcaaaataaccacaaactttcataaaaggttcttattaagcatggcatttaataagcaaaaacaactcaatcactttcatataggaggtagcctaagtacgtactcaaaatgccaacaaataaaaaaaaagttgctttaataatgccaaaacaaattaataaaacacttacaacaccggattttttttaattaaaatgtgccatttaggataaatagttaatatttttaataattatttaaaaagccgtatctactaaaatgatttttatttggttgaaaatattaatatgtttaaatcaacacaatctatcaaaattaaactaaaaaaaaaataagtgtacgtggttaacattagtacagtaatataagaggaaaatgagaaattaagaaattgaaagcgagtctaatttttaaattatgaacctgcatatataaaaggaaagaaagaatccaggaagaaaagaaatgaaaccatgcatggtcccctcgtcatcacgagtttctgccatttgcaatagaaacactgaaacacctttctctttgtcacttaattgagatgccgaagccacctcacaccatgaacttcatgaggtgtagcacccaaggcttccatagccatgcatactgaagaatgtctcaagctcagcaccctacttctgtgacgtgtccctcattcaccttcctctcttccctataaataaccacgcctcaggttctccgcttcacaactcaaacattctctccattggtccttaaacactcatcagtcatcacc
In a fourth aspect, the present invention provides the use of a lactoferrin gene as set forth in the first aspect of the invention, a recombinant expression vector as set forth in the second aspect of the invention, in the production of lactoferrin.
Specifically, the application comprises the steps of: (1) Transforming the recombinant expression vector according to the second aspect of the present invention into an explant of soybean by using an agrobacterium-mediated method to obtain an explant having the lactoferrin gene according to the first aspect of the present invention integrated in the genome; (2) Performing differentiation culture on the explant obtained in the step (1) to obtain a regenerated plant, and screening out a transgenic soybean plant expressing the lactoferrin gene of claim 1; (3) The obtained transgenic soybean plant has additional nutritive value because of the expressed lactoferrin contained in the seeds, and can be directly eaten or processed into other nutritional foods or used as an additive raw material of other foods.
More specifically, the explant is the hypocotyl of soybean seedlings.
More specifically, the differentiation culture includes cluster bud differentiation, elongation bud differentiation, and rooting differentiation.
Drawings
FIG. 1 is a flow chart of the construction of recombinant cloning vector JK01-T comprising the lactoferrin nucleotide sequence (chlF) of the present invention;
FIG. 2 is a flow chart showing the construction of recombinant expression vector JK-PT01 containing lactoferrin nucleotide sequence (chlF) of the present invention;
FIG. 3 is a schematic diagram showing the structure of a recombinant expression vector JK-PT01CK containing a lactoferrin nucleotide sequence (chlF) of the present invention;
FIG. 4 is a PCR detection of transgenic soybean plants, wherein WT is wild type plant, PC is plasmid control, NC is water control, and 1-20 are 20 positive transgenic soybean plants.
Detailed Description
The present invention is further described in terms of the following examples, which are given by way of illustration only, and not by way of limitation, of the present invention, and any person skilled in the art may make any modifications to the equivalent examples using the teachings disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
EXAMPLE 1 obtaining of lactoferrin Gene
1. Obtaining lactoferrin nucleotide sequences
The amino acid sequence of the lactoferrin is shown as SEQ ID NO. 2 (715 amino acids); the nucleotide sequence for encoding the lactoferrin is shown in SEQ ID NO. 1 (2151 nucleotides).
2. Synthesis of lactoferrin nucleotide sequence
The lactoferrin nucleotide sequence (shown as SEQ ID NO: 1) is synthesized by Nanjing Jinsri biotechnology company. The lactoferrin nucleotide sequence (shown as SEQ ID NO: 1) is connected with an NcoI cleavage site at the 5 'end and an EcoRI cleavage site at the 3' end.
