CN111454961A - Spider MaSps gene and application thereof in preparation of drug-loaded nanoparticles - Google Patents
Spider MaSps gene and application thereof in preparation of drug-loaded nanoparticles Download PDFInfo
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Abstract
The invention relates to a spider MaSps gene and application thereof in preparation of drug-loaded nanoparticles, and discloses an optimized spider MaSps gene artificially designed and modified for the first time, a transgenic expression frame for regulating fusion expression of fibroin heavy-chain protein (Fib-H) gene and spider MaSps gene by a bombyx mori fibroin heavy-chain promoter is constructed, and a piggyBac transposon arm and a fluorescence screening marker gene are connected to construct a transgenic expression vector, wherein the expression vector can be used for establishing a transgenic bombyx for efficiently expressing recombinant fused MaSps protein in a bombyx mori silk gland, so as to obtain transgenic silkworms containing the recombinant fused MaSps protein in fibroin; further obtaining the transgenic silk material which can stably secrete the recombinant MaSps protein in the fibroin layer and has the functions of promoting controlled release of the drug and endocytosis of the cells.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and relates to a spider MaSps gene and application thereof in preparation of drug-loaded nanoparticles.
Background
The fibroin belongs to amphiphilic biopolymer, and has the main properties of biocompatibility, biodegradability, controlled drug release and the like which are necessary for optimizing a drug delivery system. Silk proteins derived from spiders and insects, particularly Bombyx mori silk fibroin (BmSF), have been widely used in the creation of controlled release carrier materials for drugs and drug delivery studies. It is reported that BmSF has been successfully prepared into fibroin microspheres with the diameter of several micrometers and the drug loading efficiency of up to 21%, and the encapsulation efficiency of the drug can be improved to 100% by balancing the particle diameter from 100 to 440 mm, but the preparation process of the BmSF particles is often highly complex, and the particle preparation technology lacks scalability. In addition, the single BmSF as a delivery material has not yet achieved practical requirements in mechanical properties and the like, and the research on modification is a good way, and the modification is expected to improve the drug controlled release capacity and promote endocytosis. Researchers often need to compound BmSF with other materials to fully develop their potential and expand their application range, and to obtain a more practical biomaterial.
The main properties of silk protein as a drug delivery material are determined by the secondary structure of the silk protein, namely, the β -sheet (β -sheet) is the most basic secondary structure in the silk protein biological material including silk and spider silk protein, and the secondary structure not only stabilizes the particle structure, but also plays a leading role in drug controlled release.
Large saccular gland silk (Major amyllarte Spidro) secreted by spider large saccular glandins, MaSps), also called Dragline silk or Dragline silk (Dragline silk), is a silk protein fiber with the best known mechanical properties in nature, the spider Dragline silk contains 2 highly conserved spider silk proteins, Major ampullate gland Spidroin 1 (MaSp 1) and Major ampullate gland Spidroin 2 (MaSp ampulate Spidroin 2, MaSp2), the protein motif of MaSp1 is mainly An, (GA) n and GGX, and mainly forms a large number of β -sheets and hydrophilic 3-sheets10The MaSp2 protein motif is mainly An, (GA) n and GPGXX, and mainly forms a large number of β -sheets and β -corner structures, but because artificial large-scale and high-density feeding of spiders is very difficult, the yield of natural spidroin is extremely low, so that only different types of organisms such as escherichia coli, yeast, mammalian cells and plants are taken as hosts to artificially synthesize recombinant spidroin a genetic engineering means so as to meet the requirements of research, development and application of the recombinant spidroin.
Especially, the transgenic technology of diapause varieties of silkworms and the establishment of a high-efficiency silk gland expression system of the silkworms enable us to create novel practical variety transgenic cocoon silk materials containing spider MaSps protein through the technical systems, and β -lamella, β -corner and 3 rich in the spider MaSps protein are utilized10The spiral structure is used for improving the performance of the transgenic cocoon silk, and is beneficial to further creating transgenic silk fibroin particles with excellent drug loading and controlled release effect capacity, and the transgenic silk fibroin particles are applied to drug delivery and disease treatment research. Unfortunately, although there are few studies on the improvement of mechanical properties of silk by using the spider MaSps gene, no reports on the transgenic improvement of controlled drug release and promotion of endocytosis of silk have been found.
Therefore, based on the consideration and the research foundation, the invention utilizes the silkworm diapause variety transgenic technology system and the silkworm fibroin heavy chain expression system to respectively express the artificially modified optimized spider MaSp1 protein gene and the MaSp1 protein gene in the practical silkworm variety to improve the performances of controlled release of silk drugs and promotion of endocytosis, creates a novel MaSps gene-transferred silk material and applies the material to the research of drug delivery and treatment of intestinal cancer.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a spider MaSps gene; the second purpose of the invention is to provide a transgenic expression vector of the spider MaSps gene; the invention also aims to provide application of the spider MaSps gene in preparation of silk for promoting drug controlled release and endocytosis of silkworms through rear silk gland expression; the fourth purpose of the invention is to provide silk which is prepared by expressing the spider MaSps gene protein in the domestic silk gland and secreting the spider MaSps gene protein to a cocoon silk fibroin layer; the fifth purpose of the invention is to provide a preparation method of transgenic fibroin drug-loaded nanoparticles containing the spider MaSps protein and application of the transgenic fibroin drug-loaded nanoparticles to treatment of colon cancer.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a spider MaSps gene comprising:
the MaSp1 gene has a nucleotide sequence shown in SEQ ID NO.1 and is spliced by 12 tandem repeat MaSp1 unit sequences (shown in positions 1-105 of SEQ ID NO. 1); or
The nucleotide sequence of the MaSp2 gene is shown in SEQ ID NO.3 and is spliced by 32 tandem repeat MaSp2 unit sequences (shown in 1-75 sites of SEQ ID NO. 3).
2. The amino acids encoded by the spider MaSps gene comprise:
the amino acid sequence of the amino acid coded by the MaSp1 gene is shown in SEQ ID NO. 2; or
The amino acid sequence of the amino acid coded by the MaSp2 gene is shown in SEQ ID NO. 4.
3. And constructing a transgenic expression vector based on the spider MaSps gene.
Preferably, the transgenic expression vector is an expression cassette containing the spider MaSps gene.
Further preferably, the expression frame is a bombyx mori fibroin heavy chain protein gene promoter Fib-H, the spider MaSps gene and a termination sequence L BS containing bombyx mori light chain binding sites, namely a Fib-H-MaSps-L BS expression frame which are connected in sequence, wherein the nucleotide sequences of Fib-H and L BS are respectively shown in SEQ ID NO. 5-6.
Preferably, the transgenic expression vector takes a pBac {3 × P3-DsRedaf } vector as a skeleton vector, and a Fib-H-MaSps-L BS expression frame sequence is connected to the AscI/FseI enzyme digestion site.
4. The spider MaSps gene is applied to preparation of silkworm cocoons for promoting drug controlled release and endocytosis.
Preferably, the spider MaSps gene is expressed in the posterior silk gland of the silkworm, so that the silkworm cocoon is obtained.
Preferably, the spider MaSps gene is expressed in the posterior silk gland of the silkworm and secreted to a cocoon silk fibroin layer, so that the silkworm cocoon is obtained.
5. The spider MaSps gene is applied to preparation of silk for promoting controlled release of drugs and endocytosis.
Preferably, the spider MaSps gene is expressed in the posterior silk gland of the silkworm, and then the silk is obtained.
Preferably, the spider MaSps gene is expressed in the posterior silk gland of the silkworm and secreted to a cocoon silk fibroin layer, so that the silk is obtained.
6. The transgenic silkworm cocoon prepared by the spider MaSps gene is obtained by expressing the spider MaSps gene in the posterior silk gland of the silkworm.
