CN111454961B - Spider MaSps gene and application thereof in preparation of drug-loaded nanoparticles - Google Patents
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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 MaSp protein in the fibroin layer and has the functions of promoting controlled release of drugs and endocytosis of 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, which has the main properties of biocompatibility, biodegradability, controlled drug release and the like required by 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. Therefore, researchers often need to compound the BmSF with other materials to fully develop the potential and expand the application range of the BmSF, so as to obtain a more practical biomaterial.
The main properties of silk proteins as drug delivery materials are determined by their secondary structure. The β -sheet (β -sheet) is the most basic secondary structure in fibroin biomaterials including silk and spider silk proteins, and it is not only key to stabilize the particle structure, but also plays a dominant role in controlled drug release. In addition, the alpha-helix (alpha-helix) structure contained in silk protein is more stable and more resistant to mutation than the beta-chain in natural high molecular protein and artificially designed and synthesized beta-chain, and simultaneously the alpha-helix is one of the most common and important structural elements for protein to pass through biological membrane (transmembrane movement). Therefore, adjusting the content and the proportion of the beta-sheet layer and the alpha-spiral secondary structure in the silk protein is an important strategy for realizing the performance improvement of silk protein materials, particularly the performance in the aspects of drug controlled release, cell endocytosis promotion and the like.
The large saccular gland silk (MaSps) secreted by the large saccular gland of Aranea is also called Dragline silk or Dragline silk, and is the silk protein fiber with the best known mechanical property in nature. Spider dragline contains 2 highly conserved spider silk proteins, major ampullate Spidroin 1 (Major ampulute Spidroin 1, maSp1) and Major ampullate Spidroin 2 (Major ampulute Spidroin 2, maSp2). The protein motif of MaSp1 is mainly An, (GA) n and GGX, and mainly forms a large number of beta-sheets and hydrophilic 3 10 A helical structure; the MaSp2 protein motif is mainly based on An, (GA) n and GPGXX, and mainly forms a large number of beta-sheets and beta-turn structures. However, since it is very difficult to artificially feed spiders in large scale and high density, which results in very low yield of natural spidroin protein, only genetic engineering means is used to artificially synthesize recombinant spidroin protein by using different types of organisms such as escherichia coli, yeast, mammalian cells and plants as hosts to meet the requirements of research, development and application.
In the last two decades, the establishment and maturation of silkworm transgenic technology provides a new way for the development of new purposes and the improvement of performance of silk. Particularly, the construction of silkworm diapause variety transgenic technology and a silkworm high-efficiency silk gland expression system enables us to create a novel practical variety transgenic cocoon silk material containing spider MaSps protein through the technical systems. Use of spider MaSps protein rich in beta-sheet, beta-turn and 3 10 The spiral structure is used for improving the performance of the transgenic cocoon silk, and is favorable for 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 deliveryAnd disease treatment studies. 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 performance of the controlled release of the silk medicament and the promotion of the endocytosis of the cells, creates a novel MaSps gene-transferred silk material and applies the material to the research of medicament 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 through expression of posterior silk glands of silkworms; 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 nucleotide sequence of the MaSp1 gene is shown as SEQ ID NO.1 and is spliced by 12 MaSp1 unit sequences (shown as 1 st to 105 th sites of SEQ ID NO. 1) which are in tandem repetition; or alternatively
The nucleotide sequence of the MaSp2 gene is shown in SEQ ID NO.3 and is spliced by 32 tandem repetitive MaSp2 unit sequences (shown in 1 st to 75 th positions 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 silk fibroin heavy chain protein gene promoter Fib-H, the spider MaSps gene and a termination sequence LBS containing a bombyx mori light chain binding site which are sequentially connected, namely the Fib-H-MaSps-LBS expression frame. Wherein, the nucleotide sequences of Fib-H and LBS are respectively shown in SEQ ID NO. 5-6.
Preferably, the transgenic expression vector takes a pBac {3 xP 3-DsRedaf } vector as a skeleton vector, and a Fib-H-MaSps-LBS 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 MaSp gene is obtained by expressing the spider MaSp gene in the posterior silk gland of the silkworm.