EXAMPLE 2 construction of recombinant expression vector containing lactoferrin Gene
1. Construction of recombinant cloning vector JK01-T
The lactoferrin nucleotide sequence synthesized in example 1 was ligated into a cloning vector pEASY-T5 (Transgen, beijing, china, CAT: CT 501-01), and the procedure was followed as described in the specification of the vector pEASY-T5 by the company Transgen, to obtain a recombinant cloning vector JK01-T, the construction of which was as shown in FIG. 1 (wherein Kan represents kanamycin resistance gene; amp represents ampicillin resistance gene; pUC origin represents the replication region sequence of plasmid pUC, which can induce the double-stranded DNA replication process; lacZ is LacZ initiation codon; chIF is the nucleotide sequence of lactoferrin (SEQ ID NO: 1)).
2. Identification of recombinant cloning vector JK01-T
The recombinant cloning vector JK01-T obtained above was transformed into competent cells of E.coli T1 by heat shock (Transgen, beijin, china; cat. No. CD 501), respectively. The conversion process is as follows: (1) 50 μl of E.coli T1 competent cells and 10 μl of plasmid DNA (recombinant cloning vector JK 01-T) were mixed and then subjected to a water bath at 42℃for 30s; (2) water bath at 37 ℃ for 45min; (3) shaking on a 200rpm shaker for 1h after transformation; (4) Then spread on LB plate (tryptone 10g/L, yeast extract 5g/L, naCl 10g/L, agar 15 g/L) containing ampicillin (100 mg/L) and pH was adjusted to 7.5 with NaOH overnight; (6) White colonies were picked and cultured overnight in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, naCl 10g/L, ampicillin 100mg/L, pH 7.5 with NaOH) on a shaker at 37 ℃.
The plasmid is extracted by an alkaline method, and the specific steps are as follows: (1) The bacterial solution was centrifuged at 12000rpm for 1min, the supernatant was discarded, and the precipitated bacterial cells were suspended with 100. Mu.l of solution I (25 mM Tris-HCl,10mM EDTA (ethylenediamine tetraacetic acid), 50mM glucose, pH adjusted to 8.0) pre-chilled with ice; (2) 150 μl of freshly prepared solution II (0.2M NaOH,1% SDS (sodium dodecyl sulfate)) was added, the centrifuge tube was turned upside down 4 times, mixed, and placed on ice for 3-5min; (3) Adding 150 μl ice-cold solution III (4M potassium acetate, 2M acetic acid), immediately mixing, and standing on ice for 5-10min; centrifuging at 12000rpm at 4deg.C for 5min, adding 2 times volume of absolute ethanol into the supernatant, mixing, and standing at room temperature for 5min; (5) Centrifuging at 12000rpm at 4deg.C for 5min, removing supernatant, washing the precipitate with 70% ethanol, and air drying; (6) Mu.l of TE (10 mM Tris-HCl,1mM EDTA,PH adjusted to 8.0) containing RNase (20. Mu.g/ml) was added to dissolve the precipitate; (7) digesting RNA in a water bath at 37 ℃ for 30 min; and (8) finally storing in a refrigerator at the temperature of minus 20 ℃ for standby.
After the extracted plasmid is subjected to NcoI and EcoRI digestion identification, the positive clone is subjected to sequencing verification, and the result shows that the lactoferrin nucleotide sequence inserted in the recombinant cloning vector JK01-T is the nucleotide sequence shown in SEQ ID NO. 1, namely, the lactoferrin nucleotide sequence is correctly inserted.
3. Construction of recombinant expression vector JK-PT01CK containing lactoferrin gene
Recombinant cloning vector JK01-T (vector backbone: pCambia3301 (supplied by CAMBIA mechanism)) was digested with restriction enzymes NcoI and EcoRI, respectively, and the excised lactoferrin nucleotide sequence fragment was inserted between NcoI and EcoRI sites of cloning vector JK01-T to construct recombinant expression vector JK-PT01, the construction flow of which was shown in FIG. 2 (KanR: kanamycin gene; RB: right border; chIF: lactoferrin nucleotide sequence (SEQ ID NO: 1); pGmA1aB1b (SEQ ID NO: 3); NOs: terminator of nopaline synthase (SEQ ID NO: 4); p 35S: promoter from cauliflower mosaic virus (CaMV) (SEQ ID NO: 5): cPAT: phosphomannose isomerase gene (SEQ ID NO: 6); T35S: terminator from cauliflower mosaic virus (CaMV) (SEQ ID NO: 8); LB: left border).
