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Novel MAR (matrix attachment region) core fragment-containing animal cell expression vector

A technology of core fragments and expression vectors, which is applied to DNA/RNA fragments, cells modified by introducing foreign genetic materials, and vectors to introduce foreign genetic materials. It can solve the problems of reduced efficiency of plasmid transfected cells and achieve good stability. , improve the effect of expression

Active Publication Date: 2015-10-14
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-yielding cell lines (Kwaks TH, Otte AP2006 Trends Biotechnol24:137–142), but the MAR element is large (more than 3kb), and the efficiency of plasmid transfection cells varies with Decreased with the increase of plasmid size (Yin W et al Anal Biochem.2005Nov15;346(2):289-94), which limits the application of MAR elements in double-subunit co-expression vectors

Method used

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  • Novel MAR (matrix attachment region) core fragment-containing animal cell expression vector
  • Novel MAR (matrix attachment region) core fragment-containing animal cell expression vector
  • Novel MAR (matrix attachment region) core fragment-containing animal cell expression vector

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Introducing Afe I and EcoR V restriction enzyme cutting sites in embodiment 1pBudCE4.1

[0030] Afe I and EcoR V restriction sites were introduced into SV40 polyadenylation site and downstream of BGH polyadenylation site by point mutation method. Point mutations were performed using the Quickchange Point Mutation Kit (purchased from Agilent Technologies). Afe I restriction site is introduced.

[0031] Forward primer: 5'-GGAAAACGATTCCGAAGC GCT AC-3'

[0032] Reverse primer: 5’-CCT TCT ATG AAA GGT AGC GCT TCG-3’

[0033] Site-directed mutagenesis PCR amplification conditions are as follows: 1. 95C for 30 seconds; 2. 95C for 30 seconds; 3. 55C for 1 minute; 4. 68C for 5 minutes; 5. 2-4 repeated for 18 cycles.

[0034]Add Dpn I enzyme to digest the template plasmid, and then transform XL1-Blue competent Escherichia coli. The specific steps are as follows: 1. Freshly prepared or 100uL competent cells (purchased from Agilent Technologies) stored at -20°C were placed on ic...

Embodiment 2

[0040] In Example 2 pBudCE4.1, the Zeocin resistance gene was removed and the Kanamycin resistance gene, PGK promoter, DHFR and SV40PA were added

[0041] The kanamycin resistance gene, PGK promoter, DHFR and SV40PA were PCRed out from pCHO1.0 (purchased from Life Technologies), and loaded into pBudCE4.1 obtained in Example 1 through the NheI and AfeI sites, while Remove the Zeocin resistance gene.

[0042] Forward primer: 5'-GTG AGCGCTTTAGAAAAACTCATCGAGCATC-3'

[0043] Reverse primer: 5'-GTG GCTAGCTAA GAT ACA TTG ATG AGT TTG-3'

[0044] PCR with Primestar HS high-fidelity DNA polymerase (purchased from TAKARA company),

[0045] The specific steps are as follows: 1. 95C for 3 minutes; 2. 98C for 10 seconds; 3. 47C for 15 seconds; 4. 72C for 3 minutes; 5. Repeat 2-4 for 30 cycles; 6. 72C for 5 minutes.

[0046] The modified new carrier is named PBCM1.0, and its structure diagram is attached Figure 5 , wherein, the NheI, AfeI, FspI and EcoRV sites are the insertion sites of...

Embodiment 3

[0047] Example 3 Construction of an expression vector containing a MAR core fragment with artificial transcription factor binding sites added at both ends

[0048] The MAR core fragment (sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3) with artificial transcription factor binding sites added at both ends was synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0049] The MAR core fragment with artificial transcription factor binding sites added at both ends can be inserted at the FSP I site upstream of PBCM1.0, or the Afe I site downstream of SV40PA, or the Nhe I site upstream of PEF-1a, or the EcoRV site downstream of BGH PA point.

[0050] In the following example, the MAR core fragment with artificial transcription factor binding sites added at both ends is inserted into the FSP I site upstream of PBCM1.0 and the Nhe I site upstream of PEF-1a. The sequence of the MAR core fragment is shown in SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 3.

[0051] The MAR ...

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Abstract

The invention discloses a novel MAR (matrix attachment region) core fragment-containing animal cell expression vector. The nucleotide base sequence of the MAR core fragment of the recombinant expression vector is as shown in SEQ ID No. 1, SEQ ID No.2 or SEQ ID No. 3. The expression vector disclosed by the invention can effectively increase the yield of protein in mammalian cells and reduce production cost.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically discloses a novel animal cell expression vector containing a MAR core fragment. Background technique [0002] At present, many vectors for the production of recombinant proteins have been developed. Recombinant proteins can achieve higher yields in the expression of bacteria, eukaryotic microorganisms, insect cells, etc., but these expression systems lack Protein modification mechanism (such as glycosylation modification, etc.), the expressed animal protein tends to lack biological activity or produce inclusion bodies due to misfolding. However, the production of recombinant proteins by mammalian cell expression systems is often low, and the stability of the introduced genes will also have certain problems. Later, it was discovered that Chinese hamster ovary cells (CHO) are the best system for producing complex macromolecules such as monoclonal antibodies. Using the CHO express...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/85C12N5/10C12P21/00
Inventor 高宏海张成海张玉晶朱玲巧
Owner SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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