8. The transgenic silkworm cocoon or the transgenic silk is applied to preparing the drug-loaded nano particles.
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 nanoparticles 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 Na 2 CO 3 Boiling 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 solution 2 : ethanol: h 2 Dissolving regenerated silk fibroin in O (molar ratio 1; finally rotating at 8000 rpmInsoluble impurities were removed by rapid centrifugation for 10 min 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/mL.
Preferably, in the step (3), the drug is camptothecin, the organic solvent is acetone, and the concentration of the drug solution is 0.1mg/mL.
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 gently stirring, 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 with a sonicator at 30% amplitude for 60s.
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, 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 silkworm fibroin heavy chain promoter is constructed, and meanwhile, a piggyBac transposon arm and a fluorescence screening marker gene are connected to construct a transgenic expression vector, the expression vector can be used for establishing a transgenic silkworm which efficiently expresses and recombines the fused MaSps protein in a silkworm gland, so that transgenic cocoon silks containing the recombinated and fused MaSps protein in fibroin are obtained; transgenic silk fibroin nano-drugs (TSF-CPT-NPs) carrying Camptothecin (CPT) are prepared by using spider MaSps gene bombyx mori silk fibroin as a carrier and using a solvent volatilization method, so that the transgenic silk material which can stably secrete the recombinant MaSps protein in a fibroin layer and has the effects of promoting controlled release of drugs and endocytosis is obtained. Compared with the drug-loaded nano-particles of non-transgenic fibroin (WSF) protein, the drug-loaded nano-particles based on the transgenic silkworm fibroin protein of the spider MaSps gene obtained by the invention have the advantages that the content of the secondary structures of alpha-helix and beta-lamella is obviously improved, the more sensitive multi-stimulation responsiveness is shown, the drug controlled release capacity and the cell internalization efficiency of the particles can be effectively improved, and the drug-loaded nano-particles are expected to become an effective drug delivery system for clinical colon cancer chemotherapy. The concrete advantages are as follows:
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 (MaSp 1 or MaSp 2) 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 content of alpha-helix and/or beta-sheet in the protein secondary structure is obviously higher than that of the drug-loaded fibroin nanoparticles prepared from non-transgenic silk, and experiments prove that recombinant MaSp protein contained in the transgenic silk can effectively improve the drug controlled release capacity and the cell internalization efficiency of the particles, and has obvious treatment effect on colon cancer.
Drawings
In order to make the purpose, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a schematic structural diagram of a silkworm transgenic recombinant vector, wherein a is pBac {3 XP 3-DsRed; fibH-MaSp1-LBS }, b is pBac {3 xP 3-DsRed; fibH-MaSp2-LBS }.
FIG. 2 is SDS-PAGE and Western blotting detection diagrams of transgenic cocoon silk solutions respectively expressing MaSp1 protein and MaSp2 protein, wherein a is the SDS-PAGE detection diagram, and b and c are the Western blotting detection diagrams of recombinant MaSp1 protein and recombinant MaSp2 protein, respectively.
FIG. 3 is AFM diagram of transgenic silk fibroin nanoparticles, wherein a, b, and 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 value of 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 beta sheet 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-drugs and CT-26 cells after 24h and 48h of culture, 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 instructions (third edition, sambrook et al), or according to the conditions recommended by the manufacturer.
Example 1:
1. optimizing and constructing a manually designed and modified spider MaSps gene sequence;
according to the preference of endogenous gene expression sequence codons in the silkworm genome sequence data, the optimal design is carried out by referring to a black widow spider (Latrodectus hesperus) dragline protein gene sequence, a MaSp1 unit nucleotide sequence (shown as 1 st to 105 th bits of SEQ ID NO. 1) and a MaSp2 unit nucleotide sequence (shown as 1 st to 75 th bits of SEQ ID NO. 3) are synthesized by Shanghai bio-corporation and are respectively constructed on a pUC57-T-Simple vector (Shanghai bio-corporation) to become pUC-MaSp1 unit plasmids and pUC-MaSp2 unit plasmids. Then, the MaSp1 unit is repeatedly connected in series for 12 times by XbaI/NheI isocaudarner to obtain a pUC-MaSp1 plasmid and the MaSp2 unit is repeatedly connected in series for 32 times to obtain a pUC-MaSp2 plasmid by utilizing an XbaI cleavage site constructed at the upstream of the MaSp unit (MaSp 1 unit or MaSp2 unit) sequence and an NheI cleavage site constructed at the downstream of the MaSp unit. The nucleotide sequence of the complete spider MaSp1 gene after being connected in series is shown in SEQ ID NO.1, and the coded amino acid thereof is shown in SEQ ID NO. 2; the nucleotide sequence of the complete spider MaSp2 gene after being connected in series is shown as SEQ ID NO.3, and the coded amino acid thereof is shown as SEQ ID NO. 4.
