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Mammalian cell expression vector, expression system, preparation method and application

An expression vector and mammalian technology, applied in the field of genetic engineering, can solve problems such as the need to improve the expression level of transgenes and long MAR sequences, and achieve the effects of overcoming silencing of transgenes, improving expression levels, and lasting expression

Active Publication Date: 2019-05-17
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these MARs are still not ideal in improving the expression level of transgenes. One is that the MAR sequence is longer, such as the X-68 sequence has 3614bp, and the other is that the expression level of transgenes needs to be improved.

Method used

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  • Mammalian cell expression vector, expression system, preparation method and application
  • Mammalian cell expression vector, expression system, preparation method and application
  • Mammalian cell expression vector, expression system, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The construction of mammalian cell expression vector pIRES-TOP1, the steps are as follows:

[0041] 1) PCR amplification of TOP1 MAR sequence

[0042] Primers P1 and P2 were designed according to the TOP1 MAR sequence (GenBank: L23999.1, bases 1-2974), and the 5′ ends of the primers were respectively introduced with NruI and MluI restriction sites. The primer sequences are as follows (the underline is the enzyme cutting point):

[0043] P1: 5′-G TCGCGA GGATCCCAATAGGAGTCATT-3′;

[0044] P2: 5′-CG ACGCGT GAATTCACTTCAGGTAACAT-3'.

[0045] Genomic DNA from human peripheral blood was extracted and used as a template for PCR amplification. Primers P1 and P2 were used to amplify the TOP1 MAR sequence. The reaction system is shown in Table 1 below.

[0046] Table 1 PCR amplification system

[0047]

[0048] Reaction program: 95°C for 3min, 94°C for 40s, 56-60°C for 30s, 72°C for 40s, 4 cycles for each annealing temperature, and finally 30 cycles at 55°C for 1min, 72°C...

Embodiment 2

[0056] The construction of mammalian cell expression vector pIRES-TOP1-TOP1, the steps are as follows:

[0057] 1) PCR amplification of TOP1 MAR sequence

[0058] Primers P3 and P4 were designed according to the TOP1 MAR sequence (GenBank: L23999.1, bases 1 to 2974), and the 5′ ends of the primers were respectively introduced with XhoI restriction sites. The primer sequences are as follows (the restriction sites are underlined) point):

[0059] P3: 5′-C CTCGAG GGATCCCAATAGGAGTCATT-3′;

[0060] P4: 5′-C CTCGAG GAATTCACTTCAGGTAACAT-3'.

[0061] Genomic DNA from human peripheral blood was extracted and used as a template for PCR amplification. Primers P3 and P4 were used to amplify the TOP1 MAR gene. The reaction system and reaction conditions were basically the same as in Example 1, and P1 and P2 in Table 1 were replaced with P3 and P4.

[0062] The PCR amplification product was recovered by agarose gel electrophoresis, purified and sent to the biological company for sequ...

Embodiment 3

[0069] The construction of mammalian cell expression system, the steps are as follows:

[0070] 1) PCR amplification of EGFP gene

[0071] Referring to the Enhanced green fluorescent protein (EGFP) gene sequence of the pEGFP-C1 vector (GenBank: U55763.1, bases 613-1332), primers P5 and P6 (used to amplify EGFP gene DNA of 720bp) were designed. The NheI and AgeI restriction sites were introduced into the ' ends respectively, and the primer sequences were as follows (the restriction sites are underlined):

[0072] P5: 5′-CCG GCTAGC ATGGTGAGCAAGGGCGAGGAG-3′;

[0073] P6: 5′-CTA ACCGGT GGACTTGTACAGCTCGTCCATGC-3'.

[0074] Using the pEGFP-C1 plasmid (purchased from Clontech, USA) as a template, primers P5 and P6 were used to amplify the EGFP gene. The reaction system and reaction conditions were basically the same as in Example 1, and P1 and P2 in Table 1 could be replaced with P5 and P6.

[0075] The PCR amplification product was recovered by agarose gel electrophoresis, pu...

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Abstract

The invention discloses a mammalian cell expression vector, an expression system, a preparation method and an application, and belongs to the technical field of genetic engineering. The expression vector contains TOP1 MAR sequence (GenBank: L23999.1). By inserting the foreign target gene into the multiple cloning site of the vector, TOP1 MAR-promoter-foreign gene-Poly A, promoter-exogenous gene can be constructed ‑Poly A‑TOP1 MAR or TOP1 MAR‑promoter‑exogenous gene‑Poly A‑TOP1 MAR carrier structure, on the one hand, overcomes transgene silencing, and on the other hand, mediates the stable, efficient and lasting expression of foreign genes in host cells. Under the same conditions, compared with vectors without MAR and with 1-68 MAR, the expression vector of the present invention can significantly improve the expression level of foreign genes, and can be used for the production of recombinant proteins and the like.

Description

technical field [0001] The invention relates to a mammalian cell expression vector, and also relates to an expression system, a preparation method and an application comprising the expression vector, and belongs to the technical field of genetic engineering. Background technique [0002] Genetic engineering is based on the theory of molecular genetics, artificially extracting or synthesizing DNA fragments of genetic material of different organisms at the molecular level, cutting and splicing in vitro to form recombinant DNA, and then connecting the recombinant DNA with the carrier to form a recombinant expression vector, and It is introduced into recipient cells that do not contain the DNA, and is replicated, transcribed and translated to produce products that meet human needs or create new biological traits, and make them stably passed on to the next generation. According to the difference of target gene and expression system, it can be divided into prokaryotic genetic engi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10
CPCC12N15/85C12N2800/107
Inventor 贾岩龙郭潇王天云王稳王燕芳陈思佳田政伟徐丹华
Owner XINXIANG MEDICAL UNIV
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