The recombinant expression vector JK-PT01 is transformed into competent cells of the escherichia coli T1 by a heat shock method. The conversion process is as follows: (1) 50 μl of E.coli T1 competent cells and 10 μl of plasmid DNA (recombinant cloning vector JK 01-T) were mixed and then subjected to a water bath at 42℃for 30s; (2) water bath at 37 ℃ for 45min; (4) shaking on a 200rpm shaker for 1h after transformation; (5) Then spread on LB plate (tryptone 10g/L, yeast extract 5g/L, naCl 10g/L, agar 15 g/L) containing ampicillin (100 mg/L) and pH was adjusted to 7.5 with NaOH overnight; (6) White colonies were picked and cultured overnight on a shaker at 37℃in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, naCl 10g/L, kanamycin 50mg/L, pH 7.5 adjusted with NaOH).
Extracting the plasmid by an alkaline method, wherein the extraction method is the same as the step 2. The extracted plasmid is identified after restriction enzyme NcoI and EcoRI are used for enzyme digestion, and positive clone is sequenced, so that the result shows that the nucleotide sequence of the recombinant expression vector JK-PT01 between the NcoI site and the EcoRI site is the nucleotide sequence shown as SEQ ID NO. 1 in a sequence table, namely lactoferrin nucleotide sequence.
According to the method for constructing the recombinant expression vector JK-PT01, the p35s nucleotide sequence (SEQ ID NO: 5) cut by HindIII and NcoI enzyme-cut recombinant cloning vector JK01CK-T is inserted into the expression vector JK-PT01, and pGmA1aB1b nucleotide sequence (SEQ ID NO: 3) is replaced, so that the recombinant expression vector JK-PT01CK is obtained, and the structure of the recombinant expression vector JK-PT01CK is shown in the figure 3, wherein the recombinant expression vector JK-PT01CK comprises a promoter p35s from cauliflower mosaic virus, a lactoferrin nucleotide sequence shown as SEQ ID NO:1 and a terminator Nos of nopaline synthase. And (3) enzyme digestion and sequencing verification prove that the recombinant expression vector JK-PT01CK is the p35s nucleotide sequence (SEQ ID NO: 5) between HindIII and NcoI sites.
In addition, the nucleotide sequence of PAT is shown in SEQ ID NO. 6:
atgtctccggagaggagaccagttgagattaggccagctacagcagctgatatggccgcggtttgtgatatcgttaaccattacattgagacgtctacagtgaactttaggacagagccacaaacaccacaagagtggattgatgatctagagaggttgcaagatagatacccttggttggttgctgaggttgagggtgttgtggctggtattgcttacgctgggccctggaaggctaggaacgcttacgattggacagttgagagtactgtttacgtgtcacataggcatcaaaggttgggcctaggatccacattgtacacacatttgcttaagtctatggaggcgcaaggttttaagtctgtggttgctgttataggccttccaaacgatccatctgttaggttgcatgaggctttgggatacacagcccggggtacattgcgcgcagctggatacaagcatggtggatggcatgatgttggtttttggcaaagggattttgagttgccagctcctccaaggccagttaggccagttacccagatctga
PAT has the amino acid sequence shown as SEQ ID NO. 7
MSPERRPVEIRPATAADMAAVCDIVNHYIETSTVNFRTEPQTPQEWIDDLERLQDRYPWLVAEVEGVVAGIAYAGPWKARNAYDWTVESTVYVSHRHQRLGLGSTLYTHLLKSMEAQGFKSVVAVIGLPNDPSVRLHEALGYTARGTLRAAGYKHGGWHDVGFWQRDFELPAPPRPVRPVTQI*
the nucleotide sequence of t35S (35S in FIG. 3) is shown in SEQ ID NO: 8:
ctgaaatcaccagtctctctctacaaatctatctctctctataataatgtgtgagtagttcccagataagggaattagggttcttatagggtttcgctcatgtgttgagcatataagaaacccttagtatgtatttgtatttgtaaaatacttctatcaataaaatttctaattcctaaaaccaaaatccagtgg
EXAMPLE 3 obtaining transgenic soybean plants containing lactoferrin Gene
1. Obtaining of sterile explants: explants are prepared by selecting normal sprouting pollution-free seedlings. Cutting soybean along hypocotyl with a surgical knife in an ultra clean workbench, reserving 3-4 mm hypocotyl, placing in a sterile culture dish, adding appropriate amount of co-culture solution into the dish to facilitate peeling seed coat, cutting the hypocotyl vertically along the cotyledon hypocotyl, removing clean true leaf tissue, making 5-7 cuts axially at the joint of the cotyledon and the cotyledon hypocotyl, and making the cuts about 3-4 mm long. Each explant consists of a cotyledon connected with a hypocotyl, and two explants can be formed by one seed.
