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Bicistronic mRNA (messenger ribonucleic acid) expression vector suitable for cells of mammals and application thereof

An expression vector and mammalian technology, which is applied in the field of high-efficiency expression vectors for mammalian cells, can solve the problems of high cost and low efficiency of exogenous protein expression in animal cells, and achieve the effects of improving stability, keeping upright and timely termination, and improving production efficiency

Active Publication Date: 2012-03-28
优锐生物医药科技(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression efficiency of exogenous protein in animal cells is low and the cost is high. Therefore, it is urgent to improve the expression efficiency of exogenous protein in animal cells and reduce production costs.

Method used

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  • Bicistronic mRNA (messenger ribonucleic acid) expression vector suitable for cells of mammals and application thereof
  • Bicistronic mRNA (messenger ribonucleic acid) expression vector suitable for cells of mammals and application thereof
  • Bicistronic mRNA (messenger ribonucleic acid) expression vector suitable for cells of mammals and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The amplification of embodiment 1pCMV-intron-L-BGH PA fragment

[0035] (1) PCR amplification of pCMV promoter and intron

[0036] Plasmid pCI-neo is a eukaryotic expression plasmid vector, using the pCI-neo plasmid as a template, and taking the cytomegalovirus (CMV) early promoter / enhancer and intron sequence (SEQ ID NO: 1) as a reference, the design contains SacI PCR reaction with primers P1 / P2 with restriction sites or NheI restriction sites to amplify the early promoter / enhancer and intron sequences of cytomegalovirus (CMV) with restriction sites at both ends, PCR reaction The conditions are shown in Table 1.

[0037] Upstream primer (P1): 5′-GCG GAGCTCGGGCTATTGGCCATTGCATACGC-3' (the underline is the SacI restriction site) (SEQ ID NO: 7)

[0038] Downstream primer (P2):

[0039] 5′-TGAACTGGGAGTGGACACCTGTGGAGAGAAAGGCAAG CTAGC GC-3' (the underline is the NheI restriction site) (SEQ ID NO: 8)

[0040] Establish the following reaction system in a 100μl thin-wall...

Embodiment 2

[0069] Embodiment 2 Amplification of pCMV-H-BGHA-SV40 fragment

[0070] (1) PCR amplification of pCMV promoter and intron

[0071] Using the pCI-neo plasmid as a template, and taking the cytomegalovirus (CMV) early promoter / enhancer and intron sequence (SEQ ID NO: 1) as a reference, design primers containing an xhoI restriction site or an EcoRI restriction site P7 / P8 carry out PCR reaction, amplify cytomegalovirus (CMV) early promoter / enhancer and intron sequence with restriction site at both ends, PCR reaction conditions are shown in Table 1.

[0072] Upstream primer (P7): 5′-GCG CTCGAG GGGCTATTGGCCATTGCATACGC-3' (the underline is the xhoI restriction site) (SEQ ID NO: 13)

[0073] Downstream primer (P8): 5′-CTGGGAGTGGACATGGAGAGAAAGGCAAAGTGG GAATTC CGC-3' (the underline is the EcoRI restriction site) (SEQ ID NO: 14)

[0074] Establish the following reaction system in a 100μl thin-walled centrifuge tube:

[0075]

[0076]

[0077] The PCR product was subjected to ...

Embodiment 3

[0101] The amplification of embodiment 3DHFR-SV40PA fragment

[0102] (1) Amplification of DHFR fragments

[0103] Total cellular RNA was extracted from CHO-K1 cells subcultured conventionally, and reverse-transcribed into cDNA as a template. Using the DHFR sequence (SEQ ID NO: 23) as a reference, primers P13 / P14 were designed for PCR reaction, and one end containing SfiI was amplified. See Table 1 for the DHFR fragment at the restriction site and the PCR reaction conditions.

[0104] Upstream primer (P13): 5'GC GGGCCATCGAGGCC GCCACCATGGTTCGACCATTG-3' (the underline is the SfiI restriction site) (SEQ ID NO: 19)

[0105] Downstream primer (P14): 5'-CATCAATGTATCTTATTCAGCATCTTCCTGTTAGTC-3' (SEQ ID NO: 20)

[0106] Create the following reaction system:

[0107]

[0108]

[0109] The PCR results were subjected to agarose gel electrophoresis. The size of the PCR product was consistent with the expected base pair size, and the amplified fragments were recovered with a DNA ...

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Abstract

The invention discloses a bicistronic mRNA (messenger ribonucleic acid) expression vector which is suitable for cells of mammals and can realize high-efficient expression of exogenous genes and an application thereof. The bicistronic mRNA expression vector comprises connecting elements in the 5'-3' direction, namely (1) an open reading frame 1: containing the sequences of an early promoter / enhancer and an introne of cytomegalovirus, multiple clone sites and the sequence of a bovine growth hormone transcription terminator, which are sequentially arranged; (2) an open reading frame 2: containing the sequences of the early promoter / enhancer and the introne of the cytomegalovirus, the multiple clone sites and the sequences of the bovine growth hormone transcription terminator and a simian virus promoter, which are sequentially arranged; and (3) screening marker genes and the sequence of a simian virus 40 (SV40) terminator. The bicistronic mRNA expression vector has the high-efficient promoter, effective transcription termination signals, screening and gene amplification markers and two open reading frames, and can improve the expression efficiency of the exogenous genes and reduce the production cost.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a high-efficiency expression vector for mammalian cells. Background technique [0002] According to the type of host cell, gene expression systems can be roughly divided into prokaryotic, yeast, plant, insect and mammalian cell expression systems. Compared with other systems, the mammalian cell expression system has the advantage of being able to guide the correct folding of proteins, and provide various post-translational processing functions such as complex N-type glycosylation and accurate O-type glycosylation. The structure, physical and chemical properties and biological functions are closest to natural higher biological protein molecules. In the past 30 years since the direct transfection of naked DNA into mammalian cells, the mammalian cell expression system has not only become a production platform for various genetic engineering drugs, but also plays an important role in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85
Inventor 倪健梁辉
Owner 优锐生物医药科技(深圳)有限公司
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