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SgRNA for lowering immunogenicity, low-immunogenicity dressing and preparation method thereof

An immunogenicity and carrier technology, which is applied in the field of low immunogenicity biological dressings and its preparation, can solve the problems of high immunogenicity, reduction of immunogenicity, multiple dressing changes and scab removal, and achieve low immunogenicity, The effect of risk reduction

Pending Publication Date: 2018-10-12
武汉博杰生物医学科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the above deficiencies or improvement needs of the prior art, the present invention provides a dressing with reduced immunogenicity, a low immunogenicity dressing and a preparation method thereof, the purpose of which is to reduce the expression of immunogenic genes or silence immunogenicity by collecting Genetic pigskin is used as a dressing, thereby solving the technical problems of existing pigskin dressings with high immunogenicity, requiring multiple dressing changes to cut scabs and skin grafting

Method used

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  • SgRNA for lowering immunogenicity, low-immunogenicity dressing and preparation method thereof
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  • SgRNA for lowering immunogenicity, low-immunogenicity dressing and preparation method thereof

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preparation example Construction

[0058] The preparation method of the dressing with low immunogenicity provided by the invention is characterized in that, comprising the following steps:

[0059] (1) Preparation of transfection vector: add RNA sequence TAGG to the 5' end of the sense strand template of the sgRNA target sequence provided by the present invention, add CAAA to the 5' end of the antisense strand template, complement the cohesive end of the vector plasmid, and synthesize Double strands formed by annealing single strands of oligonucleotide chains; The CRISPR / Cas backbone plasmid was digested with restriction endonuclease BbsⅠ, and the digested plasmid was purified; the linearized plasmid was ligated with sgRNA oligonucleotide double strands, transformed, sequenced, and extracted endotoxin-free plasmid for Cell transfection. Connect the linearized transfection vector CRISPR / Cas backbone plasmid and the in vitro transcription CRISPR / Cas backbone plasmid to the double strands respectively to obtain t...

Embodiment 1

[0071] Example 1. Design and screening of GGTA1 gene sgRNA and preparation of CRISPR / Cas9 system vector

[0072] 1. Design of sgRNA

[0073] For the GGTA1 gene, the principles of sgRNA design are as follows:

[0074] (1) The length is about 20nt;

[0075] (2) sgRNA containing GG at the 3' end, with a GC content of 40%-60%;

[0076] (3) There are no SNPs in the genome sequence of the sgRNA targeting binding site;

[0077] (4) Analysis of gene-wide off-target effects, a maximum of 5 base mismatches are allowed at off-target sites.

[0078] Based on the above design principles, the target sequence set shown in Table 1 was designed.

[0079] The sequence of the sgRNA designed in Table 1 (the gray mark is the PAM sequence)

[0080]

[0081] 2. Screening of sgRNA and preparation of in vitro transcription vector

[0082] According to the above principles, 6 suitable sgRNAs were selected, and the gRNA target efficiency was further detected by in vitro enzyme digestion activit...

Embodiment 2

[0124] Embodiment two, transfection adult fibroblast

[0125] 1. Cell transfection and screening

[0126] The U6-gRNA-3 plasmid was electroporated to transfect porcine adult fibroblasts. Follow the specific steps below:

[0127] ①Observe the growth status of the cells under a microscope, select cells that grow well and have a confluence of about 80%, digest with 0.25% trypsin for 2-3min, centrifuge at 1500g for 5min, and discard the supernatant;

[0128] ②Suspend the cell pellet in 500uL DPBS buffer, centrifuge at 1500g for 5min, and thoroughly wash away the residual trypsin in the cells;

[0129] ③ Add a certain volume of electroporation buffer to suspend the above cells, divide the electroporation solution into each group of centrifuge tubes according to the electroporation system size of 50uL, and add a certain mass of corresponding electroporation plasmid to it, and mix the cell suspension with a pipette gun;

[0130] ④ Turn on the ECM2001Elector Cell ManipuLator, and s...

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Abstract

The invention discloses a SgRNA for lowering immunogenicity, a low-immunogenicity dressing and a preparation method thereof. The sgRNA for lowering immunogenicity has a length from 18nt and 22nt; the3' end of the target sequence on the GGTA1 gene of the sgRNA contains GG, and the GC content is 40%-60%; The sgRNA does not have SNPs in the genome sequence of the target combination site; the sgRNA is compared with a pig GGTA1 gene to carry out the whole gene off-target effect analysis, and maximum 5 base mismatches are allowed in the off-target site. The low-immunogenicity dressing is pig skin,the pig skin is derived from a GGTA1 gene knockout pig. The sgRNA for lowering immunogenicity and the low-immunogenicity dressing have the advantages of low immunogenicity, even no immunogenicity, canbe used as a human skin substitute and reduce the risk of complications.

Description

technical field [0001] The invention belongs to the technical field of biological materials, and more specifically relates to a biological dressing with low immunogenicity and a preparation method thereof. Background technique [0002] In the treatment of severe deep burns and skin defects, wound dressings are needed to promote rapid wound healing. As a new type of wound dressing, biological wound dressings are gaining momentum in the market, mainly divided into animal skin dressings (such as autologous skin, allogeneic skin, heterogeneous skin, etc.) and non-animal skin dressings (such as collagen, chitosan, honey bio dressings, etc.). Autologous skin is the best in curative effect, but the source of skin is extremely limited. The permeability and adhesion of allogeneic skin are similar to those of burn patients, which can effectively reduce antigenicity and rejection. However, it mainly comes from corpse skin, which has extremely limited sources, high price, and extreme...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85A01K67/027A61L27/36A61L27/60
CPCA01K67/0276A01K2217/075A01K2227/108A61L27/362A61L27/3687A61L27/60A61L2430/40C12N15/113C12N15/8509C12N2310/10C12N2510/00C12N2800/80C12N2810/10
Inventor 姚杰毕延震代佳丽
Owner 武汉博杰生物医学科技有限公司
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