2. Preparation of agrobacterium: the recombinant expression vector JK-PT01CK obtained in example 2 was introduced into Agrobacterium EHA105 by freeze thawing, and a small amount of strain was inoculated on LB plates with an inoculating loop or sterile gun head, and LB solid medium contained 50mg/L of the corresponding antibiotic. Culturing for 1-2 d under the constant temperature of 28 ℃ to obtain the monoclonal. After 2d, the monoclonal was picked up and inoculated in 5ml of YEP medium (containing the corresponding antibiotic) and grown overnight at 220rpm at 28℃for about 12 h. When the bacterial liquid is activated to a saturated state for the first time, 1ml of bacterial liquid is extracted from the bacterial liquid, inoculated into a triangular shaking flask containing 100ml of YEP (containing corresponding antibiotics) and activated for the second time under the conditions of 28 ℃ and 220 rpm. When the agrobacterium is fully activated to the OD 600=1.0, the bacterial liquid is centrifuged for 10min at 4000rpm and 4 ℃, the supernatant is discarded to collect the sediment, and the bacterial body precipitated at the bottom of the tube is suspended by the equal volume of co-culture liquid, and the OD600 is about 0.5-0.8 at the moment for standby.
3. Co-culturing the explant and agrobacterium: when the explant is prepared, every 40-60 pieces of the explant are placed in a 100ml triangular flask, about 50ml of re-suspended agrobacterium liquid is added into each flask, and the bacteria liquid needs to permeate the explant. And (3) infecting for 30-35 min under dark or weak light conditions, and shaking the triangular flask once every 5min to fully contact the agrobacterium with the explant. After infection is completed, the excess agrobacterium liquid is carefully poured out, a layer of sterile filter paper is paved on a co-culture medium, the impregnated explant is paved on the filter paper downwards towards one side of the axis, and co-culture is carried out for 3d under the condition of darkness or low light at 24 ℃.
4. Resistance screening and regeneration: after co-culturing for 3d, the explant is subjected to the stages of resistance cluster bud induction, elongation bud induction, rooting and the like to obtain a regenerated plant; and respectively adding a proper resistance screening agent in the clustered bud induction and elongation stage, and adding a proper concentration of agrobacterium bacteriostat and IBA (IBA) to induce rooting according to the practical requirement in an experiment in the rooting stage, wherein the clustered bud induction and elongation bud induction stage is carried out once every two weeks for the explant, and a new incision is prepared on the back of the explant during the subculture so that the explant can absorb nutrients better. The elongated buds are elongated to 4-6 cm and can induce rooting, and after rooting, the sealing film opening on the culture dish is uncovered for hardening seedlings for 1-3 days; transplanting the plants to a pot plant or a field for growth to obtain transgenic plants.
EXAMPLE 4 detection of lactoferrin Gene in transgenic soybeans
After the transgenic plants obtained in example 3 are flowering and fruiting, seeds are harvested, the harvested seeds are sown in a greenhouse, and when the plants grow to 4-6 leaf periods, whether lactoferrin is successfully transferred into soybean plants is detected.
1. Soybean plants transformed with lactoferrin gene were verified by conventional PCR using the Whole gold company 2X EasyTaq PCR SuperMix (China, beijin, cat: AS 111-11).
