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Promoter for driving specific expression of gene in human erythrocyte system and application

A human red blood cell, driving gene technology, applied in the field of genetic engineering, can solve the problems of limited improvement of HBG level, unstable treatment effect, and increased platelet reactivity.

Active Publication Date: 2021-01-15
GUIZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These drugs all act on the gene expression that inhibits HBG expression, and reactivate HBG expression, but the curative effect is low, the treatment effect is unstable, the long-term prognosis of patients is poor, the increase of HBG level is limited, and the platelet reactivity can also be increased [11-13] And other issues

Method used

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  • Promoter for driving specific expression of gene in human erythrocyte system and application
  • Promoter for driving specific expression of gene in human erythrocyte system and application
  • Promoter for driving specific expression of gene in human erythrocyte system and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] 1. Predict the promoter core regions of AHSP and GYPA from http: / / www.genomatix.de / , and finally select the fragments AHSP-439bp; GYPA-416bp and GYPA-1378bp.

[0049] 2. Select a single restriction site on the upstream and downstream of the backbone plasmid promoter, the upstream is Vsp I, and the downstream is Hind III. The upstream and downstream primers were designed according to the NCBI gene sequence and the predicted promoter region, and restriction sites and protective bases were added at the 5' end (Table 1).

[0050] Table 1 Cloning primer information of the promoter

[0051] Primer name sequence SEQ ID NO: AHSP 439bp FP GCATTAATGGCTCTTGCCTTCTTGCATTTC 4 AHSP 439bp RP TCCAAGCTTCTGGGTAGAGAAAAGGGTAGA 5 GYPA 416bp FP GCATTAATTAGCAACCTGTTCCTTGCAG 2 GYPA 416bp RP GCCAAGCTTGACTGGAAGAGGAAATACTACTC 3 GYPA 1378bp FP CCATTAATGAGGCATTCTGGATTCTTGTCC 6 GYPA 1378bp RP GCTAAGCTTCAGACTGGAAGAGGAAATAC 7

[0052] 3. ...

Embodiment 2

[0106]2.1 Design more fragments of the AHSP promoter, which are respectively p-AHSP-EGFP (358bp), numbered 1004; p-AHSP-EGFP (367bp), numbered 1005; p-AHSP-EGFP (529bp), numbered 1006. The primer information is shown in Table 4:

[0107] Table 4 Primer Information

[0108]

[0109] 2.2. Repeat the above plasmid construction steps (results see figure 1 ) and the transfection protocol to compare the optimal promoter length. Such as Figure 3F with Figure 3G As shown, the 1004 plasmid was not active in both HEL and Ramos cells, so no statistical analysis was performed. Although 1005 and 1006 are expressed in the erythroid, they are more active in Ramos cells and do not have erythroid specificity ( Figure 3H ).

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Abstract

The invention provides a promoter for driving specific expression of a gene in a human erythrocyte system and application, and relates to the technical field of gene engineering. The promoter has a nucleotide sequence as shown in SEQ ID NO. 1. The promoter has functions of specifically interfering expression of a certain gene in erythroid and researching specific expression of the certain gene inthe erythroid. EGFP expression plasmids driven by promoters of different lengths are constructed by gene recombination technology, after the plasmids are introduced into HEL and Ramos cells, fluorescence expression is observed, and results show that there is no difference in activities of 1001 and 1003 in HEL cells, but the activity of 1003 is significantly higher than that of 1001 in Ramos cells,confirming that GYPA-416bp (1001) has erythroid specificity. The promoter provided by the invention has important clinical significance in directional gene therapy of erythroid system diseases and research of erythroid genes.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a promoter for driving gene specific expression in human erythrocyte system and its application. Background technique [0002] The promoter is the core component of gene function, which is usually located upstream of the gene transcription start site, and plays an important role in recruiting transcription factors and RNA polymerase. Promoters have the characteristic of selective expression during cell development, and cells will specifically express some promoters according to their developmental stages to drive cells to perform specific functions. Therefore, the promoter specifically regulates gene expression [1] , carrier transport [2] and specific interference [3] can have a wide range of applications. [0003] The carrier rate of abnormal β-globin gene in Guizhou Province is as high as 5.4% [4] , a high incidence area of ​​β-thalassemia. The gol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/10C12N15/85A61K48/00A61P7/00A61P7/06
CPCC07K14/4702C12N15/85A61K48/0058A61P7/00A61P7/06
Inventor 张鹏刘含何志旭范安然周艳华杨红兰
Owner GUIZHOU MEDICAL UNIV
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