Novel animal cell expression vector containing MAR (Matrix Attachment Region) core fragment

A core fragment and expression vector technology, which can be applied to DNA/RNA fragments, cells modified by introducing foreign genetic material, and using vectors to introduce foreign genetic material. To achieve good stability and improve the effect of expression

Active Publication Date: 2019-04-05
SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Putting the MAR element into the vector can increase the yield of cell lines and the ratio of high-yield cell lines (Kwaks TH, Otte AP2006 Trends Biotechnol 24:137–142), but the MAR element is large (more than 3kb), and the efficiency of plasmid transfected cells Decreases with increasing plasmid size (Yin W et al Anal Biochem. 2005Nov15;346(2):289-94), thus limiting the use of MAR elements in dual-subunit co-expression vectors

Method used

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  • Novel animal cell expression vector containing MAR (Matrix Attachment Region) core fragment
  • Novel animal cell expression vector containing MAR (Matrix Attachment Region) core fragment
  • Novel animal cell expression vector containing MAR (Matrix Attachment Region) core fragment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Introduction of Afe I and EcoR V restriction sites in pBudCE4.1

[0032] The Afe I and EcoR V restriction sites were introduced into the SV40 polyadenylation site and downstream of the BGH polyadenylation site by point mutation method. Use Quickchange Point Mutation Kit (purchased from Agilent Technologies) for point mutation. Introduce Afe I restriction site.

[0033] Forward primer: 5’-GGAAAACGATTCCGAAGC GCT AC-3’

[0034] Reverse primer: 5’-CCT TCT ATG AAA GGT AGC GCT TCG-3’

[0035] The PCR amplification conditions for site-directed mutagenesis are as follows: 1, 95°C for 30 seconds; 2, 95°C for 30 seconds; 3, 55°C for 1 minute; 4, 68°C for 5 minutes; 5, 2-4 repeat 18 cycles.

[0036] Add Dpn I enzyme to digest the template plasmid, and then transform XL1-Blue competent E. coli. The specific steps are as follows: 1. 100uL competent cells (purchased from Agilent Technologies), freshly prepared or stored at -20°C, are placed on ice, and the cells are gently suspend...

Embodiment 2

[0042] Example 2 Remove Zeocin resistance gene from pBudCE4.1 and add kanamycin resistance gene, PGK promoter, DHFR and SV40PA

[0043] The kanamycin resistance gene, PGK promoter, DHFR and SV40PA were PCR out of pCHO1.0 (purchased from LifeTechnologies), and loaded into pBudCE4.1 obtained in Example 1 through NheI and AfeI sites, and removed at the same time Zeocin resistance gene.

[0044] Forward primer: 5’-GTG AGCGCTTTAGAAAAACTCATCGAGCATC-3’

[0045] Reverse primer: 5'-GTG GCTAGCTAA GAT ACA TTG ATG AGT TTG-3'

[0046] Primestar HS high-fidelity DNA polymerase for PCR (purchased from TAKARA),

[0047] The specific steps are as follows: 1, 95°C for 3 minutes; 2, 98°C for 10 seconds; 3, 47°C for 15 seconds; 4, 72°C for 3 minutes; 5, 2-4 repeat 30 cycles; 6, 72°C for 5 minutes.

[0048] The modified new carrier is named PBCM1.0, and its structure diagram is attached Figure 5 Among them, the NheI, AfeI, FspI and EcoRV sites are the insertion sites of MAR core fragments with artificial t...

Embodiment 3

[0049] Example 3 Construction of an expression vector containing MAR core fragments with artificial transcription factor binding sites added at both ends of MAR core fragments with artificial transcription factor binding sites added at both ends (sequences such as SEQ ID NO: 1, SEQ ID NO: 2 Or SEQ IDNO: 3) synthesized by Shanghai Shenggong Biological Engineering Co., Ltd.

[0050] The MAR core fragment with artificial transcription factor binding sites at both ends can be inserted at the upstream FSPI site of PBCM1.0, or the downstream Afe I site of SV40PA, or the upstream Nhe I site of PEF-1a, or the downstream EcoRV site of BGH PA .

[0051] In the following example, the MAR core fragment with artificial transcription factor binding sites at both ends is inserted at the upstream FSPI site of PBCM 1.0 and the upstream Nhe I site of PEF-1a. The MAR core fragment sequence is as SEQ ID NO:1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0052] The MAR core fragment with artificial transcription fa...

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PUM

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Abstract

The invention discloses a novel animal cell expression vector containing a MAR (Matrix Attachment Region) core fragment. A nucleotide base sequence of the MAR core fragment of the recombinant expression vector is shown as SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. The expression vector can be used for effectively improving the yield of protein in mammalian cells and the production cost is reduced.

Description

[0001] This application is a divisional application of the invention patent application No. 201410129979.2 "A new type of animal cell expression vector containing MAR core fragments" filed on April 1, 2014. Technical field [0002] The present invention relates to the field of biotechnology, and more specifically, discloses a novel animal cell expression vector containing MAR core fragments. Background technique [0003] At present, many vectors for the production of recombinant proteins have been developed. Recombinant proteins can obtain higher yields in the expression of bacteria, eukaryotic microorganisms, insect cells, etc., but these expression systems lack similar mammalian cells. Protein modification mechanisms (such as glycosylation modification, etc.), expressed animal proteins tend to lack biological activity or produce inclusion bodies due to folding errors. However, the production of recombinant protein by mammalian cell expression system is often low, and the stabili...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/67C12N5/10
Inventor 高宏海张成海张玉晶朱玲巧
Owner SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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