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A recombinant sequence specifically expressing human beta globin in erythroid cells and its application

A technology of β-globin and erythroid cells, applied in the field of genetic engineering, can solve the complex problems of β-globin expression and regulation

Active Publication Date: 2022-04-01
GUIZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above multi-center clinical studies have shown that more and more patients with β-thalassemia can benefit from gene therapy vectors with increased β-globin genes, indicating that the hematopoietic stem cell transplantation treatment strategy based on lentiviral vectors with increased β-globin genes is expected to be a cure for β-thalassemia. A new measure of thalassemia, but because the regulation of β-globin expression is very complex, how to obtain highly efficient β-globin expression sequence in vitro is crucial for the gene therapy of β-thalassemia

Method used

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  • A recombinant sequence specifically expressing human beta globin in erythroid cells and its application
  • A recombinant sequence specifically expressing human beta globin in erythroid cells and its application
  • A recombinant sequence specifically expressing human beta globin in erythroid cells and its application

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Experimental program
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Effect test

Embodiment 1

[0046] Construction of promoter plasmids with different lengths and determination of the most active promoter

[0047] 1. Predict the core region of the β-globin promoter from the website http: / / www.genomatix.de / , and combine the literature search results with the previous promoter lengths in different vectors, and finally select the first 152bp and 266bp of β-globin exon 1 and 422bp fragments.

[0048] 2. Select a single enzyme cutting site on the upstream and downstream of the pEGFP-N1 backbone plasmid promoter, the upstream is Vsp I, and the downstream is NheI. Design upstream and downstream primers according to the NCBI gene sequence and the predicted promoter region, and add restriction sites and protective bases at the 5' end, synthesize the primer sequence, the upstream primer FP (Forward Primer), and the common downstream primer named Exon- RP (Reverse Primer), as shown in Table 1.

[0049] Table 1 List of primers for amplification of the 3 promoter fragments

[005...

Embodiment 2

[0132] Using the screened promoter length to construct a plasmid without the LCR (HS) locus control region of the β-globin gene

[0133]1. Predict the enhancer core region of β globin from http: / / www.genomatix.de / website, combine the literature search results with the length of promoters in different vectors in the past, and start the amplification from the promoter with the best activity (No. 1 Exon 266bp) to the β-globin enhancer without β-globin locus control region LCR (HS), named: pβp(Exon266 bp)-βglobin-βE-713, number 8019.

[0134] 2. Select a single restriction site in the backbone plasmid, upstream is Vsp I, downstream is NotI, design upstream and downstream primers according to the NCBI gene sequence and the predicted promoter region, and add a restriction site and protection at the 5' end base.

[0135] Table 5 The primer sequence information for the amplification of the promoter protected by the present invention

[0136]

[0137] 3. Extracting normal human b...

Embodiment 3

[0161] The effectiveness of the β-globin recombinant plasmid without LCR was detected, the recombinant plasmid was electrotransfected into K562 cells, and untransfected and blank transfected cells were set as the control group, the relative expression of β-globin mRNA was detected by qPCR, and the β-globin was detected by WB globin protein expression content, the results are as follows Figure 5 C. Figure 5 As shown in D, compared with the control group, there was no significant change in the expression of β-globin in both the mRNA level and the protein level compared with the blank group, and the difference was not statistically significant.

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Abstract

The invention provides an erythroid-specific human beta globin gene promoter, a recombinant sequence for expressing human beta globin and applications thereof, belonging to the technical field of genetic engineering. The present invention provides an erythroid-specific human β-globin gene promoter, the nucleotide sequence of which is shown in SEQ ID NO: 1, which has the characteristics of high-efficiency and erythroid-specific activation of functional gene expression. The present invention also provides a recombinant sequence specifically expressing human β-globin in erythroid cells, which is human β-globin gene locus control region HS3~1-β-globin gene promoter-β-globin gene-β-globin Gene enhancers. By constructing a recombinant vector expressing the recombinant sequence, it was verified in erythroid cells that the recombinant sequence can specifically express the human β-globin gene with high efficiency, which provides a basis and experimental reference for gene therapy of β-thalassemia, and is useful for the treatment of thalassemia. It is of great significance, and at the same time provides method support for the study of the expression regulation of β globin.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant sequence for specifically expressing human beta globin in erythroid cells and its application. Background technique [0002] β-thalassemia (Thalassemia) is a blood disorder caused by mutations in the gene cluster encoding β-globin. The Mediterranean Sea, Africa, Southeast Asia, the Indian mainland, Southwest my country, and South China are high-incidence areas. China's Guangxi, Guangdong, Fujian, Taiwan, Hong Kong, Yunnan, Guizhou, Hainan and other provinces are high-incidence areas. Thalassemia is a common genetic disease, which not only affects the life and health of patients, but also affects the health of the next generation. [0003] β-thalassemia is caused by mutations in the β-globin gene cluster located in subband 5, subband 4 (11p15.4) of the short arm of chromosome 11. The β-globin gene cluster contains five structural genes, β, δ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/12C12N15/85C12N15/867A61K48/00A61K38/42A61P7/06
CPCC07K14/805C12N15/85C12N15/86A61K48/0058A61K48/0008A61P7/06C12N2800/107C12N2740/15043C12N2830/008A61K38/00C12N2740/16043C12N2830/15
Inventor 张鹏杨红兰何志旭刘含周艳华范安然
Owner GUIZHOU MEDICAL UNIV
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