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Human and mammal cell attachment expression vector and application thereof

An expression vector and mammalian technology, applied in the field of genetic engineering, can solve problems such as diseases and insertion mutations, and achieve high-efficiency expression and good safety performance

Inactive Publication Date: 2012-10-03
河南普诺易生物制品研究院有限公司 +1
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Integrating vectors can cause insertion mutations or cause diseases due to the position effect of the inserted gene

Method used

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  • Human and mammal cell attachment expression vector and application thereof
  • Human and mammal cell attachment expression vector and application thereof
  • Human and mammal cell attachment expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Construction of Human and Mammalian Cell Attachment Expression Vectors

[0024] 1.1 Materials and methods

[0025] 1.1.1 pEGFP-C1 vector ( figure 1 ) is an expression vector for human and mammalian cells, which contains the reporter gene EGFP, purchased from Clontech Corporation of the United States.

[0026] Chinese hamster ovary (CHO) cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0027] 1.1.2 Synthesis of MAR sequence

[0028] The 2200bp human β-interferon MAR sequence (GenBank: M83137.1) and the characteristic elements (such as AT-rich sequence) containing the 2150bp MAR sequence (GenBank: L22754.1) of human β-globin were cut and spliced ​​into 387bp , artificially synthesized a short MAR sequence as shown in Seq ID No.1.

[0029] 1.1.3 Enzyme digestion reaction

[0030] Use KpnI and BamHI to double-digest MAR characteristic sequence and pEGFP-C1 vector, the enzyme digestion system is as follows: plasmid 25μl (1...

Embodiment 2

[0036] The screening of embodiment 2 stable monoclonal cell lines

[0037] 1.1 The endotoxin-free pEGFP-C1-MAR plasmid was transfected into CHO cells by Suohua-sofast cationic polymer transfection method, and the CHO cells were cultured in G418 DMEM complete medium with a final concentration of 800 μg / ml.

[0038] 1.2 After positive clones were formed in the CHO cell transfection positive group, and all the cells in the control wells died, they were transferred to DMEM complete medium containing 400 μg / ml G418 for culture.

[0039] 1.3 When the CHO cells reach 70-90%, collect the cells, count them by hemocytometer, dilute with serum-free DMEM medium until the number of cells is about 1 cell in each well, and then add Appropriate amount of complete medium.

[0040] 1.4 Observe the growth of the monoclonal cells, and maintain the selection pressure with DMEM medium at half the concentration of G418. When the monoclonal cell density reaches 1 / 3 of the inner volume of the well, t...

Embodiment 3

[0042] Embodiment 3 plasmid reduction experiment

[0043] 1.1 Utilize the Hirt lysis method to extract the episomal plasmids in two stable monoclonal cells: add 2ml of Hirt lysis buffer to the collected cells, let stand at room temperature for 20min; add 1 / 4 volume (0.5ml) of 5mol / L NaCl, overnight at 4°C. The next day, centrifuge at 15000rpm for 40min. Take the supernatant, add RNase A (10mg / mL) 125μl in 37℃ water bath for 60min; extract twice with phenol chloroform. Take the supernatant, add 5ml -20°C pre-cooled isopropanol and place at -20°C overnight. The next day, centrifuge at 12000rpm for 30min. After washing twice with absolute ethanol and drying in the air, add 10 μl of elution buffer to dissolve the attached body plasmid.

[0044] 1.2 Under sterile conditions, take 250 μl of freshly prepared competent cells and add 10 μl of episomal plasmids for transformation. During recovery, 800 μl of SOC solution preheated at 37°C was added, placed on a shaker at 37°C at 250 r...

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Abstract

The invention provides a human and mammal cell attachment expression vector. The expression vector contains a nuclear matrix attachment region sequence, wherein the nuclear matrix attachment region sequence is shown as Seq ID No. 1 or is a nucleotide sequence which is homologous with the sequence by over 95 percent. The vector can mediate stable, high-efficiency and lasting expression of exogenous genes in mammal cells, has high safety performance, can be used for gene therapy and has broad market prospect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a human and mammalian cell attachment expression vector and its application. Background technique [0002] Gene therapy technology refers to the introduction of normal genes or therapeutic DNA sequences into target cells in a certain way to correct gene defects or exert therapeutic effects, so as to achieve the purpose of treating diseases. Since the first successful clinical trial of gene therapy in the early 1990s, thousands of gene therapy clinical trials have been completed so far, in which gene expression vectors play a key role in gene therapy. According to the way the vector enters the host cell, it is divided into viral vector and plasmid vector. Because the transfection efficiency of viral vectors is higher than that of plasmid vectors, it is widely used in clinics. However, when viral vectors (such as adenovirus, adeno-associated virus, and retroviral vectors) transf...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10
Inventor 王天云林艳刘中何李照熙赵俊玲张靖高建辉王小引杨全中王俐
Owner 河南普诺易生物制品研究院有限公司
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