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siRNA sequence for targeted inhibition of human alpha-globin gene expression

A technology of gene expression and globin, which is applied in the fields of molecular biology and biomedicine, can solve problems such as red blood cell destruction, unbalanced synthesis ratio of α and non-α globin chains, and influence on hemoglobin synthesis, so as to facilitate inhibition and improve synthesis imbalance Effect

Inactive Publication Date: 2015-04-01
广东省计划生育科学技术研究所
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the synthesis of β-globin chain is insufficient or cannot be synthesized at all, the synthesis ratio of α-globin chain and non-α-globin chain is unbalanced, which affects the synthesis of normal hemoglobin; in addition, due to the relative excess of α-globin chain, the remaining α-globin chain Protein chains form inclusion bodies in erythrocytes, resulting in oxidative damage to erythrocyte membranes, resulting in destruction of erythrocytes and ineffective hematopoiesis of bone marrow

Method used

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  • siRNA sequence for targeted inhibition of human alpha-globin gene expression
  • siRNA sequence for targeted inhibition of human alpha-globin gene expression

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Example 1 siRNA sequence design

[0022] According to the bioinformatics method, the human α-globin mRNA sequence (V00489.1) was obtained from genebank, and the secondary structure of the human α-globin 1 mRNA sequence was predicted by RNA structure analysis software, combined with the siRNA design principles, the comprehensive analysis was carried out. The siRNA sequence was designed according to the requirements of GC content, free energy at both ends, secondary structure of the mRNA action site, and base preference of siRNA.

[0023] According to the software, the designed siRNA series were evaluated, and the BLAST software provided by NCBI GenBank (written by the National Library of Medicine and the National Institutes of Health) was further used to analyze the homology of the selected target sequences and exclude non-specific siRNAs. Inhibiting the possibility of other gene fragments, a set of siRNA sequences with the best specificity was finally screened out, wh...

Embodiment 2

[0028] Example 2 Construction and Identification of RNAi Interference Vector pLKO.1

[0029] 1. Entrust Shanghai Bioengineering Company to synthesize the above siRNA sequence.

[0030] 2. Construction of gene recombinant lentiviral vector

[0031] First use the pLKO.1 vector with EcoR I and Nco I digestion, and then the two fragments of the synthetic siRNA sequence with the same EcoR I and Nco I digestion, and then slow annealing of the two fragments, using EcoR I and Nco The I restriction site was connected to the carrier, and the ligation reaction mixture was transformed into Escherichia coli DH5α, and several single clones were picked and inoculated in LB medium containing ampicillin, and shaken overnight at 37°C.

[0032] 3. Identification

[0033] Take 5 mL of the bacterial liquid, use the QIAGEN plasmid mini-extraction kit to extract a small amount of the recombinant vector plasmid, use EcoR I and Nco After I performed double digestion, the recombin...

Embodiment 3

[0035] Example 3 Lentivirus packaging and titer determination

[0036] Using the gene recombinant lentiviral vector constructed in Example 2, the lentiviral packaging was carried out in HEK 293T cells, and the terminal dilution method was used to verify the lentiviral packaging and detect the infectious titer of the virus.

[0037] 1. Lentiviral packaging

[0038] HEK 293T cells were routinely cultured in DMEM high-glucose medium containing 10% FBS. One day before transfection, take cells in good logarithmic phase, trypsinize and adjust the cell concentration to 5×10 5 / mL, inoculate 2 mL of cell suspension in each well of a 6-well plate.

[0039]During the transfection, observe that the cells were 80-90% confluent. According to the instructions of LipofectaminTM2000, co-transfect the three plasmids of psPAX2, pMD2.G and lentiviral vector into 293T cells. After 48 hours of transfection, the supernatant of the cells was collected and centrifuged at 3000 r / min at 4 °C. For ...

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Abstract

The invention provides a siRNA sequence for targeted inhibition of human alpha-globin gene expression. The siRNA sequence is a HBA2 siRNA1 sequence and comprises a sense strand and an antisense strand; the nucleotide sequence of the sense strand is shown by SEQ ID No.1; and the nucleotide sequence of the antisense strand is shown by SEQ ID No.2. The siRNA sequence can specifically and efficiently inhibit the expression of the alpha-globin gene so as to improve the synthesis imbalance of alpha / beta globin of beta thalassemia sufferers and lay a foundation for establishing a medicine and technology system for treating beta thalassemia through siRNA. Moreover, the siRNA-mediated RNAi technology is efficient, specific and convenient and has a good application prospect.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and biomedicine. More specifically, it relates to a siRNA sequence that targets and inhibits the expression of human α-globin gene. Background technique [0002] Thalassemia (thalassemia, referred to as thalassemia) is one of the most common single-gene genetic diseases in the world, which is the most harmful to human health. The molecular genetic basis of β-thalassemia (β-thalassemia, referred to as β-thalassemia) is due to the deletion or mutation of the β-globin gene, which leads to hemolytic anemia caused by the disorder of β-globin chain synthesis. Because the synthesis of β-globin chain is insufficient or cannot be synthesized at all, the synthesis ratio of α-globin chain and non-α-globin chain is unbalanced, which affects the synthesis of normal hemoglobin; in addition, due to the relative excess of α-globin chain, the remaining α-globin chain Protein chains form inclusion bod...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/713A61P7/06
Inventor 赵文忠李铭臻王柏贤周冰燚徐珊珊朱志勇顾恒
Owner 广东省计划生育科学技术研究所
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