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Spider MaSps gene and application of spider MaSps gene in preparation of drug-loaded nanoparticles

A gene and spider technology, which is applied to the spider MaSps gene and its application in the preparation of drug-carrying nanoparticles, can solve the problems such as the controlled release of transgenic improved silk drugs and the research report on promoting cell endocytosis, etc. Controlled release ability, improved cell internalization efficiency, and obvious therapeutic effect

Active Publication Date: 2020-07-28
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But unfortunately, so far, although there are a small number of studies on the mechanical properties of silk improved by transgenic spider MaSps genes, there have been no reports on the controlled release of silk drugs and the promotion of endocytosis in transgenic silk.

Method used

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  • Spider MaSps gene and application of spider MaSps gene in preparation of drug-loaded nanoparticles
  • Spider MaSps gene and application of spider MaSps gene in preparation of drug-loaded nanoparticles
  • Spider MaSps gene and application of spider MaSps gene in preparation of drug-loaded nanoparticles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] 1. Optimization and construction of artificially designed and modified spider MaSps gene sequence;

[0063] According to the codon preference of the endogenous gene expression sequence in the silkworm genome sequence data, the present invention refers to the black widow spider (Latrodectus hesperus) towed silk protein gene sequence to optimize the design, and Shanghai Shenggong Company synthesized the MaSp1 unit nucleotide sequence (as shown in the 1st-105th position of SEQ ID NO.1) and the MaSp2 unit nucleotide sequence (as shown in the 1st-75th position of SEQID NO.3) and were respectively constructed in the pUC57-T-Simple vector (Shanghai Sangon) became pUC-MaSp1 unit and pUC-MaSp2 unit plasmids. Then, using the XbaI restriction site upstream and the NheI restriction site downstream of the MaSps unit (MaSp1 unit or MaSp2 unit) sequence respectively, the MaSp1 unit was repeated 12 times in series with the XbaI / NheI homologous enzyme to obtain the pUC-MaSp1 plasmid , ...

Embodiment 2

[0067] 1. Preparation of silkworm with transgenic spider MaSps gene

[0068] The commercial diapause silkworm strain 302 (preserved by the State Key Laboratory of Silkworm Genome Biology, Southwest University) was used as the original material, and the parental silkworm eggs were treated with low-temperature catalysis at 16°C to relieve the diapause of the offspring silkworm eggs; 400ng / μL (w / v) mixture of recombinant vector pBac{3×P3-DsRed; Fib-H-MaSp1-LBS} and auxiliary plasmid pHA3PIG (preserved by the State Key Laboratory of Silkworm Genome Biology, Southwest University) (1: 1, v / v) Injected into 207 G0 generation silkworm eggs released from diapause, sealed with non-toxic glue and placed in an environment of 25°C and 85% relative humidity to accelerate green incubation, and hatched to obtain 84 G0 generation ants Silkworms were raised with mulberry leaves until they turned into moths, and 25 G0 generation silkworm moths were obtained, and 15 rounds of G1 generation silkwo...

Embodiment 3

[0073] The steps for preparing silk fibroin drug-loaded nanoparticles using the WC, TC-M1 and TC-M2 protein samples obtained in Example 2 above are as follows:

[0074] 1) After the transgenic silkworm cocoon TC-M1 (or TC-M2) in Example 2 is degummed with a weak alkaline solution, it is completely dissolved in a lithium bromide solution, and then dialyzed with deionized water to obtain the transgenic regenerated silk fibroin TSF-M1 (or TSF-M2);

[0075] 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;

[0076] 3) Weigh an appropriate amount of camptothecin (CPT), and completely dissolve it in acetone to obtain an acetone solution in which CPT is dissolved (concentration is 0.1 mg / ml);

[0077] 4) Add the hydrated regenerated silk fibroin obtained in step (2) dropwise to the acetone solution dissolved in CPT obtained in step (3) under vortex conditions, at this time the solution becomes...

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Abstract

The invention relates to a spider MaSps gene and an application of the spider MaSps gene in preparation of drug-loaded nanoparticles. The artificially designed and modified optimized spider MaSps geneis disclosed for the first time; a transgenic expression cassette in which fusion expression of a silk fibroin heavy chain protein (Fib-H) gene and a spider MaSps gene is regulated and controlled bya bombyx mori silk fibroin heavy chain promoter is constructed; a piggyBac transposon arm and a fluorescent selection marker gene are connected to construct a transgenic expression vector; the expression vector can be used for establishing transgenic bombyx mori capable of efficiently expressing recombinant fusion MaSps protein in bombyx mori silk glands, so that transgenic cocoon silk containingthe recombinant fusion MaSps protein in silk fibroin is obtained; and further a transgenic silk material which can stably secrete the recombinant MaSps protein in a silk fibroin layer and has the effects of promoting controlled release of drugs and endocytosis is obtained.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a spider MaSps gene and its application in preparing drug-loaded nanoparticles. Background technique [0002] Silk protein is an amphiphilic biopolymer, which possesses the main properties such as biocompatibility, biodegradability and controlled drug release necessary for an optimized drug delivery system. Silk proteins derived from spiders and insects, especially Bombyx mori silk fibroin (BmSF), have been widely used in the creation of drug-controlled release carrier materials and drug delivery research. According to reports, BmSF has been successfully made into silk fibroin microspheres with a diameter of several microns and a drug loading efficiency of up to 21%, and the drug encapsulation efficiency can be increased to 100% by weighing the particle diameter from 100 to 440 mm, but these The preparation process of BmSF particles is often highly complex, and the particle pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435A61K9/14A61K9/51A61K31/4745A61K47/42A61P35/00
CPCC07K14/43518A61K9/146A61K9/5169A61K31/4745A61K47/42A61P35/00
Inventor 肖波龙定沛张雪卿
Owner SOUTHWEST UNIVERSITY
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