Human beta-globin gene and recombinant adeno related viral vector thereof

A technology of β-globin and viral vectors, applied in the field of biomedicine, can solve the problems of small vector capacity, insufficient site-specific integration, and size limitation of target gene fragments, etc.

Active Publication Date: 2009-01-21
DONGGUAN ZHENGXING BEITE MEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has recently been found that this type of vector has many obvious defects in the application, including a very small number of virus receptors on some cell membranes, insufficient site-specific integration of recombinant AAV vectors, and AAV capsid components and transgene products causing host immunity. reaction, the carrier capacity is small (Curr.Top.Microbiol.Immunol.1996, 218:1-23; Proc.Natl.Acad.Sci USA, 1990,87:2211-2215), the size of the loaded target gene fragment is limited and many more

Method used

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  • Human beta-globin gene and recombinant adeno related viral vector thereof
  • Human beta-globin gene and recombinant adeno related viral vector thereof
  • Human beta-globin gene and recombinant adeno related viral vector thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of recombinant adeno-associated virus vector pAAV-HS234-hβ-globin

[0033] (1) HS234 fragment:

[0034] According to the existing fragment sequences of HS2, HS3 and HS4 (Nucl Acid Res 1994.22: 1006-1011; EMBO J 1993.12: 1077-1085; Nucl Acid Res 1990.9: 2159-2167; Nucl Acid Res 1991.19: 1413-1419; PNAS 1995.92: 67 6732) designed primers ①, ② and ③ respectively.

[0035] Primer ①P5: 5'-ttaaataagcttcagtttttccttagttcc-3' (as shown in SEQ ID NO.5),

[0036] P3: 5'-tctagaatatgtcacattctgtct-3' (as shown in SEQ ID NO.6);

[0037] Primer ②P5: 5'-tggtgtgccagatgtgtctatcag-3' (as shown in SEQ ID NO.7),

[0038] P3: 5'- gatatc gctgctatgctgtgcctccccca-3' (as shown in SEQ ID NO.8);

[0039] EcoRV

[0040] Primer ③P5: 5'-ggaccccagtacacaagaggggacgca-3' (as shown in SEQ ID NO.9),

[0041] P3: 5'- gatatc ggaatgggaggggagagtctctg-3' (shown in SEQ ID NO. 10).

[0042] EcoRV

[0043] From the human β-globin gene Genomic DNA extracted from normal human periph...

Embodiment 2

[0053] Example 2 Obtaining of Purified Recombinant Adeno-Associated Virus Vector rAAV / β-globin

[0054] HEK293 cells were used as packaging cells, subcultured with DMEM medium and 10% newborn bovine serum, the cells grew to 80%-85% confluent, and the pAAV-HS234-β-globin prepared in Example 1 was purified by calcium phosphate co-precipitation method Co-transfect HEK293 cells with helper plasmids pAAV2-RC and pHelper, each plasmid 10μg / dish (10cm), take 5ml of 1.5mol / L CaCl 2 , add water to 20ml, mix well, add 2xBuffer A (50mM Hepes, 1.5mM Na 2 HPO 4 , 280mM NaCl, pH 7.05) 25ml, mix well and let it stand for 5 minutes, when a white cloud appears, the transfection of 293 cells is started, 2ml / dish, and continue to be placed in a carbon dioxide incubator with 5% CO 2 , 37°C and saturated humidity for 65-70 hours, collect the cells and store them in a -70°C refrigerator. Next, purify, take out the frozen cells from -70°C and put them in a 37°C water bath for 30 minutes, freeze a...

Embodiment 3

[0056] Example 3 Detection of the expression of the introduced exogenous β-globin gene in K562 cells by PCR and RT-PCR

[0057] Under sterile conditions, take 12×10 6K562 cells suspended in RPMI 1640 culture medium containing 10% newborn bovine serum and growing well were divided into control groups without pretreatment (8×10 6 cells) and group pretreated with hydroxyurea (4×10 6 Cells), the unpretreated control group added an appropriate amount of solution 1×PBS for dissolving hydroxyurea; 2 , 37°C, and cultured in saturated humidity for 4 hours, collect the cells respectively, and wash the cells once with 1×PBS. 16 μl of RPMI 1640 culture solution was added to the control group, and 16 μl (equivalent to 50 MOI) of the recombinant adeno-associated virus vector rAAV / β-globin obtained in Example 2 were added to the experimental group and the pretreatment group, and cultured under the above conditions, every 10- Shake once every 15 minutes. After 2 hours of transfection, wash...

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Abstract

The invention discloses a human beta globin gene containing a human beta globin gene promoter, a recombinant adeno-associated virus vector containing the human beta globin gene, and a method for preparing the recombinant vector. The recombinant adeno-associated virus vector inserts an HS2 segment, an HS3 segment and an HS4 segment of a human beta globin gene cluster enhancer core sequence and a human beta globin gene sequence containing the human beta globin gene promoter into repeated sequence ITRs on the reverse terminal of an adeno-associated virus. The recombinant vector has high transfection efficiency; and mediated exogenous genes can be expressed in vivo for a long time, have good safety and can be used for gene therapy of beta Mediterranean anemia.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a human β-globin gene containing a human β-globin gene promoter and a recombinant adeno-associated virus vector containing it, as well as a preparation method of the recombinant vector and gene therapy for β-thalassemia in the application. Background technique [0002] Beta thalassemia (thalassemia) is a group of inherited blood disorders characterized by reduced or absent synthesis of the beta globin peptide chain of hemoglobin. The incidence of the disease is as high as 10% in the Mediterranean Basin and Southeast Asia. The detection rate in my country's Guangdong and Guangxi regions can reach 3-4%. Severe β-thalassemia seriously threatens the lives of children. At present, clinical treatment mainly relies on blood transfusion, and long-term blood transfusion can easily cause excessive iron load in the body. Excessively absorbed iron exceeds the capacity of serum transferrin to dep...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/861A61K48/00A61K35/12A61P7/06A61K35/28
Inventor 谭孟群
Owner DONGGUAN ZHENGXING BEITE MEDICINE TECH CO LTD
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