CN115386561A - Human lysozyme protein gene, construction method of expression vector thereof and application thereof - Google Patents
Human lysozyme protein gene, construction method of expression vector thereof and application thereof Download PDFInfo
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
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- 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
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
The invention relates to a human lysozyme protein gene, a construction method of an expression vector thereof and application thereof, wherein the PTF102 vector is connected with a human lysozyme gene fragment artificially synthesized after corn codon optimization, and an independent transgenic event for expressing the human lysozyme protein is obtained through an agrobacterium-mediated corn immature embryo transformation experiment. Through the analysis of biochemical components of an offspring sample, the human lysozyme protein is found to be stably expressed in endosperm and can be accumulated in the endosperm of mature grains. The human lysozyme protein after codon optimization has bacteriostatic activity, has obvious bacteriostatic action on gram-positive bacteria and has no obvious bacteriostatic action on gram-negative bacteria.
Description
Technical Field
The invention relates to creation and application of a corn transgenic material for expressing human lysozyme protein.
Technical Field
Lysozyme (also known as muramidase or N-acetylmuramidase hydrolase) dissolves bacteria by breaking the beta-1, 4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in bacterial cell wall peptidoglycan, breaking down the cell wall insoluble mucopolysaccharide into soluble glycopeptides, resulting in the escape of the contents of the broken cell wall. Lysozyme can also be directly combined with virus protein with negative charge, and forms double salt with DNA, RNA and apoprotein, so as to inactivate virus. Lysozyme has many other functions, including reproduction, growth stimulation, antiviral, immunomodulation, anti-inflammatory and anti-tumor. Therefore, lysozyme is widely used in the fields of feed, food, clinical medicine, biology, and the like.
The lysozyme is added into the feed as an antibiotic substitute in the daily feed for the poultry, and has the advantages of enhancing the body resistance of the poultry and promoting the reproduction of intestinal beneficial bacteria, along with no toxic or side effect, no pesticide residue and the like on human beings and the poultry. Research shows that the feed added with a certain amount of lysozyme additive can improve the growth, development and reproductive performance of pigs, chickens and other livestock and poultry and improve the meat quality, but the dosage is limited. And a research on constructing a human lysozyme expression vector by using a mammary bioreactor of an animal, establishing a transgenic sow expressing recombinant human lysozyme protein in milk, and feeding the milk to protect piglets from being infected by enterotoxigenic escherichia coli and improve the intestinal performance of the piglets.
Corn (Zea mays) is one of the highest yielding gramineous crops in the world. Besides being used as food, corn is also an important feed source for livestock and poultry, transgenic offspring is obtained by constructing a human lysozyme protein expression vector and by means of an agrobacterium-mediated corn immature embryo transformation technology, and a series of physiological and biochemical expressions are further analyzed, so that the significance of obtaining the transgenic corn ear with antibacterial activity is great.
Disclosure of Invention
One of the purposes of the invention is to provide a human lysozyme protein gene optimized by a corn codon.
The second object of the present invention is to provide an expression vector containing the gene and a method for constructing the same.
The invention also aims to provide a corn transgenic material for expressing the human lysozyme protein.
The fourth purpose of the invention is to provide the human lysozyme protein expressed by the corn transgenic material, and the protein can generate bacteriostatic activity and has obvious inhibition effect on gram-positive bacteria.
In order to achieve the purpose, the invention adopts the following technical scheme:
a human lysozyme protein gene is a human lysozyme protein gene optimized by a corn codon, and the gene is a nucleotide sequence shown in SEQ ID NO. 1; meanwhile, the signal peptide of 27 kD-gamma-alcohol soluble protein and the endoplasmic reticulum retention signal are respectively added at the front end and the rear end of the optimized lysozyme gene sequence.
