DNA composite hydrogel and preparation method thereof

A technology of composite hydrogel and polymer compound, applied in the field of DNA composite hydrogel and its preparation, can solve the problems of limited reuse of hydrogel template, high cost of X-DNA synthesis, limited types of proteins, etc. The effect of protein expression cost, excellent mechanical properties and low price

Pending Publication Date: 2021-04-09
苏州珀罗汀生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Most of the existing protein preparation methods use cells as hosts to synthesize in living cells, but they have many disadvantages: cumbersome operations, long cycle times, and extremely limited types of synthesized proteins, etc.
(2) Using a strain that has knocked out the gene encoding endonuclease E (RNase E) to prepare cell extracts delayed the degradation of mRNA molecules, and the protein levels expressed from LETs amplified by PCR became comparable to conventional plasmid-based methods. The response is comparable, but the problem is that the strain grows slowly and takes time
(3) By designing special primers, DNA linear fragments are formed into circular DNA molecules similar to plasmids, which improves the resistance of target gene fragments to exonucleases, but is expensive
(4) Luo's research group applied the DNA hydrogel system to CFPS, mainly linking linear template DNA molecules with X-shaped DNA (X-DNA) to generate DNA hydrogels for combined cell-free transcription and translation systems. Glue; in a wheat germ cell-free system, this method resulted in a 300-fold increase in protein yield compared to soluble DNA templates, however X-DNA synthesis is expensive
Although the clay hydrogel is cheap, it has thixotropy and is easily broken during the shaking process, which limits the reuse of the hydrogel template.

Method used

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  • DNA composite hydrogel and preparation method thereof
  • DNA composite hydrogel and preparation method thereof
  • DNA composite hydrogel and preparation method thereof

Examples

Experimental program
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preparation example Construction

[0040] In the preparation of the DNA composite hydrogel, a clay solution is added, and the solution can be connected with a polymer compound containing a functional group through hydrogen bonding and coordination, which helps to improve the mechanical properties of the DNA composite hydrogel .

[0041] In the polymer compound containing a functionalized group, the functionalized group can be selected from at least one of an acrylate bond, an amide bond, a maleimide bond, a vinylsulfone group, an amino group, an azido group or a mercapto group or a combination of several.

[0042] The backbone of the polymer compound containing functional groups can be polyethylene glycol or polypeptide; preferably polyethylene glycol backbone, which has good biocompatibility, low cytotoxicity and low price.

[0043] Polymer compounds containing functional groups can be linear or multi-arm polyethylene glycol maleimide, linear or multi-arm polyethylene glycol vinyl sulfone, linear or multi-arm...

Embodiment 1

[0051] Example 1 Preparation of DNA Composite Hydrogel with PCR Product as DNA Template

[0052] 1. Preparation of thiol-modified DNA (HS-DNA) template

[0053] The pIJ8660-GFP plasmid was obtained by DNA full-sequence synthesis, PCR primers were designed according to the GFP gene, and Thiol-Modifier C6 S-S (Table 1) with functional group sulfhydryl was modified at the primer 5' end, and the modified primers were combined with pIJ8660-GFP The plasmids were mixed for PCR reaction to obtain the PCR product HS-DNA of the target gene GFP, which was used as a template for cell-free protein synthesis (CFPS) in this embodiment.

[0054] Table 1

[0055]

[0056] 2. Preparation of DNA composite hydrogels

[0057] (1) Configuration of solution 1: dissolve the clay with pure water, and configure it into a clay solution with a mass fraction of 4%;

[0058] (2) Configuration of solution 2: add four-arm polyethylene glycol mercapto (number average molecular weight: 20,000) to the abo...

Embodiment 2

[0072] Example 2 Preparation of DNA Composite Hydrogel Using Plasmid as DNA Template

[0073] 1. Preparation of DNA composite hydrogel

[0074] (1) Configuration of solution 1: dissolve the clay with pure water, and configure it into a clay solution with a mass fraction of 4%;

[0075] (2) Configuration of solution 2: add four-arm polyethylene glycol mercapto (number average molecular weight: 20,000) to the above solution 1, and mix evenly, wherein the mass fraction of four-arm polyethylene glycol mercapto is 15%;

[0076] (3) Configuration of solution 3: dissolve polyethylene glycol diacrylate (number-average molecular weight: 575) with 2x PBS (pH=7), and configure a polyethylene glycol diacrylate solution with a mass fraction of 1%;

[0077] (4) Configuration of solution 4: add pIJ8660-GFP plasmid to the above solution 3, mix well, and the final concentration of pIJ8660-GFP plasmid is 200ng / μL;

[0078] (5) Take 5 μL each of the above solution 2 and solution 4, mix them ev...

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Abstract

The invention relates to the technical field of biology, in particular to DNA composite hydrogel and a preparation method thereof. The preparation method comprises the following steps of uniformly mixing 0.5-4% by mass of clay, 0.2-30% by mass of a functional group-containing high-molecular compound and 10-400 ng/mu L of a DNA template at a relatively low temperature, and polymerizing at a relatively high temperature to form the DNA composite hydrogel. The DNA composite hydrogel provided by the invention can protect the DNA template, and can also improve the local effective concentration of the DNA template in acellular protein expression, thereby improving the protein yield. Meanwhile, the hydrogel can be repeatedly used for multiple times, so that the reaction cost is further reduced.

Description

technical field [0001] The application relates to the field of biotechnology, in particular to a DNA composite hydrogel for cell-free protein expression and a preparation method thereof. Background technique [0002] Most of the existing protein preparation methods use cells as hosts to synthesize in living cells, but they have many disadvantages: cumbersome operations, long cycle times, and extremely limited types of proteins to be synthesized. Cell-free protein synthesis (Cell-free Protein Synthesis, CFPS) system is an in vitro gene expression system that uses exogenous DNA or mRNA as a template, artificially adds the required raw materials and energy substances, and synthesizes proteins under the condition of cell extracts, which can break through the limitations of cells. Express various proteins conveniently and quickly. In detail, CFPS uses DNA as a template to transcribe corresponding mRNA under the action of RNA polymerase and transcription factors; using mRNA as a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/10C12Q2527/125
Inventor 杨修竹贺亮庄淼杜静张忆恒其他发明人请求不公开姓名
Owner 苏州珀罗汀生物技术有限公司
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