7. The transgenic silk prepared by the spider MaSps gene is obtained by expressing the spider MaSps gene in the posterior silk gland of the silkworm.
8. The transgenic silkworm cocoon or the transgenic silk is applied to the preparation of drug-loaded nanoparticles.
Preferably, the drug loaded by the drug-loaded nanoparticles is camptothecin.
Preferably, the drug-loaded nanoparticles can be used for preparing colon cancer resisting drugs.
9. The drug-loaded nano particle is prepared by utilizing the transgenic silkworm cocoon or the transgenic silk.
Preferably, the drug loaded by the drug-loaded nanoparticles is camptothecin.
Preferably, the drug-loaded nanoparticles can be used for preparing colon cancer resisting drugs.
10. The preparation method of the drug-loaded nano particle comprises the following specific steps:
(1) degumming the transgenic silkworm cocoons to obtain transgenic silk fibroin;
(2) then completely dissolving the transgenic silk fibroin in water to obtain a transgenic silk fibroin solution;
(3) dissolving hydrophobic drug in organic solvent to obtain drug solution;
(4) and then encapsulating the transgenic silk fibroin solution in a drug solution to obtain emulsion, performing ultrasonic treatment, removing the organic solvent, and performing post-treatment to obtain the drug-loaded nano particle.
Preferably, the specific method of step (1) is: and (3) degumming the transgenic silkworm cocoons by using a weak base solution, completely dissolving the transgenic silkworm cocoons by using a ternary solution, and dialyzing the solution by using deionized water to obtain the transgenic silk fibroin.
Further preferably, the specific steps are as follows: silkworm cocoon in weak base solution Na2CO3Boiling for 3 times (0.5%, w/v) for 20 minutes each time, and thoroughly rinsing with deionized water to remove sericin; subsequently, the raw silk fibroin was completely dried at 40 ℃ for 24 hours in a ternary CaCl solution2: ethanol: h2Dissolving regenerated silk fibroin in O (molar ratio of 1: 2: 8) at 78 ℃ for 2 hours; finally, the insoluble impurities were removed by centrifugation at 8000 rpm for 10 minutes and dialyzed against distilled water for at least 3 days (MWCO 1400 Da).
Preferably, in the step (2), the concentration of the transgenic silk fibroin solution is 10mg/m L.
Preferably, in the step (3), the drug is camptothecin, the organic solvent is acetone, and the concentration of the drug solution is 0.1mg/m L.
Preferably, in step (4), the emulsion is prepared by the following method: dropwise adding the transgenic fibroin solution (water phase) into the drug solution (oil phase), and stirring gently, wherein the volume ratio of the transgenic fibroin solution to the drug solution is 1: 5.
preferably, in the step (4), the specific method of ultrasonic treatment is as follows: after vortexing for 30s, the emulsion was deposited in an ice bath and sonicated using a sonicator at 30% amplitude for 60 s.
Preferably, in the step (4), the organic solvent is removed by: the emulsion was stirred in a fume hood for 3 hours.
Preferably, in the step (4), the post-treatment is performed by the following specific method: centrifuging at 15000g for 15min, discarding supernatant, washing with ultrapure water for 3 times, lyophilizing, and storing at-20 deg.C under sealed condition.
The invention has the beneficial effects that:
the invention discloses an optimized spider MaSps gene (comprising a MaSp1 gene and a MaSp2 gene) which is artificially designed and modified, constructs a transgenic expression frame which is formed by regulating and controlling fusion expression of a fibroin heavy chain protein (Fib-H) gene and the spider MaSps gene by a bombyx mori fibroin heavy chain promoter, and is simultaneously connected with a piggyBac transposer arm and a fluorescence screening marker gene to construct a transgenic expression vector, wherein the expression vector can be used for constructing transgenic bombyx mori which efficiently expresses recombinant fusion MaSps protein in a bombyx so as to obtain transgenic cocoon fibers containing the recombinant fusion MaSps protein in fibroin, the transgenic fibroin nano-drugs (TSF-CPT-NPs) carrying Camptothecin (CPT) are prepared by using a solvent volatilization method by taking the transgenic bombyx mori fibroin protein as a vector, so that the transgenic silk fibroin nano-drugs (TSF-CPT-NPs) which can secrete the recombinant MaSps protein stably in a fibroin layer and have the effects of promoting drug controlled release and endocytosis, the transgenic nano-particle containing the transgenic MASps protein can be effectively improved, the clinical drug-carrying nano-particle has the advantages of the transgenic nano-TSF-CPF-CPT nano-NPs, the obtained transgenic nano-carrying agent can be effectively improved, the clinical drug-carrying efficiency of the transgenic nano-carrying nano-silk-protein-activating and the clinical:
1. according to the method, the spider MaSps gene sequence is optimally designed according to the preference of endogenous gene expression sequence codons in the silkworm genome sequence data, so that the artificially modified MaSps gene (MaSp1 or MaSp2) is more favorable for expression in living silkworm individuals;
2. the invention utilizes a fibroin heavy chain expression system to regulate and control the fusion expression of the MaSp gene and the fibroin heavy chain Fib-H gene in silk glands at the rear part of silkworms, and the MaSp gene and the fibroin heavy chain Fib-H gene are stably and uniformly distributed in silk of silk along with the silking and cocooning of the silkworms, so that the MaSp protein and the fibroin are firmly combined, and the MaSp protein cannot be lost due to degumming even in the silk reeling and raw silk refining processes;
3. the drug-loaded fibroin nanoparticles prepared from the transgenic silk expressing MaSp1 or MaSp2 protein have the advantages that the α -helix and/or β -sheet content in the protein secondary structure of the drug-loaded fibroin nanoparticles is/are obviously higher than that of the drug-loaded fibroin nanoparticles prepared from non-transgenic silk, experiments prove that recombinant MaSps protein contained in the transgenic fibroin can effectively improve the drug controlled release capacity and the cell internalization efficiency of the particles, and the drug-loaded fibroin nanoparticles have obvious treatment effect on colon cancer.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a schematic diagram of a silkworm transgenic recombinant vector structure, wherein a is pBac {3 × P3-DsRed, FibH-MaSp 1-L BS }, and b is pBac {3 × P3-DsRed, FibH-MaSp 2-L BS }.
FIG. 2 is SDS-PAGE and Westernblotting detection maps of transgenic cocoon silk solutions expressing MaSp1 protein and MaSp2 protein, respectively, wherein a is the SDS-PAGE detection map, and b and c are the Western blotting detection maps of recombinant MaSp1 protein and recombinant MaSp2 protein, respectively.
FIG. 3 is an AFM image of transgenic silk fibroin nanoparticles, wherein a, b, c are WSF-CPT-NPs, TSF-M1-CPT-NPs and TSF-M2-CPT-NPs in sequence.
Fig. 4 is a graph of controlled drug release curves for different silk fibroin nanoparticles, wherein a is the CPT release curve in buffers of different pH values (7.4, 6.8, 5.5, and 4.5); b is the CPT release curve in GSH buffer solution with different concentrations and pH 7.4; c is the CPT release profile in different concentrations of hydrogen peroxide at ph 7.4; d is the drug release profile of different NPs cultured in buffer at pH 5.5 with or without GSH; e is the drug release profile in different concentrations of hydrogen peroxide buffer at pH 5.5.
Fig. 5 is a representation of particle secondary structures of different silk fibroin nanoparticles, wherein a is the secondary structure content under different pH buffer conditions, and b is the β fold structure content in buffers with pH7.4, 6.8, and 5.5.
FIG. 6 is a graph of cytotoxicity experimental results of different silk fibroin nanoparticles, wherein a and b are antitumor cell performances of WSF-CPT-NPs or TSF-M1-CPT-NPs nano-drug and CT-26 cell after 24h and 48h, respectively.