The gene sequence of SEQ ID NO.1 is:
tctagaATGAGGGTCCTCCTTGTGGCCCTTGCGCTTCTCGCGCTTGCCGCGTCTGCCACCTCTAAGGTGTTCGAGAGGTGCGAGCTGGCCCGCACCTTAAAGAGGCTCGGCATGGACGGCTACCGCGGCATGTCTCTTGCGAACTGGATGTGCCTCGCGAAGTGGGAGTCTGGCTACAACACCAGGGCGACCAACTACAACGCCGGCGACCGCAGTACCGACTACGGCATCTTCCAGATCAACTCTAGGTACTGGTGCAACGACGGCAAGACCCCTGGCGCCGTGAACGCCTGCCACCTCAGCTGCTCCGCGCTCCTTCAGGACAACATCGCGGACGCCGTGGCCTGCGCCAAGCGGGTCGTGAGGGACCCGCAGGGCATCCGGGCCTGGGTCGCGTGGAGGAACCGGTGCCAGAACCGGGACGTCAGGCAATATGTCCAGGGCTGCGGCGTCCACGACGAGCTTTGAAGAAACTATGTGCTGTAGTATAGCCGCTGGCTAGCTAGCTAGTTGAGTCATTTAGCGGCGATGATTGAGTAATAATGTGTCACGCATCACCATGGGTGGCAGTGTCAGTGTGAGCAATGACCTGAATGAACAATTGAAATGAAAAGAAAAAAGTATTTTCCAAAAAAAAAAAAAAAAAAgagctc。
the invention relates to a construction method of an expression vector of a human lysozyme protein gene, which adopts PTF102 as a transgenic vector, and a sequence shown in SEQ ID NO.1 is connected into the PTF102 vector to obtain the expression vector of the human lysozyme protein gene; wherein, the PTF102 vector is modified, and the expression of a target gene is driven and terminated by a corn 27 kD-gamma-prolamin promoter and a terminator respectively.
The invention relates to an application of a human lysozyme protein gene in constructing a genetic material of a transgenic plant of the human lysozyme protein, which adopts a corn hybrid PB x PA to construct the genetic material of the transgenic plant of the human lysozyme protein. Specific identification primers are designed through NCBI Primer-blast, and the corn transgenic material expressing the human lysozyme protein is identified.
The invention relates to an application of a transgenic corn material for expressing human lysozyme protein constructed by using the human lysozyme protein gene in the aspect of inhibiting gram-positive bacteria, which expresses the human lysozyme protein in corn endosperm and utilizes the protein to regulate the growth of the gram-positive bacteria.
The invention relates to a corn transgene for expressing human lysozyme protein, which has the following gene sequence:
tctagaATGAGGGTCCTCCTTGTGGCCCTTGCGCTTCTCGCGCTTGCCGCGTCTGCCACCTCTAAGGTGTTCGAGAGGTGCGAGCTGGCCCGCACCTTAAAGAGGCTCGGCATGGACGGCTACCGCGGCATGTCTCTTGCGAACTGGATGTGCCTCGCGAAGTGGGAGTCTGGCTACAACACCAGGGCGACCAACTACAACGCCGGCGACCGCAGTACCGACTACGGCATCTTCCAGATCAACTCTAGGTACTGGTGCAACGACGGCAAGACCCCTGGCGCCGTGAACGCCTGCCACCTCAGCTGCTCCGCGCTCCTTCAGGACAACATCGCGGACGCCGTGGCCTGCGCCAAGCGGGTCGTGAGGGACCCGCAGGGCATCCGGGCCTGGGTCGCGTGGAGGAACCGGTGCCAGAACCGGGACGTCAGGCAATATGTCCAGGGCTGCGGCGTCCACGACGAGCTTTGAAGAAACTATGTGCTGTAGTATAGCCGCTGGCTAGCTAGCTAGTTGAGTCATTTAGCGGCGATGATTGAGTAATAATGTGTCACGCATCACCATGGGTGGCAGTGTCAGTGTGAGCAATGACCTGAATGAACAATTGAAATGAAAAGAAAAAAGTATTTTCCAAAAAAAAAAAAAAAAAAgagctc
1-6bp:XbaI
10-63bp endoplasmic reticulum retention signal
64-453bp Lysozyme ORF
454-465bp signal peptide
468-647bp
648-653bp:SacI
The invention obtains 4 genes expressing human lysozyme protein:
GTTACCTTTGGTGCATTTGTTGG
GTCATTGGTACAAACCATCAGGG
GTTACTTAATCCACTGGCAGTGG
GGCTCTTAGTCAGCTAGTTGTGG
the invention uses PTF102 carrier to connect with artificial synthetic human lysozyme gene segment after corn codon optimization, and obtains 4 independent transgenic events expressing human lysozyme protein through agrobacterium-mediated transformation of corn embryo.
