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Two-cistron expression vector suitable for HEK293 cell, and preparation method, expression system and application thereof

An expression vector and bicistronic technology, which is applied in the field of genetic engineering and can solve the problems of inability to screen stably transformed cell lines and not including screening markers.

Active Publication Date: 2017-08-18
河南普诺易生物制品研究院有限公司 +1
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Problems solved by technology

[0006] The Chinese invention patent with the publication number CN102994536A discloses a bicistronic gene transfer body for expression and its preparation method. The expression vector of the combined promoter CAG, suppressor protein gene FSTN, internal ribosome entry site IRES, green fluorescent protein AcGFP gene, and Rabbit globin polyA signal region, but its MAR sequence is derived from bovine and is not suitable for human-derived HEK293 cell line. Moreover, the expression vector does not include a selection marker, so it cannot be screened for stably transformed cell lines
The Chinese invention patent with the publication number CN106520832A discloses a bicistronic expression vector, but different combinations of MAR elements have different effects on the expression of transgenes (Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells.SciRep.2017 ), the backbone structure of the vector is related to its transfected host cells (Cell compatibility of aneposimal vector mediated by the characteristic motifs of matrix attachment regions. Curr Gene Ther, 2016), the vector suitable for CHO cell expression may not be suitable for HEK293 cells (Evaluating post- transcriptional regulatory elements for enhancing transientgene expression levels in CHO K1 and HEK293 cells. Protein Expr Purif. 2010), so it is of great significance to develop expression vectors suitable for HEK293 cells

Method used

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  • Two-cistron expression vector suitable for HEK293 cell, and preparation method, expression system and application thereof
  • Two-cistron expression vector suitable for HEK293 cell, and preparation method, expression system and application thereof
  • Two-cistron expression vector suitable for HEK293 cell, and preparation method, expression system and application thereof

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Experimental program
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Effect test

Embodiment 1

[0048] The construction of a bicistronic expression vector suitable for HEK293 cells, the specific steps are as follows:

[0049] 1. Construction of pIRES-H1 vector

[0050] The purpose of this step is to pIRES-neo carrier (its structure is as follows figure 1 Shown) the selection marker gene was replaced by the NPT resistance weakening gene.

[0051] 1) Artificially synthesized NPT resistance weakening gene

[0052] The NPT resistance weakening gene (as shown in SEQ ID NO.3) was designed and artificially synthesized, which was specifically handed over to General Biogene (Anhui) Co., Ltd. to complete.

[0053] In order to facilitate cloning and ensure sequence integrity, when synthesizing the weakened NPT resistance gene, AAC CCCGGGATAATTCCTGCAGCCAAT sequence was introduced at the 5′ end, where AAC was the protective base, CCCGGG was the XmaI restriction site, and ATAATTCCTGCAGCCAAT was the partial sequence on the vector pIRES-Neo (GenBank: U89673.1, bases 1892-1909); the 3...

Embodiment 2

[0077] The construction of the eukaryotic cell expression system, the specific steps are as follows:

[0078] 1. Construction of a bicistronic expression vector containing an exogenous gene of erythropoietin (EPO)

[0079] 1) Synthetic EPO sequence

[0080] According to the EPO sequence published by NCBI (GenBank: JN849371.1, bases 1 to 582), the EPO sequence was artificially synthesized (EcoRI and BamHI restriction sites were introduced at the 5' end and 3' end respectively), and the details were handed over to General Biotech. Gene (Anhui) Co., Ltd. completed.

[0081] 2) Construction of a bicistronic expression vector containing the EPO sequence

[0082] The artificially synthesized EOP sequence was digested with EcoRI / BamHI double-enzyme primers, and pIRES-neo and pIRES-H2 plasmid DNA were simultaneously digested with EcoRI / BamHI double-enzyme. The digestion results were identified by agarose gel electrophoresis, and the digested EPO sequence fragments and pIRES-neo and...

Embodiment 3

[0089] Construction of bicistronic expression vector pIRES-Neo-EPO suitable for HEK293 cells.

[0090] Refer to the method in Example 2 to construct the pIRES-Neo-EPO expression vector containing the EPO exogenous gene, which differs from pIRES-H2-EPO as follows:

[0091] 1) Different screening markers; pIRES-Neo-EPO screening marker is 1906-2709 on the vector (GenBank: U89673.1, base 1906-2709); pIRES-H2-EPO screening marker sequence is as SEQ ID NO.2 shown;

[0092] 2) pIRES-Neo-EPO does not contain the MAR sequence at both ends, and pIRES-H2-EPO contains the human X-29 MAR sequence before the CMV promoter, and contains the β-globin MAR sequence downstream of poly A.

[0093] Construct the pIRES-Neo-EPO eukaryotic cell expression system, and its construction method is the same as that in Example 2.

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Abstract

The invention relates to a two-cistron expression vector suitable for an HEK293 cell, and a preparation method, an expression system and application thereof, which belong to the technical field of genetic engineering. The two-cistron expression vector suitable for an HEK293 cell comprises the following elements: a first nuclear matrix combined region sequence-promoter-target gene-internal ribosome entry site sequence-screened marker genes-poly A-second nuclear matrix combined region sequence. According to the vector, by utilizing the optimized MAR sequence, the transgenic expression of the HEK293 cell can be significantly improved, the transgenic silencing is overcome, and the efficient long-term expression of the transgene in a host cell can be realized. Meanwhile, the simultaneous expression of the target gene and the screened marker genes can be realized, the false positive cell clone caused by the existence of different expression cassettes can be reduced, the positive cell clone screening rate is increased, and the problem of imbalanced expression of the target gene and the screened marker gene of the traditional vector can be solved.

Description

technical field [0001] A bicistronic expression vector suitable for HEK293 cells and its preparation method, expression system and application belong to the technical field of genetic engineering. Background technique [0002] The development of genetic engineering technology in the 1970s transformed the use of animal organs, tissues or human blood to extract protein drugs into the production of genetic engineering. Pharmaceutical proteins, including monoclonal antibodies, peptides, and proteins, have become an important part of the pharmaceutical industry and will become an important area of ​​future pharmaceutical development. [0003] 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 an...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/65
Inventor 王天云林艳井长勤李琴王建华郭梦龙
Owner 河南普诺易生物制品研究院有限公司
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