A fusion gene betatrcp-cypa capable of inhibiting HIV-1 and its construction method

A technology of betatrcp-cypa and HIV-1, applied in the field of fusion genes, can solve the problems of inability to clear the virus, virus rebound, etc.

Active Publication Date: 2020-05-05
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 cells, long-term uninterrupted medication is required. Once the drug is stopped, the virus will rebound.

Method used

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  • A fusion gene betatrcp-cypa capable of inhibiting HIV-1 and its construction method
  • A fusion gene betatrcp-cypa capable of inhibiting HIV-1 and its construction method
  • A fusion gene betatrcp-cypa capable of inhibiting HIV-1 and its construction method

Examples

Experimental program
Comparison scheme
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), DNASTAR was used to optimize the N-terminal sequence of betaTrCP gene. After optimization, it was entrusted to Shanghai Sangon Bioengineering Co., Ltd. to synthesize the primers. Primers were designed with Premier5.0 software, and B1 and B2 were used as primers to amplify the end of betaTrCPN, and PrimeSTAR HS DNA polymerase was added to the 50µl system. Amplification conditions were: pre-denaturation at 98°C for 15s, then 98°C for 10s, 58°C for 15s, and then 72°C for 60s, 28 cycles, using CypAcDNA plasmid as template, using B3 and B4 as primers to amplify the CypA part, adding PrimeSTAR HS DNA polymerase enzyme etc. to 50µl system. The amplification conditions were: 98°C for 15s, then 98°C for 10s, 58°C for 15s, and then 72°C for 30s, 28 cycles. The results were to amplify the target bands with sizes of about 800bp and 500bp, resp...

experiment example 2

[0044] Western blot detection of the expression of betaTrCP-CypA and CypA recombinant proteins

[0045] The 293T cells were cultured, and the 293T cells were grown at 1 × 10 the day before transfection. 6 The density of cells / well was plated in 6-well plates. Day 2 with Entranster TM -D4000 Transfection Reagent 2µg each of pcDNA3.1his / betaTrCP – CypA and pcDNA3.1his / CypA (ratio of 3:1) were transfected into each well respectively, and 293T cells were transfected with pcDNA3.1his empty vector as a control. The medium was replaced with fresh medium on the 2nd day after transfection, and the cells were collected 48 hours later, and the total cell protein was extracted. BCA protein concentration assay kit was used for protein quantification and Western blot detection. After the protein was subjected to 12% SDS-PAGE, it was transferred to PVDF membrane, and after blocking, his-tag monoclonal antibodies (pcDNA3.1his / betaTrCP-CypA and pcDNA3.1hisCypA containing His-tag) were adde...

experiment example 3

[0047] Degradation analysis of HIV-1Gag by betaTrCP-CypA 293T cells were seeded into six-well plates one day before transfection and treated with plasmid pSPA X 2 Co-transfected with pcDNA3.1his / betaTrCP-CypA and plasmid pcDNA3.1his. Refer to Entranster 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 0.4µg with pcDNA3.1 / CypA2µg, (3) control group pSPA X 2 0.4µg and pcDNA3.1his2µg, harvest the cells 48 hours after transfection: aspirate the culture medium, wash once with 1 mL of pre-cooled PBS, and aspirate the PBS; lyse the cells with Biyuntian IP and western cell lysis buffer, lyse the cells PMSF was added to the solution so that the final concentration of PMSF was 1 mmol / L; 100 µL of cell lysate was mixed with the harvested cells, and the supernatant was collected after centrifugation at 12 000 r / min at 4°C for 30 min. The BCA pro...

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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 which has the effect of inhibiting HIV-1 and a construction method thereof. 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 of HIV (HIV-1) as the pathogen causing AIDS (AIDS) in 1984, a large number of top scientists around the world have devoted themselves to conquering this disease. Treatments such as HAART have significantly reduced AIDS incidence and mortality. However, because it can only inhibit the replication of HIV-1 and cannot clear the virus infected in the cells, long-term uninterrupted medication is required. Once the drug is stopped, the virus rebounds. Long-term use of large amounts of antiviral drugs has brought a series of problems to patients, such as some drugs h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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