Compared with the prior art, the invention has the following obvious substantive characteristics and remarkable advantages:
1. the invention confirms that the lysozyme protein is accumulated at the position of 14kDa through SDS-PAGE analysis and Western blot experiment;
2. the invention proves that the human lysozyme protein optimized by the maize codon has obvious inhibition effect on the growth of gram-positive bacteria through in vitro protein induction experiments;
3. the human lysozyme protein subjected to codon optimization has bacteriostatic activity and has an obvious bacteriostatic action on gram-positive bacteria.
Drawings
FIG. 1 is a schematic diagram of the expression vector of the lysozyme transgene of the present invention.
FIG. 2 is a graph showing the Southern blot copy number detection analysis results of the transgenic positive events of the present invention.
FIG. 3 is SDS-PAGE analysis chart of total protein, prolamin and non-prolamin of endosperm of mature grains of the invention.
FIG. 4 is a Western blot analysis of total endosperm protein and non-prolamin protein of mature kernels of the invention.
FIG. 5 is a graph of in vitro protein induction and purification of the present invention.
FIG. 6 is a graph of protein bacteriostatic activity detected by agar diffusion method according to the invention.
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for the specific experimental conditions not specified in the examples below are generally carried out according to conventional conditions, such as those described in Molecular Cloning (A Laboratory Manual,3rd ed.) or Plant Molecular Biology-Laboratory Manual (Plant Molecular Biology-A Laboratory Manual, eds., memory S.Clark, springer-verlag Berlin Heidelberg, 1997), or according to the conditions recommended by the manufacturer.
Example one
In this example, a PTF102 transgenic vector for human lysozyme protein was constructed.
The fragments were synthesized by the organism company by maize codon optimization according to the gene sequence of human lysozyme protein. The vector PTF102 was selected as a vector for Agrobacterium transformation of maize embryos.
A corn 27 kD-gamma-prolamin promoter and a terminator are selected in an experiment to drive the expression of a human lysozyme gene, and a signal peptide of the 27 kD-gamma-prolamin and an endoplasmic reticulum retention signal are respectively added to the front end and the rear end of the gene, so that the target gene can be expressed in the endosperm of corn, as shown in figure 1. The EHA105 strain was shock transformed. Selecting young embryos pollinated for 8-12 days by the PBPA corn strain, taking the young embryos with the size of about 1.5mm as a receptor material, and carrying out young embryo transformation, wherein the specific process comprises the following steps:
1. infecting with Agrobacterium for 10min, and co-culturing at 20 deg.C for 3 days;
2. recovery culture at 28 ℃ for 7 days-selection culture (bialaphos concentration 1.5 mg/l) at 28 ℃ for 14 days;
3. screening and culturing (bialaphos concentration is 3 mg/l) at 28 deg.C for 14 days for 3-5 rounds;
4. obtaining resistant callus-dark regeneration culture at 28 deg.C for 14-21 days;
5. performing light regeneration culture at 28 deg.C for 14-21 days to obtain positive seedling;
6. transferred into pots, pollinated and offspring obtained.
