Preparation and application of ptc stable cell line using optimized gene codon extension system

A codon and translation system technology, applied in the field of biopharmaceuticals, can solve problems such as high cost, cumbersome construction process, and low virus rescue efficiency

Active Publication Date: 2021-10-01
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Firstly, since the transcription and processing of tRNA are different from those of proteins, how to achieve efficient and stable expression of orthogonal prokaryotic tRNA in eukaryotic cells is still an international problem; secondly, according to the traditional method, stable expression of three different exogenous The engineering cells of genetic elements need to go through three rounds of gene transfection or virus transduction and the corresponding screening process of three different antibiotics. Due to the poor state of the cells under the pressure of multiple antibiotics, it is difficult to survive, and the antibiotics used for simultaneous screening are expensive. , resulting in cumbersome cell line construction process, low success rate and high cost; currently, the engineered cells that stably integ...

Method used

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  • Preparation and application of ptc stable cell line using optimized gene codon extension system
  • Preparation and application of ptc stable cell line using optimized gene codon extension system
  • Preparation and application of ptc stable cell line using optimized gene codon extension system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] Example 1: Construction and acquisition of Tol2-RS-IRES-*GFP-12tRNA-puro transposable vector

[0128] In order to ensure the expression of bio-orthogonal tRNA and aminoacyl-tRNA synthetase, the screening efficiency of positive cells and the integration efficiency of genes, it is necessary to combine multiple copies of promoter-tRNA, pylRS and single-point mutation GFP reporter gene in tandem Cloned into a transposable vector.

[0129] Therefore, the inventor has designed such as figure 1 In the Tol2-RS-IRES-*GFP-12tRNA-puro transposon vector shown in the middle left panel, the aminoacyl tRNA synthetase initiated by the CAG promoter was first introduced into the Tol2-puro transposon vector, and the internal ribosome entry site was used to The mutant GFP reporter gene with premature stop codon was introduced by dot connection; at the same time, 12 copies of the tRNA promoted by the PolIII promoter were designed, and the multiple copies of the tRNA fragments in series ...

Embodiment 2

[0130] Example 2: Screening of Vero-Tol2-NAEK stable cell lines

[0131] (1) Preparation of cell suspension:

[0132] Vero cells (ATCC, CCL-81) were cultivated to a confluence of 70 with complete medium (MEM, Gibco, 11095080; 10% fetal bovine serum, PAN, P30-3302; 1% penicillin / streptomycin, Macgene, CC004). When % to 80%, the cells were digested and collected, rinsed three times with Opti-MEM (Gibco, 31985070) to wash away the antibiotics and serum in the medium, then resuspended with Opti-MEM, and the cells were blown up and down to make them free of clumping. Take a small amount of suspension for cell count and calculate the total cell number.

[0133] (2) Prepare the mixture of cells and plasmids:

[0134] Mix a certain amount of cells with plasmid DNA, and mix thoroughly to make the final concentration reach 1×10 in each tube of 100 μl mixture. 6 Cells and 10 μg plasmid DNA (the ratio of transposable vector to transposase plasmid DNA is 3:2), in which the cell volume...

Embodiment 3

[0143] Embodiment 3: Identification of Vero-Tol2-ncAA stable cell line (taking Vero-Tol2-NAEK as an example)

[0144] The stable cell line Vero-Tol2-NAEK constructed in the present invention contains tRNA derived from Methanosarcina pasteurii and pyrrolysyl-tRNA synthetase (MbPylRS), in protein expression in stable cell lines, with premature stop codons (including TAG, TAA, and TGA) as sense codons, capable of incorporation of the unnatural amino acid NAEK into protein. Next, the inventors examined the incorporation possibility of NAEK and the production performance of the mutant protein.

[0145] (1) Synthesis and identification of unnatural amino acid NAEK:

[0146] The chemical synthesis reaction formula of unnatural amino acid NAEK is as follows:

[0147]

[0148] As described in the above formula, 2.3 mL of raw material 1 (2-bromoethanol) was dissolved in a mixed solution of 90 mL of acetone and 15 mL of water, 3.12 g of NaN3 was added, and the mixture was heated ...

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Abstract

The invention belongs to the field of biopharmaceuticals, and in particular relates to a preparation method and application of a PTC stable cell line. Based on gene codon extension/PTC extension technology, use orthogonal unnatural amino acid (UAA) and tRNA/aminoacyl tRNA synthetase to read through premature stop codon (PTC, UAG/UAA/UGA), and introduce non- Natural amino acids. The invention further relates to the use of the stable cell lines, eg for packaging replication deficient (PTC) virus vaccines.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a stable cell line with premature termination codons (Premature termination codons, PTC), its preparation method and its application. The PTC stable cell line of the present invention can be based on gene codon expansion technology, utilizes orthogonal unnatural amino acids (non-canonical Amino Acids, ncAAs) and tRNA / aminoacyl tRNA synthetase to read through the premature stop codon (comprising TAG, TAA and TGA), and site-specific introduction of unnatural amino acids into proteins. The present invention further relates to the use of PTC stable cell lines, such as packaging replication-deficient virus vaccines and the like. Background technique [0002] PTC Technology and Its Application Bottleneck [0003] After several years of research, people have a more comprehensive understanding of the translation mechanism of prokaryotic ribosomes. The crystal and electron...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12N7/01A61K39/125A61P31/14
CPCA61K35/76A61K39/12A61K2039/5254A61P31/14C12N7/00C12N15/85C12N2770/32321C12N2770/32334
Inventor 夏青杨琦王宇
Owner PEKING UNIV
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