The primers used for PCR detection (fragment size 527 bp) were:
primer 1 (ChlF-F1): aaaggagaagcggatgctat (SEQ ID NO: 9)
Primer 2 (ChlF-R1): gcctcattgttgtttccatc (SEQ ID NO: 10)
Conditions of PCR reaction:
2. verification of soybean plants transfected with lactoferrin Gene Using PCR
The specific method for detecting the lactoferrin gene copy number is as follows:
(1) Taking 100mg of each leaf of a soybean plant and a wild soybean plant into which a lactoferrin nucleotide sequence is transferred, grinding the soybean plant and the wild soybean plant into homogenates in a mortar by using liquid nitrogen, and taking 3 repeats of each sample;
(2) Genomic DNA of the above samples was extracted using EasyPure Plant Genomic DNA Kit (RNase A-containing) (Transgen, beijing, china, cat: EE 111-01) and the specific method was referred to the product specifications;
(3) The genomic DNA concentration of the above samples was determined using a NanoDrop 2000 (Thermo Scientific, USA);
(4) Adjusting the concentration of the genomic DNA of the sample to the same concentration value, wherein the concentration value ranges from 80 ng/mu l to 100 ng/mu l;
(5) The copy number of the sample is identified by using a TransStart Green fluorescence quantitative PCR method, and the sample with the identified known copy number is used as a standard substance. Samples of wild type soybean plants were also used as controls, 3 replicates per sample, and their average was taken.
The following primers were used to detect lactoferrin nucleotide sequences:
primer 3 (CF 2): TCCAATTGGCACGCTACGA (SEQ ID NO: 11);
primer 4 (CR 2): ACGGCCGCCTCAATAGG (SEQ ID NO: 12);
probe 1 (CP 1): CCTTCTTAAATTGGACTGGGCCTCCTGA (SEQ ID NO: 13).
The following primers were used to detect the 18S nucleotide sequence for internal control leveling.
Primer 5 (CF 3): CCCCAAACCGATTTTACTTTCA (SEQ ID NO: 14);
primer 6 (CR 3): AGGCCAAGGGTGAGTTTGATT (SEQ ID NO: 15);
probe 2 (CP 2): ACGTATCTTTGGAACCCTCCCCCTGG (SEQ ID NO: 16).
The PCR reaction system is as follows:
the PCR reaction conditions were:
the data were analyzed using SDS2.3 software (Applied Biosystems).
As shown in FIG. 4, there is a PCR detection chart of the transformant of the present invention, wherein WT is a wild type plant, PC is a plasmid control, NC is a water control, and 1 to 20 positive transformants are 20 positive transformants; it was shown that the lactoferrin nucleotide sequence synthesized in example 1 of the present invention was integrated into the genome of the soybean plants examined, and that the soybean plants that were successfully transgenic all obtained single copy lactoferrin genes.
EXAMPLE 5 detection of lactoferrin content in transgenic Soybean seeds
Taking 0.05g of soybean seeds transferred into lactoferrin nucleotide sequences as samples, grinding, adding 500 μl of the extraction buffer, centrifuging at 12000rpm for 10min, diluting the supernatant with the extraction buffer 10000 times, and taking 100 μl of diluted supernatant for ELISA detection. The ratio of lactoferrin in the sample to fresh weight of leaf blade was detected and analyzed by ELISA (enzyme-linked immunosorbent assay) kit (Abcam company).
The results of the lactoferrin content determination in the transgenic soybean seeds are shown in table 1. The average expression level of lactoferrin in soybean seeds transferred with the nucleotide sequence of lactoferrin is 2.21mg/g, and the result shows that the lactoferrin has higher expression level and stability in soybean.