As a result: 1000 immature embryos are selected as receptor materials, and 4 transgenic positive events are obtained after transformation and screening. And identifying each event after obtaining, extracting the genome of each event plant by a TPS method, designing a PCR primer of a target gene according to the gene sequence of the human lysozyme, and detecting whether the target gene contains a target fragment through PCR amplification so as to judge whether the event is a transgenic positive event.
Example two
In this example, the genome of leaves of a positive event for the transgene of human lysozyme was extracted in large quantities. The method comprises the following steps:
1. firstly, adding 10mL of a large amount of genome extract into a 50mL centrifuge tube, and marking a centrifuge tube cover and a centrifuge tube wall correspondingly;
2. taking 1-3g of whole fresh tender leaf, removing veins, and placing in a refrigerator at-80 deg.C for use; or putting the mixture into liquid nitrogen for quick freezing, grinding the mixture into whitened powder, and directly carrying out an experiment; putting the powder into a 50mL centrifuge tube (containing DNA large extraction solution) with a corresponding number, adding 20uL of 10mg/mL RNase mother solution, turning upside down, uniformly mixing, and standing at room temperature for 10min;
3. phenol was added to each tube in equal amounts to the extract: chloroform: isoamyl alcohol (volume ratio 25;
4. sucking all supernatants into a new 50mL centrifuge tube, writing numbers, adding isopropanol with the volume 0.7 times of the total volume into the centrifuge tube, slightly inverting the centrifuge tube to generate flocculent precipitate, and immediately carrying out the next step; or placing at-20 deg.C for 30min to precipitate genome DNA completely;
5. picking out the flocculent precipitate, transferring to a 2mL EP tube, and washing with 70% ethanol for 3 times;
6. sucking up ethanol, drying the precipitate at 65 ℃, adding 1mL of 1 × TE solution to dissolve the precipitate, adding 20uL of 10mg/mL RNAase, and digesting RNA at 37 ℃ for 1 h;
7. and (3) electrophoresis detection: adding 3uL of staining solution into 1uL of sample to perform agarose gel electrophoresis, and judging whether RNA is completely digested;
8. after determining that the RNA degradation in the sample was complete, phenol was added to it in an equal volume to the solution: chloroform: isoamyl alcohol (volume ratio 25;
9. the supernatant was transferred to another new 2mL EP tube (taking care not to suck down the lower impurities), added 7/10 volume of isopropanol and 1/10 volume of sodium acetate (PH = 5.2), gently mixed until a flocculent precipitate appeared;
10. the precipitate was transferred to a new 2mL EP tube, washed 3 times with 70% ethanol to remove the ethanol, dried at 37 deg.C, and dissolved in 100. Mu.L of 1 XTE for Southern blot experiments.
EXAMPLE III
In this example, southern blot copy number analysis of human lysozyme transgene positive events.
In order to study the copy number of the transgenic maize, this example digested some transgenic positive plants with Hind III enzyme and Southern blot analysis; this experiment performed Southern blot analysis on 4 positive events for copy number. The method comprises the following steps:
1. the enzyme digestion system is 50 μ L:1-2 mul of restriction enzyme (the volume of the restriction enzyme is less than 1/10 of the volume of the enzyme digestion system), 5 mul of 10 xbuffer, a proper amount of genome DNA (10 mug-15 mug), and ddH2O is added to make the volume constant to 50 mul; water bath at 37 ℃, and enzyme digestion is carried out for 8h;
2. preparing low-concentration macroporous agarose gel (without EB), adding 10 mu L staining solution into a system completely digested by enzyme, and performing electrophoresis for 13-18h at a constant low temperature at 25-36V;
