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DNA hydrogel, its preparation method and application

A technology of hydrogel and cross-linking reaction, applied in the field of DNA hydrogel, can solve the problems of high cost of X-DNA synthesis, complicated operation and high cost, and achieve the effect of improving protein expression and synthesis efficiency and reducing cost.

Active Publication Date: 2021-08-17
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

At present, 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.
(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) Applying 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; This approach resulted in 300-fold higher protein yields compared to soluble DNA templates in a wheat germ cell-free system, however X-DNA synthesis is expensive

Method used

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  • DNA hydrogel, its preparation method and application
  • DNA hydrogel, its preparation method and application
  • DNA hydrogel, its preparation method and application

Examples

Experimental program
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preparation example Construction

[0064] figure 1 It is a flowchart of the preparation method of the DNA hydrogel of the present application, which may include the following steps:

[0065] Step S11 is to provide linear DNA with modified functional groups. The functionalized group is, for example, acrylamide, sulfhydryl, amino or azido, etc., which can be modified at the 3′ end and / or 5′ end of the linear DNA. For example, linear DNA modified with a functional group can be prepared by modifying the functional group to the 5' end of the primer, followed by PCR amplification. Linear DNA modified with functional groups can be, for example, Acrydite-DNA, HS-DNA, H 2 N-DNA, N 3 - DNA etc.

[0066] Step S12 is to provide a polymer compound containing functional groups. The functionalized groups contained in the polymer compound can be selected from acrylate bonds, succinamide bonds, acrylamide bonds or mercapto groups, wherein the functionalized groups contain at least two; preferably, both ends of the polymer ...

Embodiment 1

[0091] (1) Preparation of Acrydite-DNA template:

[0092] The pIJ8660-GFP plasmid was obtained by DNA full-sequence synthesis, and PCR primers were designed according to the GFP gene. The sequence of the forward primer sfGFP-FOR is shown in SEQ ID No.1, and the sequence of the reverse primer sfGFP-REV is shown in SEQ ID No.2. And the functional group 6-methacrylamidohexylphosphate (Acrydite) was modified at the 5' end of the primer (Table 1), and the modified primer was mixed with the pIJ8660-GFP plasmid for PCR reaction to obtain the target Acrydite-DNA, the PCR product of the gene GFP, was used as a template for cell-free protein synthesis (CFPS) in this example.

[0093] Table 1

[0094]

[0095] (2) Preparation of DNA hydrogel:

[0096] Add 0.2 μL PEGDA (number average molecular weight 575) or 0.5 μL PEGDA (number average molecular weight 575), 5.7 μL Crydite-DNA (concentration 185 ng / μL), 2 μLAPS (concentration 0.01 mg / ml), 1% TEMED In the buffer solution HEPES-KOH ...

specific Embodiment 2

[0130] Specific embodiment 2: Cell-free protein expression from DNA hydrogels

[0131] (1) Preparation of HS-DNA template:

[0132] The pIJ8660-GFP plasmid was obtained by DNA full-sequence synthesis, and PCR primers were designed according to the GFP gene. The sequence of the forward primer sfGFP-FOR is shown in SEQ ID No.1, and the sequence of the reverse primer sfGFP-REV is shown in SEQ ID No.2. And modify the Thiol-Modifier C6 S-S (Table 2) of the functional group thiol at the 5' end of the primer, mix the modified primer with the pIJ8660-GFP plasmid and carry out the PCR reaction to obtain the PCR product HS-DNA of the target gene GFP, use As a template for cell-free protein synthesis (CFPS) in this example.

[0133] Table 2

[0134]

[0135] (2) Preparation of DNA hydrogel:

[0136] Add 0.2 μL PEGDA (number average molecular weight 575) or 0.5 μL PEGDA (number average molecular weight 575), 5.7 μL HS-DNA (concentration 185 ng / μL), 2 μLAPS (concentration 0.01 mg / m...

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Abstract

The application discloses a DNA hydrogel, its preparation method, and its application in protein synthesis. The DNA hydrogel is formed by cross-linking the linear DNA modified with the functional group and the polymer compound containing the functional group, which can significantly increase the protein expression level when applied to a cell-free protein synthesis system and can be reused, effectively Reduce the cost of protein synthesis.

Description

technical field [0001] The present application relates to the field of hydrogels, in particular to a DNA hydrogel, its preparation method and application. Background technique [0002] Proteins are of great scientific and practical significance as potential therapeutic biologics, drug targets, and biocatalysts. Due to the complexity of proteins, there is an increasing need to prepare and evaluate them in a simple manner. At present, 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. The 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 , convenient...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08J3/075C08L99/00C08L35/02C07K1/00
CPCC07K1/00C08J3/075C08J2399/00C08J2435/02
Inventor 杨修竹贺亮庄淼
Owner 苏州珀罗汀生物技术有限公司
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