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Use of chick beta actin gene intron-1

An actin and intron technology, applied in the introduction of foreign genetic material, virus/phage, organic chemistry, etc. using vectors, can solve problems such as insufficient strength

Active Publication Date: 2011-07-13
QINGDAO HUINUODE BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the promoters used in conventional expression vectors are not robust enough for high levels of gene expression in these fast and high density growing cell lines
Additionally, there are not many vectors that are universally available for most types of cell lines

Method used

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  • Use of chick beta actin gene intron-1
  • Use of chick beta actin gene intron-1
  • Use of chick beta actin gene intron-1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Sequencing of the 5'flanking region of chicken β-actin gene

[0068] The 5'flanking region of the chicken beta actin gene was from Dr. N Fregien (ATCC 37507) (Fregien N and Davidson N, 1986) and was sequenced by commercial service provider Laragen Inc. The complete sequence is as follows:

[0069] CACCGGTGTTATTGCTGCTCGGTGCGTGCATGCACATCAGTGTCGCTGC

[0070] AGCTCAGTGCATGCACGCTCATTGCCCATCGCTATCCCTGCCTCTCCTGC

[0071] TGGCGCTCCCCGGGAGGTGACTTCAAGGGGACCGCAGGACCACCTCGG

[0072] GGGTGGGGGGAGGGCTGCACACGCGGACCCCGCTCCCCCTCCCCAACA

[0073] AAGCACTGTGGAATCAAAAAGGGGGGAGGGGGGATGGAGGGGCGCGT

[0074] CACACCCCCGCCCCACACCCTCACCTCGAGGTGAGCCCCACGTTCTGCT

[0075] TCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTA

[0076] TTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCG

[0077] CGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGG

[0078] AGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCT

[0079] TTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCG

[0080] CGGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCG...

Embodiment 2

[0163] Example 2: Construction of mammalian expression vector

[0164] The 5'flanking regulatory elements of the full-length chicken beta actin gene are from Dr. N Fregien (ATCC37507) (Fregien N and Davidson N, 1986). It is located, sequenced and characterized by restriction enzymes, and matches the published sequence (Kost et al., 1983). The 1.494kb chicken actin gene promoter fragment was digested by Pst I and Hind III, and purified by SDS gel. The 1.494kb Pst I / Hind III promoter fragment was further digested with HinfI to obtain 1.006kb intron 1, and was modified with phosphorylated Pst I / HinfI adaptor so that the intron 1 (SEQ No: 1) was in Pst I at the 5'end and Hind III at the 3'end.

[0165] Natural expression vector based on chicken beta actin promoter ( figure 1 ) (SEQ ID NO: 3) is the pBR322-based vector backbone opened by inserting the 1.272 kb Xho I / Hind III fragment containing the 5'flanking regulatory element of the full-length chicken β-actin gene of intron 1 into ...

Embodiment 3

[0176] Example 3: GC content analysis of intron 1 of chicken β-actin gene

[0177] The following lists the intron 1 of the chicken β-actin gene (SEQ ID No: 1):

[0178] CTGCAGTGACTCGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCG

[0179] CGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCA

[0180] CAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCT

[0181] TGGTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAA

[0182] GGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGC

[0183] GTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCG

[0184] GCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGT

[0185] GTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGG

[0186] GCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGT

[0187] GAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCAC

[0188] CCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCC

[0189] GTGCGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGC

[0190] AGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCT

[0191] CGGGGGAGGGGCGCGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGC

[0192] GGCGAGCCGCAGCCATTGCCTTT...

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Abstract

A method to use chick beta actin gene intron-1 or functional equivalent as a gene expression enhancer eiement or a gene expression 'hot spot' sequence for constructing or reconstructing a mammalian expression vector for extremely high expression of recombinant proteins is disclosed. Composition of a set of extremely strong gene expression vectors is also disclosed.

Description

[0001] Related application [0002] This application claims the priority of the U.S. Provisional Application Serial No. 60 / 897,394 filed on January 25, 2007, the contents of which are incorporated herein by reference. Invention field [0003] The present invention relates to the use of chicken beta actin gene intron 1 (Intron-1) as a gene expression enhancer or gene expression "hot spot" on the 5'or 3'flanking region of a mammalian gene expression promoter. Construct a new mammalian expression vector or reconstruct an existing gene expression vector for the extremely high level of expression of recombinant protein and the production of mammalian cell lines that produce extremely high levels of recombinant protein. Background of the invention [0004] The recombinant protein can be prepared by first introducing an expression vector encoding the recombinant protein into a host cell, and then expressing the recombinant protein in the host cell. Traditional host cells include primitive...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04
CPCC12N2830/42C12N2830/00C12N2830/46C12N15/85C12N15/11C12N15/67
Inventor 米祖·惠
Owner QINGDAO HUINUODE BIOLOGICAL TECH CO LTD
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