Fig. 7 is a comparison graph of phagocytosis efficiency of CT-26 cells on different silk fibroin nanoparticles, wherein a and b are the phagocytosis rate and the average fluorescence intensity, respectively.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
The experimental procedures, for which specific conditions are not indicated in the examples, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (third edition, sambrook et al), or according to the conditions recommended by the manufacturers.
Example 1:
firstly, artificially designing and modifying optimization and construction of a spider MaSps gene sequence;
the invention carries out optimization design according to the preference of endogenous gene expression sequence codons in silkworm genome sequence data by referring to black widow spider (L atrodectus hesperus) dragline silk protein gene sequences, a MaSp1 unit nucleotide sequence (shown as SEQ ID NO.1, 1 st to 105 th sites) and a MaSp2 unit nucleotide sequence (shown as SEQ ID NO.3, 1 st to 75 th sites) are synthesized by Shanghai bio-corporation and are respectively constructed on a pUC57-T-Simple vector (Shanghai bio-corporation) to become pUC-MaSp1 unit and pUC-MaSp2 unit plasmids, then the pUC-865Sp unit is repeatedly connected in series by utilizing pUC 865Sp unit (MaSp1 unit or MaSp 634 unit) upstream and NheI enzyme cutting site downstream respectively, the pUC-MaSp 23 unit is repeatedly connected in series for 12 times to obtain pUC-MaSp1, the MaSp-Sp-26 unit is repeatedly connected in series by utilizing pUC I/NheI isocaue, the MaSp unit is repeatedly connected in series to obtain the pUC-Sp-pUC-Ma-MaSp-11 unit, the Ma-Sp-11 gene sequence is repeatedly connected in series after the Ma-Sp encoding sequence, the Ma gene sequence is connected in series, the Ma-Sp-11.
Secondly, constructing a silkworm transgenic recombinant vector
According to the requirements of the current publicly known transgenic technology based on the piggyBac transposable vector, silkworm transgenic recombinant vectors pBac { 3P-DsRed; Fib-H-MaSp-BS } (a in figure 1) and pBac { 3P-DsRed; Fib-H-MaSp-0 BS } (b in figure 1) are constructed, the detailed preparation method is as follows, pUC-MaSp and pUC-MaSp vector plasmids constructed by BamHI/XbaI double digestion are respectively recovered to obtain corresponding MaSp and MaSp gene fragments, then the MaSp and MaSp gene fragments are connected into pS 1{ Fib-H-MaSp-2 BS } vector (pS 3{ Fib-H-EGFP-4 BS } vector disclosed in China patent with CN number) which is cut by BamHI, the MaSp and MaSp gene fragments are respectively recovered to obtain intermediate vectors pSsRS-H-MaSp-6 BS } and pSpSpSB 7{ Fib-Sp-0 } which are used for replacing EGFP gene sequences of the MaSp, and obtain the genes of MaSp and the FisSp gene fragments which are respectively connected into a national fluorescent protein expression system of MasRS-Sp, and the Sp-Sp gene fragments of the Sp-Sp gene fragments, and the Sp gene fragments of the FisSp, and the FisSp gene fragments of the FisSp, and the FisSp gene fragments are constructed by the Sp, and the Sp gene fragments, and the FisSp gene fragments of the FisSp, and the FisSp gene fragments are connected into the Sp, and the Sp gene fragments of the silk protein of the Sp gene of the Sp, and the silk protein of the silk-Sp, and the silk.
Example 2:
preparation of transgenic silkworm with spider MaSps gene
Commercial diapause silkworm strain 302 (national emphasis of silkworm genome biology at southwest university) was usedLaboratory preservation) as raw material, subjecting parent silkworm eggs to low-temperature incubation treatment at 16 ℃ to remove diapause of offspring silkworm eggs, then taking 10n L concentration 400 ng/mu L (w/v) of a mixed solution (1: 1, v/v) of a recombinant vector pBac {3 × P3-DsRed, Fib-H-MaSp 1-L BS } and an auxiliary plasmid pHA3PIG (preservation in key laboratory of genome biology of southwest silkworm university), injecting the mixed solution into 207G 0 generation silkworm eggs which are removed from diapause, sealing the mixed solution with non-toxic glue, placing the sealed solution into an environment at 25 ℃ and 85% relative humidity, carrying out incubation and hatching to obtain 84G 0 generation silkworm, feeding the silkworm with mulberry leaves to a moth to obtain 25G 0 generation moth, carrying out backcrossing or selfing to obtain 15 rings of G1 generation silkworm eggs, and using the silkworm eggs to obtain 15 rings of G1 generation silkworm eggs
Observing by an electric macroscopic fluorescence microscope, obtaining 1 moth ring emitting red fluorescence by screening, and obtaining 10 positive transgenic silkworms emitting red fluorescence at eyes in total, wherein the transformation efficiency is 6.67 percent. And feeding the obtained positive transgenic silkworms to cocoon picking and further carrying out self-selection and purification to obtain the transgenic line which can express the MaSp1 protein in the silkworm cocoons and is stably inherited.
Using a commercial diapause silkworm strain 302 as an original material, subjecting parent silkworm eggs to low-temperature hatching treatment at 16 ℃ to remove diapause of offspring silkworm eggs, then injecting a mixed solution of 10n L recombinant vector pBac {3 × P3-DsRed, Fib-H-MaSp 2-L BS } and auxiliary plasmid pHA3PIG with the concentration of 400 ng/mu L into 173G 0 generation silkworm eggs which are subjected to diapause removal, sealing with non-toxic glue, placing in an environment with the relative humidity of 85% at 25 ℃ for hatching to obtain 65G 0 generation silkworm, feeding the silkworm with mulberry leaves to the silkworm moth to obtain 55G 0 generation silkworm moths, backcrossing or selfing the obtained silkworm moths to obtain 21G 1 generation silkworm eggs, and using the silkworm eggs to obtain 55G 1 generation silkworm eggs
Observing by an electric macroscopic fluorescence microscope, obtaining 2 moth rings emitting red fluorescence by screening, and obtaining 10 positive transgenic silkworms emitting red fluorescence at eyes in total, wherein the transformation efficiency is 9.52 percent. Feeding the obtained positive transgenic silkworm to cocoon picking and further selfing and purifying to obtain the silkworm cocoonStably inherited transgenic line expressing the MaSp2 protein in silkworm cocoons.
Second, expression detection of recombinant MaSps protein in transgenic cocoon silk
The expression of MaSp1 protein and MaSp2 protein in transgenic cocoon silk is detected by SDS-PAGE and Western blotting, and the specific operation steps are that transgenic cocoons (TC-M1) expressing MaSp1 protein and transgenic cocoons (TC-M2) expressing MaSp2 protein are randomly screened and cut into 1 × 1cm2Size, 0.02M Na2CO3Boiling the solution for 30min, rinsing with ultra pure water from Milli-Q system (Millipore, Billerica, Mass.) 3 times to remove sericin, and then rinsing with 60% (w v) of 500. mu. L-1) Dissolving degummed cocoon about 30mg in lithium thiocyanate (L iSCN) solution, detecting protein concentration with dicyandiamide acid (BCA) protein detection kit (Beyosine Biotech), adding β -galactosidase (2-ME) to protein sample, and performing SDS-PAGE (15%, w v)-1Bio-Rad) gel for electrophoretic separation; 0.1% of SDS-PAGE gel (w v)-1) Coomassie Brilliant blue R-250, 10% (vv)-1) Acetic acid, 50% (vv)-1) Western Blotting was performed using polyclonal Anti-MaSp1 antibody (Anti-MaSp1) and MaSp2 antibody (Anti-MaSp2) purchased from ZoonbioBiotechnology Co., L td (Nanjing, China). In the assay using EC L + Western Blotting detection reagents (Beyotime, Jiangsu, China) and operating according to the manufacturer's instructions, immunoreactive protein hybridization signals were detected using a chemiluminescent imaging system (Clinx Chemisce series, Shanghai, China). the results of SDS-PAGE gel staining showed that specific recombinant MaSp1 protein (a, triangle in FIG. 2) and specific recombinant MaSp 733 protein (a, arrow in FIG. 2) and that specific recombinant MaSp 7342 protein (a, arrow in FIG. 2) and that specific recombinant MaSp1 protein in MaSp-M2) and that of recombinant MaSp-M2 protein (TC-M6342 and that of MaSp-M2 protein were detected as negative controls, respectively, as well as the results of Western Blotting of the specific recombinant MaSp1 protein hybridization (TC-M) and the recombinant MaSp-M-PAGE proteins (TC-M6342, respectively, as well as shown in FIG. 36, as a bar, a bar for the results of the recombinant MaSp-PAGE proteins of the recombinant MaSp-M-PAGE proteins.
