Fusion gene betaTrCP-CypA with HIV-1 inhibition effect and construction method thereof

A technology of fusion gene and construction method, applied in gene therapy, fusion polypeptide, chemical instruments and methods, etc., can solve problems such as inability to clear viruses, virus rebound, etc.

Active Publication Date: 2017-07-21
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since it can only inhibit the replication of HIV-1 and cannot clear the virus infected in ce

Method used

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  • Fusion gene betaTrCP-CypA with HIV-1 inhibition effect and construction method thereof
  • Fusion gene betaTrCP-CypA with HIV-1 inhibition effect and construction method thereof
  • Fusion gene betaTrCP-CypA with HIV-1 inhibition effect and construction method thereof

Examples

Experimental program
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Effect test

experiment example 1

[0041] Construction of pcDNA3.1his / betaTrCP-CypA According to the N-terminal sequence of human betaTrCP gene (GenBank accession number: NM_003939), the N-terminal sequence of betaTrCP gene was optimized by using DNASTAR and others. After optimization, Shanghai Sangon Bioengineering Co., Ltd. was commissioned to synthesize, and Premier5.0 software was used to design primers, and B1 and B2 were used as primers to amplify the part of the betaTrCPN end, and PrimeSTAR HS DNA polymerase was added to a 50 µl system. Amplification conditions are: pre-denaturation at 98°C for 15s, then 98°C for 10s, 58°C for 15s, and 72°C for 60s, 28 cycles, use CypAcDNA plasmid as template, use B3 and B4 as primers to amplify CypA part, add PrimeSTAR HS DNA polymerase enzyme to 50µl system. Amplification conditions are: pre-denaturation at 98°C for 15s, then 98°C for 10s, 58°C for 15s, and 72°C for 30s, 28 cycles. As a result, the target bands were amplified, and the sizes were about 800bp and 500bp r...

experiment example 2

[0044] Western blot detection of expression of betaTrCP-CypA, CypA recombinant protein

[0045] 293T cells were cultured, and the day before transfection, 293T cells were mixed with 1×10 6 The density of cells / well was cultured in 6-well plates. Day 2, with Entranster TM -D4000 Transfection Reagent Transfer 2 µg of pcDNA3.1his / betaTrCP-CypA and pcDNA3.1his / CypA (ratio 3:1) into each well respectively, and transfect 293T cells with pcDNA3.1his empty vector as a control. The fresh medium was replaced on the second day after transfection, and the cells were collected 48 hours later to extract the total protein of the cells. The BCA protein concentration assay kit was used for protein quantification and then detected by Western blot. After the protein was subjected to 12% SDS-PAGE, it was transferred to PVDF membrane, and after blocking, his-tag monoclonal antibody (pcDNA3.1his / betaTrCP-CypA and pcDNA3.1hisCypA with His tag) were added for incubation, and HRP-labeled goat anti...

experiment example 3

[0047] Degradation analysis of HIV-1Gag by betaTrCP-CypA 293T cells were inoculated into six-well plates one day before transfection, and plasmid pSPA X 2 Co-transfection with pcDNA3.1his / betaTrCP-CypA and plasmid pcDNA3.1his. Refer toEntranster TM -D4000 Transfection Reagent instructions. The experiment was divided into 3 groups: (1) Experimental group pSPA X 2 0.4µg and pcDNA3.1his / betaTrCP-CypA2µg; (2) CypA control group pSPA X 2 0.4 µg with pcDNA3.1 / CypA2 µg, (3) control group pSPAX 2 0.4µg and pcDNA3.1his2µg, collect the cells 48 hours after transfection: absorb the culture medium, wash once with 1 mL pre-cooled PBS, and absorb the PBS; lyse the cells with Biyuntian IP and western cell lysate, and lyse Add PMSF to the solution so that the final concentration of PMSF is 1 mmol / L; take 100 µL of cell lysate and mix with the harvested cells, centrifuge at 12 000 r / min at 4°C for 30 min, collect the supernatant, and use the BCA protein concentration assay kit for protein ...

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Abstract

The invention relates to a fusion gene betaTrCP-CypA with HIV-1 inhibition effect and a construction method thereof. The fusion gene betaTrCP-CypA can inhibit HIV-1 virus, and has a gene sequence shown as SEQ ID NO:1. The gene construction method includes the steps of: performing artificial synthesis to obtain an optimized betaTrCP gene N-terminal DNA; using the betaTrCP gene N-terminal DNA sequence as the template, and adopting B1 and B2 as the primers to amplify a betaTrCP gene N-terminal DNA fragment; taking CypAcDNA plasmid as the template, and using B3 and B4 as the primers to amplify the CypA part so as to obtain a CypA DNA fragment; respectively taking 1microL of a betaTrCP gene N-terminal part PCR product fragment and 1microL of a CypA DNA PCR product fragment as the template, using B1 and B4 as the primers to perform PCR amplification of the fusion gene betaTrCP-CypA, wherein the middle part is DNA containing 5 serine/glycine, thus obtaining a target band, and cloning the fusion gene into an appropriate expression vector for expression. The fusion gene betaTrCP-CypA constructed by the method provided by the invention can degrade HIV-1Gag, thereby reaching the effect of inhibiting HIV-1.

Description

technical field [0001] The invention relates to a fusion gene, in particular to a fusion gene betaTrCP-CypA capable of inhibiting HIV-1 and its construction method. Background technique [0002] Human immunodeficiency virus (Human immunodeficiency virus, HIV), commonly known as AIDS (Acquiredme deficiency syndrome, AIDS) virus, induces human acquired immunodeficiency syndrome. Since the first report in 1984 that HIV (HIV-1) was the cause of AIDS (AIDS), a large number of top scientists in the world have devoted themselves to conquering this disease. Therapies such as HAART have significantly reduced AIDS incidence and mortality. However, since it can only inhibit the replication of HIV-1 and cannot clear the virus infected in the cells, it needs long-term uninterrupted medication, and once the drug is stopped, the virus will rebound. Long-term and large-scale use of antiviral drugs has brought a series of problems to patients, such as some drugs have side effects such as m...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/10A61K48/00A61P31/18
CPCA61K48/005C07K14/47C07K2319/00C12N9/90C12N15/10C12Y502/01008C12Q2531/113
Inventor 孟祥平杨建英乔晓岚白雪飞梁高峰冯文坡郑军周为
Owner HENAN UNIV OF SCI & TECH
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