Sphingosine kinase 1 and its fusion protein and application

A technology of sphingosine kinase and fusion protein, which is applied in the field of biopharmaceuticals, can solve the problems of drug resistance and long-term medication, and achieve weight loss and significant hypoglycemic effects

Active Publication Date: 2019-02-19
北京华奥玄德生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The other is to inject the SPHK1 gene into cells for gene therapy, such as gene therapy using adenovirus as a carrier. However, this method is easy to produce drug resistance in the body, and it takes a long time to treat metabolic diseases such as diabetes. medication, which limits the use of this method

Method used

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  • Sphingosine kinase 1 and its fusion protein and application
  • Sphingosine kinase 1 and its fusion protein and application
  • Sphingosine kinase 1 and its fusion protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of fusion protein SPHK1-Fc

[0039]1. Construction of lentiviral expression vector pCDH-SPHK1-L-Fc containing fusion protein SPHK1-Fc

[0040] Wherein, sphingosine kinase 1 or its active amino acid sequence comprises such as SEQ ID NO: 1;

[0041] MDPAGGPRGVLPRPCRVLVLLNPRGGKGKALQLFRSHVQPLLAEAEISFTLMLTERRNHARELVRSEELGRWDALVVMSGDGLMHEVVNGLMERPDWETAIQKPLCSLPAGSGNALAASLNHYAGYEQVTNEDLLTNCTLLLCRRLLSPMNLLSLHTASGLRLFSVLSLAWGFIADVDLESEKYRRLGEMRFTLGTFLRLAALRTYRGRLAYLPVGRVGSKTPASPVVVQQGPVDAHLVPLEEPVPSHWTVVPDEDFVLVLALLHSHLGSEMFAAPMGRCAAGVMHLFYVRAGVSRAMLLRLFLAMEKGRHMEYECPYLVYVPVVAFRLEPKDGKGVFAVDGELMVSEAVQGQVHPNYFWMVSGCVEPPPSWKPQQMPPPEEPL;

[0042] Its coding nucleotide sequence is shown in SEQ ID NO:5;

[0043] ATGGACCCAGCGGGCGGCCCCCGGGGCGTGCTCCCGCGGCCCTGCCGCGTGCTGGTGCTGCTGAACCCGCGCGGCGGCAAGGGCAAGGCCTTGCAGCTCTTCCGGAGTCACGTGCAGCCCCTTTTGGCTGAGGCTGAAATCTCCTTCACGCTGATGCTCACTGAGCGGCGGAACCACGCGCGGGAGCTGGTGCGGTCGGAGGAGCTGGGCCGCTGGGACGCTCTGGTGGTCATGTCTGGAGACGGGCTGAT...

Embodiment 2

[0066] Example 2 Preparation of Lentiviral Particles Carrying Plasmid pCDH-SPHK1-L-Fc

[0067] Inoculate 293T cells (Lab217 Embryo Engineering Laboratory of Northeast Agricultural University) with a cell confluence of more than 90% into 150mm culture dishes, and inoculate 1.2×10 cells per dish. 7 1 cell, add 20ml DMEM medium containing 10% FBS, 37°C, 5% CO 2 Cultivate in saturated humidity. 2 hours before transfection, the original medium was discarded and replaced with 18ml serum-free DMEM medium. Mix the p-SPHK1-L-Fc plasmid prepared above with lentiviral-packaged helper plasmids pHelper1 and pHelper2 (Lab217 Embryo Engineering Laboratory of Northeast Agricultural University) in equal proportions, and refer to Lipofectamin 2000 transfection kit (purchased from Invitrogen) instructions to transfect 293T cells. Discard the supernatant containing the transfection mixture 6-8 hours after transfection, add 20ml of new DMEM medium containing 5% FBS to each dish, and store at ...

Embodiment 3

[0068] Example 3 Screening and verification of lentivirus infection of CHO-S cells and positive monoclonal

[0069] 3.1 Lentivirus infection of CHO-S cells

[0070] Suspended FreeStyle CHO-S cells (purchased from Thermo scientific company) were mixed with 2×10 5 Cells / mL were inoculated in a 125 mL shake flask (purchased from Corning) containing 30 mL of CD-SFM medium (CD FortiCHO medium+8mM glutamine+1×HT supplement, both purchased from Thermo scientific company), 120rpm, 8% CO2, cultured at 37°C until the logarithmic growth phase, the cells were diluted with CD-SFM medium to 4×10 4 cells / mL of cell suspension. Take 0.5 mL of cell suspension, add the above-mentioned lentiviral particles according to the multiplicity of infection (MOI) of 80, and centrifuge at 32°C and 800 g for 30 min. Discard the supernatant, re-add 0.5mL CD-SFM medium to resuspend the cells, transfer to a 24-well plate, and store at 37°C, 5% CO 2 After culturing for 48-72 hours under certain condition...

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PUM

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Abstract

The invention belongs to the technical field of biomedicine and particularly relates to sphingosine kinase 1 and its fusion protein and application, namely sphingosine kinase 1 and application of an active amino acid sequence having its activity in the preparation of protein drugs to prevent and / or treat obesity, hyperlipemia or diabetes mellitus. The invention also provides a protein drug comprising sphingosine kinase 1 or an ammino acid sequence having its activity. The invention also provides application of the protein drug, an encoding gene, an expression construction and a host cell in the preparation of drugs to prevent and / or treat obesity, hyperlipemia or diabetes mellitus. It is discovered herein that sphingosine kinase 1 and its fusion protein have significant glucose-lowering and weight-reducing effects and are applicable to the preparation of protein drugs to control obesity and other metabolic diseases as well as diabetes mellitus.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals. Specifically, the present invention relates to the use of sphingosine kinase 1, more specifically, the present invention relates to a fusion protein of sphingosine kinase 1, especially a fusion protein containing sphingosine kinase 1 and FC sequences and its use. Background technique [0002] Obesity and type 2 diabetes mellitus (T2DM) are one of the major public health issues plaguing modern society. Obesity and its accompanying insulin resistance are key factors in the pathogenesis of type 2 diabetes. According to surveys, 80-90% of type 2 diabetes patients are overweight or obese (Zou Dajin et al., Shanghai Medicine, 2014, 37(9), 729-734). Therefore, effective control of blood sugar and body weight has always been a focus topic in related research fields. According to statistics, the global incidence of type 2 diabetes has reached 400 million, accounting for 90%-95% of all diabetic patient...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/867C12N5/10A61K38/45A61P3/04A61P3/10
CPCA61K38/45A61P3/04A61P3/10C12N9/1205C12N15/86C12Y207/01091C07K2319/30C12N2740/16043C12N2740/15043A61K38/2013A61K47/68A61K47/60C07K14/55C07K2319/02
Inventor 段海峰解晶胡显文弓景波薛冰华张群伟肖秀孝崔美兰庞如梦王瑞于婷婷
Owner 北京华奥玄德生物医药科技有限公司
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