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Polypeptides capable of realizing targeted editing of GS gene, and proteins thereof

A targeting and gene technology, applied in the field of genetic engineering, can solve the problems of inhibiting cell growth, cell apoptosis, affecting protein expression, etc., and achieve the effect of strong specificity, efficient target identification, and rapid screening.

Active Publication Date: 2016-06-01
CHENGDU KANGHONG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During the growth and metabolism of cells, free ammonium ions will be continuously produced. Studies have shown that too much ammonium ions will seriously inhibit cell growth, trigger cell apoptosis, and affect protein expression.

Method used

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  • Polypeptides capable of realizing targeted editing of GS gene, and proteins thereof
  • Polypeptides capable of realizing targeted editing of GS gene, and proteins thereof
  • Polypeptides capable of realizing targeted editing of GS gene, and proteins thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Target Screening Results

[0036] The applicant screened a large number of targets in the GS gene sequence of CHO cells, some of which are listed in Table 1.

[0037] Table 1. Preliminary optimized target sequence information for GS gene

[0038]

Embodiment 2

[0039] Construction and cell transfection of embodiment 2 short peptide coding sequence

[0040] According to the target information obtained in Example 1, carry out short peptide construction, and its specific construction process is as follows:

[0041] 1) Construction of plasmids containing TALE modules: commercially purchased TALE modules, a total of four single module plasmids (plasmid modules are: pMD-TALE-A (a TALE module plasmid that recognizes base A), pMD-TALE- T (TALE module plasmid that recognizes base T) pMD-TALE-C (TALE module plasmid that recognizes base C) pMD-TALE-G (TALE module plasmid that recognizes base G)), 16 double module plasmids (by Two single modules were assembled by enzyme-cut ligation). Each single module contains a repeat variable double residue (RVD) unit domain that specifically recognizes a single nucleotide base, specifically: NI recognizes A, NG recognizes T, HD recognizes C, NN recognizes G, and module domains Set SpeI site on one side, a...

Embodiment 3

[0088] Identification of embodiment 3TALEN plasmid activity

[0089] 1) Centrifuge the transfected CHO-S cells to harvest the cells, perform DNA extraction according to the Qiagen Genome Extraction Kit, and set aside.

[0090]2) Using the extracted genomic DNA as a template for PCR reaction. Among them, target 1 corresponds to the CHO-S cell genomic DNA primer pair after vector transfection is primer 1 (5'-CCATGAGATTTCATAGCTG-3'), primer 2 (5'-GCAGATGGTCAGAGAATAAC-3'), and the peptides obtained from targets 2 and 3 After the sequence vector was transfected, the genomic DNA primer pair was primer 3 (5'-GACCTTGTCATTTTTCTGGCT-3') and primer 4 (5'-TGGAATTCCCACTAGAAAGAAC-3'). Target 1 corresponds to the PCR reaction conditions of CHO-S cell genomic DNA after vector transfection:

[0091] PCR reaction system:

[0092] 10× Taq enzyme buffer 2 μl

[0093] Primer 1 (10 μM) 0.8 μl

[0094] Primer 2 (10 μM) 0.8 μl

[0095] dNTP1.6μl

[0096] DNA template 50-100ng

[0097] r-Taq e...

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Abstract

The invention discloses a pair of short peptides with nucleotide base coding sequences represented by SEQ ID NO.1 and SEQ ID NO.2 respectively. The short peptides are capable of identifying two adjacent nucleotide sequence segments on host cell GS gene with high efficiency and specificity, and realizing targeted editing of GS gene with accuracy and high efficiency. The short peptides are high in identification efficiency, specificity, and accuracy. Cell strains obtained via gene targeted modification can be applied to industrialized production, stable and high-yield monoclone rapid screening of foreign proteins can be realized with eukaryotic expression vectors, wherein GS is taken as the screening marker.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a polypeptide and protein for targeted editing of GS gene. Background technique [0002] Targeted genome editing technology refers to the targeted modification of genomic DNA to change specific genes in the genome by targeting exogenous sequences to specific sites on chromosomes, achieving the purpose of site-specific insertion of a gene or DNA element, or knockout of a specific gene sequence , in order to more accurately and deeply understand the pathogenesis of diseases and explore gene functions. Early targeted genome editing technology mainly relied on gene homologous recombination (gene targeting) and somatic cell nuclear transfer technology to complete the transformation of specific genes. The main technical means in this period include: gene recombination technology (Recombineering), Nucleotide-mediated gene repair (single-stranded oligonucleotides), the breakthrough of...

Claims

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Application Information

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IPC IPC(8): C07K14/00C07K19/00C12N15/11C12N15/62C12N15/85
Inventor 柯潇雷刚高小平
Owner CHENGDU KANGHONG BIOTECH
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