Construction of stable cell line carrying orthogonal tRNA/aminoacyl tRNA synthetase

A cell line and synthetase technology, applied in genetically modified cells, retroRNA viruses, cells modified by introducing foreign genetic material, etc., can solve problems such as the bottleneck of industrial application of gene codon expansion technology

Inactive Publication Date: 2019-02-01
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 3) The bottleneck of industrial app

Method used

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  • Construction of stable cell line carrying orthogonal tRNA/aminoacyl tRNA synthetase
  • Construction of stable cell line carrying orthogonal tRNA/aminoacyl tRNA synthetase
  • Construction of stable cell line carrying orthogonal tRNA/aminoacyl tRNA synthetase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Construction and acquisition of double lentiviral vectors

[0058] 1. Obtaining the Vector Skeleton

[0059] The backbone of the double lentiviral vector is the lentiviral vector pSD31 (Zhang Jing. et al. RNA, 2007, 13: 1375-1383.), wherein the sv40 promoter promotes the puromycin resistance gene protein puro Rexpression.

[0060] 2. Primer design for SOE PCR

[0061] The inventors used SOE PCR to splicing the DNA fragments of the internal ribosome entry sequence (IRES) and the puromycin resistance gene / hygromycin B resistance gene to obtain IRES-puro and IRES- hygro fragment, the specific primers are shown in the table below.

[0062] Table 1: List of SOE PCR primers

[0063]

[0064] 3. Transformation of Lentiviral Vectors

[0065] On the basis of pSD31, the sv40 promoter and puromycin resistance gene fragments were replaced by IRES-puro and IRES-hygro fragments by BamHI and xbal double digestion, respectively, so as to obtain puromycin resistance an...

Embodiment 2

[0068] Example 2 Construction and acquisition of pXH-12tRNA-blasticidn vector

[0069] In order to ensure the expression level of tRNA, it is necessary to clone multiple copies of the tandemly expressed promoter-tRNA into a suitable vector. In the present invention, the pXH blank vector is used as the backbone, and the zeomycin-polyA sequence is introduced into the back of the SV40 promoter through the EcoRI restriction site, so that it has bleomycin resistance. After that, 12 copies of the promoter-tRNA sequence were cloned into the pXH-zeo vector using the SalI restriction site. In order to avoid the probability of recombination of the repeated sequences, 4 different tRNA promoters were used: 7sk / hu6 / H1 / mu6. Finally, the vector bjmu-12t-zeo for screening tRNA was obtained.

[0070] 1. Obtaining the Vector Skeleton

[0071] The backbone of the pXH-12tRNA-zeo vector is the vector pXH, which is a shuttle vector obtained by transforming the PUC19 vector. The pXH sequence is...

Embodiment 3

[0083] Example 3 Screening of stable cell lines

[0084] (1) Packaging and transduction of lentivirus, including the following steps:

[0085] a. HEK 293T cell plating: use medium A, components (DMEM+10% FBS, 1×NEAA, without sodium pyruvate), cell digestion count, and the number of cells seeded in each well of a six-well plate is 4×10 5 cells / well.

[0086] b. Lentiviral packaging: transfection is carried out at a cell density of 70% to 80%. The formulations of plasmids and transfection reagents are shown in Table 3-1. 6 hours after transfection, medium B (DMEM + 3% FBS, 1 x NEAA, With Sodium Pyruvate) was changed. Continue to cultivate. The virus solution was harvested at 48 hours and 72 hours after transfection, and filtered through a PVDF membrane syringe filter with a pore size of 0.45 μm.

[0087] Table 3-1. Plasmid ratio for lentiviral packaging

[0088] Plasmids / Transfection Reagents

Dosage per well

Opti-MEM

200μl

Transfer vector

...

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Abstract

The present invention relates to a method for constructing a stable cell line carrying an orthogonal tRNA/aminoacyl tRNA synthetase, and the method stably integrates an orthogonal tRNA/aminoacyl tRNAsynthetase gene into cell genomes by double lentiviral stable transduction and plasmid stable transfection. The invention further relates to the stable cell line obtained by the method and the use ofthe stable cell line in the expression of a protein containing non-natural amino acids, and by utilizing unique reactive groups on the non-natural amino acids, the purpose of high-efficiency and specific protein modification can be achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for constructing a stable cell line carrying an orthogonal tRNA / aminoacyl tRNA synthetase. Background technique [0002] 1) Gene codon expansion technology [0003] In recent years, genetic code expansion technology has developed rapidly. Using amber stop codons as sense codons, and by introducing corresponding orthogonal tRNAs and aminoacyl tRNA synthetases, the designed unnatural amino acids can finally be introduced into proteins. According to the properties of unnatural amino acids, special functions can be imparted to proteins. So far, this technology has successfully expressed hundreds of unnatural amino acids on the surface of proteins. The unnatural amino acids involved include azide, alkynyl, keto, aldehyde, alkenyl, amide, nitro , phosphate, sulfonate and other diverse functional groups, can carry out a variety of bioorthogonal reactions, such as: click chemistry, ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10A61K38/02A61P31/12A61P35/00A61P37/02
CPCA61K38/00C12N5/00C12N15/86C12N2500/32C12N2510/02C12N2740/15043C12P21/00
Inventor 周德敏王妍刘思沫徐欢王伟玲吴一鸣司龙龙陈袆周雪莹
Owner PEKING UNIV
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