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Artificially synthesized MAR (Matrix Attachment Region) fragment, expression vector, expression system and application thereof

A technology for artificially synthesizing and expressing vectors, applied in the directions of DNA/RNA fragments, vectors, and the introduction of foreign genetic material using vectors, which can solve the problems of increasing the difficulty of vector construction and reducing the efficiency of plasmid transfection, and achieve stable and long-term expression, reducing The effect of expressing differences and improving stability

Pending Publication Date: 2018-04-03
XINXIANG MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the MAR element is relatively large, and the commonly used MAR, such as the invention patent of Publication No. CN106520832A, discloses a bicistronic expression vector, expression system, preparation method and application. The β-globin MAR sequence (GenBank: L22754 .1, bases 840-2998), X-29 sequence (GenBank: EF694970.1, bases 1-3337), β-interferon MAR sequence (GenBank: M83137.1, bases 1-2201 bases), MAR 1-68 (GenBank: EF694965.1, 1st to 3614th base) are all above 2000bp in size, which will increase the difficulty of vector construction on the one hand, and the transfection efficiency of the plasmid will vary with the size of the vector increased and decreased, which limits the application of MAR elements in mammalian cell expression systems

Method used

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  • Artificially synthesized MAR (Matrix Attachment Region) fragment, expression vector, expression system and application thereof
  • Artificially synthesized MAR (Matrix Attachment Region) fragment, expression vector, expression system and application thereof
  • Artificially synthesized MAR (Matrix Attachment Region) fragment, expression vector, expression system and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] 1) Design and synthesis of artificial MAR fragments.

[0028] The MAR sequence is an AT-rich DNA sequence. Although it has no consensus sequence, the MAR sequence has typical characteristic motifs such as A-box, T-box, ARS, Unwinding sequence, Curved DNA, Oligo A-T tracts, A- tracts, T-tracts, Stem loops, Curved DNA, SATB1, ATF site, Topoisomerase II, CEBP, FAST, Hox, NMP4, GSH, etc. (as shown in Table 1).

[0029] Table 1 Characteristic motifs of MAR

[0030]

[0031] In Table 1, Y is T / C, W is A / T, R is G / A, N is A / T / G / C, K is G / T, S is G / C, and M is A / C.

[0032]According to literature reports (Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells. Nat. Methods. 2007; Positional effects of the matrix attachment region on transgene expression in stably transfected CHO cells. Cell Biol. Int. 2010);

[0033] (Molecular characterization of a human matrix attachment region that improves transgene expression in CHO cells....

Embodiment 2

[0036] An expression vector containing synthetic MAR-1 upstream of the CMV promoter was constructed.

[0037] For the convenience of cloning, NruI (TCGCGA) and MluI (ACGCGT) restriction sites were respectively inserted at the 5' and 3'-ends of the synthesized MAR-1 fragment.

[0038] The synthesized MAR-1 sequence was digested with NruI / MluI, and the pIRES-Neo plasmid DNA vector (purchased from Clontech Biological Company) was simultaneously digested with NruI / MluI. The digestion results were identified by agarose gel electrophoresis, and the digested MAR sequence fragment and pIRES-Neo linear plasmid DNA were recovered from the gel.

[0039] The double enzyme digestion system of MAR-1 sequence is: MAR-1 sequence 10μL (1μg / μL), 10×NE Buffer 3.1 3μL, NruI / MluI (10U / μL) each 1.0μL, make up water to 30μL; enzyme digestion conditions are : 37°C, enzyme digestion for 3min.

[0040] The double enzyme digestion system of pIRES-Neo plasmid is: pIRES-Neo plasmid 5 μL (1 μg / μL), 10×NE...

Embodiment 3

[0043] The construction method is the same as that in Example 2, except that the fragment is replaced by the MAR-2 fragment (sequence shown in SEQ ID NO.2), and the correctly constructed plasmid is named pIRES-MAR1-2.

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Abstract

The invention relates to an artificially synthesized MAR (Matrix Attachment Region) fragment, an expression vector, an expression system and application thereof and belongs to the technical field of biology. The novel artificially synthesized MAR fragment respectively comprises MAR-1, MAR-2 and MAR-3. The MAR fragment disclosed by the invention is a novel MAR fragment designed and synthesized according to common sequence characteristics on the basis of analyzing a beta-globin MAR sequence, an X-29 sequence, a beta-interferon MAR sequence and an MAR 1-68 sequence. Researches find out that, whenthe MAR-1, MAR-2 and MAR-3 are simultaneously used for expressions of foreign proteins in CHO (Chinese Hamster Ovary) cells, the MAR-1 has the best effect, the expression amount of the foreign proteins can be improved by 5.04-5.47 times in a stable cell pool compared with that of a system without the MAR fragment, preferably, 8 copied tandem repeat DNA fragments composed of the MAR fragments havethe ability of well improving the expression amount.

Description

technical field [0001] The invention relates to artificially synthesized MAR fragments, expression vectors, expression systems and applications thereof, belonging to the field of biotechnology. Background technique [0002] With the development of genetic engineering technology, the number and types of recombinant proteins produced by genetic engineering are constantly increasing, and have become an important part of the pharmaceutical industry. The expression systems of recombinant drug proteins mainly include E.coli, yeast and non-humanized mammalian cell lines. E.coli is suitable for expressing proteins with small molecular weight and relatively simple structure, and the yeast expression system is suitable for expressing proteins with large molecular weight, complex structure and less glycosylation. The complex structure or glycosylation is very important for the activity of proteins. Since non-humanized mammalian cells have post-translational modifications (post-transla...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/85
CPCC12N15/11C12N15/85C12N2830/46
Inventor 王天云郭潇贾岩龙李琴田政伟王稳徐丹华
Owner XINXIANG MEDICAL UNIV
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