CN115992181A - Method for producing chimeric silk by silkworm for alfalfa silver vein moth nuclear polyhedrosis virus - Google Patents
Method for producing chimeric silk by silkworm for alfalfa silver vein moth nuclear polyhedrosis virus Download PDFInfo
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a method for producing chimeric silk by silkworm of alfalfa silver vein moth nuclear polyhedrosis virus, which constructs a silk containing FibL-MaSp-c-polyA FibL The plasmid pFAST-FibL-MaSp-c of the fragment is transformed to contain Acbacmid DH10Ac escherichia coli, recombinant Acbacmid-FibL-MaSp-c is obtained by screening, recombinant virus AcNPV-FibL-MaSp-c is obtained by transfecting Sf-9 cells, 5-year-old silkworm larvae are inoculated, fresh mulberry leaves which are immersed or sprayed with antibiotic liquid medicine after being air-dried are fed for 1 day, then fresh mulberry leaves are fed to mature silkworms, and fresh mulberry leaves immersed or sprayed with ecdysone liquid medicine are fed to the mature silkworms; or directly spraying ecdysone liquid on mature silkworms, transferring the mature silkworms to a cluster tool, collecting cocoons after cocoons are planted, drying the cocoons, and reeling silk to obtain embedded silk, thereby meeting the requirements of various biological materials on the diversity of silk proteins.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a method for producing chimeric silk containing golden silk woven web spider large pot-shaped gland silk by silkworms for a nocturnal polyhedrosis virus of alfalfa silver vein moth.
Background
The spider silk has excellent mechanical property and small immunogenicity, and has wide application prospect in the fields of textile, biomedical materials and national defense industry. Unlike typical silking insects, spiders have multiple silk-producing glands, which produce primary ampullate gland silk, secondary ampullate gland silk, whip silk, glue gland silk, grape gland silk, tubular gland silk, and polymeric gland mucin, respectively. Different spider silk or spider silk proteins have different biological functions and also have obvious differences in properties. The spider silk protein has 3 functional areas, and the two ends of the amino acid sequence respectively form an N-terminal domain and a C-terminal domain, and the spider silk protein consists of non-repeated amino acid sequences; the middle region of the spider silk protein amino acid sequence is highly repetitive of simple amino acids. The properties of spider silk proteins and the fibrous nature of spider silk are largely dependent on the structural properties of the repeat sequence. The major ampullate gland filaments (MaSp), also known as index filaments or dragline filaments, are the filaments of the spider silk that have the best mechanical strength. The main ampullate gland filaments mainly comprise MaSp-1 and MaSp-2. The main characteristic motifs of the repeat region of MaSp-1 are (GA) n (n: 6-14) or (A) n (n: 6-14) and GGX, which form the beta-sheet crystal structure and 3, respectively 10 The helix structure, the former is related to the tensile strength of the wire, and the latter is related to the ductility. MaSp-2 has a characteristic motif GPX, QQ, GSG, and can form a beta-turn helix structure, which is related to the elasticity of the filament.
In the wild state, the daily silk yield of the spider is less than 1mg, the silk yield is very small, and the requirements of people on the spider silk are difficult to meet. In addition, spiders are non-social animals and have life habits that are mutually disabled, and so far no spider silk has been available for large-scale population rearing. In recent years, with the progress of biotechnology, attempts have been made to express spider silk proteins in bacteria, yeasts, mammalian culture cells, insect cells, even through transgenic animals and plants, using genetic engineering techniques, and to manufacture artificial spider silk or other biological materials from purified recombinant proteins, and considerable progress has been made in the related studies. The prior art discloses a single polyprotein molecule for improving mechanical and mechanical properties and an application method thereof, wherein the single polyprotein molecule is used as a single polyprotein molecule, one of 4 tandem protein sequences type 1-type 4 of a spider large ampullate gland silk protein I molecule is used as a single polyprotein molecule, an exogenous gene vector corresponding to the single polyprotein sequence is constructed, the single polyprotein sequence is integrated into a silkworm genome by utilizing a molecular biology technology, and finally, a novel silkworm variety with excellent composite silk performance capable of being inherited stably is obtained, and composite silk is produced by utilizing the novel silkworm variety. The prior art obtains chimeric silk containing spider silk protein components through natural spinning capability of silkworms, improves the mechanical properties of silk fibers to a certain extent, and is limited by the technology of introducing exogenous genes into silkworm eggs and artificially incubating the silkworm eggs, so that genetic modification of silkworms by introducing genes into silkworm eggs through microinjection is basically limited to diversified silkworm varieties without practical production value. As common knowledge, practical varieties in production are all binarization, and the spawning is the older spawn, and diapause can be relieved by long-time low-temperature stimulation (refrigeration) or instant pickling (hydrochloric acid) treatment or combination of refrigeration and pickling, so that embryo development is promoted. The optimal period for egg microinjection is several hours after the laying, whereas the instant pickling treatment is usually around 24 hours after the laying of the eggs. Silkworm eggs after microinjection die due to pickling treatment, so that the prior art mostly chooses varieties which do not need pickling treatment, and therefore, people still hope to produce chimeric silks containing spider silk proteins by utilizing silkworms of conventional varieties through a plurality of novel strategies and technologies.
