Intelligent hydrogel formed from single protein

A hydrogel and intelligent technology, which is applied in the direction of resisting vector-borne diseases, non-active ingredients of polymer compounds, prostheses, etc., to achieve the effects of sensitive temperature response, avoiding use and residue, and simple and mature preparation process

Active Publication Date: 2020-10-16
HUAANTANG BIOTECH GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no smart thermosensitive hydrogels formed from proteins with biocatalytic activity have been reported so far.

Method used

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  • Intelligent hydrogel formed from single protein
  • Intelligent hydrogel formed from single protein
  • Intelligent hydrogel formed from single protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Construction of genetically engineered bacteria

[0050] In this example, an expression vector of the target protein was constructed by cloning the target gene shown in SEQ ID No. 2, and the expression vector was introduced into E. coli to realize expression production. The specific operation of this embodiment includes: obtaining the target gene sequence from NCBI, inserting the NcoI restriction enzyme site at the N end of the target sequence, and inserting the XhoI restriction enzyme site at the C end to perform in vitro gene synthesis. Using two restriction enzyme sites, NcoI and XhoI, it is connected to the pET28a expression vector by means of restriction enzyme linkage. Then, the successfully constructed pET28a-LplA expression vector was transformed into Escherichia coli BL21 (DE3) cells to construct recombinant Escherichia coli engineering bacteria.

Embodiment 2

[0051] Example 2 Fermentation of genetically engineered bacteria

[0052] The successfully constructed Escherichia coli engineering bacteria were cultured in 4mL LB medium containing kanamycin (50μg / mL) at 37℃, 220r / min to OD 600 When it is 1.8 to 2.0, prepare seed liquid. The seed solution was transferred to 200mL LB medium containing kanamycin (50μg / mL) with 1% inoculum, and cultivated to OD at 37℃, 220r / min 600 At 0.7, add 0.2mM IPTG at 30℃ to induce the expression of the target protein, and collect the bacteria after 12h.

[0053] The components of the LB medium are: tryptone 10g / L, sodium chloride 10g / L and yeast powder 5g / L.

Embodiment 3

[0054] Example 3 Preparation of LPLA protein

[0055] The preparation method of LPLA protein is as follows:

[0056] (1) After resuspending the collected bacteria with 10 mL of Tris-HCl (50 mM, pH 7.5) buffer solution, use a homogenizer to disrupt the cells;

[0057] (2) Centrifuge the cell lysis solution at 10000×g for 30 min. After centrifugation, the supernatant was filtered through a 0.45μM filter membrane, and then loaded on a column filled with Ni-Sepharose, and used Buffer containing different imidazole concentrations. Gradient elution

[0058] a. Buffer A and Buffer B wash away non-specifically bound proteins;

[0059] b. Use Buffer C to elute the target protein;

[0060] (3) Collect the target protein, and use an ultrafiltration tube with a cut-off volume of 30kDa to concentrate the volume of the target protein solution to less than 1 mL;

[0061] (4) Add Tris-HCl (50mM, pH 7.5) buffer solution in batches, change the target protein solution system to 50mM, pH 7.5 Tris-HCl buffer...

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Abstract

The invention belongs to the field of biological materials, and particularly relates to intelligent hydrogel formed by single protein and having catalytic activity. The intelligent hydrogel is formedby diluting lipoic acid ligase protein (LplA) from escherichia coli into a buffer solution and controlling physical and chemical conditions of the system. The intelligent hydrogel has the advantages that the temperature sensitivity and reversibility are realized, the critical temperature range is 8-80 DEG C, hydrogel is formed when the critical temperature is higher than the critical temperature,the solution state is recovered when the temperature is reduced, the reversible conversion process can be repeated for more than 30 times, and meanwhile, the temperature response is sensitive, the conversion temperature range is within 2 DEG C, and the temperature response time is short (within 3 minutes). The hydrogel can also control the gel state by adjusting the pH and the ion concentration. The intelligent hydrogel can be widely applied to the fields of tissue engineering and medical cosmetology such as three-dimensional cell culture, drug sustained release, targeted therapy drug preparation, beauty skin care products and temperature sensing.

Description

Technical field: [0001] The present invention belongs to the field of biological materials. Specifically, the present invention relates to a smart hydrogel with catalytic activity formed by a single protein. Background technique: [0002] Hydrogel is a dispersion system in which the dispersion medium in the voids of a three-dimensional network structure is water. Smart hydrogel refers to a type of special hydrogel whose shape changes between solution and gel with changes in environmental factors such as temperature, solution characteristics, ion concentration and pH value. Generally speaking, smart hydrogels will undergo corresponding changes based on external physical or chemical stimuli. [0003] Among the many smart hydrogels, because the temperature-sensitive smart hydrogel does not introduce other substances, it only needs temperature changes, is easy to operate, and is environmentally friendly, which has attracted much attention. Smart thermosensitive hydrogel is a special ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08J3/075C08L89/00A61K8/04A61K8/64A61K9/06A61K47/42A61L27/22A61L27/52
CPCC08J3/075A61K9/06A61K47/42A61K8/042A61K8/64A61L27/52A61L27/227C08J2389/00Y02A50/30
Inventor 曾安平张心怡任杰聂晶磊
Owner HUAANTANG BIOTECH GRP CO LTD
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