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Beta-globin recombinant lentiviral vector and application thereof

A technology of recombinant lentivirus and globin, applied in the field of molecular biology

Active Publication Date: 2017-07-25
济南赛尔生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows for creating highly effective and stable viral systems that are able to deliver genes into red blood cell (RBC) neurons or bone marrow stroma cells through insertion within them. These systems have been developed from different sources such as pseudorabies strain BCG-19, which has two copies of beta globin protein - one behind each others called CYF1A 1 , another overlapping version of this molecule found naturally occurring in certain plants like rubber trees. By controllably introducing these sequences into various types of mammals' embryonic bodies it becomes possible to produce therapeutic agents against diseases caused by inherited abnormalities associated with alpha fetuses.

Problems solved by technology

This patents describes various technical problem areas associated with studying beta - gamma hemoglobodies (HBgs). These include understanding the functions of beta-hemolysin and alpha subunit(a type called αsubhydroxyglutaminyl transferring carboxypeptid receptor involved in signal transmission. Additionally, identifying key players controlling certain aspects during cellular processes like embryonic growth and neuronal communication involves integrative loop components containing several essential parts. To improve efficiency and accuracy, we propose introducing new ones by combining them together.

Method used

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  • Beta-globin recombinant lentiviral vector and application thereof
  • Beta-globin recombinant lentiviral vector and application thereof
  • Beta-globin recombinant lentiviral vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Acquisition of specific β-globin gene promoter sequence

[0038] 1. Use anticoagulant-containing anticoagulant tubes to collect normal human peripheral blood, and use the Chelex-100 method to extract total DNA (see the blood DNA extraction kit instructions).

[0039] 2. Design specific primers according to the existing Primer Premier 5 software as follows:

[0040] Upstream primer 5'- GGGTACCCC GCTCCAGATAGCCATAGAAG-3'

[0041] KpnI

[0042] Downstream primer 5'- CTGCAG AAGCAATAGATGGCTCTGCCC-3'

[0043] PstI

[0044] 3. Carry out PCR amplification according to the reaction system in Table 1.

[0045] Table 1

[0046]

[0047]

[0048] 4. Use LightCycler 96 PCR instrument (Bio-Rad, USA) to carry out PCR reaction. The reaction system is 94°C pre-denaturation for 5 minutes; After 10 min, the reaction was terminated at 4°C.

[0049] 5. Use 1% agarose gel electrophoresis to detect and recover the PCR amplification product (see the instructions of th...

Embodiment 2

[0051] Example 2: Acquisition of exon Ⅰ, intron IVS-1 and exon Ⅱ sequences of β-globin gene

[0052] 1. Collect peripheral blood from normal people, and extract total DNA using the Chelex-100 method (see the instructions of the blood DNA extraction kit).

[0053] 2. Use Primer Premier 5 software to design specific primers as follows:

[0054] Upstream primer 5'- CTGCAG ACATTTGCTTCTGACACA-3'

[0055] PstI

[0056] Downstream primer 5'- AAGCTT CCTGAAGTTCTCAGGATC-3'

[0057] Hind III

[0058] 3. Carry out PCR amplification according to the reaction system in Table 1.

[0059] 4. Bio-Rad C1000 PCR instrument (Bio-Rad, USA) was used for PCR reaction. The reaction system was pre-denaturation at 94°C for 5min; After renaturation for 10 minutes, the reaction was terminated at 4°C.

[0060] 5. Use 1% agarose gel electrophoresis to detect and recover the PCR amplification product (see the instructions of the DNA fragment recovery kit from the agarose gel).

[0061] 6. The PCR...

Embodiment 3

[0062] Example 3: Acquisition of β-globin gene intron IVS-2 sequence

[0063] 1. Collect peripheral blood from normal people, and extract total DNA using the Chelex-100 method (see the instructions of the blood DNA extraction kit).

[0064] 2. Use Primer Premier 5 software to design specific primers as follows:

[0065] Upstream primer 5'- AAGCTT GTGAGTCTATGGGACGCTT-3'

[0066] Hind III

[0067] Downstream primer 5'- CCCGGG CTGTGGGAGGAAGATAAGAG-3'

[0068] AvaI

[0069] 3. Carry out PCR amplification according to the reaction system in Table 1.

[0070] 4. Bio-Rad C1000 PCR instrument (Bio-Rad, USA) was used for PCR reaction. The reaction system was pre-denaturation at 94°C for 5min; After renaturation for 10 minutes, the reaction was terminated at 4°C.

[0071] 5. Use 1% agarose gel electrophoresis to detect and recover the PCR amplification product (see the instructions of the DNA fragment recovery kit from the agarose gel).

[0072] 6. Digest with RsaI / RsaI (GT^A...

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Abstract

The invention discloses a beta-globin recombinant lentiviral vector. A gene sequence of the vector comprises a modified beta-globin full-length gene, a beta-globin gene control region HS2-HS3, a beta-globin gene 5' promoter, a beta-globin gene 3' enhancer, a chromatin insulator CTCF and pSIN4-CMV-K2M plasmids; and a nucleotide sequence is as shown in SEQ ID No.1. The invention further discloses an application of the recombinant lentiviral vector in transfection of hematopoietic stem/progenitor cells to obtain normal function beta-globin. An experiment proves that specific, efficient and stable expression of the lentiviral vector in erythroid cells can be achieved by using a vector expression system, pseudoviral particles with relatively high virus titer are obtained and the normal function beta-globin is obtained after transfection of the hematopoietic stem/progenitor cells.

Description

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Claims

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Application Information

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Owner 济南赛尔生物科技股份有限公司
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