Example 3:
the steps of preparing the fibroin drug-loaded nanoparticles by using the WC, TC-M1 and TC-M2 protein samples obtained in the above example 2 are as follows:
1) degumming the transgenic silkworm cocoon TC-M1 (or TC-M2) in the embodiment 2 by using a weak alkali solution, completely dissolving the degummed silkworm cocoon with a lithium bromide solution, and dialyzing the degummed silkworm cocoon with deionized water to obtain transgenic regenerated silk fibroin TSF-M1 (or TSF-M2);
2) dissolving TSF-M1 (or TSF-M2) protein in secondary water, and hydrating at room temperature to obtain hydrated TSF-M1 (or TSF-M2) protein;
3) weighing appropriate amount of Camptothecin (CPT), and dissolving in acetone completely to obtain acetone solution (concentration of 0.1mg/ml) containing CPT;
4) dropwise adding the hydrated regenerated silk fibroin obtained in the step (2) into the acetone solution dissolved with the CPT obtained in the step (3) under a vortex condition, wherein the solution becomes turbid into emulsion to obtain emulsion containing the CPT; the volume ratio of the hydrated TSF-M1 (or TSF-M2) protein to the acetone solution dissolved with the CPT is 1: 5;
5) immediately after vortexing the emulsion for 30s, it was placed in an ice bath and sonicated at 30% amplitude for 60s, 2s each, 2s apart.
6) Stirring the emulsion obtained in the step (5) in a fume hood for 3 hours, and evaporating the organic solvent acetone in the emulsion;
7) centrifuging the emulsion from which the organic solvent acetone is removed at the centrifugation rate of 13000g for 15min, discarding the supernatant, and collecting the precipitate;
8) and (3) washing the collected regenerated fibroin drug-loaded nanoparticles TSF-M1-CPT-NPs (or TSF-M2-CPT-NPs) with mil-Q water for 3 times, freezing at-80 ℃ overnight, freeze-drying in a freeze-dryer for 24h, and finally placing in a refrigerator at-20 ℃ for later use.
Fibroin drug-loaded nanoparticles WSF-CPT-NPs prepared using non-transgenic wild silk fibroin cocoons (WC) in example 2 were used as negative controls in the same manner as described above.
Example 4:
the detection steps of the particle morphology characterization and the surface charge are as follows:
the lyophilized nanoparticles of WSF-CPT-NPs, TSF-M1-CPT-NPs (or TSF-M2-CPT-NPs) obtained in example 3 were suspended in secondary water and mixed in a volume ratio of 1: 1000, then dripping the diluted suspension liquid on a mica sheet, air-drying for 4 hours, and observing the morphology of the particles by an Atomic Force Microscope (AFM). As shown in FIG. 3, from the AFM scanning electron micrographs of the WSF-CPT-NPs (a in FIG. 3), the TSF-M1-CPT-NPs (b in FIG. 3) and the TSF-M2-CPT-NPs (c in FIG. 3), it can be seen that 3 kinds of nanoparticles are spherical, are densely packed, have relatively uniform particle size distribution, and are also the relatively obvious characteristics of the silk fibroin nanoparticles. The WSF-CPT-NPs nano-particle measured by a particle size analyzer has the particle size of 139.5 +/-3.5 nm and the surface charge of-24.6 +/-0.3 mV; the particle size of the TSF-M1-CPT-NPs nano particle is 167.6 +/-2.1 nm, and the surface charge is-23.9 +/-0.1 mV; the particle diameter of the TSF-M2-CPT-NPs nano particle is 182 +/-1.8, and the surface charge is-23.4 +/-0.2 mV.
Example 5:
drug-loaded particle drug controlled release characterization experiment, which is to study the cumulative release curve of nanoparticles in vitro buffer solution by dialysis bag method, WSF-CPT-NPs and TSF-M1-CPT-NPs nanoparticles (200 μ g/M L) obtained in example 3 are respectively filled into a pre-treated dialysis bag (MWCO. 1400Da), and are put into a 50M L centrifuge tube containing 30ml of different release media of Tween-80 (0.3%, w/v) for further dialysis, the tube is put into a constant temperature oscillation box (120rpm) at 37 ℃ for oscillation, a fixed volume of release media is collected at a predetermined time point, and is replaced by a new release media, finally fluorescence is measured by an enzyme-labeling instrument at an excitation wavelength of 360nm and an emission wavelength of 430nm, the content of CPT in the release media is analyzed, and FIG. 4 is a drug controlled release graph of the nanoparticles of WSF-CPT-NPs and TSF-M1-CPT-NPs, and shows that the pH value decreases and GSH or GSH decreases2O2The drug release rate of the two particles is greatly improved by increasing the concentration, but the cumulative release amount of TSF-M1-CPT-NPs under the same condition is obviously higher than that of WSF-CPT-NPs, and different stimuli (acidity, GSH and H2O2) are proved to cause the change of the secondary structure of the silk fibroin nano-particles. These results indicate that the transgenic silk fibroin nanoparticles are superior to non-transgenic wild silk fibroin nanoparticles as a drug delivery system with significantly multi-stimulus responsiveness.