Disclosure of Invention
The invention aims to provide a method for producing chimeric silk containing golden silk woven web spider large pot-shaped gland silk by silkworms, which not only obtains silk with improved mechanical properties, but also avoids excessive reduction of silk length, and is applicable to all silkworm varieties. The invention obtains chimeric silk containing spider silk protein by expressing gold silk woven mesh spider gland silk through recombinant noctiluca californica nuclear polyhedrosis virus mediated by rear silk gland of silkworm, and the related technical proposal is not reported.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for producing chimeric silk from silkworm by means of a california nocturnal polyhedra virus, comprising the steps of:
(1) FibL-MaSp-c-polyA FibL Cloning the fragment into the multiple cloning site of pFAST-Bac-Dual to construct plasmid pFAST-FibL-MaSp-c; the FibL-MaSp-c-polyA FibL The sequence of the fragment is SEQ ID NO. 1;
(2) Transforming plasmid pFAST-FibL-MaSp-c into Escherichia coli containing Acbacmid DH10Ac, coating on an LB agar medium plate for culture, picking white colonies, and extracting recombinant Acbacmid-FibL-MaSp-c DNA, wherein the LB agar medium preferably contains tetracycline, kanamycin, gentamicin, IPTG and X-gal; the temperature of the culture is 37 DEG C
(3) Transfecting recombinant Acbacmid-FibL-MaSp-c DNA into Spodoptera frugiperda cultured cells, culturing until the cells are ill, inoculating the cultured cells again from the cell culture supernatant, culturing until the cells are ill, collecting the cell culture supernatant, and centrifugally purifying to obtain recombinant Autographa californica baculovirus particle AcNPV-FibL-MaSp-c, wherein the cultured cells are preferably Sf9 cultured cells, and the culture temperature is 26-27 ℃;
(4) Inoculating silkworm larvae with cell culture supernatant or recombinant Spodoptera frugiperda baculovirus particles AcNPV-FibL-MaSp-c collected in the step (3), and then feeding the silkworm larvae to the cocooning frame; then cocooning, cocoon picking and silk reeling are carried out to obtain chimeric silk, cocooning is carried out under the environment of 25 ℃, cocooning is carried out after 7 days, preferably, the silkworm larva is 5-year-old silkworm larva, the silkworm larva is fed to mature silkworms, then ecdysone treatment is carried out, and cocooning is carried out; further preferably, the silkworm larvae are treated once by antibiotics in the process of raising the silkworm larvae to the mature silkworm.
The method for producing chimeric silk by silkworm for preparing the noctuid nuclear polyhedrosis virus of alfalfa is further described as follows:
(1) Synthesis of silk fibroin light chain Gene promoter control of family with coding signal peptide sequence at 5 'end and tailing signal at 3' end, and filature spider silk protein expression cassette FibL-MaSp-c-polyA FibL The sequence of the polypeptide is shown as SEQ ID NO. 1;
(2) FibL-MaSp-c-polyA FibL Cloning of fragments into pFAST-Bac Tm The Dual multiple cloning site construction of plasmid pFAST-FibL-MaSp-c;
(3) Converting the strain pFAT-FibL-MaSp-c into a strain containing Acbacmid DH10Ac, coating the strain on LB agar medium plates containing 10 mug/ml, 50 mug/ml, 7 mug/ml, 40 mug/ml and 100 mug/ml of tetracycline, kanamycin, gentamicin, IPTG and X-gal respectively, culturing the strain at 37 ℃, picking white colonies, and extracting recombinant Acbacmid-FibL-MaSp-c DNA;
(4) Transfecting recombinant Acbacmid-FibL-MaSp-c DNA into Spodoptera frugiperda Sf9 cultured cells, culturing at 26-27 ℃ until the cells are ill, inoculating the cultured cells with cell culture supernatant again, collecting cell culture supernatant after the cells are ill, and centrifugally purifying to obtain recombinant Autographa californica rod-shaped virus particles AcNPV-FibL-MaSp-c;
(5) Inoculating recombinant virus AcNPV-FibL-MaSp-c to 5-year-old silkworm larva, feeding fresh mulberry leaves immersed or sprayed with antibiotic liquid medicine (500 mg/L) after air drying at 24-25deg.C for 1 day, and feeding fresh mulberry leaves to mature silkworm; preferably, the dosage proportion of the antibiotic liquid medicine and the mulberry leaf is 5-7L to 100Kg;
(6) Feeding mature silkworm with fresh mulberry leaf soaked or sprayed with ecdysone liquid medicine for 1 time; or spraying ecdysone liquid directly onto mature silkworm for 1 time;
(7) Transferring the mature silkworms to a cluster tool, camping cocoons at 25 ℃ and collecting cocoons after 7 days;
(8) And (3) drying the cocoons, and reeling to obtain the embedded silk containing the golden silk woven mesh spider gland silk.
In the technical proposal, in the step (5), the quantity of the recombinant virus AcNPV-FibL-MaSp-c inoculated by the 5-instar silkworm larvae is 10 4 ～10 7 Copying/silkworm; antibiotic medicine liquidIs ciprofloxacin or norfloxacin or florfenicol liquid medicine; the concentration of the ecdysone medicine liquid is 22.5mg/L, and the dosage proportion of the antibiotic medicine liquid and the mulberry leaf is 5-7L to 100Kg; if the method of spraying mature silkworm is selected, the dosage of ecdysone liquid medicine is used to moisten the body surface of silkworm.
In the method of the invention, the FibL-MaSp-c-polyA is constructed FibL A preferred embodiment of the expression cassette is according to SEQ ID NO:1 sequence adopts a full chemical synthesis method. FibL-MaSp-c-polyA FibL Expression cassettes can also be prepared by the strategy of PCR cloning. FibL-MaSp-c-polyA FibL Cloning of fragments into pFAST-Bac Tm Construction of the Dual multiple cloning site in the plasmid pFAST-FibL-MaSp-c, fibL-MaSp-c-polyA FibL Cloning of fragments into pFAST-Bac Tm The multiple cloning site of Dual (Invitrogen company) can be connected by enzyme digestion, or by seamless cloning; white colonies were picked and recombinant Acbacmid-FibL-MaSp-c DNA was extracted using light F (SEQ ID NO: 2) and light R: (SEQ ID NO: 3) primer pair Acbacmid-FibL-MaSp-c for PCR identification; in the preparation of recombinant viruses, purified AcNPV-FibL-MaSp-c DNA was confirmed by PCR amplification and sequencing of the combined PCR products by TCID 50 Or detecting the virus titer in the supernatant by quantitative PCR, if the obtained virus titer in the supernatant of the cultured cells is lower, further inoculating the cultured cells to obtain a cell culture supernatant with high virus titer, and purifying the virus particles from the diseased cell culture supernatant by ultracentrifugation to further improve the virus titer.
In the invention, the silkworm variety is preferably a practical silkworm variety for silk cocoon breeding, for example, the silkworm variety of 'Jingsong X bright moon', and also can be selected from silkworm breeds such as '75 new'. The present invention adopts a new method to successfully solve the problem, and is applicable to all silkworm varieties, in particular to silk cocoon breeding practical silkworm varieties. The growth period of the inoculated 5-year-old silkworm larvae is 1-3 days after 5-year-old molting. The collected cell culture supernatant or centrifugally purified virus puncture inoculator can be dipped by a No. 4 insect needle when the virus is inoculatedSilkworm larva, the optimized scheme is that according to 10 6 Copy/silkworm injection of recombinant virus AcNPV-FibL-MaSp-c.