3. preparing a film transfer device, adding 400mL of ddH2O and 20 mu L of EB dye into a square groove of the device, uniformly mixing, soaking the gel subjected to electrophoresis in the solution, taking a picture after 10min, developing, and washing for 2 times by using ddH 2O;
4. adding 500mL of 0.2M hydrochloric acid into the other square groove of the device, gently putting the gel into the device, and soaking for 10min;
5. pouring out the hydrochloric acid solution, adding 500mL of denatured liquid, soaking for 15min, and repeating once; shearing a nylon membrane (11 × 12) and filter paper (12 × 12) during denaturation, and soaking the nylon membrane in alkali-transfer solution for 5-10min;
6. assembling a film transfer device: pouring a little alkali transfer liquid on a film transfer table, laying 1 layer of filter paper, laying a nylon film in the middle of the filter paper, pressing the nylon film by using a black skin pad, and finally placing gel in the middle, wherein each layer is laid by using a glass rod to remove bubbles, so as to ensure that a closed environment for film transfer is formed;
7. starting a vacuum pump for vacuum extraction, adding a small amount of alkali-transfer liquid at 100mbar, pre-pumping for 5min, then adding a proper amount of alkali-transfer liquid into a film-transferring device, and paying attention to not overflow gel for 75min;
8. after the film is turned, the power supply is turned off; taking out the nylon membrane by using a pair of tweezers, soaking the nylon membrane in a proper amount of neutralizing solution for 10min, then placing the nylon membrane on filter paper for about 25-35min, airing, and carrying out ultraviolet crosslinking and fixing;
9. putting the nylon membrane into a hybridization tube (the side which is contacted with the gel faces inwards), adding 10mL of pre-hybridization solution, and hybridizing for 2h in a hybridization furnace at 42 ℃;
10. adding 1 × TE solution to 40 μ L of prepared probe about 250ng, sealing with sealing film, boiling in water bath for 10min, immediately ice-cooling for 5min after reaction, and instantly separating;
11. adding the denatured probe into preheated 10mL of hybridization solution, and gently blowing, beating and uniformly mixing; guiding the uniformly mixed hybridization solution into a hybridization tube by using a glass rod, carrying out hybridization furnace at 50 ℃, and incubating at a low rotation speed for 16-20 hours, wherein the optimal time is 18-19 hours;
12. after hybridization, 10mL of membrane washing solution I (preheated at 25 ℃) is added, and the hybridization is carried out in a hybridization oven at 25 ℃ for 15min, and the process is repeated;
13. adding 10mL of membrane washing solution II (preheated at 65 ℃), performing hybridization at 65 ℃ for 30min, and repeating the steps;
14. preparing membrane washing liquid at the temperature of 20mL and 25 ℃, washing for 5-10min at the temperature of 10mL each time, and washing twice;
15. adding 10mL of Blocking Solution (the Blocking Solution is used for diluting 10 Xblocking Solution in the kit to 1X by maleic acid), and at 25 ℃ for 30-40min;
16. adding 10mL antibody solution, keeping out of the sun, and keeping at 25 deg.C for 30-60min;
17. adding 20mL of membrane washing solution, each time being 10mL, at 25 ℃ for 10min;
18. adding 10mL of detection solution, and keeping at 25 ℃ for 5-10min;
19. spreading the preservative film on a horizontal experiment table, preferably without folds, uniformly dripping the CSPD color developing solution on the preservative film, contacting one surface of the inner side of the nylon film with the preservative film, and developing and storing;
20. the results show a low background of hybridization and a clear copy number of the sample, as shown in FIG. 2. The copy numbers of the four positive events of the transgene were found to be 5, 6, 4, 2, respectively, by statistics.