2. Construction of silkworm transgenic recombinant vector
Constructing a silkworm transgenic recombinant vector pBac {3 xP 3-DsRed according to the requirements of the publicly known transgenic technology of the bombyx mori based on the piggyBac transposable vector; fib-H-MaSp1-LBS } (a in FIG. 1) and pBac {3 XP 3-DsRed; fib-H-MaSp2-LBS } (b in FIG. 1). The detailed preparation method is as follows: respectively recovering pUC-MaSp1 and pUC-MaSp2 vector plasmids constructed by BamHI/XbaI double enzyme digestion to obtain corresponding MaSp1 and MaSp2 gene fragments, then connecting the vector fragments into a pSL { Fib-H-EGFP-LBS } vector (see a Chinese patent with the publication number of CN 1912116A) subjected to the same enzyme digestion, namely replacing the EGFP gene sequence with the MaSp1 and the MaSp2 to obtain intermediate vectors pSL { Fib-H-MaSp1-LBS } and pSL { Fib-H-MaSp2-LBS }; subsequently, fib-H-MaSp1-LBS and Fib-H-MaSp2-LBS sequences are respectively recovered by carrying out double enzyme digestion on pSL { Fib-H-MaSp1-LBS } and pSL { Fib-H-MaSp2-LBS } vectors through AscI/FseI, and then are connected into a pBac {3 xP 3-DsRedaf } vector subjected to the same enzyme digestion (stored in national key laboratory of silkworm genome biology at southwest university), so that a transgenic recombinant vector pBac {3 xP 3-DsRed is obtained; fib-H-MaSp1-LBS } (a in FIG. 1) and pBac {3 XP 3-DsRed; fib-H-MaSp2-LBS } (b in FIG. 1). The constructed transgenic recombinant vector uses an expression cassette of red fluorescent protein (DsRed) started by an eye and nerve specific promoter 3 xP 3, the red fluorescent protein is used as a screening marker, and then a silkworm fibroin heavy chain expression system of MaSp1 or MaSp2 gene constructed after expression optimization is contained, the system utilizes a silkworm fibroin heavy chain promoter (FibH) to start the specific expression of the MaSp1 or MaSp2 gene in the silkworm silk gland and is fused and expressed with the fibroin heavy chain (the silkworm fibroin heavy chain promoter is referred to Chinese patent with the publication number of CN 2111916A).
Example 2:
1. preparation of spider MaSps gene-transferred silkworm
The method comprises the following steps of (1) taking a commercial diapause silkworm strain 302 (stored in a key laboratory of silkworm genome biology country of southwest university) as an original material, and carrying out low-temperature incubation treatment on parent silkworm eggs at 16 ℃ to release diapause of offspring silkworm eggs; then taking 10nL of recombinant vector pBac {3 xP 3-DsRed with the concentration of 400 ng/muL (w/v); injecting a mixed solution (1, v/v) of Fib-H-MaSp1-LBS } and an auxiliary plasmid pHA3PIG (stored in a key laboratory of the national silkworm genome biology of southwest university) into 207G 0 generation silkworm eggs which are released from diapause, sealing the mixed solution by using non-toxic glue, placing the sealed mixed solution in an environment with the temperature of 25 ℃ and the relative humidity of 85%, carrying out accelerated incubation to obtain 84G 0 generation silkworm eggs, then feeding the silkworm with mulberry leaves to a moth culture to obtain 25G 0 generation silkworm moths, carrying out backcrossing or selfing on the obtained silkworm moths to obtain 15 circles of G1 generation silkworm eggs, and carrying out backcrossing or selfing on the silkworm moths to obtain 15 circles 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 performing self-selection and purification to obtain the transgenic line which can express the MaSp1 protein in the silkworm cocoons and is stably inherited.