In the invention, in order to reduce bacterial septicemia caused by bacteria polluted by inoculated wounds when virus is inoculated, the antibiotics are ciprofloxacin or norfloxacin or florfenicol; the method for using ecdysone preferably comprises soaking or spraying fresh Sang Sheliang with ecdysone liquid medicine when humidity is high, and feeding silkworm; when the climate is drier, the silkworm can be directly sprayed with ecdysone liquid.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:
1. the technology of the invention can be used for obtaining a large amount of chimeric silk containing golden silk woven web spider large pot-shaped gland silk by utilizing the high-level protein synthesis capability of silkworm silk glands and the biological habit of spinning cocoons of silkworms.
2. In the prior art, spider silk proteins can be expressed by escherichia coli, yeast, animal cells or transgenic animals and plants, and in order to further obtain spider silk fibers, the recombinant proteins are purified through complicated steps and further realized through artificial spinning, so that the process is time-consuming and expensive, the current technical level is difficult to produce in a large scale, and the mechanical properties of the prepared spider silk fibers are still much lower than those of natural spider silk. By utilizing the technology of the invention, the capability of high-efficiency synthesis of protein by the silk gland tissue of the silkworm and the natural capability of silk spinning and cocooning of the silkworm can be directly utilized to obtain the chimeric silk containing the golden silk woven mesh spider large pot-shaped gland silk on a large scale, and the obtained chimeric silk can gather the advantages of silk and spider index silk.
3. Silk protein materials have been widely used in various fields, and after repeated doubling of the repeated units of the spider silk protein gene, expression has been achieved in escherichia coli, yeast, animal cells or transgenic animals and plants by using genetic engineering technology, but because the amino acid sequence of spider silk protein is highly repeated, the expression level is often extremely low, and the molecular weight of the expression product is lower than that of natural products. Therefore, the cost for purifying the recombinant spider silk protein is high, and mass production is difficult; according to the invention, the recombinant alfalfa silver vein noctuid nuclear polyhedrosis virus is used for mediating the expression of golden silk mesh spider large pot-shaped gland silk protein in the rear silk gland of the silkworm, and the recombinant large pot-shaped gland silk protein enters a cocoon layer through spinning to form chimeric silk, so that the silk protein material is prepared without complex purification steps, and the mass production is convenient.
Drawings
FIG. 1 is an identification of recombinant Acbacmid-FibH-MaSp-c in example one. Recombinant Acbacmid-FibL-MaSp-c DNA was extracted using primers LightF (SEQ ID NO: 2) and LighR: (SEQ ID NO: 3) PCR identification was performed. The PCR products were separated by electrophoresis on a 1% agarose gel. M, DNA standard molecular weight; lane CK, wild Bacmid; lanes 1 and 2, recombinant Acbacmid-FibL-MaSp-c.
FIG. 2 shows the relative expression levels of MaSp-c in silk glands of an AcNPV-FibL-MaSp-c infected family by RT-qPCR in example one. At 10 6 Copy recombinant virus AcNPV-FibL-MaSp-c was inoculated with "Jingpong X bright moon" 5-year old silkworms, the posterior silk glands at days 3, 4,5 after inoculation were taken, total RNA was extracted, reverse transcribed into cDNA, and the relative expression level of MaSp-c was detected by quantitative PCR using primers qc-F (SEQ ID NO: 4) and qc-R (SEQ ID NO: 5). And simultaneously detecting the expression of the eukaryotic initiation factor 4A of the reference gene by using the primer eIF4-1 (SEQ ID NO: 6) and the primer eIF4-2 (SEQ ID NO: 7).
FIG. 3 shows the Western blot detection of MaSp-c expressed in the posterior silk gland 3 days after infection of silkworm (Cynanchum candidum. Times. Bright moon) with AcNPV-FibL-MaSp-c recombinant virus in example I. CK, control silk gland (uninfected); lane 1,5 age day 3 injection 10 4 Copy AcNPV-FibL-MaSp-c virus; lane 2, day 4 5 injection 10 4 Copy AcNPV-FibL-MaSp-c virus; lane 3,5 age day 2 injection 10 6 Copy AcNPV-FibL-MaSp-c virus; lane 4,5 age day 3 injection 10 6 Copy AcNPV-FibL-MaSp-c virus; lane 5, day 4 5 injection 10 6 Copy AcNPV-FibL-MaSp-c virus; lane 6, day 25 injection 10 5 Copy AcNPV-FibL-MaSp-c virus; lane 7, day 3 5 injection 10 5 Copy AcNPV-FibL-MaSp-c virus; lane 8, day 4 5 injection 10 6 Copy AcNPV-FibL-MaSp-c virus. The primary antibody is an anti-MaSp-c antibody, and the secondary antibodyThe antibodies were HRP-labeled goat anti-rabbit IgG.
FIG. 4 shows the detection of MaSp-c in cocoon filaments by Western blot hybridization in example one. Lane M, standard molecular weight DNA, lane Ck, silk of non-infected virus AcNPV-FibL-MaSp-c silkworms; lane 1, silk infected with AcNPV-FibL-MaSp-g silkworm. The primary antibody is MaSp-c, and the secondary antibody is HRP-labeled goat anti-rabbit IgG.
FIG. 5 shows the immunohistochemical detection of MaSp-c secretory expression in AcNPV-FibL-MaSp-c infected silk glands in example two. A, silk gland of control silkworm infected with AcNPV-FibL-MaSp-c virus; b and C, 5-year-old silkworms are infected with the virus AcNPV-FibL-MaSp-C for 48 hours and 72 hours. The primary antibody is an anti-MaSp-c antibody, and the secondary antibody is HRP-labeled goat anti-rabbit IgG.
FIG. 6 shows Western blot detection of MaSp-c expressed in the posterior silk gland 3 days after infection of silkworm (75 New) with AcNPV-FibL-MaSp-c recombinant virus in example two.
