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CYBB (cytochrome B-245 beta chain) lentiviral vector, lentiviral vector-transfected stem cells and preparation method and application of lentiviral vector-transfected stem cells

A lentiviral vector and lentiviral technology, applied in the field of genetic engineering, can solve problems such as safety concerns, inability to express for a long time, foreign genes cannot be expressed in appropriate regions, etc., to ensure safety, improve expression efficiency and expression amount , the effect of increasing the amount of expression

Pending Publication Date: 2019-07-05
BEIJING MEIKANG JIMIAN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although since the 1990s, attempts have been made to apply adenoviral vectors to gene therapy for hemophilia, but so far there has been no positive report of life-long sustained expression of coagulation factors in animal experiments, and the difficulty can be attributed to the immunity induced by the vectors. response, exogenous gene cannot be expressed continuously and efficiently, and exogenous gene cannot be expressed in the appropriate area, etc.
In the past 10 years, clinical gene therapy methods mostly use retroviral vectors (gamma-oncoretroviral vectors), whose viral characteristics are chimeric insertion into the promoter region of oncogenes, which have a mechanism of silencing, and their safety is questionable, and they cannot be expressed for a long time

Method used

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  • CYBB (cytochrome B-245 beta chain) lentiviral vector, lentiviral vector-transfected stem cells and preparation method and application of lentiviral vector-transfected stem cells
  • CYBB (cytochrome B-245 beta chain) lentiviral vector, lentiviral vector-transfected stem cells and preparation method and application of lentiviral vector-transfected stem cells
  • CYBB (cytochrome B-245 beta chain) lentiviral vector, lentiviral vector-transfected stem cells and preparation method and application of lentiviral vector-transfected stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Construction of the lentiviral vector carrying the CYBB gene

[0074] Synthesize the normal CYBB gene sequence (amino acid sequence as shown in SEQ ID NO.2, nucleic acid sequence as shown in SEQ ID NO.3) through the whole gene synthesis into the TYF-EF1α lentiviral vector (NHP / TYF lentivirusvector system), located behind the human EF1α (hEF1α) promoter sequence (the nucleic acid sequence is shown in SEQ ID NO.1), by sequencing and double enzyme digestion (5' cloned at the BamHI site, 3' cloned at the SpeI site , the optimal reaction conditions refer to the original NEB factory recommendations) and other methods to identify the obtained products, and obtain the correctly connected lentiviral vector carrying the CYBB gene under the activation of hEF1α. like figure 1 Shown is the NHP / TYF lentiviral vector system, including viral packaging plasmids (NHP, EF-VSV-G) and vector plasmids (pTYF-EF-CYBB), where the packaging plasmids include pNHP and pHEF-VSV-G(env) ,...

Embodiment 2

[0075] Example 2 lentiviral packaging

[0076] In this example, a multi-plasmid packaging system was used to package the lentiviral vector carrying the CYBB gene into a complete lentivirus through 293T cells. The specific steps are:

[0077] (1) Cultivate the 293T cell line for 17-18 hours, add fresh DMEM containing 10% FBS;

[0078] (2) Add DMEM, pNHP, pHEF-VSV-G and the lentiviral vector constructed in Example 1 in sequence in a sterile centrifuge tube, and vortex;

[0079] (3) Add Superfect Transfection Reagent (QIAGEN) into the centrifuge tube and let stand at room temperature for 7-10min;

[0080] (4) Add the lentiviral vector-Superfect mixture in the centrifuge tube dropwise to the 293T cells, vortex well, and store at 37°C, 5% CO 2 Cultivate for 4-5 hours;

[0081] (5) Remove the cell culture medium, wash the cells, and add the culture medium to continue culturing;

[0082] (6) Return the culture medium to 5% CO 2 After culturing overnight in an incubator, the tra...

Embodiment 3

[0083] Example 3 Purification and Concentration of Lentivirus

[0084] The purification and concentration process of lentivirus, specifically:

[0085] (1) Lentivirus purification

[0086] The packaged lentivirus was centrifuged at 1000g for 5min to remove cell debris, and the obtained supernatant was filtered with a 0.45μm low protein binding filter, and stored at -80°C after aliquoting;

[0087] (2) Lentivirus concentration

[0088] Add the lentivirus supernatant to the Centricon filter tube and centrifuge at 2500g for 30min; shake the filter tube and centrifuge at 400g for 2min to collect the concentrated virus into a collection cup.

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Abstract

The invention provides a CYBB (cytochrome B-245 beta chain) lentiviral vector, lentiviral vector-transfected stem cells and a preparation method and application of the lentiviral vector-transfected stem cells. The lentiviral vector comprises a hEF1alpha promoter and CYBB tandem co-expression. The lentiviral vector carries CYBB gene; under promotion of the hEF1alpha promoter, the carried CYBB genecan be expressed safely and efficiently differentiated or non-differentiated stem cells; in addition, the stems can act as a latent transport vector, and the expression quantity of the CYBB gene in transgenic cells is increased.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a lentiviral vector, a lentiviral vector-transfected stem cell and its preparation method and application, in particular to a CYBB lentiviral vector, a lentiviral vector-transfected stem cell, its preparation method and application. Background technique [0002] Chronic granulomatous disease (CGD) is a hereditary primary immunodeficiency disease caused by the loss of NADPH oxidase function in neutrophils (neutrophilicgranulocytes) and monocytes (monocytes), which is characterized by repeated infections in patients , inflammation and autoimmunity. NADPH oxidase is composed of p47phox, p40phox, p67phox and qp91-phox, and the loss of function of any part will cause CGD. Most of the disease is sex-linked recessive inheritance, and a few are autosomal recessive inheritance. There is often a family history, and it is more common in children. Among them, the qp91-phox subun...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N7/01C12N5/10A61K35/28A61P37/02
CPCC12N15/86C12N7/00C12N9/0036A61K35/28A61P37/02C12Y106/99001C12N2740/15021C12N2740/15043C12N2740/16043C12N2740/16051C07K14/80A61P37/00A61K48/005A61K38/44C12N2740/15032C12N2740/15052C12Y106/03
Inventor 王硕
Owner BEIJING MEIKANG JIMIAN BIOTECH CO LTD
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