The commercial diapause silkworm strain 302 is used as an original material, and the parent silkworm eggs are subjected to low-temperature incubation treatment at 16 ℃ to relieve the diapause of the offspring silkworm eggs; then taking 10nL recombinant vector pBac {3 xP 3-DsRed with the concentration of 400 ng/muL; the mixed solution of Fib-H-MaSp2-LBS } and helper plasmid pHA3PIG is injected into 173G 0 generation silkworm eggs for diapause relief, sealed by nontoxic glue, and placed in an environment with 25 deg.C and 85% relative humidity for incubationHatching to obtain 65G 0 generation silkworm, breeding silkworm with mulberry leaf to transform moth to obtain 55G 0 generation silkworm moth, backcrossing or selfing to obtain 21G 1 generation silkworm eggs, and breeding with silkworm moth to obtain silkworm egg
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. And feeding the obtained positive transgenic silkworms to cocoon picking and further selfing for purification to obtain the transgenic line which can express the MaSp2 protein in the silkworm cocoons and can be stably inherited.
2. Expression detection of recombinant MaSps protein in transgenic cocoon silk
The expression of the MaSp1 protein and the MaSp2 protein in the transgenic cocoon silks is detected by SDS-PAGE and Western blotting. The specific operation steps are as follows: randomly screening transgenic silkworm cocoon (TC-M1) expressing MaSp1 protein and transgenic silkworm cocoon (TC-M2) expressing MaSp2 protein, and cutting into pieces of 1 × 1cm 2 Size, 0.02M Na 2 CO 3 Boiling the solution for 30min, and rinsing with ultrapure water from Milli-Q system (Millipore, billerica, MA) for 3 times to remove sericin; then 60% (w v) of 500. Mu.L -1 ) About 30mg of degumming cocoon is dissolved in lithium thiocyanate (LiSCN) solution; protein concentration was measured with a dicyandiamide acid (BCA) protein detection kit (Beyosine Biotech), and then beta-galactosidase (2-ME) was added to the protein sample and SDS-PAGE (15%, w v -1 Bio-Rad) gel for electrophoretic separation; 0.1% (w v) of SDS-PAGE gel after electrophoresis -1 ) Coomassie Brilliant blue R-250, 10% (vv) -1 ) Acetic acid, 50% (vv) -1 ) And (4) dyeing with methanol. Western blotting was performed using polyclonal Anti-MaSp1 antibody (Anti-MaSp 1) and MaSp2 antibody (Anti-MaSp 2) purchased from Zoonbio Biotechnology Co., ltd (Nanjing, china). Immunoreactive protein hybridization signals were detected using a chemiluminescent imaging system (Clinx ChemiScope series, shanghai, china) using an ECL + Western Blotting detection reagent (Beyotime, jiangsu, china) and operating according to the manufacturer's instructions. The result of examination of SDS-PAGE gel showed that the protein was negativeCompared with the control non-transgenic silkworm cocoon (WC) sample, specific recombinant MaSp1 protein (a in figure 2, marked by triangles) and specific recombinant MaSp2 protein (a in figure 2, marked by arrows) are detected in the TC-M1 and TC-M2 protein samples respectively. Further Western blotting hybridization using the MaSp1 antibody and the MaSp2 antibody respectively shows that the specific difference bands in the TC-M1 and TC-M2 protein samples are respectively recombinant MaSp1 protein (b in FIG. 2) and recombinant MaSp2 protein (c in FIG. 2).
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 transgenic silkworm cocoon TC-M1 (or TC-M2) by using a lithium bromide solution, and dialyzing the degummed transgenic silkworm cocoon by using 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 0.1 mg/ml) containing CPT;
4) Dropwise adding the hydrated regenerated silk fibroin obtained in the step (2) into the acetone solution dissolved with CPT obtained in the step (3) under a vortex condition, wherein the solution becomes turbid into emulsion to obtain emulsion containing CPT; the volume ratio of the hydrated TSF-M1 (or TSF-M2) protein to the acetone solution dissolved with the CPT is 1;
5) Immediately after vortexing the emulsion for 30s, it was placed in an ice bath and sonicated at 30% amplitude for 60s, 2s each, at 2s intervals.