FIG. 7 shows SDS-PAGE of fibroin in example II. Lane M, standard molecular weight DNA; lane 1-4, 5-year old silkworms infected with AcNPV-FibL-MaSp-c virus; CK, control silk fibroin.
FIG. 8 shows the Western blot detection of MaSp-c in silk in example two. Lane M, standard molecular weight DNA; lane 1-4, 5-year old silkworms infected with AcNPV-FibL-MaSp-c virus; CK, control silk fibroin.
Detailed Description
The noctilucent nuclear polyhedrosis virus (AcNPV) is a model species of baculovirus, the main host of which is insect, and the baculovirus plays an important role in controlling the population quantity of insects. With the intensive research, baculoviruses have been widely used for research on the development of biopesticides, expression of foreign proteins and gene delivery to vertebrate cells. Baculoviruses are of a wide variety and vary in host domain, infectivity, and pathogenicity from one baculovirus to another. The specific preparation operation of the invention is a conventional method in the field, and can also be operated according to commodity specifications, such as synthesis sequence, cloning, transformation, transfection, infection, silkworm breeding, cocooning, cocoon picking, silk reeling and the like.
The invention is further described below with reference to the accompanying drawings and examples:
embodiment one: method for producing chimeric silk containing golden silk woven web spider large pot-shaped gland silk protein by using silkworm of Jing Pinus and Haoyue variety
1. Chemical synthesis expression cassette FibL-MaSp-c-polyA FibL Sequences, respectively added on both sides of the sequencesXhoI andSphi site, and then entrusted with commercial company to perform chemical synthesis, the synthesized sequence is shown as SEQ ID NO. 1.
2. Construction of plasmid pFAST-FibL-MaSp-c: fibL-MaSp-c-polyA FibL Cloning of fragments into pFAST-Bac Tm Dual (Invitrogen company product)XhoI andSphthe I site constructs the plasmid pFAST-FibL-MaSp-c.
3. Screening of recombinant Bacmid AcBacmid-FibL-MaSp-c: pFAT-FibL-MaSp-c was transformed into E.coli containing Acbacmid DH10Ac, and then spread on LB agar medium plates containing 10 [ mu ] g/ml, 50 [ mu ] g/ml, 7 [ mu ] g/ml, 40 [ mu ] g/ml and 100 [ mu ] g/ml, respectively, tetracycline, kanamycin, gentamicin, IPTG and X-gal, after culturing at 37℃for 12 hours, white colonies were picked up, inoculated in LB medium containing 10 [ mu ] g/ml, 50 [ mu ] g/ml and 7 [ mu ] g/ml of tetracycline, kanamycin and gentamicin, shaking-cultured for 8 hours, recombinant Acbacmid-FibL-MaSp-c DNA was extracted, PCR identification was performed using primers light F (SEQ ID NO: 2) and light R (SEQ ID NO: 3), agarose gel electrophoresis results of amplified products were shown in FIG. 1, a specific band corresponding to the molecular weight of approximately 2 was amplified from the Accmid-FicmL-MaSp-c DNA, and NO successful construction of the recombinant Bacmid DNA was not shown.
4. Construction of recombinant virus AcNPV-FibL-MaSp-c: recombinant Acbacmid-FibL-MaSp-c DNA 2. Mu.g was mixed with liposome Lipofectamine 2000 (Invitrogen company), cells were cultured by transfection of Spodoptera frugiperda Sf9, cultured at 27℃for 4 days, and then the cell culture supernatant was taken and inoculated again with the cultured cells, and after the cells developed, the cells and cell culture supernatant were collected.
5. Purification of AcNPV-FibL-MaSp-c virions and determination of viral copy number: centrifuging the cell culture supernatant in the step 4 at 8000 rpm for 10 minutes at 4 ℃ for 2 times; centrifuging the supernatant at 30000 rpm for 30 min, collecting precipitate, dissolving the precipitate with phosphate buffer to obtain recombinant virus stock solution, and preserving at-20deg.C; taking a stock solution of the virus, measuring TCID50 of the stock solution, and calculating the copy number of the virus in the stock solution.
6. RT-qPCR detection of the relative expression level of MaSp-c in the silk gland of AcNPV-FibL-MaSp-c infected domestic animals: at 10 6 Copy recombinant virus AcNPV-FibL-MaSp-c was inoculated with "Jingpong X bright moon" 5-year old silkworms, the posterior silk glands at days 3, 4,5 after inoculation were taken, total RNA was extracted, reverse transcribed into cDNA, and the relative expression level of MaSp-c was detected by quantitative PCR using primers qc-F (SEQ ID NO: 4) and qc-R (SEQ ID NO: 5). And simultaneously detecting the expression of the eukaryotic initiation factor 4A of the reference gene by using the primer eIF4-1 (SEQ ID NO: 6) and the primer eIF4-2 (SEQ ID NO: 7). As a result, as shown in FIG. 2, acNPV-FibL-MaSp-c entered the posterior tissue of the silk gland and transcribed MaSp-c, the transcription level of which was significantly increased with infection by the virus.
7. Western blot detection of MaSp-c expressed in the posterior silk gland 3 days after infection of silkworm (Cynanchum. RTM.) with recombinant virus AcNPV-FibL-MaSp-c: day 3 and 4 of 5 years of injection 10 respectively 4 Copy, 5 th, day 2, 3, 4 days of injection 10, respectively 6 Copy, 5 th day 2 and 3 rd day injections 10 respectively 5 Copy and day 4 injection at 5 years 10 6 The copied virus was subjected to Western blot detection 3 days later from the posterior silk gland tissue, and the primary antibody was anti-MaSp-c antibody and the secondary antibody was goat anti-rabbit IgG labeled with HRP. As a result, as shown in FIG. 3, specific bands representing MaSp-c expression were detected by the recombinant virus vaccinated.
