Regulatory sequences, expression vectors and expression systems for improving cell transgenic expressions in mammals and applications thereof

A technology for regulating sequences and expression vectors, applied in the field of genetic engineering, can solve the problems of long combined promoter sequences and the need to improve the expression level of transgenes, and achieve the effect of lasting expression and improving the expression level.

Pending Publication Date: 2019-10-18
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these combined promoters are still not ideal in improving the expression level of transgenes. One is that the combined promoter sequence is longer, and the other is that the expression level of transgenes needs to be improved.

Method used

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  • Regulatory sequences, expression vectors and expression systems for improving cell transgenic expressions in mammals and applications thereof
  • Regulatory sequences, expression vectors and expression systems for improving cell transgenic expressions in mammals and applications thereof
  • Regulatory sequences, expression vectors and expression systems for improving cell transgenic expressions in mammals and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The design of embodiment 1 control element

[0037] The -34 to +48 position of the CMV promoter is its core sequence, including the TATA element and its initiator element, the sequence of which is shown in SEQ ID NO.1.

[0038]Use the website: http: / / www.cbil.upenn.edu / cgi-bin / tess / tess, http: / / trap.molgen.mpg.de / cgi–bin / trap_form.cgi to analyze the TFREs sequence of the promoter CMV, The design sequence is shown in SEQ ID NO.2, including NGEE'NE'E'NNNE'ENE' sequence (N=NFκB (GGGACTTTCC), E=E-box (CACGTG), G=GC-box (GGGGCGGGG), " '" represents the corresponding reverse sequence).

[0039] The hCMV-IE enhancer is shown in SEQ ID NO.3.

Embodiment 2p

[0061] Example 2 Construction of pIRES-regulatory sequence-HEF1-α-EGFP

[0062] The construction of expression vectors pIRES-hCPE-HEF1-α-EGFP, pIRES-SEE-HEF1-α-EGFP, pIRES-hCMVE-HEF1-α-EGFP, taking pIRES-hCPE-HEF1-α-EGFP as an example, the steps are as follows:

[0063] 1) The hCPE-HEF1-α fragment was artificially synthesized. For the convenience of cloning, MluI and EcoRI restriction sites were added to the 5' and 3' of the synthetic sequence, respectively.

[0064] 2) The digestion system of hCPE-HEF1-α sequence is: 10×M buffer 2μL, 10U / μL MluI, EcoRI enzyme 0.5μL each, 1.289μg / μL EGFP amplification product 0.78μL, make up water to 20μL. After mixing well, incubate at 37°C for 6h.

[0065] The digestion system of pIRES-HEF1-α-EGFP plasmid is: 10×M buffer 2μL, 10U / μL MluI, EcoRI enzyme 0.5μL each, 0.81μg / μL plasmid pIRES-HEF1-α-EGFP 1.23μL, make up to 20μL with water . After mixing well, incubate at 37°C for 3h.

[0066] Take the digested pIRES-HEF1-α-EGFP mother carrier ...

Embodiment 3

[0081] Example 3 Construction of the expression vector pIRES-regulatory sequence-HEF1-α-EPO containing regulatory sequences and EPO sequences

[0082] 1. Use EcoRI and BamHI to double-digest the PCR amplification product of human EPO sequence (correct sequence verified by sequencing), and simultaneously use EcoRI and BamHI to double-digest pIRES-hCPE-HEF1-α-EGFP plasmid DNA. The digestion results were identified by agarose gel electrophoresis, and the digested human EPO sequence fragment and pIRES-hCPE-HEF1-α-EGFP linear plasmid DNA were recovered from the gel.

[0083] The enzyme digestion system for human EPO sequence is: 10×M buffer 2 μL, 10 U / μL EcoRI, BamHI enzyme 0.5 μL each, 0.78 μg / μL EPO amplification product 1.39 μL, make up water to 20 μL. After mixing well, incubate at 37°C for 6h.

[0084] The digestion system of pIRES-hCPE-HEF1-α-EGFP plasmid is: 10×M buffer 2μL, 10U / μL EcoRI, BamHI enzyme 0.5μL each, 0.854μg / μL plasmid pIRES-hCPE-HEF1-α-EGFP 1.17μL, Make up to...

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Abstract

The invention relates to regulatory sequences, expression vectors and expression systems for improving cell transgenic expressions in mammals and applications thereof, and belongs to the technical field of genetic engineering. According to sequence characteristics of cytomegalovirus (CMV) promoters and enhancers, 3 kinds of regulatory sequences are invented for synthetic promoters combined with biological information and experiments, namely a human CMV promoter core sequence (hCPE), a synthetic regulatory element (SEE) and a human cytomegalovirus immediate early enhancer (hCMVE), and the threekinds of regulatory sequences are separately inserted into the upstream of a promoter of an expression vector to drive high-efficiency, continuous and stable expressions of exogenous genes. Under thesame condition, compared with expression vectors without the above-mentioned regulatory sequences, the expression vectors of the invention can obviously improve expression levels of the exogenous genes, and can be used for the production of recombination proteins, etc.

Description

technical field [0001] The invention relates to a control sequence, an expression carrier, an expression system and an application thereof for improving transgene expression of mammalian cells, and belongs to the technical field of genetic engineering. Background technique [0002] Genetic engineering is based on the theory of molecular genetics. It introduces recombinant expression vectors into recipient cells for replication, transcription and translation to produce products that meet human needs or create new biological traits, and make them stable. passed on to the next generation. Since the mammalian cell expression system is capable of correct folding and post-translational modification of recombinant proteins, the mammalian cell expression system is the preferred system for protein expression of complex recombinant proteins, especially those with complex structures or glycosylation. However, mammalian expression systems generally have low expression levels, and trans...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/67
CPCC12N15/113C12N15/85C12N15/67C12N2310/113C12N2800/107
Inventor 王天云贾岩龙王稳王小引郭潇王燕芳井长勤王芳林艳
Owner XINXIANG MEDICAL UNIV
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