Expression vector for animal cell as well as preparation method and application of expression vector

An expression vector, animal cell technology, applied in the field of genetic engineering, can solve the problem of low target protein expression, achieve the effect of increasing yield and reducing translation efficiency

Active Publication Date: 2015-06-10
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The technical effects of this patented technology include improved production levels by adding specific genes or parts thereto during cell culture processes instead of relying on other methods like increasing methionine synthesis (which can be expensive). This results from utilizing a strong promoter for driving the transcription process rather than just making it harder to produce new proteins due to its low activity level compared to existing techniques such as cloning.

Problems solved by technology

Technologies described involve improving the ability of producing highly effective amounts of desired products from cloned animal tissue sources called germ line fractions through fusion techniques involving specific parts of the parent molecule's own genomes. By combining multiple different components like other gene segments within certain regions of the recipients, even if some genes were deleted during culture process, the transformed product could remain stable overtime without losing any key structural features found naturally occurring ones. Additionally, modifying the expression rate of each component allows for efficient expression of various genes while minimizing changes caused by external influences including pigment accumulation, oxidative stress, and reduced sensitivity due to decreased transferring efficacy. Overall, these technical means improve the quality and quantity of produced materials derived from CHOs cells.

Method used

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  • Expression vector for animal cell as well as preparation method and application of expression vector
  • Expression vector for animal cell as well as preparation method and application of expression vector
  • Expression vector for animal cell as well as preparation method and application of expression vector

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Embodiment 1 of the present invention provides an expression vector for animal cells, such as figure 1 As shown, the expression vector inserts the dhfr gene and the SV40-neo fragment into the pIRES vector digested by PvuII and BamHI, respectively, wherein the SV40-neo fragment is formed by connecting the SV40 early promoter gene and the neo gene, and the dhfr gene is located at Between the adjacent Xma I and Not I restriction sites; the SV40-neo fragment is located between the PvuII and BamHI restriction sites.

[0048] The above expression vector can be obtained by the following steps:

[0049] Step S101: using the pSV2-dhfr plasmid as a template and using SEQ ID No: 2 and SEQ ID No: 3 in the sequence table as primers for PCR amplification, wherein the primers shown in SEQ ID No: 2 contain an Xma I restriction site, The primer shown in SEQ ID No: 3 contains a Not I restriction site, and the pSV2-dhfr plasmid is from ATCC37146;

[0050] Step S102: The PCR product obta...

Embodiment 2

[0054] Embodiment 2 of the present invention provides an application of the expression vector of Embodiment 1, comprising the following steps:

[0055] Step S201: Insert the target gene into the multiple cloning site of the expression vector obtained in Example 1 to obtain a recombinant vector;

[0056] Step S202: using dhfr-deficient CHO cells as hosts to transfect expression vectors;

[0057] The day before transfection, inoculate 1X106 dhfr-deficient CHO cells in a 60mm culture dish, add 5ml serum-containing, antibiotic-free DMEM medium, 37°C, 5% CO 2 Culture until the cells are required to be 40%-80% confluent at the time of transfection. Dilute 5 μg of recombinant plasmid with OPTI-MEM1 medium to a total volume of 250 μl. After dilution, invert the centrifuge tube several times to mix the mixture. Add 10 μl transfection reagent LIPOFECTAMINE2000 to the mixture, and pipette several times with the pipette. Incubate the mixture at room temperature for 5-10 min. While inc...

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Abstract

The invention relates to the field and particularly relates to an expression vector for an animal cell. The expression vector sequentially contains a promoter with strong expression inductivity, a multiple cloning site for gene integration, a ribosome entry site sequence, a dihydrofolate reductase gene, a SV40 early stage promoter gene without an enhanser, a neo gene and an anti-drug gene from upstream. The invention further provides a preparation method of the expression carrier, a recombinant carrier and application. According to the expression carrier provided by the invention, expression of the neo gene is weakened by using the promoter without the promoter; a dhfr gene as a co-amplification gene is located at the downstream of the ribosome entry site IRES, so that the translation efficiency of the dhfr gene is reduced; the expression quantity of a target protein is increased by combining G418 pressurized screening and pressure screening of the dihydrofolate reductase gene.

Description

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Claims

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Application Information

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Owner SHENZHEN INST OF ADVANCED TECH
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