8. Recombinant virus AcNPV-FibL-MaSp-c (stock solution) is inoculated to silkworm: conventional rearing of silkworms of the variety "Jingsong X Haoyue" to 5-year old silkworms (autumn silkworms), inoculating 10 per silkworm 6 And (3) copying the virus. Silkworm vaccinated with virus is fed with the antibiotic ciprofloxacin: the ciprofloxacin solution with the concentration of 500mg/L is prepared and uniformly sprayed on the leaves of the mulberry leaves according to the concentration of 6L/100Kg, and the front and back surfaces of the mulberry leaves are wet. Spraying ciprofloxacin solution, drying, feeding silkworm for 1 day, and feeding fresh folium Mori at 24deg.C to mature silkworm.
9. Silkworm is added with ecdysone: preparing 22.5mg/L ecdysone liquid medicine, uniformly spraying 6L/100Kg of ecdysone liquid medicine on leaf surfaces of the mulberry leaves, wetting front and back surfaces of the mulberry leaves, airing, and feeding the mulberry leaves with the mature silkworms for 1 time; and then moving to a cluster tool, and cocooning at 25 ℃ for 7 days, and then collecting cocoons.
10. Drying fresh cocoons, storing, and obtaining embedded silk through silk reeling after conventional degumming of the stored dried cocoons before silk reeling; the protein solution obtained by dissolving chimeric silk in lithium bromide solution is added into a dialysis membrane, and Western blot detection is carried out after dialysis for 72 hours, and the result is shown in FIG. 4, wherein MaSp-c signals can be observed, which indicates that MaSp-c is contained in cocoon filaments.
The strong elongation curve of the above chimeric silks was routinely tested and their mechanical properties were evaluated against silks not infected with the virus AcNPV-FibL-MaSp-c silks. As shown in Table 1, the chimeric silk of the present invention has improved strength and elongation, but the single silk has reduced length, but compared with the prior art, the present invention has significantly improved that the single silk has been kept at 60% or more of the length of the uninfected silk.
Embodiment two: method for producing chimeric silk containing golden silk woven net spider large pot-shaped gland silk protein by using 75 new silkworm variety
1. Preparation of recombinant virus AcNPV-FibL-MaSp-c: steps 1-5 of example one are implemented;
2. secretion expression detection of MaSp-c in the posterior silk gland of the silkworm of the "75 New" variety infected with the recombinant virus AcNPV-FibL-MaSp-c: inoculation of 5 th instar silkworms 10 6 After 48 and 72 hours of copying of the virus, the postsilk gland is cut into slices, and the secretory expression of MaSp-c in the AcNPV-FibL-MaSp-c infected silk gland is detected through immunohistochemistry, wherein the primary antibody is an anti-MaSp-c antibody, and the secondary antibody is an HRP-labeled goat anti-rabbit IgG. As a result, as shown in FIG. 6, a brown signal representing MaSp-c was observed in the lumen of the infected silk gland, indicating that MaSp-c was expressed and secreted into the lumen of the gland.
Western blot detection of recombinant virus AcNPV-FibL-MaSp-c expressed in the posterior silk gland 3 days after infection with 75 New silkworms: inoculation of 5 th instar silkworms 10 6 Copy diseaseAfter 48 and 72 hours of toxicity, the expression of MaSp-c was detected by Western blot from the postharvest silk gland, and the results are shown in FIG. 7, wherein a specific signal band representing MaSp-c was observed in the virus-infected silk gland sample, indicating that MaSp-c was successfully expressed.
4. Recombinant virus AcNPV-FibL-MaSp-c is inoculated to silkworm: raising 75 new variety silkworm to 5 years old, inoculating 10 each silkworm 6 And (3) copying the virus. The silkworm inoculated with the virus is fed with the antibiotic florfenicol: the florfenicol solution with the concentration of 500mg/L is prepared and uniformly sprayed on the leaves of the mulberry leaves according to the concentration of 6L/100Kg, and the front and back surfaces of the mulberry leaves are wet. Spraying fresh Sang Sheliang of florfenicol solution, drying, feeding to silkworm for 1 day, and feeding to mature silkworm with fresh folium Mori at 24deg.C.
5. Spraying ecdysone on silkworm body: preparing ecdysone liquid medicine of 22.5mg/L, and spraying on the body surface of silkworm in step 5 until wetting is achieved. The step 9 of clustering, cocoon harvesting and cocoon harvesting is the same as in the first embodiment.
6. And (5) drying the fresh cocoons and storing the cocoons. Before reeling silk, storing the dried cocoons, degumming, and reeling silk to obtain the embedded silk containing golden silk woven spider large pot-shaped gland silk protein. SDS-PAGE and Western blot routine detection of chimeric fibroin, the results of which are shown in FIG. 8, a band representing MaSp-c can be observed in cocoon silk samples of virus-infected silkworms; western blot detection results show that a signal band representing MaSp-c can be observed in cocoon silk samples of the silkworms infected by viruses, which indicates that the silkworm cocoon silk contains the MaSp-c. The single cocoon silk length of infected silkworms is at least 58% of that of uninfected silkworms.
The prior art can obtain chimeric silk containing spider silk protein components through a piggyBac mediated silkworm transgenic technology, and the mechanical properties of silk fibers are improved to a certain extent, but the content of spider silk protein in the chimeric silk is very limited; substitution of the heavy chain gene of silk protein of the silkworm with repeated doubling of the main ampullate gland silk protein gene of the spider has been achieved by TALEN-mediated homologous end recombination, and the genetically modified silkworm produced by this method has significantly increased levels of spider silk protein in chimeric silks produced by the silkworm, and decreased strength despite increased extensibility of such chimeric silks. In addition, genetic modification of silkworms is currently limited to silkworms of a variety of practical production value due to the technical limitations of gene transfer into silkworm eggs by microinjection. The technology of the invention can be used for obtaining the chimeric silk with excellent properties of silk and spider silk by utilizing the advantage of high production performance of practical silkworm varieties. The key, the existing method for producing spider silk chimeric silk by silkworms greatly reduces silk length, and experimental research shows that the silk length obtained by recombination is up to 41% of the original silk length. In the prior art, a spider silk protein gene is introduced into a silkworm genome by a silkworm transgenic method, and the transgenic silkworm is screened and identified by a more complex procedure, and a transgenic pure line is obtained by hybridization screening for 1-2 years; the technology of the invention can obtain recombinant viruses in a short time, and the chimeric silk containing spider silk proteins can be obtained by inoculating 5-year-old silkworms with the viruses for about one week, thereby achieving remarkable technical progress.