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 without the organic solvent acetone at 13000g for 15min, discarding the supernatant, and collecting the precipitate;
8) 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 WSF-CPT-NPs, TSF-M1-CPT-NPs (or TSF-M2-CPT-NPs) nanoparticles 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 micrograph 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 all 3 nanoparticles are spherical, are densely packed, and have uniform particle size distribution, which is also a more obvious characteristic 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: the cumulative release profile of nanoparticles in vitro buffer was studied using the dialysis bag method. The WSF-CPT-NPs and TSF-M1-CPT-NPs nanoparticles (200. Mu.g/mL) obtained in example 3 were each filled into pre-treated dialysis bags (MWCO =1 400Da) and placed into 30mL centrifuge tubes containing Tween-80 (0.3%, w/v) in different release media, 50mL for further dialysis. The tube was placed in an incubator (120 rpm) at 37 ℃ and shaken. A fixed volume of release medium is collected at a predetermined point in time and replaced with a new release medium. And finally, measuring fluorescence under the excitation wavelength of 360nm and the emission wavelength of 430nm by using an enzyme-labeling instrument, and analyzing the content of CPT in the release medium. FIG. 4 shows the WSF-CPT-NPs and TSF-M1-CPT-NPs nanoparticlesControlled drug release profiles of the particles, the results showing a decrease in pH and GSH or H 2 O 2 The 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 the fact that different stimuli (acidity, GSH and H2O 2) can cause the change of the secondary structure of the silk fibroin nanoparticles is proved. 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, while 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, disulfide bonds (S — S) are very susceptible to cleavage, making disulfide bonds the necessary sites for a reduction stimulus-responsive delivery system. Thus, the targeted controlled release of anticancer drugs based on pH stimulus responsive drug delivery systems is the key to the treatment of tumors. Initially, we evaluated CPT release profiles of WSF-CPT-NPs or TSF-CPT-NPs by incubating them in buffers at different pH values (7.4, 6.8, 5.5 and 4.5) for seven consecutive days, as shown by a in FIG. 4, the drug release rates of both particles increased greatly with decreasing pH, 41.7% of CPT was released from TSF-CPT-NPS in a buffer solution at pH7.4, whereas the cumulative amount of WSF-CPT-NPs released 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 is increased with the increase of GSH concentration under the same pH (7.4) condition. After TSF-CPT-NPs are cultured in buffer solutions (0.1mM, 1mM and 10 mm) containing GSH at different concentrations for 7 days, the cumulative drug release rates of the TSF-CPT-NPs are 43.3%,59.8% and 73.7%, respectively, which are obviously higher than that of the 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 1 mM) 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 was 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-responsive ability (c in FIG. 4). Next, we investigated the synergistic effect of both stimuli by testing the drug release profile of different NPs cultured in buffer pH 5.5 with or without GSH (d in fig. 4). When the concentration of GSH in buffer solution (pH 5.5) is 10mM, the drug release rate of TSF-CPT-NPs is obviously accelerated, and the cumulative drug release rate after 7 days reaches 87.7%, which is higher than that of WSF-CPT-NPs (72.2%). 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 1 mm), the cumulative release percentage of TSF-CPT nanoparticles reached 62.1%, 86.2% and 94.7%, respectively, which was much higher than the cumulative release percentage of WSF-CPT nanoparticles at the same concentration of H2O2 (50.3%, 70.4% and 83.1%) (fig. 4, e).
Characterization experiment of particle secondary structure: and (3) characterizing the secondary structure of the particles in different release media by using a circular dichrograph (MOS-500 CD, 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 content of the beta-sheet structure of the particles. FIG. 5 is a graph representing the secondary structure of different particles, and the CD map results show that the beta-sheet content of TSF-M1-CPT-NPs is higher than that of WSF-CPT-NPs.