TABLE 1 expression level of lactoferrin in transgenic soybean seeds
The solutions involved in this experiment were as follows:
extraction buffer: 0.05M Tris,0.5M NaCl,pH 7.4;0.05% tween 20;
wash buffer PBST:8g/L NaCl,0.2g/L KH 2 PO 4 ，2.9g/L Na 2 HPO 4 ·12H 2 O,0.2g/LKCl,0.5ml/L Tween 20 (Tween-20), pH 7.4;
stop solution: 1M HCl.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (5)
1. The construction method of the recombinant expression vector for expressing the lactoferrin gene is characterized by comprising the following steps: (1) The lactoferrin nucleotide sequence is connected to a cloning vector pEASY-T5 to obtain a recombinant cloning vector JK01-T; (2) Respectively enzyme-cutting a recombinant cloning vector JK01-T by using restriction endonucleases NcoI and EcoRI, inserting the cut lactoferrin nucleotide sequence fragment between NcoI and EcoRI sites of the recombinant cloning vector JK01-T, and constructing a recombinant expression vector JK-PT01; (3) Cutting recombinant cloning vector JK01CK-T by HindIII and NcoI, inserting the cut p35s promoter sequence into the recombinant expression vector JK-PT01, and replacing pGmA1aB1b nucleotide sequence on the recombinant expression vector JK-PT01 to obtain recombinant expression vector JK-PT01CK; the nucleotide sequence of pGmA1aB1b is shown as SEQ ID NO. 3;
the recombinant expression vector comprises the following elements in sequence from 5 'to 3': the p35s promoter, the lactoferrin nucleotide sequence, and terminator Nos of nopaline synthase;
the nucleotide sequence of the lactoferrin is shown as SEQ ID NO. 1;
the p35s promoter is a promoter p35s from cauliflower mosaic virus, and the nucleotide sequence of the promoter p35s is shown as SEQ ID NO. 5;
the nucleotide sequence of the terminator Nos of nopaline synthase is shown as SEQ ID NO. 4.
2. Use of a recombinant expression vector according to claim 1 for the production of lactoferrin.
3. The use according to claim 2, characterized by the steps of: (1) Transforming the recombinant expression vector into an explant of soybean by using an agrobacterium-mediated method to obtain the explant integrated with the lactoferrin gene in the genome; (2) Performing differentiation culture on the explant obtained in the step (1) to obtain a regenerated plant, and screening out a transgenic soybean plant expressing the lactoferrin gene of claim 1; (3) The obtained transgenic soybean plant has additional nutritive value because of the expressed lactoferrin contained in the seeds, and can be directly eaten or processed into other nutritional foods or used as an additive raw material of other foods.
4. The use according to claim 3, wherein the explant is the hypocotyl of a soybean seedling.
5. The use according to claim 3, wherein said differentiation culture comprises clustered shoot differentiation, elongated shoot differentiation, rooting differentiation.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10323137A (en) * | 1997-03-24 | 1998-12-08 | Allergen Free Technol Kenkyusho:Kk | Addition of lactoferrin function to food plant and food plant having the same function |
| WO2004060392A1 (en) * | 2002-12-20 | 2004-07-22 | Ventria Bioscience | Lactoferrin protein with iron for treating iron deficiency and anemia |
| CN101628941A (en) * | 2008-07-18 | 2010-01-20 | 中国农业大学 | Bovine lactoferrin antibacterial peptide fusion protein, coding gene and application thereof |
| CN105274137A (en) * | 2015-11-13 | 2016-01-27 | 长沙中科晶博生物科技有限公司 | Method of producing lactoferrin by using Artemisia apiacea |
| CN110114460A (en) * | 2016-10-28 | 2019-08-09 | Nrl制药股份有限公司 | Lactoferrin/albumin fusion protein matter and preparation method thereof |
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| US6991824B2 (en) * | 2000-05-02 | 2006-01-31 | Ventria Bioscience | Expression of human milk proteins in transgenic plants |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10323137A (en) * | 1997-03-24 | 1998-12-08 | Allergen Free Technol Kenkyusho:Kk | Addition of lactoferrin function to food plant and food plant having the same function |
| WO2004060392A1 (en) * | 2002-12-20 | 2004-07-22 | Ventria Bioscience | Lactoferrin protein with iron for treating iron deficiency and anemia |
| CN101628941A (en) * | 2008-07-18 | 2010-01-20 | 中国农业大学 | Bovine lactoferrin antibacterial peptide fusion protein, coding gene and application thereof |
| CN105274137A (en) * | 2015-11-13 | 2016-01-27 | 长沙中科晶博生物科技有限公司 | Method of producing lactoferrin by using Artemisia apiacea |
| CN110114460A (en) * | 2016-10-28 | 2019-08-09 | Nrl制药股份有限公司 | Lactoferrin/albumin fusion protein matter and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 植物生物反应器制药的现状及展望;陈金梅;姜路壹;洪治;;生物技术通报(10);全文 * |
| 植物生物反应器在分子医药农业中的应用;张丹凤;余自青;吴锁伟;饶力群;万向元;;中国生物工程杂志(01);第86-89页 * |
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