SEQ ID NO: 1：
CTCGAGGTACGGTTCGTAAAGTTCACCTGCGGCTATATTCCGACTCGCCAAGTTACGTCAGTCGTATTGTAATGAGCGATTTAGTGGGCAACTTCATTCTGTTAATTTTGTGTCACGGTGCGCGCGCATCGTAAAACTTCACTCTCATAGATTTTTCATAACGCGCCTAAAGAAGTATAACTTCAATAATTTAAATTTAAAAAAAAACATGCATAGAATAATTATATGAATTATTTAAAATGTCATTTACCGACATTGACATAACAGACGACGTTAACACTACAAAACATTTTAATTCCACATTGTTACATATTCAACAGTTAAATTTGCGTTAATTCTCGATGCGAACAAATATAAGAACAATCGGATCAATTAGATCGCTTTGTTTCGAACAACACTTAGTTTAACTAGAGGCGTACACCTCAAGAAATCATCTTCATTAGAAACTAAACCTTAAAATCGCAATAATAAAGCATAGTCAATTTTAACTGAAATGCAAAGTCTTTTGAACGTTAGATGCTGTCAGCGTTCGTTGGTACAGTTGTTTGATATTTATTTTAATTGTCTTTTTATATATAAATAGTGGAACATTAATCACGGAATCCTGTATAGTATATACCGATTGGTCACATAACAGACCACTAAAATGAAACCTATCTTCCTCGTTCTGCTGGTGGCTACATCTGCCTATGCCGCCCCATGGTCTTCGACGGAGTTGGCCGACGCTTTTATCAACGCTTTCCTCAATGAAGCCGGAAGAACTGGCGCTTTCACCGCCGACCAACTCGACGATATGTCTACCATTGGTGACACCCTGAAAACAGCTATGGATAAGATGGCCAGATCCAACAAATCATCTCAATCGAAGCTCCAGGCTCTGAATATGGCTTTCGCTTCATCAATGGCTGAAATCGCTGCCGTGGAACAAGGTGGATTGAGCGTTGCTGAAAAAACAAACGCTATTGCCGATTCCCTCAATTCGGCTTTCTACCAAACAACTGGAGCCGTTAACGTCCAGTTCGTCAATGAAATAAGAAGTCTCATCTCAATGTTCGCTCAGGCCAGCGCTAACGAAGCTAGCTACGGCGGTGGATACGGCGGTGGACAAGGCGGTCAATCTGCTGGTGCTGCCGCTGCCGCTGGTGCTGGACAAGGTGGTTACGGTGGACTGGGCGGTCAAGGTGCTGGTAGTGCCGCTGCCGCTGCCGCTTCAGGAGCAGGTCAAGGTGGTTATGGTGGAGTGGGAAACCAGGGTGCTGGAAGAGGCGCCGGAGCCGCTGCCGCTGCCGCTGGCGGTGCTGGTCAAGGTGGTTACAATGGTGGACAAGGACCTTCTGCCGCTGCCGCTGCCGCTGCCAGCGGAGCTGGCCAGGGCGGTTACGGAGGCCCTGGTTCCCAAGGTGCTGGACAAGGAGCTGGAGCTGCCGCTGCCGCTGCCGGTGGAGCTGGACAAGGCGGTTACGGAGGCTTGGGTGGACAGGGAGCTGGAAGAGGCGGTGCTGCCGCTGCCGCTGCCGCTGCCGGTGTGGCTGGACAAGGTGGTCTGGGTTCGCAGGGTGCTGGAAGAGGTGGACTCGGCGGTCAGGGTGCAGGCGCTGCCGCTGCCGCTGGAGGCGCCGGACAGGGTGGATACGGTGGTCTGGGACAAGGTGCTGGTCAAGGAGCTGGAGTCGCCGCTGCCGCTGCCGCTGGAGGCGCTGGCCAAGGTGGATACGGCGGTTTCGGTTCCCAGGGAGCAGGAAGAGGTGGTCAAGGTGGACAAGGTTCGGCCGCTGCCGCTGGCGGTGCTGGGCAAAGAGGTTACGGAGGCCAGGGTGCTGGTCAGGGTGGATTGGGCGGTGGAGAACAGGGAGCTGGCGAAGAAGGTTCTGGTGCCAGCGCTGGCGCTGGTGCCGCTGCCGGAAGAGGCGCTGGCGGTGGAGGCAAGGGTGGACTGGGCGGTCAAGGTGGTAGTGCTGCCGCTGCCGCTGCCGGTGGAGCTGGGCAAGGCGGTTTGGGAGGCTCAAGAGGTGCTGGACAAGGTGCTGGAGCTGCCGCTGCCGCTGCCGGTGGAGCTGGTCAGGGCGGTTATGGAGGCCTGGGCTCACAAGGAGCTGGTAGAGGTGGACAAGGCGCTGGTGCTGCCGCTGCCGCTGCCGGCGGTGCTGGCCAAGGTGGTTACGGTGGACTGGGCGGTCAGGGCGTTGGTAGAGGTGGTCTGGGTGGTCAAGGTGCAGGTGCTGCCGCTGCCGTCGGTGCTGGACAGGGCGGTTACGGAGGCGTGGGATCTGGTGCTTCGGCTGCCAGTGCTGCCAGATCTAGATTGTCGAGTCCTCAAGCTTCATCTAGAGTGAGCTCCGCTGTTTCGAACCTCGTCGCCAGTGGTCCAACAAATTCAGCTGCCCTGTCGAGTACTATTTCAAACGTGGTTTCTCAAATAGGAGCTTCTAATCCTGGACTGAGCGGCTGCGACGTTTTGATACAGGCTCTGTTGGAAGTCGTGTCAGCCTTGATCCAAATTCTCGGTTCATCTAGCATCGGACAGGTCAATTACGGCTCAGCGGGACAGGCTACGCAAATAGTGGGACAGTCAGTCTACCAGGCTTTAGGATAAATAAGAACTGTAAATAATGTATATATATAATTATATAAAAGATATATATAACCATATACAAACATATATATCATTATAAGACAATCTACCTATATAAAAACAGACTAAAATTAATAATTATGTATACTTTAATTGTGTTTAGGACATTTTATGCAAATTGTGTTTGCGTTAGGATTTTTTTTGGAAGTTTTTTAGATTATTTATGAATATATAAATAAATATACGTTAATATAATATATATTATATAAATCAACGACACGGCTTTTCATTTTGGTGATGATCAATCTTATTGTTCTTCTAATTGATTTTTTTGTACAATAAAGATGTATCCAGTTTTCCAGATAAAGAATTTAGTTTGTTATTTCTGGCCCCATTAAAATAAGTACGGTATTCGACAATAGCATGC
SEQ ID NO: 2（lightF）
ATGAAACCTATCTTCCTCGTTCTG
SEQ ID NO: 3 （lightR）
TCCTAAAGCCTGGTAGACTGACTG
SEQ ID NO: 4(qc-F)
AGAGTGAGCTCCGCTGTTTC
SEQ ID NO: 5(qc-R)
TCAAGGCTGACACGACTTCC
SEQ ID NO: 6 eIF4-1
GAATGGACCCTGGGACACTT
SEQ ID NO: 7 eIF4-2
CTGACTGGGCTTGAGCGATA
Sequence listing
<110> university of Suzhou
<120> a method for producing chimeric silk from silkworm for nuclear polyhedrosis virus of Spodoptera frugiperda
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2999
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ctcgaggtac ggttcgtaaa gttcacctgc ggctatattc cgactcgcca agttacgtca 60
gtcgtattgt aatgagcgat ttagtgggca acttcattct gttaattttg tgtcacggtg 120
cgcgcgcatc gtaaaacttc actctcatag atttttcata acgcgcctaa agaagtataa 180
cttcaataat ttaaatttaa aaaaaaacat gcatagaata attatatgaa ttatttaaaa 240
tgtcatttac cgacattgac ataacagacg acgttaacac tacaaaacat tttaattcca 300
cattgttaca tattcaacag ttaaatttgc gttaattctc gatgcgaaca aatataagaa 360
caatcggatc aattagatcg ctttgtttcg aacaacactt agtttaacta gaggcgtaca 420
cctcaagaaa tcatcttcat tagaaactaa accttaaaat cgcaataata aagcatagtc 480
aattttaact gaaatgcaaa gtcttttgaa cgttagatgc tgtcagcgtt cgttggtaca 540
gttgtttgat atttatttta attgtctttt tatatataaa tagtggaaca ttaatcacgg 600
aatcctgtat agtatatacc gattggtcac ataacagacc actaaaatga aacctatctt 660