To further explore the release mechanism, as shown in a of fig. 5, we performed circular dichroism measurements of the secondary structure content of particles under different pH buffer conditions. It was found from the circular dichroism chart that the content of beta sheet structure of the particles showed a sharp decrease tendency with the increase of the acidity of the buffer solution, as shown in b in fig. 5, the beta sheet structure of TSF-CPT-NPs in the buffers with pH7.4, 6.8 and 5.5 was about 66.1%,60.5% and 47.2%, respectively, higher than the content of beta sheet in the corresponding WSF-CPT-NPs (56.5%, 50.3% and 40.2%). The secondary structure of silk fibroin determines the main property of drug release, and different stimuli (acidity, GSH and H2O 2) can cause the change of the secondary structure of silk fibroin nanoparticles, further explaining that the beta folding content of TSF-CPT-NPs is higher than that of WSF-CPT-NPs, which explains why the stimulation effect of TSF-CPT-NPS on drug release is more sensitive and stronger than that of WSF-CPT-NPs.
Example 6:
cytotoxicity experiments with drug-loaded particles: CT-26 cells (purchased from iCell Bioscience Inc (Shanghai, P.R. China)) were cultured at 2X 10 4 The density of cells/well was inoculated in 96-well plates and the plates were incubated overnight in an incubator at 37 ℃. After 24h, the culture medium is removed, a serum-free culture medium containing WSF-CPT-NPs or TSF-M1-CPT-NPs is used for replacing the culture medium (wherein the content of CPT is 0.1-64 mu M), the non-drug-added group is used as a negative control group, 1% (w/v) of Triton X-100 is added as a positive control group, and the negative control group and the positive control group are placed into an incubator to be cultured for 24h and 48h respectively. After the incubation is finished, the particles are washed clean by PBS containing calcium and magnesium, then 0.5mg/mL MTT is added for incubation for 4h, then the MTT is discarded, 50 mu L DMSO is added, then shaking is carried out for 15min by using a shaking table at 150rpm, and the OD value of each hole is measured at the wavelength of 570 nm. FIG. 6 shows the antitumor cell performance of WSF-CPT-NPs or TSF-M1-CPT-NPs nano-drugs after being cultured with CT-26 cells for 24h and 48h, and the results show that TSF-M1-CPT-NPs nanoparticles for colon cancer treatment have obvious inhibition effect on colon cancer CT-26 cells, show better treatment effect and are superior to WSF-CPT-NPs nanoparticles.
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.
Drug-loaded particle endocytosisAnd (3) efficiency experiment: CT-26 cells were cultured at 2X 10 5 The density of each well was inoculated into 12-well culture plates for overnight culture. In order to realize fluorescence visualization detection, drug CPT in WSF-CPT-NPs and TSF-M1-CPT-NPs nanoparticles is replaced by coumarin-6 (COU) as a model drug according to the steps in example 3, corresponding nanoparticles WSF-COU-NPs and TSF-M1-COU-NPs are prepared and respectively dispersed in serum-free culture medium to form particle suspension, and then the particle suspension is added into cells. After co-culturing with the cells for 0.5, 1 or 3h, respectively, the cells were washed 3 times with PBS solution, trypsinized, and resuspended in flow cytometer buffer. The result of a comparative experiment of the CT-26 cells on the phagocytosis efficiency of the TSF-M1-COU-NPs nanoparticles and the WSF-COU-NPs nanoparticles by analyzing the obtained cell suspension by using a flow cytometer system is shown in FIG. 7, which shows that the phagocytosis efficiency of the TSF-M1-COU-NPs nanoparticles by the CT-26 cells is remarkably higher than that of the WSF-COU-NPs nanoparticles, and the content of the alpha-helix structure of the TSF-M1-COU-NPs nanoparticles is higher, so that the phagocytosis efficiency of the cells on the TSF-M1-COU-NPs nanoparticles 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 phagocytosis rate of the cells treated by the TSF-COU-NPs is 41.5%, 63.0% and 98.7% respectively, which is obviously higher than the uptake rate of the cells treated by the WSF-COU-NPs under the same conditions (27.5%, 48.7% and 64.6%). As shown in b in FIG. 7, the mean fluorescence intensity of the TSF-COU-NPs-treated cells after 0.5, 1 and 3h incubation was 1.5, 1.3 and 1.5 times that of the WSF-COU-NPs-treated cells, respectively. The results show that compared with WSF-COU-NPs, the TSF-COU-NPs treatment group has obviously enhanced green fluorescence intensity and obviously increased phagocytosis, which is caused by the fact that TSF-COU-NPs contain more alpha-helix and beta-sheet structures.