The tumor microenvironment has the characteristics of acidity, high active oxygen content, abnormal redox state and the like. The pH of normal tissues and blood is 7.4, and the glycolysis rate and the carbon dioxide concentration of tumor tissues are higher due to hypoxia, so that a slightly acidic microenvironment (pH 6.0-7.0) is presented, and cellular endosomes (pH 5.5), lysosomes (pH < 5.5) and other organelles present stronger acidity. In addition, it has been shown from the results of the study that a reducing agent such as Glutathione (GSH) is present in tumor cells at a concentration approximately 3 times that of normal cells. In the presence of GSH, the disulfide bond (S — S) is very susceptible to cleavage, making the disulfide a necessary site for a reductive stimulus-responsive delivery system. Therefore, the targeted controlled release of anticancer drugs based on a pH stimulus responsive drug delivery system becomes the key to treat tumors. Initially, we evaluated CPT release profiles of WSF-CPT-NPs or TSF-CPT-NPs by incubating them in buffers of different pH values (7.4, 6.8, 5.5 and 4.5) for seven consecutive days, as shown by a in FIG. 4, with decreasing pH, the drug release rates of both particles increased significantly, 41.7% of CPT was released from TSF-CPT-NPS in a buffer solution of pH7.4, whereas the cumulative release of WSF-CPT-NPs under the same conditions was less than 40%. In an acidic buffer (pH 5.5,4.5), the cumulative release of TSF-CPT-NPs reached 51.1% and 60.3%, respectively, which was higher than the cumulative release of WSF-CPT-NPs (44.2% and 48.7%). These results indicate that both WSF and TSF-based NPs release drug-loaded anticancer drugs in a controlled manner and exhibit significant pH sensitivity. As can be seen from b in fig. 4, the drug release rate increases with the increase of GSH concentration under the same pH (7.4) condition. After TSF-CPT-NPs were cultured in buffers containing GSH at different concentrations (0.1mM,1mM and 10mM) for 7 days, the cumulative drug release rates of TSF-CPT-NPs reached 43.3%, 59.8% and 73.7%, respectively, which were significantly higher than that of WSF-CPT-NPs (39.7%, 51.1% and 59.2%). In addition, we also incubated the nanoparticles in buffers (pH 7.4) containing different concentrations of hydrogen peroxide (10 μm, 100 μm and 1mM) and further tested the release behavior of the drug in the hydrogen peroxide-containing buffer. About 50.4%, 62.3% and 81.5% of CPT were released from TSF-CPT-NPs, respectively, higher than WSF-CPT-NPs (44.5%, 56.9% and 64.5%), indicating that TSF-CPT-NPs have a stronger ROS-response ability (c in FIG. 4). Next, we investigated the synergistic effect of the two stimuli by testing the drug release profiles of different NPs cultured in buffer at pH 5.5 with or without GSH (d in fig. 4). When the GSH concentration in the buffer solution (pH 5.5) is 10mM, the drug release rate of the TSF-CPT-NPs is obviously accelerated, and the cumulative drug release rate after 7 days reaches 87.7 percent, which is higher than that of the WSF-CPT-NPs (72.2 percent). In addition, the combination of the acidic environment and the GSH has obvious synergistic effect on the release rate of the NPs. Similarly, after incubation in buffers with pH 5.5 and different concentrations of H2O2(10 μ M, 100 μ M and 1mm), the cumulative release percentages of TSF-CPT nanoparticles reached 62.1%, 86.2% and 94.7%, respectively, which were much higher than the cumulative release percentages of WSF-CPT nanoparticles at the same concentration of H2O2 (50.3%, 70.4% and 83.1%) (fig. 4, e).
And (3) a secondary structure characterization experiment of the particles, namely characterizing the secondary structures of the particles in different release media by using a circular dichrograph (MOS-500CD, USA), wherein the scanning wavelength range of a CD spectrum is 190-250 nm, and further analyzing the CD spectrum by using CD Pro software to obtain the β -sheet structure content of the particles, wherein as shown in a secondary structure characterization diagram of different particles in figure 5, the CD spectrum result shows that the β folding content of TSF-M1-CPT-NPs is higher than that of WSF-CPT-NPs.
As shown in a in FIG. 5, in order to further explore the release mechanism deeply, we performed circular dichroism measurements on the secondary structure content of particles under buffer conditions of different pH values, it was found from the circular dichroism plot that the β folded structure content of particles shows a sharp decrease trend as the acidity of the buffer solution increases, as shown in b in FIG. 5, the β folded structure content is about 66.1%, 60.5% and 47.2% respectively in the buffers of pH7.4, 6.8 and 5.5, higher than the β folded content (56.5%, 50.3% and 40.2%) in the corresponding WSF-CPT-NPs, the secondary structure of silk fibroin determines the main property of drug release, and different stimuli (acidity, GSH and H2O2) cause the change of the secondary structure of the nanoparticle of silk fibroin, further explaining that the β folded content of WSF-CPT-NPs is higher than that of TSF-CPT-NPs respond to the strong stimulation and the stronger stimulation of the drug release.
Example 6:
of drug-loaded particlesCytotoxicity experiments CT-26 cells (purchased from iCell Bioscience Inc (Shanghai, p.r. china)) were plated at 2 × 104The density of each hole is inoculated in a 96-hole plate, the hole plate is placed in an incubator at 37 ℃ for culture overnight for 24 hours, the culture medium is removed, a serum-free culture medium containing WSF-CPT-NPs or TSF-M1-CPT-NPs is used for replacing the serum-free culture medium (wherein the content of CPT is 0.1-64 mu M), the non-added medicine is used as a negative control group, 1% (w/v) of Triton X-100 is added as a positive control, the mixture is placed in the incubator for culture for 24 hours and 48 hours respectively, after the incubation is finished, the particles are washed clean by PBS containing calcium and magnesium, 0.5mg/M L MTT is added for incubation for 4 hours, then the MTT is discarded, 50 mu L DMSO is added, then shaking is carried out for 15 minutes by 150rpm of a shaking table, the OD value of each hole is measured at the wavelength of 570nm, the WSF-CPT-NPs or TSF-M1-CPT-NPs nano medicine and CT-26 cells are cultured for 24 hours and 48 hours, the anti-tumor cell therapy effect of the WSF-CPT-NPs is better than that of the CPF-26 cells is shown in colon cancer treatment, and the anti-CPF-CPT nano-NPs.
MTT method test results show that the two kinds of particles have killing effects on CT-26 cells in different degrees after being co-cultured with the cells for 24 hours and 48 hours respectively, and are dose-dependent and time-dependent. As shown in a-b in FIG. 6, the anti-colon cancer activity of TSF-CPT-NPs was significantly higher than that of WSF-CPT-NPs, and the IC50 values of TSF-CPT-NPs at 24 hours and 48 hours were 2.31. mu.M and 0.52. mu.M, respectively, which were 1.7 times and 1.4 times the corresponding IC50 values (6.13. mu.M and 1.24. mu.M) of WSF-CPT-NPs, respectively. This may be associated with increased cellular uptake and drug release of TSF-CPT-NPs.