cctcgttctg ctggtggcta catctgccta tgccgcccca tggtcttcga cggagttggc 720
cgacgctttt atcaacgctt tcctcaatga agccggaaga actggcgctt tcaccgccga 780
ccaactcgac gatatgtcta ccattggtga caccctgaaa acagctatgg ataagatggc 840
cagatccaac aaatcatctc aatcgaagct ccaggctctg aatatggctt tcgcttcatc 900
aatggctgaa atcgctgccg tggaacaagg tggattgagc gttgctgaaa aaacaaacgc 960
tattgccgat tccctcaatt cggctttcta ccaaacaact ggagccgtta acgtccagtt 1020
cgtcaatgaa ataagaagtc tcatctcaat gttcgctcag gccagcgcta acgaagctag 1080
ctacggcggt ggatacggcg gtggacaagg cggtcaatct gctggtgctg ccgctgccgc 1140
tggtgctgga caaggtggtt acggtggact gggcggtcaa ggtgctggta gtgccgctgc 1200
cgctgccgct tcaggagcag gtcaaggtgg ttatggtgga gtgggaaacc agggtgctgg 1260
aagaggcgcc ggagccgctg ccgctgccgc tggcggtgct ggtcaaggtg gttacaatgg 1320
tggacaagga ccttctgccg ctgccgctgc cgctgccagc ggagctggcc agggcggtta 1380
cggaggccct ggttcccaag gtgctggaca aggagctgga gctgccgctg ccgctgccgg 1440
tggagctgga caaggcggtt acggaggctt gggtggacag ggagctggaa gaggcggtgc 1500
tgccgctgcc gctgccgctg ccggtgtggc tggacaaggt ggtctgggtt cgcagggtgc 1560
tggaagaggt ggactcggcg gtcagggtgc aggcgctgcc gctgccgctg gaggcgccgg 1620
acagggtgga tacggtggtc tgggacaagg tgctggtcaa ggagctggag tcgccgctgc 1680
cgctgccgct ggaggcgctg gccaaggtgg atacggcggt ttcggttccc agggagcagg 1740
aagaggtggt caaggtggac aaggttcggc cgctgccgct ggcggtgctg ggcaaagagg 1800
ttacggaggc cagggtgctg gtcagggtgg attgggcggt ggagaacagg gagctggcga 1860
agaaggttct ggtgccagcg ctggcgctgg tgccgctgcc ggaagaggcg ctggcggtgg 1920
aggcaagggt ggactgggcg gtcaaggtgg tagtgctgcc gctgccgctg ccggtggagc 1980
tgggcaaggc ggtttgggag gctcaagagg tgctggacaa ggtgctggag ctgccgctgc 2040
cgctgccggt ggagctggtc agggcggtta tggaggcctg ggctcacaag gagctggtag 2100
aggtggacaa ggcgctggtg ctgccgctgc cgctgccggc ggtgctggcc aaggtggtta 2160
cggtggactg ggcggtcagg gcgttggtag aggtggtctg ggtggtcaag gtgcaggtgc 2220
tgccgctgcc gtcggtgctg gacagggcgg ttacggaggc gtgggatctg gtgcttcggc 2280
tgccagtgct gccagatcta gattgtcgag tcctcaagct tcatctagag tgagctccgc 2340
tgtttcgaac ctcgtcgcca gtggtccaac aaattcagct gccctgtcga gtactatttc 2400
aaacgtggtt tctcaaatag gagcttctaa tcctggactg agcggctgcg acgttttgat 2460
acaggctctg ttggaagtcg tgtcagcctt gatccaaatt ctcggttcat ctagcatcgg 2520
acaggtcaat tacggctcag cgggacaggc tacgcaaata gtgggacagt cagtctacca 2580
ggctttagga taaataagaa ctgtaaataa tgtatatata taattatata aaagatatat 2640
ataaccatat acaaacatat atatcattat aagacaatct acctatataa aaacagacta 2700
aaattaataa ttatgtatac tttaattgtg tttaggacat tttatgcaaa ttgtgtttgc 2760
gttaggattt tttttggaag ttttttagat tatttatgaa tatataaata aatatacgtt 2820
aatataatat atattatata aatcaacgac acggcttttc attttggtga tgatcaatct 2880
tattgttctt ctaattgatt tttttgtaca ataaagatgt atccagtttt ccagataaag 2940
aatttagttt gttatttctg gccccattaa aataagtacg gtattcgaca atagcatgc 2999
<210> 2
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atgaaaccta tcttcctcgt tctg 24
<210> 3
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
tcctaaagcc tggtagactg actg 24
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
agagtgagct ccgctgtttc 20
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
tcaaggctga cacgacttcc 20
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
gaatggaccc tgggacactt 20
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
ctgactgggc ttgagcgata 20
Claims (10)
1. A method for producing chimeric silk from silkworm by means of a california nocturnal polyhedra virus, comprising the steps of:
(1) FibL-MaSp-c-polyA FibL Cloning the fragment into the multiple cloning site of pFAST-Bac-Dual to construct plasmid pFAST-FibL-MaSp-c; the FibL-MaSp-c-polyA FibL The sequence of the fragment is SEQ ID NO. 1;
(2) Converting plasmid pFAST-FibL-MaSp-c into Escherichia coli containing Acbacmid DH10Ac, coating on an LB agar medium plate for culture, and picking white bacterial colony to extract recombinant Acbacmid-FibL-MaSp-c DNA;
(3) Transfecting recombinant Acbacmid-FibL-MaSp-c DNA into Spodoptera frugiperda cultured cells, culturing until cell onset, inoculating the cultured cells again with cell culture supernatant, culturing until cell onset, collecting cell culture supernatant, and centrifuging and purifying to obtain recombinant Autographa californica rod-shaped virus particles AcNPV-FibL-MaSp-c;