Example four
In this example, SDS-PAGE of total protein and non-prolamin protein of human lysozyme transgenic material was performed. The method comprises the following steps:
1. peeling and degerming the seeds, and leaving endosperm for later use;
2. grinding endosperm with liquid nitrogen to a flour end stage;
3. putting the ground kernel endosperm powder into an EP tube, putting the tube into a freeze dryer, and freezing and pumping the tube to dry;
4. putting 50mg of frozen and pumped dry kernel endosperm powder into an EP tube, adding 1ml of petroleum ether, uniformly mixing by vortex, and incubating for 1 hour at room temperature by a shaking table;
5.12,000rpm, centrifugating for 15 minutes and discarding the supernatant;
6. adding 1ml of petroleum ether, swirling and mixing uniformly, centrifuging at 12,000rpm for 15 minutes, and discarding the supernatant;
7. putting the obtained precipitate into a freeze dryer, and freezing and drying;
8. adding 1ml of sodium borate buffer solution and 20 mul of mercaptoethanol; after the mixture was stirred and mixed, the mixture was placed in a constant temperature shaking table at 37 ℃ and incubated overnight (12 hours);
9.12000rpm, centrifugal 15 minutes, transfer about 900 u l of supernatant to the new tube, the supernatant is total protein;
10. taking 300 mu l of total protein solution, adding 700 mu l of absolute ethyl alcohol, uniformly mixing the solution in a swirling manner, and incubating the solution for 2 hours at room temperature by using a shaking table;
centrifuging at 11.12000rpm for 15min, and sucking all supernatant into a new tube, wherein the supernatant is prolamin and the precipitate is non-prolamin;
12. washing the precipitate twice with 70% ethanol at 12,000rpm, and centrifuging for 5min; air-drying until the edge is transparent and the tube has no ethanol smell, adding 200 μ l IPG solution, and uniformly dissolving;
13. putting the supernatant into a freeze dryer, freeze-drying, adding 200. Mu.l of IPG solution, and uniformly flicking and dissolving;
14. taking 300 mu l of total protein solution, putting the total protein solution into a freeze dryer, freezing and drying the total protein solution, adding 200 mu l of IPG solution, and uniformly flicking and dissolving the IPG solution;
15. adding 4 μ l each of the dissolved total protein, prolamin and non-prolamin to 1 μ l of 5 xSDS protein loading buffer mixed with 1M DTT, denaturing at 99 ℃ for 10 minutes, and immediately inserting the protein sample on ice;
SDS-PAGE electrophoresis verifies that the accumulation gel is 5 percent, the separation gel is 12.5 percent after 80V electrophoresis is carried out for half an hour, and the electrophoresis time is about 2 hours;
17. taking off the protein gel, placing the protein gel in Coomassie brilliant blue, dyeing for 4 hours at room temperature by a shaking table, decoloring by using decoloring solution until the background is transparent, and performing gel shooting by using a Bio-Rad electrophoresis imaging instrument.
The results showed that protein accumulation occurred around 14kDa in total protein and non-prolamin, as shown in FIG. 3.
EXAMPLE five
In this example, western blot detection of total protein and non-prolamin of human lysozyme transgenic material was performed.
The method of the four methods of the embodiment is used for extracting the total protein and the non-prolamin protein of the human lysozyme transgenic material respectively. The method comprises the following steps:
1. mu.l of each protein was taken from each of the 4 events, and 1. Mu.l of 5 XSDS protein loading buffer mixed with 1M DTT was added, and after denaturation at 99 ℃ for 10 minutes, protein samples were immediately inserted on ice.
SDS-PAGE electrophoresis, the accumulation gel is 5%, after 80V electrophoresis for half an hour, the separation gel is 12.5%, and the electrophoresis time is about 2 hours.
The film is rotated for 1h at 3.200 mA. Sealing with TBST at room temperature for 1h.
4. The lysozyme antibody and the Actin antibody (ABClonal) were diluted in 5% milk at a ratio of 1/1000, respectively. Hybridization was performed at room temperature for 1h.
5.TBST washing membrane 6 times, each time for 5min.
6. Hybridization was performed with the corresponding secondary antibody for 1h at room temperature.
TBST washing membrane 6 times, each time for 5min.
8. Chromogenic substrate was added and the appearance was monitored using a TANON chemiluminescence imager.
The results showed that the internal reference Actin was present in 4 events, and it was found that lysozyme protein was detected near 14kDa in total protein and non-prolamin, as shown in FIG. 4, which is consistent with the results of SDS-PAGE.