Experiment of endocytosis efficiency of drug-loaded particle cells, CT-26 cells are treated with 2 × 105The density of each well was inoculated into 12-well culture plates for overnight culture. To achieve fluorescence visualization, the drugs CPT in WSF-CPT-NPs and TSF-M1-CPT-NPs nanoparticles were changed to coumarin-6 (COU) as model drugs according to the procedure in example 3, and the corresponding nanoparticles WSF-COU-NPs and TSF-M1-COU-NPs were prepared and dispersed in serum-free medium to form particle suspensions, respectively, and then the particle suspensions were added to the cells. After co-culturing with cells for 0.5, 1 or 3h, respectively, the cells were washed 3 times with PBS solution, trypsinized, and in flow cytometer bufferThe cell suspension obtained by analyzing the cell suspension by using a flow cytometer system shows that the phagocytic efficiency of TSF-M1-COU-NPs nanoparticles and WSF-COU-NPs nanoparticles by CT-26 cells is remarkably higher than that of the WSF-COU-NPs nanoparticles, as shown in FIG. 7, the content of α -helix structures of the TSF-M1-COU-NPs nanoparticles is higher, and the phagocytic efficiency of cells is remarkably enhanced, as shown in a in FIG. 7, after the particles and the cells are incubated for 0.5h, 1h and 3h respectively, the phagocytic rate of the cells treated by TSF-COU-NPs is 41.5%, 63.0% and 98.7%, as shown in a in FIG. 7, the phagocytic rate of the cells treated by TSF-COU-NPs is remarkably higher than that of the cells treated by WSF-COU-NPs (27.5%, 48.7% and 64.6%, as shown in a FIG. 7, as shown in a graph, the fluorescent intensity of the TSF-COF-COU-NPs treated by CT-NPs treated by TSF-1-COF-COU-NPs is remarkably higher than that of the cell-NPs treated by the TSF-COF-COU-NPs treated by the same conditions (27.5-COU-7), and COU-1.7), and COF-NPs) are remarkably increased, as shown in the fluorescent intensity after the fluorescent intensity of the fluorescence intensity of the.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Sequence listing
<110> university of southwest
<120> spider MaSps gene and application thereof in preparation of drug-loaded nanoparticles
<141>2020-04-22
<160>6
<170>SIPOSequenceListing 1.0
<210>1
<211>1332
<212>DNA
<213>Artificial Sequence
<400>1
ggaggagcag gtcaaggagg acaaggagga tacggacgtg gtggttacgg acaaggaggt 60
gccggacaag gtggagcagg agcagcagca gctgcagcag cagcagctag aggaggagca 120
ggtcaaggag gacaaggagg atacggacgt ggtggttacg gacaaggagg tgccggacaa 180
ggtggagcag gagcagcagc agctgcagca gcagcagcta gaggaggagc aggtcaagga 240
ggacaaggag gatacggacg tggtggttac ggacaaggag gtgccggaca aggtggagca 300
ggagcagcag cagctgcagc agcagcagct agaggaggag caggtcaagg aggacaagga 360
ggatacggac gtggtggtta cggacaagga ggtgccggac aaggtggagc aggagcagca 420
gcagctgcag cagcagcagc tagaggagga gcaggtcaag gaggacaagg aggatacgga 480
cgtggtggtt acggacaagg aggtgccgga caaggtggag caggagcagc agcagctgca 540
gcagcagcag ctagaggagg agcaggtcaa ggaggacaag gaggatacgg acgtggtggt 600
tacggacaag gaggtgccgg acaaggtgga gcaggagcag cagcagctgc agcagcagca 660
gctagaggag gagcaggtca aggaggacaa ggaggatacg gacgtggtgg ttacggacaa 720
ggaggtgccg gacaaggtgg agcaggagca gcagcagctg cagcagcagc agctagagga 780
ggagcaggtc aaggaggaca aggaggatac ggacgtggtg gttacggaca aggaggtgcc 840
ggacaaggtg gagcaggagc agcagcagct gcagcagcag cagctagagg aggagcaggt 900
caaggaggac aaggaggata cggacgtggt ggttacggac aaggaggtgc cggacaaggt 960
ggagcaggag cagcagcagc tgcagcagca gcagctagag gaggagcagg tcaaggagga 1020
caaggaggat acggacgtgg tggttacgga caaggaggtg ccggacaagg tggagcagga 1080
gcagcagcag ctgcagcagc agcagctaga ggaggagcag gtcaaggagg acaaggagga 1140
tacggacgtg gtggttacgg acaaggaggt gccggacaag gtggagcagg agcagcagca 1200
gctgcagcag cagcagctag aggaggagca ggtcaaggag gacaaggagg atacggacgt 1260
ggtggttacg gacaaggagg tgccggacaa ggtggagcag gagcagcagc agctgcagca 1320
gcagcagcta gc 1332
<210>2
<211>444
<212>PRT
<213>Artificial Sequence
<400>2
Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gly Gly Tyr
1 5 10 15
Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala
20 25 30
Ala Ala Ala Ala Arg Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr
35 40 45
Gly Arg Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Gly
50 55 60
Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly Gln Gly
65 70 75 80
Gly Gln Gly Gly Tyr Gly Arg Gly Gly Tyr Gly Gln Gly Gly Ala Gly
85 90 95
Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly
100 105 110
Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gly Gly Tyr Gly
115 120 125
Gln Gly Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala
130 135 140
Ala Ala Ala Arg Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly
145 150 155 160
Arg Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Gly Ala
165 170 175
Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly Gln Gly Gly
180 185 190
Gln Gly Gly Tyr Gly Arg Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln
195 200 205
Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly
210 215 220
Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gly Gly Tyr Gly Gln
225 230 235 240
Gly Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala
245 250 255
Ala Ala Arg Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg
260 265 270
Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala
275 280 285
Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly Gln Gly Gly Gln
290 295 300
Gly Gly Tyr Gly Arg Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly
305 310 315 320
Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala
325 330 335
Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gly Gly Tyr Gly Gln Gly
340 345 350
Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala
355 360 365
Ala Arg Gly Gly Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gly
370 375 380
Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Gly Ala Ala Ala
385 390 395 400
Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly Gln Gly Gly Gln Gly
405 410 415
Gly Tyr Gly Arg Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly
420 425 430
Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ser
435 440
<210>3
<211>2592
<212>DNA
<213>Artificial Sequence
<400>3
ggtggagccg gaccaggaag acaacaggca tatggaccag gaggatcagg agccgctgca 60
gcagcagccg ccgccgctag aggtggagcc ggaccaggaa gacaacaggc atatggacca 120
ggaggatcag gagccgctgc agcagcagcc gccgccgcta gaggtggagc cggaccagga 180
agacaacagg catatggacc aggaggatca ggagccgctg cagcagcagc cgccgccgct 240
agaggtggag ccggaccagg aagacaacag gcatatggac caggaggatc aggagccgct 300
gcagcagcag ccgccgccgc tagaggtgga gccggaccag gaagacaaca ggcatatgga 360
ccaggaggat caggagccgc tgcagcagca gccgccgccg ctagaggtgg agccggacca 420
ggaagacaac aggcatatgg accaggagga tcaggagccg ctgcagcagc agccgccgcc 480
gctagaggtg gagccggacc aggaagacaa caggcatatg gaccaggagg atcaggagcc 540
gctgcagcag cagccgccgc cgctagaggt ggagccggac caggaagaca acaggcatat 600
