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Expression vector, expression system, preparation method and application of mammal cell attachment on basis of cHS4 element

An expression vector and mammalian technology, applied in the field of genetic engineering and gene therapy, can solve the problems of transgene silencing, low copy number, poor vector stability, etc., achieve lasting expression, good safety, and overcome the effect of transgene silencing

Active Publication Date: 2017-03-29
XINXIANG MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the first generation of MAR-mediated episomal expression vector pEPI was confirmed, a number of improved studies have been carried out, such as inserting regulatory elements or reducing bacterial sequences to make up for the lack of vectors, but transgene silencing often occurs in the research
Although the vector can remain attached to the host cells after transfection, the stability of the vector is poor and the copy number is low. Studies have shown that about 90% of the vector molecules are gradually lost during passage.

Method used

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  • Expression vector, expression system, preparation method and application of mammal cell attachment on basis of cHS4 element
  • Expression vector, expression system, preparation method and application of mammal cell attachment on basis of cHS4 element
  • Expression vector, expression system, preparation method and application of mammal cell attachment on basis of cHS4 element

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The construction of the episomal expression vector pES4, the steps are as follows:

[0038] 1) Synthesis of cHS4 sequence

[0039] The cHS4 sequence was synthesized according to the literature (Saunders F, Sweeney B, Antoniou, et al. Chromatin function modifying elements in an industrial antibody production platform-comparison of UCOE, MAR, STAR and cHS4 elements.2015, PLoS One 10:e0120096.) for directional cloning And the identification of subsequent vectors, the restriction sites of BspE I and BsrG I were respectively introduced at the 5' end of the sequence. Since the pES4 vector was constructed using seamless cloning technology, the company added 15 bp homologous sequences to both ends of the sequence when synthesizing the cHS4 sequence. The synthetic cHS4 sequence is as follows:

[0040]

[0041] Wherein, the parts in italics represent homologous sequences, and the underlined parts are enzyme cutting sites.

[0042] 2) Construction of pES4 plasmid vector

[...

Embodiment 2

[0051] The construction of mammalian cell expression system, the steps are as follows:

[0052] 1) Synthesis of IFN-β sequence

[0053] According to the IFN-β sequence published by NCBI (GenBank: BC096151.2, bases 1 to 564), the company synthesized IFN-β (as shown in SEQ ID NO: 3, with 15 bp vectors added to both ends of the sequence) Homologous sequence, and IFN-β is inserted downstream of eGFP), which was completely consistent with the sequence registered in GenBank by sequencing analysis.

[0054] 2) Construction of the episomal expression vector pES4-IFN-β containing the IFN-β sequence

[0055] Digest the pES4 plasmid with BsrG I. The enzyme digestion system is: 1 μg / μL pES4 plasmid 10 μL, 10×NE buffer 2 μL, BsrG I enzyme 0.5 μL, add triple distilled water to 20 μL; mix well, and digest in a 37°C water bath 8h. After the enzyme digestion, 1.5% agarose gel electrophoresis was performed to recover the digested pES4 linear plasmid DNA.

[0056] 10 μL In-Fusion Cloning lig...

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Abstract

The invention discloses an expression vector, an expression system, a preparation method and application of a mammal cell attachment on the basis of a cHS4 element, and belongs to the technical field of gene engineering and gene therapy. A chicken hypersensitive site 4 (cHS4) is a classic insulator element, has functions of enhancer interdicting and heterochromatin barrier, can lower the main reason-position effect of silence of transgenosis to the lowest, and can further lower the probability of abnormal expression, caused by an exogenous promoter and an enhancer, of neighboring genes. According to the expression vector, the expression system, the preparation method and the application of the mammal cell attachment on the basis of the cHS4 element, a cHS4 sequence is introduced into a multiple cloning site, namely the downstream position of eGFP, of a carrier, on the one hand, the silence of the transgenosis can be overcome, and on the other hand, exogenous genes can be mediated to be stably, efficiently and constantly expressed in mammal cells under the condition of no selective pressure; and safety is good.

Description

technical field [0001] The invention relates to a cHS4 element-based mammalian cell attachment expression vector, and also relates to an expression system, preparation method and application comprising the attachment expression vector, and belongs to the technical field of genetic engineering and gene therapy. Background technique [0002] With the continuous development of molecular biology and biotechnology, people have a deeper understanding of the pathogenesis of genetic diseases, and on this basis, a large number of disease-causing genes have been discovered. There is even more scope for treating diseases by replacing faulty genes in damaged cells. Gene therapy refers to the direct introduction of exogenous normal genes into the target cells of the diseased part of the patient through gene transfer technology, and by controlling the expression of the target gene, inhibiting, correcting, replacing or compensating for defective or abnormal genes, a method of treating gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66
CPCC12N15/66C12N15/85C12N2800/107C12N2830/40
Inventor 赵春澎王天云王小引张玺徐光华付笑笑高向征白可可
Owner XINXIANG MEDICAL UNIV
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