A pair of short peptides, proteins and polynucleotides, host cells and applications thereof

A polynucleotide, host cell technology, applied in the field of genetic engineering, can solve the problem of human IL2RG gene targeting and other problems

Inactive Publication Date: 2013-07-03
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] However, there is no report on targeting the human IL2RG gene at present, so there is an urgent need in this field to carry out research on the efficient targeting of the human IL2RG gene

Method used

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  • A pair of short peptides, proteins and polynucleotides, host cells and applications thereof
  • A pair of short peptides, proteins and polynucleotides, host cells and applications thereof
  • A pair of short peptides, proteins and polynucleotides, host cells and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] The design of embodiment 1 TALENs module sequence

[0072] 1. NCBI downloaded the genome sequence of human IL2RG (No. NC_000023.10), and selected exon2 as the target.

[0073] 2. Design primers and PCR amplify the target site fragments on the genome, and sequence them.

[0074] The design of PCR primers and sequencing primers is shown in Table 1

[0075]

[0076] Table 1

[0077] 3. Design TALENs recognition sequence

[0078] Determine the TALENs recognition sequence based on the sequence obtained by sequencing and the following principles

[0079] 1) The base at position 0 is T (referring to the base preceding the first position of the recognition sequence)

[0080] 2) The last base is T

[0081] 3) The length of the recognition sequence is between 14-19

[0082] 4) The length of the spacer sequence between the two recognition sequences is controlled between 12-21, preferably between 14-21, and the position of the designed target sequence is as follows fi...

Embodiment 2

[0085] Example 2 Connection between TALENs recognition modules and construction of recombinant vector

[0086] 1. Obtaining the identification module

[0087] Synthesis of four recognition modules NI, NG, HD, and NK sequences (SEQ ID NO: 14-17) that recognize bases A, T, C, and G are shown in Table 3

[0088]

[0089] table 3

[0090] ●Connect the four fragments into the pEASY-B vector (purchased from Beijing Quanshijin Company), the connection method is as follows:

[0091] 1) Take 3 μl of PCR primers

[0092] 2) Add 1μl pEASY-B vector

[0093] 3) 25°C, 7min

[0094] 4) Transfect DH5α competent cells, spread kanamycin plate

[0095] 5) Pick clones, extract plasmids in small quantities, digest, and sequence

[0096] Finally, the recognition templates NI, NG, HD and NK connected to the vector pEASY-B were obtained

[0097] 2. Identify connections between modules

[0098] ●Connection strategy

[0099] Take the connection of 19 recognition modules as an example to ...

Embodiment 3

[0172] The transfection of embodiment 3 plasmids

[0173] 1. Add 300 μl of gelatin to each space of the 6-well plate, shake it back and forth, so that the gelatin covers the bottom of the entire well, and place it in 5% CO 2 10 minutes in the incubator.

[0174] 2. Aspirate the culture medium in the T25 bottle of cultured 293T cells, wash it once with PBS, add 1ml of 0.25% trypsin, shake back and forth to make it evenly cover the bottom of the bottle, and place in 5% CO 2 5 minutes in the incubator.

[0175] ●After digestion, add 1ml 10% DMEM to neutralize trypsin, transfer the digested cells to a 15ml centrifuge tube, count the cells, and centrifuge at 1200rpm for 5 minutes.

[0176] ●Use an appropriate amount of 10% DMEM to resuspend the cells, take 2 million 293T cells and place them in a 6-well plate covered with gelatin, and add 2ml of fresh 10% DMEM.

[0177] ●Transfect when the density of 293T cells reaches 80-90%, no need to change the medium.

[0178] ●The const...

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Abstract

The present invention provides a pair of short peptides, proteins and polynucleotides, a host cell and application thereof, and provides propose an artificial synthetic method for transcriptional activator-like effector nuclease (TALEN) gene of human cell endogenous IL2RG gene efficient targeting, and a method for orientation targeting by using a recombinant plasmid containing the gene. The TALEN comprises a pair of transcription activator-like effector (TALEs) proteins and DNA incision enzyme catalytic subunit respectively fused with the proteins, and can respectively recognize two adjacent sites on human IL2RG gene exon 2. On the basis of designing a TALEs amino acid sequence, a nucleotide sequence encoding the TALEN is synthesized and a vector containing the nucleotide sequence is constructed. By using the TALEN plasmid to transfect cells, the efficiency of cell targeting can be greatly improved.

Description

technical field [0001] This patent relates to the field of genetic engineering, in particular to a synthesis and targeting method of a transcriptional activator-like effector nuclease (TALEN) that can efficiently target the human IL2RG gene. Background technique [0002] IL2RG, also known as interleukin-2 receptor subunit gamma, is a subunit of cytokine receptors and is a complex of at least six interleukin receptors (IL-2, IL-4, IL-7, component of IL-9, IL-15 and IL-21). The IL2RG gene is generally located on the X chromosome of mammals. [0003] Mutations in the IL2RG gene cause X-linked severe combined immunodeficiency disease (X-SCID). More than 200 different IL2RG gene mutations have been identified in the genomes of human X-linked severe combined immunodeficiency disease (X-SCID) patients, and most of these mutations involve changes in one or several DNA bases. After IL2RG gene mutation, related interleukin receptors lose their function, important signaling molecule...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/55C12N15/63C12N5/10A61K38/46A61K48/00A61P37/00
Inventor 肖磊赵金龙
Owner ZHEJIANG UNIV
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