ggaccaggag gatcaggagc cgctgcagca gcagccgccg ccgctagagg tggagccgga 660
ccaggaagac aacaggcata tggaccagga ggatcaggag ccgctgcagc agcagccgcc 720
gccgctagag gtggagccgg accaggaaga caacaggcat atggaccagg aggatcagga 780
gccgctgcag cagcagccgc cgccgctaga ggtggagccg gaccaggaag acaacaggca 840
tatggaccag gaggatcagg agccgctgca gcagcagccg ccgccgctag aggtggagcc 900
ggaccaggaa gacaacaggc atatggacca ggaggatcag gagccgctgc agcagcagcc 960
gccgccgcta gaggtggagc cggaccagga agacaacagg catatggacc aggaggatca 1020
ggagccgctg cagcagcagc cgccgccgct agaggtggag ccggaccagg aagacaacag 1080
gcatatggac caggaggatc aggagccgct gcagcagcag ccgccgccgc tagaggtgga 1140
gccggaccag gaagacaaca ggcatatgga ccaggaggat caggagccgc tgcagcagca 1200
gccgccgccg ctagaggtgg agccggacca ggaagacaac aggcatatgg accaggagga 1260
tcaggagccg ctgcagcagc agccgccgcc gctagaggtg gagccggacc aggaagacaa 1320
caggcatatg gaccaggagg atcaggagcc gctgcagcag cagccgccgc cgctagaggt 1380
ggagccggac caggaagaca acaggcatat ggaccaggag gatcaggagc cgctgcagca 1440
gcagccgccg ccgctagagg tggagccgga ccaggaagac aacaggcata tggaccagga 1500
ggatcaggag ccgctgcagc agcagccgcc gccgctagag gtggagccgg accaggaaga 1560
caacaggcat atggaccagg aggatcagga gccgctgcag cagcagccgc cgccgctaga 1620
ggtggagccg gaccaggaag acaacaggca tatggaccag gaggatcagg agccgctgca 1680
gcagcagccg ccgccgctag aggtggagcc ggaccaggaa gacaacaggc atatggacca 1740
ggaggatcag gagccgctgc agcagcagcc gccgccgcta gaggtggagc cggaccagga 1800
agacaacagg catatggacc aggaggatca ggagccgctg cagcagcagc cgccgccgct 1860
agaggtggag ccggaccagg aagacaacag gcatatggac caggaggatc aggagccgct 1920
gcagcagcag ccgccgccgc tagaggtgga gccggaccag gaagacaaca ggcatatgga 1980
ccaggaggat caggagccgc tgcagcagca gccgccgccg ctagaggtgg agccggacca 2040
ggaagacaac aggcatatgg accaggagga tcaggagccg ctgcagcagc agccgccgcc 2100
gctagaggtg gagccggacc aggaagacaa caggcatatg gaccaggagg atcaggagcc 2160
gctgcagcag cagccgccgc cgctagaggt ggagccggac caggaagaca acaggcatat 2220
ggaccaggag gatcaggagc cgctgcagca gcagccgccg ccgctagagg tggagccgga 2280
ccaggaagac aacaggcata tggaccagga ggatcaggag ccgctgcagc agcagccgcc 2340
gccgctagag gtggagccgg accaggaaga caacaggcat atggaccagg aggatcagga 2400
gccgctgcag cagcagccgc cgccgctaga ggtggagccg gaccaggaag acaacaggca 2460
tatggaccag gaggatcagg agccgctgca gcagcagccg ccgccgctag aggtggagcc 2520
ggaccaggaa gacaacaggc atatggacca ggaggatcag gagccgctgc agcagcagcc 2580
gccgccgcta gc 2592
<210>4
<211>864
<212>PRT
<213>Artificial Sequence
<400>4
Gly Gly Ala Gly Pro Gly Arg Gln Gln Ala Tyr Gly Pro Gly Gly Ser
1 5 10 15
Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly Pro
20 25 30
Gly Arg Gln Gln Ala Tyr Gly Pro Gly Gly Ser Gly Ala Ala Ala Ala
35 40 45
Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly Pro Gly Arg Gln Gln Ala
50 55 60
Tyr Gly Pro Gly Gly Ser Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala
65 70 75 80
Arg Gly Gly Ala Gly Pro Gly Arg Gln Gln Ala Tyr Gly Pro Gly Gly
85 90 95
Ser Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly
100 105 110
Pro Gly Arg Gln Gln Ala Tyr Gly Pro Gly Gly Ser Gly Ala Ala Ala
115 120 125
Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly Pro Gly Arg Gln Gln
130 135 140
Ala Tyr Gly Pro Gly Gly Ser Gly Ala Ala Ala Ala Ala Ala Ala Ala
145 150 155 160
Ala Arg Gly Gly Ala Gly Pro Gly Arg Gln Gln Ala Tyr Gly Pro Gly
165 170 175
Gly Ser Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala
180 185 190
Gly Pro Gly Arg Gln Gln Ala Tyr Gly Pro Gly Gly Ser Gly Ala Ala
195 200 205
Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly Pro Gly Arg Gln
210 215 220
Gln Ala Tyr Gly Pro Gly Gly Ser Gly Ala Ala Ala Ala Ala Ala Ala
225 230 235 240
Ala Ala Arg Gly Gly Ala Gly Pro Gly Arg Gln Gln Ala Tyr Gly Pro
245 250 255
Gly Gly Ser Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly
260 265 270
Ala Gly Pro Gly Arg Gln Gln Ala Tyr Gly Pro Gly Gly Ser Gly Ala
275 280 285
Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly Pro Gly Arg
290 295 300
Gln Gln Ala Tyr Gly Pro Gly Gly Ser Gly Ala Ala Ala Ala Ala Ala
305 310 315 320
Ala Ala Ala Arg Gly Gly Ala Gly Pro Gly Arg Gln Gln Ala Tyr Gly
325 330 335
Pro Gly Gly Ser Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly
340 345 350
Gly Ala Gly Pro Gly Arg Gln Gln Ala Tyr Gly Pro Gly Gly Ser Gly
355 360 365
Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly Pro Gly
370 375 380
Arg Gln Gln Ala Tyr Gly Pro Gly Gly Ser Gly Ala Ala Ala Ala Ala
385 390 395 400
Ala Ala Ala Ala Arg Gly Gly Ala Gly Pro Gly Arg Gln Gln Ala Tyr
405 410 415
Gly Pro Gly Gly Ser Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg
420 425 430
Gly Gly Ala Gly Pro Gly Arg Gln Gln Ala Tyr Gly Pro Gly Gly Ser
435 440 445
Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly Pro
450 455 460
Gly Arg Gln Gln Ala Tyr Gly Pro Gly Gly Ser Gly Ala Ala Ala Ala
465 470 475 480
Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly Pro Gly Arg Gln Gln Ala
485 490 495
Tyr Gly Pro Gly Gly Ser Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala
500 505 510
Arg Gly Gly Ala Gly Pro Gly Arg Gln Gln Ala Tyr Gly Pro Gly Gly
515 520 525
Ser Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly
530 535 540
Pro Gly Arg Gln Gln Ala Tyr Gly Pro Gly Gly Ser Gly Ala Ala Ala
545 550 555 560
Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly Pro Gly Arg Gln Gln
565 570 575
Ala Tyr Gly Pro Gly Gly Ser Gly Ala Ala Ala Ala Ala Ala Ala Ala
580 585 590
Ala Arg Gly Gly Ala Gly Pro Gly Arg Gln Gln Ala Tyr Gly Pro Gly
595 600 605
Gly Ser Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala
610 615 620
Gly Pro Gly Arg Gln Gln Ala Tyr Gly Pro Gly Gly Ser Gly Ala Ala
625 630 635 640
Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly Pro Gly Arg Gln
645 650 655
Gln Ala Tyr Gly Pro Gly Gly Ser Gly Ala Ala Ala Ala Ala Ala Ala
660 665 670
Ala Ala Arg Gly Gly Ala Gly Pro Gly Arg Gln Gln Ala Tyr Gly Pro
675 680 685
Gly Gly Ser Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly
690 695 700
Ala Gly Pro Gly Arg Gln Gln Ala Tyr Gly Pro Gly Gly Ser Gly Ala
705 710 715 720
Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly Pro Gly Arg
725 730 735
Gln Gln Ala Tyr Gly Pro Gly Gly Ser Gly Ala Ala Ala Ala Ala Ala
740 745 750
Ala Ala Ala Arg Gly Gly Ala Gly Pro Gly Arg Gln Gln Ala Tyr Gly
755 760 765
Pro Gly Gly Ser Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly
770 775 780
Gly Ala Gly Pro Gly Arg Gln Gln Ala Tyr Gly Pro Gly Gly Ser Gly
785 790 795 800
Ala Ala Ala Ala Ala Ala Ala Ala Ala Arg Gly Gly Ala Gly Pro Gly
805 810 815
Arg Gln Gln Ala Tyr Gly Pro Gly Gly Ser Gly Ala Ala Ala Ala Ala
820 825 830
Ala Ala Ala Ala Arg Gly Gly Ala Gly Pro Gly Arg Gln Gln Ala Tyr
835 840 845
Gly Pro Gly Gly Ser Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ser
850 855 860
<210>5
<211>2304
<212>DNA
<213>Artificial Sequence
<400>5
aagcttgttg tacaaaactg ccacacgcat ttttttctcc actgtaggtt gtagttacgc 60