(4) Inoculating silkworm larvae with cell culture supernatant or recombinant Spodoptera frugiperda baculovirus particles AcNPV-FibL-MaSp-c collected in the step (3), and then feeding the silkworm larvae to the cocooning frame; and then cocooning, cocoon harvesting and silk reeling to obtain the embedded silk.
2. The method for producing chimeric silk from silkworm by the nuclear polyhedrosis virus of Spodoptera frugiperda according to claim 1, wherein the LB agar medium contains tetracycline, kanamycin, gentamicin, IPTG and X-gal.
3. The method for producing chimeric silk from silkworm by the alfalfa silver vein moth nuclear polyhedrosis virus according to claim 1, wherein in the step (2), the temperature of the culture is 37 ℃.
4. The method for producing chimeric silk from silkworm by nuclear polyhedrosis virus of Spodoptera frugiperda according to claim 1, wherein in the step (3), the cultured cells are Sf9 cultured cells at a temperature of 26 to 27 ℃.
5. The method for producing chimeric silk from silkworm by the nuclear polyhedrosis virus of Spodoptera frugiperda according to claim 1, wherein the silkworm larva is a 5-year-old silkworm larva.
6. The method for producing chimeric silk from silkworm by means of the nocturnal polyhedra californica nuclear polyhedrosis virus as claimed in claim 1, wherein the silkworm is raised to mature silkworm, then treated with ecdysone and then cocooning is carried out.
7. The method for producing chimeric silk from silkworm by alfalfa silver vein moth nuclear polyhedrosis virus according to claim 6, wherein the silkworm larva is treated once with antibiotics during the period from the raising to the mature silkworm.
8. The method for producing chimeric silk from silkworm by the nuclear polyhedrosis virus of Spodoptera frugiperda according to claim 1, wherein cocoons are collected after 7 days at 25 ℃.
9. Chimeric silk produced by the method for producing chimeric silk from silkworm by means of the alfalfa silver vein moth nuclear polyhedrosis virus according to claim 1.
10. Nuclear polyhedrosis virus of noctuid, and FibL-MaSp-c-polyA FibL The application of the fragment in the production of chimeric silk by silkworms.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210482505.0A CN115992181A (en) | 2022-05-05 | 2022-05-05 | Method for producing chimeric silk by silkworm for alfalfa silver vein moth nuclear polyhedrosis virus |
| PCT/CN2022/105424 WO2023213010A1 (en) | 2022-05-05 | 2022-07-13 | Method for producing chimeric silk using bombyx mori by means of autographa californica nuclear polyhedrosis virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210482505.0A CN115992181A (en) | 2022-05-05 | 2022-05-05 | Method for producing chimeric silk by silkworm for alfalfa silver vein moth nuclear polyhedrosis virus |
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| Publication Number | Publication Date |
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| CN115992181A true CN115992181A (en) | 2023-04-21 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210482505.0A Pending CN115992181A (en) | 2022-05-05 | 2022-05-05 | Method for producing chimeric silk by silkworm for alfalfa silver vein moth nuclear polyhedrosis virus |
Country Status (2)
| Country | Link |
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| CN (1) | CN115992181A (en) |
| WO (1) | WO2023213010A1 (en) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1362520A (en) * | 2001-01-02 | 2002-08-07 | 成都天友发展有限公司 | Constitution process of blank expression system for transferring spider's dragline protein gene into Bombyx mori |
| JP2014502140A (en) * | 2010-09-28 | 2014-01-30 | ザ ユニバーシティー オブ ノートルダム | Chimeric spider silk and its use |
| BR112018067899A2 (en) * | 2016-04-28 | 2019-04-24 | Spiber Inc. | modified fibroin, nucleic acid, expression vector, host, and product. |
| CN114957485B (en) * | 2022-05-05 | 2023-11-10 | 苏州大学 | High-strength silk containing multiple spider gland silk proteins and preparation method thereof |
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2022
- 2022-05-05 CN CN202210482505.0A patent/CN115992181A/en active Pending
- 2022-07-13 WO PCT/CN2022/105424 patent/WO2023213010A1/en unknown
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| WO2023213010A1 (en) | 2023-11-09 |
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