Example six
In this example, the in vitro induction activity of human lysozyme protein after codon optimization of maize was examined.
In order to detect whether the human lysozyme protein optimized by the corn codon has bacteriostatic activity, a prokaryotic expression system is constructed to induce protein expression in vitro. Thereafter, the activity of lysozyme protein was examined by the agar diffusion method. The method comprises the following steps:
1. construction of the vector: the human lysozyme protein optimized by the maize codon is connected into an expression vector, and the selected expression vector is a P-cold vector with a His label;
2. transforming the constructed vector into an expression strain BL-21;
3. selecting positive strains (available bacterial liquid PCR) transformed with plasmids, and preserving the strains;
4. selecting positive clones to be cultured in 5ml of culture medium at 37 ℃ and 220rpm overnight;
5. inoculated into 150ml of fresh LB medium at 1;
6. add final concentration of 0.5mM IPTG (1M), 16 ℃,180rpm, overnight;
centrifuging at 7.4 deg.C and 13200rpm for 10min to collect thallus, and re-suspending the thallus with 15ml precooled PBS;
8. ultrasonically breaking cells;
centrifuging at 4 deg.C for 10min at 9.13200rpm, and collecting supernatant;
10. sample denaturation, SDS-PAGE gel running, coomassie brilliant blue staining;
11. and (3) purification: eluting the target protein with the His label by using magnetic beads, and storing at 4 ℃ for later use;
12. the bioactivity of lysozyme protein by agar diffusion method: uniformly punching holes on agar plates respectively containing staphylococcus aureus and escherichia coli by using a puncher with the aperture of 5mm, dripping 200uL of lysozyme protein subjected to in-vitro induction and purification into the holes, simultaneously using a standard substance as a positive control and PBS as a negative control, culturing for 12h in a 37 ℃ thermostat, and observing the condition of a bacteriostatic zone.
As shown in FIG. 5, the results show that the human lysozyme protein induced in vitro and optimized by maize codon has an inhibitory effect on the growth of Staphylococcus aureus (gram-positive bacteria), as shown in FIG. 6.
In the embodiment, the PTF102 vector is connected with the artificially synthesized humanized lysozyme gene fragment after the codon optimization of corn, and the independent transgenic event for expressing the humanized lysozyme protein is obtained through an agrobacterium-mediated corn immature embryo transformation experiment. Through the analysis of biochemical components of an offspring sample, the human lysozyme protein is found to be stably expressed in endosperm and can be accumulated in the endosperm of mature grains. The human lysozyme protein after codon optimization has bacteriostatic activity, has obvious bacteriostatic action on gram-positive bacteria and has no obvious bacteriostatic action on gram-negative bacteria.
The embodiments of the present invention have been described with reference to the accompanying drawings, but the present invention is not limited to the embodiments, and various changes and modifications can be made according to the purpose of the invention, and any changes, modifications, substitutions, combinations or simplifications made according to the spirit and principle of the technical solution of the present invention shall be equivalent substitutions, as long as the purpose of the present invention is met, and the present invention shall fall within the protection scope of the present invention without departing from the technical principle and inventive concept of the present invention.
Sequence listing
<110> university at Shanghai
<120> human lysozyme protein gene, construction method of expression vector thereof and application thereof
<141> 2022-05-09
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> Gene sequence: ()
<220>
<223> GTTACCTTTGGTGCATTTGTTGG
<400> 1
tctagagagc tc 17
<210> 2
<211> 23
<212> DNA
<213> Gene sequence ()
<220>
<223> GTCATTGGTACAAACCATCAGGG
<400> 2
<210> 3
<211> 23
<212> DNA
<213> Gene sequence ()
<220>
<223> GTTACTTAATCCACTGGCAGTGG
<400> 3
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<212> DNA
<213> Gene sequence ()
<220>
<223> GGCTCTTAGTCAGCTAGTTGTGG
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Claims (5)
1. A human lysozyme protein gene is characterized in that: is a human lysozyme protein gene optimized by a corn codon, and the gene is a nucleotide sequence shown in SEQ ID NO. 1; meanwhile, the signal peptide of 27 kD-gamma-alcohol soluble protein and the endoplasmic reticulum retention signal are respectively added at the front end and the rear end of the optimized lysozyme gene sequence.