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 (7)
1. The spider MaSps gene is characterized by comprising the following components:
the nucleotide sequence of the MaSp1 gene is shown in SEQ ID NO. 1; or
The nucleotide sequence of the MaSp2 gene is shown in SEQ ID NO. 3.
2. The protein encoded by the spider MaSps gene of claim 1, which is:
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. The use of the spider MaSps gene of claim 1 in 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. The use of the spider MaSps gene of claim 1 in the preparation of drug-loaded nanoparticles.
7. The preparation method of the drug-loaded nanoparticles is characterized by comprising the following specific steps:
(1) Firstly, the transgenic silkworm cocoon obtained by expressing the spider MaSps gene of claim 1 in the silk gland at the back of the silkworm is degummed 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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| 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 |
| 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 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105400817A (en) * | 2015-10-23 | 2016-03-16 | 浙江大学 | Method for simultaneously synthesizing and secreting black widow spider dragline silk protein 1 and protein 2 through bombyx mori |
| 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 |
| CN108290935A (en) * | 2015-08-10 | 2018-07-17 | 赛威克斯材料科学公司 | The composition and method for dragging a spider silk for manufacturing synthesis |
-
2020
- 2020-04-22 CN CN202010323570.XA patent/CN111454961B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108290935A (en) * | 2015-08-10 | 2018-07-17 | 赛威克斯材料科学公司 | The composition and method for dragging a spider silk for manufacturing synthesis |
| CN105400817A (en) * | 2015-10-23 | 2016-03-16 | 浙江大学 | Method for simultaneously synthesizing and secreting black widow spider dragline silk protein 1 and protein 2 through bombyx mori |
| 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 |
Non-Patent Citations (11)
| Title |
|---|
| Acidic Residues Control the Dimerization of the N-terminal Domain of Black Widow Spiders" Major Ampullate Spidroin 1;Joschka Bauer等;《Scientific reports.》;20160929;第1-9页 * |
| Latrodectus geometricus isolate SJ001 major ampullate spidroin 1 (MaSP1) gene, partial cds;Amaike Campen,S.等;《GenBank Database》;20200119;Accession No.MN045866.1 * |
| Latrodectus geometricus major ampullate spidroin 2 (MaSp2) gene, partial cds;Ayoub,N.A.等;《GenBank Database》;20160726;Accession No.EU177657.1 * |
| major ampullate spidroin 1 locus 1, partial;Ayoub,N.A等;《GenBank Database》;20160726;Accession No. ABY67403.1 * |
| major ampullate spidroin 2, partial [Latrodectus hesperus];Ayoub,N.A.等;《GenBank Database》;20160726;Accession No. ABY67409.1 * |
| Multiple recombining loci encode MaSp1, the primary constituent of dragline silk, in widow spiders (Latrodectus: Theridiidae);Nadia A Ayoub等;《Molecular biology and evolution》;20080228;第25卷(第2期);第277-286页 * |
| Understanding the Native Spider Silk Spinning Process Using Recombinant Silk Proteins;Stengel, Dillan.;《University of California, San Diego ProQuest Dissertations》;20211231;全文 * |
| 利用黑寡妇蜘蛛牵引丝基因培育超强度蚕丝家蚕新品种;钟伯雄等;《中国蚕学会第十二届家柞蚕遗传育种与良种繁育学术研讨会论文集》;20160701;第134页 * |
| 家蚕后部丝腺基因表达差异的分析与蚕丝性能改良的研究;尤征英;《万方》;20160127;第1-72页 * |
| 家蚕条件基因打靶技术的建立及应用研究进展;龙定沛等;《蚕业科学》;20180131(第1期);第1-15页 * |
| 蜘蛛大壶状腺丝蛋白基因的克隆和原核表达;潘红春等;《生物工程学报》;20070515;第23卷(第03期);第89-94页 * |
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