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A group of efficient expression carriers for humanized antibody of mammal cell

An expression vector and high-efficiency expression technology, which is applied in the field of high-efficiency expression vectors for a group of mammalian cell human antibodies for production, and can solve the problem of no commercial vectors

Inactive Publication Date: 2004-05-19
梁米芳 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The more successful full antibody expression system is the CHO cell expression system. Although there are many vector systems for this expression system, there are no commercial vectors available so far.

Method used

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  • A group of efficient expression carriers for humanized antibody of mammal cell
  • A group of efficient expression carriers for humanized antibody of mammal cell
  • A group of efficient expression carriers for humanized antibody of mammal cell

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0057] Example 1: The primers used in vector construction and application were designed by ourselves and synthesized in MWG (Germany) commercial company. The sequence numbers of all primers are as follows:

[0058] 1. BssHII-F

[0059] 5′ TATCCGCGCGCACTCCCCAGGTGCAACTGCTCGAGTCTGGG-3′

[0060] 2. XbaI-R

[0061] 5′-TGCTCTAGATTATTTTCCCGGACTTAAGGAGAG-3′

[0062] 3. BstEII-HR

[0063] 5′-GGAGACGGTGACCAGGGTTCCCTC-3′

[0064] 4. XhoI-KR

[0065] 5′-CTTGATCTCGAGCTTGGTCCCCTGGTC-3′

[0066] 5. Hind III R

[0067] 5′-CGCAAGCTTATTGAATCAAAGGATATATGAC-3′

[0068] 6. XohI-LR

[0069] 5′-TAGGACCTCGAGCTTGGTCCCCTCCGCC-3′

[0070] 7. SpeI-LR

[0071] 5′-GGACTAGTCCTATGAACATTCTGTAGG-3′

[0072] 8. SpeI-KR

[0073] 5′-GGACTAGTCCTAACACTTCTCCCCTGTTGA-3′

[0074] 9. EcoRV-VL2

[0075] 5′-CCGGATATCGCCCTCACGCAGCCTGCCTCCGTG-3′

[0076] 10. EcoRV-VL5

[0077] 5′-CCGGATATCGTG-CTCACGCCGCCCTCAGTG-3′

[0078] 11. EcoRV-VK1a

[0079] 5′-CCGGATATCGTGCTCACCCAGTCTCCA-3′ ...

example 2

[0082] Example 2: Obtaining the elements of the antibody expression cassette in the vector system: 1. The main elements of the backbone vector EF-1a, DHFR gene and IRES gene are from vectors EF-VH, EF-VL, pSV2-DHFR and IRES respectively. 2. The antibody light and heavy chain constant region genes in the vectors VH-dhfr1, VH-IRES-shfr1, VK-dhfr1, and VH-IRES-dhfr1 are from the vectors pTA-IgGH and pTA-K developed by the inventor Professor Bautz's laboratory. 3. The antibody genes in the vectors VH-dhfr2, VH-dhfr3, VH-IRES-dhfr2, VH-IRES-dhfr3, VK-dhfr2, VK-dhfr3, VH-IRES-dhfr2, VH-IRES-dhfr3 are from the inventor Liang Mi Fang and Professor Bautz's laboratory jointly developed vectors pAc-K-CH3, pAc-K-Fc and pAc-L-CH3.

example 3

[0083] Example 3. Construction of expression vector: as attached figure 1 And attached figure 2 , is a schematic diagram of the backbone vector and different expression cassettes of the series of vectors in the present invention, which mainly consists of the following key steps:

[0084]1) Use BssHII and XbaI double enzymes and the carrier EF-VH, electrophoresis to recover the carrier band, this vector contains the EF-1a promoter and BGHpolyA tail and the new enzyme gene (NEO), about 5468 base pairs (bp),

[0085] 2) Using the carrier pTA-IgGH as a template, use primers BssHII-F and XbaI-R to amplify the heavy chain constant region gene containing the intron gene by PCR method, and recover the fragment by electrophoresis, about 1796bp, containing the VH gene cloning site BssHII and BstEII.

[0086] 3) Using the vector pAc-K-CH3 as a template, use primers BssHII-F and XbaI-R to amplify the heavy chain constant region gene without intron gene by PCR method, and recover the fr...

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Abstract

A group of 12 carriers with efficient expression for preparing the humanized antibody of mammalian is prepared by directional combination of different elements, that is, the eukaryotic cell expression elements including strong promoter EF-1 alpha, the gene in light-or heavy-chain constent region of the antibody molecular for human genom DNA or message RNA, intrinsic ribosomal recognition site and dihydrofolate reductase gene. It can be used to clone antibody virable region or Fab gene and introduce it into CHO cell to obtain permanently efficiency expression to different antibody genes.

Description

Background technique [0001] Since the advent of B lymphocyte hybridoma cell fusion technology in 1975, monoclonal antibodies, as a new type of product used in basic research, laboratory diagnosis, clinical treatment and prevention, have been widely recognized for their application value and development prospects. The research and development of molecular biology and molecular immunology have led to the production and development of genetically engineered antibodies. With the rise of phage antibody gene library technology in the late 1980s and early 1990s and the development of the entire field of genetic engineering antibody technology research, breakthroughs have been made in the research of monoclonal antibodies, and their significance and practical application prospects have increasingly shown. As a new type of specific therapeutic biological drug or passive immune vaccine, human genetically engineered antibody has attracted more and more attention from the investment commu...

Claims

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Application Information

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IPC IPC(8): A61K39/395C07K16/00C07K16/40C12N15/12C12N15/53C12N15/85
Inventor 梁米芳李德新爱克哈德・博茨爱丽丝・奎滋
Owner 梁米芳
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