2. The human lysozyme protein gene of claim 1, wherein: the gene sequence of SEQ ID NO.1 is:
tctagaATGAGGGTCCTCCTTGTGGCCCTTGCGCTTCTCGCGCTTGCCGCGTCTGCCACCTCTAAGGTGTTCGAGAGGTGCGAGCTGGCCCGCACCTTAAAGAGGCTCGGCATGGACGGCTACCGCGGCATGTCTCTTGCGAACTGGATGTGCCTCGCGAAGTGGGAGTCTGGCTACAACACCAGGGCGACCAACTACAACGCCGGCGACCGCAGTACCGACTACGGCATCTTCCAGATCAACTCTAGGTACTGGTGCAACGACGGCAAGACCCCTGGCGCCGTGAACGCCTGCCACCTCAGCTGCTCCGCGCTCCTTCAGGACAACATCGCGGACGCCGTGGCCTGCGCCAAGCGGGTCGTGAGGGACCCGCAGGGCATCCGGGCCTGGGTCGCGTGGAGGAACCGGTGCCAGAACCGGGACGTCAGGCAATATGTCCAGGGCTGCGGCGTCCACGACGAGCTTTGAAGAAACTATGTGCTGTAGTATAGCCGCTGGCTAGCTAGCTAGTTGAGTCATTTAGCGGCGATGATTGAGTAATAATGTGTCACGCATCACCATGGGTGGCAGTGTCAGTGTGAGCAATGACCTGAATGAACAATTGAAATGAAAAGAAAAAAGTATTTTCCAAAAAAAAAAAAAAAAAAgagctc。
3. a method for constructing an expression vector of a human lysozyme protein gene of claim 1, wherein the method comprises the following steps: PTF102 is used as a transgenic vector, and a sequence shown in SEQ ID NO.1 is connected into the PTF102 vector to obtain an expression vector of the human lysozyme protein gene; wherein, the PTF102 vector is modified, and the expression of a target gene is driven and terminated by a corn 27 kD-gamma-prolamin promoter and a terminator respectively.
4. An application of the human lysozyme protein gene of claim 1 in the construction of genetic material of transgenic plants of human lysozyme protein, which is characterized in that: the corn hybrid variety PB x PA is adopted to construct the gene material of the transgenic plant of the human lysozyme protein.
5. The application of the human lysozyme protein gene of claim 1 in constructing transgenic corn material for expressing human lysozyme protein in the aspect of inhibiting gram-positive bacteria, which is characterized in that: human lysozyme protein is expressed in corn endosperm, and the protein is used for regulating the growth of gram-positive bacteria.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0251730A2 (en) * | 1986-06-30 | 1988-01-07 | Takeda Chemical Industries, Ltd. | Production of human lysozyme |
| CN101979591A (en) * | 2010-10-15 | 2011-02-23 | 洋浦华氏禾元生物科技有限公司 | Method for producing human lysozyme by using rice as bioreactor |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0251730A2 (en) * | 1986-06-30 | 1988-01-07 | Takeda Chemical Industries, Ltd. | Production of human lysozyme |
| CN101979591A (en) * | 2010-10-15 | 2011-02-23 | 洋浦华氏禾元生物科技有限公司 | Method for producing human lysozyme by using rice as bioreactor |
Non-Patent Citations (1)
| Title |
|---|
| 夏清风;侯英敏;曹芳;金朝霞;: "人溶菌酶基因在毕赤酵母中的表达及其抗菌活性检测", 大连工业大学学报, no. 05, pages 346 - 349 * |
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