gaaaacaaaa tcgttctgtg aaaattcaaa caaaaatatt ttttcgtaaa aacacttatc 120
aatgagtaaa gtaacaattc atgaataatt tcatgtaaaa aaaaaatact agaaaaggaa 180
tttttcatta cgagatgctt aaaaatctgt ttcaaggtag agatttttcg atatttcgga 240
aaattttgta aaactgtaaa tccgtaaaat tttgctaaac atatattgtg ttgttttggt 300
aagtattgac ccaagctatc acctcctgca gtatgtcgtg ctaattactg gacacattgt 360
ataacagttc cactgtattg acaataataa aacctcttca ttgacttgag aatgtctgga 420
cagatttggc tttgtatttt tgatttacaa atgttttttt ggtgatttac ccatccaagg 480
cattctccag gatggttgtg gcatcacgcc gattggcaaa caaaaactaa aatgaaacta 540
aaaagaaaca gtttccgctg tcccgttcct ctagtgggag aaagcatgaa gtaagttctt 600
taaatattac aaaaaaattg aacgatatta taaaattctt taaaatatta aaagtaagaa 660
caataagatc aattaaatca taattaatca cattgttcat gatcacaatt taatttactt 720
catacgttgt attgttatgt taaataaaaa gattaatttc tatgtaattg tatctgtaca 780
atacaatgtg tagatgttta ttctatcgaa agtaaatacg tcaaaactcg aaaattttca 840
gtataaaaag gttcaacttt ttcaaatcag catcagttcg gttccaactc tcaagatgag 900
agtcaaaacc tttgtgatct tgtgctgcgc tctgcaggtg agttaattat tttactatta 960
tttcagaagg tggccagacg atatcacggg ccacctgata ataagtggtc gccaaaacgc 1020
acagatatcg taaattgtgc catttgattt gtcacgcccg ggggggctac ggaataaact 1080
acatttattt atttaaaaaa tgaaccttag attatgtaac ttgtgattta tttgcgtcaa 1140
aagtaggcaa gatgaatcta tgtaaatacc tgggcagact tgcaatatcc tatttcaccg 1200
gtaaatcagc attgcaatat gcaatgcata ttcaacaata tgtaaaacaa ttcgtaaagc 1260
atcattagaa aatagacgaa agaaattgca taaaattata accgcattat taatttatta 1320
tgatatctat taacaattgc tattgccttt ttttcgcaaa ttataatcat tttcataacc 1380
tcgaggtagc attctgttac attttaatac attggtatgt gattataaca cgagctgccc 1440
actgagtttc tcgccagatc ttctcagtgg gtcgcgttac cgatcacgtg atagattcta 1500
tgaagcactg ctcttgttag ggctagtgtt agcaaattct ttcaggttga gtctgagagc 1560
tcacctaccc atcggagcgt agctggaata ggctaccagc taataggtag ggaaaacaaa 1620
gctcgaaaca agctcaagta ataacaacat aatgtgacca taaaatctcg tggtgtatga 1680
gatacaatta tgtactttcc cacaaatgtt tacataatta gaatgttgtt caacttgcct 1740
aacgccccag ctagaacatt caattattac tattaccact actaaggcag tatgtcctaa 1800
ctcgttccag atcagcgcta acttcgattg aatgtgcgaa atttatagct caatatttta 1860
gcacttatcg tattgattta agaaaaaatt gttaacattt tgtttcagta tgtcgcttat 1920
acaaatgcaa acatcaatga ttttgatgag gactattttg ggagtgatgt cactgtccaa 1980
agtagtaata caacagatga aataattaga gatgcatctg gggcagttat cgaagaacaa 2040
attacaacta aaaaaatgca acggaaaaat aaaaaccatg gaatacttgg aaaaaatgaa 2100
aaaatgatca agacgttcgt tataaccacg gattccgacg gtaacgagtc cattgtagag 2160
gaagatgtgc tcatgaagac actttccgat ggtactgttg ctcaaagtta tgttgctgct 2220
gatgcgggag catattctca gagcgggcca tacgtatcaa acagtggata cagcactcat 2280
caaggatata cgagcgattt cagc 2304
<210>6
<211>333
<212>DNA
<213>Artificial Sequence
<400>6
agttacggag ctggcagggg atacggacaa ggtgcaggaa gtgcagcttc ctctgtgtca 60
tctgcttcat ctcgcagtta cgactattct cgtcgtaacg tccgcaaaaa ctgtggaatt 120
cctagaagac aactagttgt taaattcaga gcactgcctt gtgtgaattg ctaattttta 180
atataaaata acccttgttt cttacttcgt cctggataca tctatgtttt ttttttcgtt 240
aataaatgag agcatttaag ttattgtttt taattacttt tttttagaaa acagatttcg 300
gattttttgt atgcatttta tttgaatgta cta 333
Claims (10)
1. A spider MaSps gene comprising:
the MaSp1 gene has a nucleotide sequence shown in SEQ ID NO. 1; or
The nucleotide sequence of the MaSp2 gene is shown in SEQ ID NO. 3.
2. The spider MaSps gene encoding amino acids of claim 1, comprising:
the amino acid sequence of the amino acid coded by the MaSp1 gene is shown in SEQ ID NO. 2; or
The amino acid sequence of the amino acid coded by the MaSp2 gene is shown in SEQ ID NO. 4.
3. A transgenic expression vector constructed based on the spider MaSps gene of claim 1.
4. Use of the spider MaSps gene of claim 1 for the preparation of silkworm cocoons for promoting controlled drug release and endocytosis.
5. Use of the spider MaSps gene of claim 1 in the preparation of silk for promoting controlled drug release and endocytosis.
6. A transgenic silkworm cocoon prepared by using the spider MaSps gene according to claim 1, wherein the spider MaSps gene is expressed in the posterior silk gland of silkworms.
7. A transgenic silk prepared by using the spider MaSps gene of claim 1, wherein the spider MaSps gene is expressed in the posterior silk gland of silkworms.
8. Use of the transgenic silkworm cocoon of claim 6 or the transgenic silk of claim 7 for the preparation of drug-loaded nanoparticles.
9. A drug-loaded nanoparticle prepared from the transgenic silkworm cocoon of claim 6 or the transgenic silk of claim 7.
10. The preparation method of the drug-loaded nanoparticles of claim 9, which is characterized by comprising the following specific steps:
(1) degumming the transgenic silkworm cocoons to obtain transgenic silk fibroin;
(2) then completely dissolving the transgenic silk fibroin in water to obtain a transgenic silk fibroin solution;
(3) dissolving hydrophobic drug in organic solvent to obtain drug solution;
(4) and then encapsulating the transgenic silk fibroin solution in a drug solution to obtain emulsion, performing ultrasonic treatment, removing the organic solvent, and performing post-treatment to obtain the drug-loaded nano particle.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113563446A (en) * | 2021-07-22 | 2021-10-29 | 浙江超丝生物科技有限公司 | Single polyprotein molecule for improving mechanical properties and application method thereof |
| CN114907467A (en) * | 2022-05-13 | 2022-08-16 | 四川轻化工大学 | Recombinant spider silk protein fused with carbon end, preparation method thereof and drug-loaded microspheres based on recombinant spider silk protein |
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| CN105400815A (en) * | 2015-10-23 | 2016-03-16 | 浙江大学 | Method for synthesizing and secreting black widow spider dragline silk protein 1 through bombyx mori silk gland bioreactor |
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| CN102399786A (en) * | 2010-09-10 | 2012-04-04 | 冈本株式会社 | Dragline protein |
| CN103232977A (en) * | 2013-05-16 | 2013-08-07 | 西南大学 | Application of phiC31 recombinase system and piggyBac transposon and fixed point transgenetic system of silkworm and preparation method of fixed point transgenetic system |
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| CN113563446A (en) * | 2021-07-22 | 2021-10-29 | 浙江超丝生物科技有限公司 | Single polyprotein molecule for improving mechanical properties and application method thereof |
| CN114907467A (en) * | 2022-05-13 | 2022-08-16 | 四川轻化工大学 | Recombinant spider silk protein fused with carbon end, preparation method thereof and drug-loaded microspheres based on recombinant spider silk protein |
| CN114907467B (en) * | 2022-05-13 | 2023-09-15 | 四川轻化工大学 | Recombinant spider silk protein fused with carbon ends, preparation method thereof and drug-loaded microsphere based on recombinant spider silk protein |
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