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An insulin-like growth factor binding protein 7 eukaryotic expression plasmid, and a constructing method and application thereof

A technology for expressing plasmids and growth factors, applied in peptide/protein components, biochemical equipment and methods, applications, etc., can solve the problems of easy inactivation, high clearance rate, large molecular weight, etc., and achieve improved bioavailability and low price , the effect of low molecular weight

Inactive Publication Date: 2015-07-22
XIANGYA HOSPITAL CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although IGFBP7 is expected to become a new targeting factor for the treatment of cholangiocarcinoma, its protein half-life is short, its clearance rate is high, it is insoluble in water, its molecular weight is large, it is easy to inactivate, easy to degrade, poor stability, and low bioavailability for non-injection administration. Problems such as low purification level and high price (1 g can reach more than 100,000 yuan) greatly limit its clinical application

Method used

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  • An insulin-like growth factor binding protein 7 eukaryotic expression plasmid, and a constructing method and application thereof
  • An insulin-like growth factor binding protein 7 eukaryotic expression plasmid, and a constructing method and application thereof
  • An insulin-like growth factor binding protein 7 eukaryotic expression plasmid, and a constructing method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Construction of eukaryotic expression plasmid pIRES-ZsGreen1-IGFBP7

[0024] 1 Materials and methods

[0025] 1.1 Materials

[0026] Main reagents: restriction endonuclease (MBI, NEB), T4DNA ligase (Takara), pMD-19T-Simple kit (Takara), pIRES-ZsGreen1 plasmid (Yrbio), plasmid mini-extraction kit (YRBIO), DNA gel recovery kit (YRBIO), dNTP, Taq enzyme and buffer (NEB), agarose (Spain), DNA Marker (full gold), yeast powder (Oxide), tryptone (Oxide).

[0027] Main instruments: ultra-clean workbench (Suzhou Group Antai Air Technology Co., Ltd.), micro pipette (German Eppendorf Company), electric heating constant temperature blast drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.), electronic balance (German Precision Instrument Factory ), PCR instrument (German Eppendorf Company), horizontal electrophoresis tank (Beijing Liuyi Instrument Factory), gel imaging system (US KODAK Company), constant temperature water bath (Haier Company), microwave oven (LG), Aqu...

Embodiment 2

[0135] Effect of pIRES-ZsGreen1-IGFBP7 expression plasmid on proliferation and cycle distribution of RBE cells

[0136] 1 Materials and methods

[0137] 1.1 Materials

[0138] Reagents: Lipofectamine 2000 (Invitrogen), Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan), RNA extraction reagent (TRIgol, Dingsheng), reverse transcription kit (RevertAid TM First Strand cDNA Synthesis Kit#K1621, Ferments), SYBR Green Realtime PCR Master Mix (QPK-201Toyobo, Japan), KGI Annexin V-PE Cell Apoptosis Detection Kit (NANJING KEYGEN BIOTECH.CO, LTD)

[0139] Instrument: CO 2 Incubator (Sanyo), 3K-18 refrigerated centrifuge (Sigma, Germany), UV spectrophotometer (PerKinElmer, Lambda3A), automatic microplate reader (AWARENS, USA), 2400 PCR instrument (PerKinElmer), gel image analyzer (Q55olw: Leiea, Heidelberg, Germany), fluorescent PCR instrument MX300OP (stratagene company, the United States), flow cytometer (Cytomics FC 500Beckman Coulter)

[0140] 1.2 Method

[0141] 1.2.1 ...

Embodiment 3

[0176] Application of pIRES-ZsGreen1-IGFBP7 expression plasmid in preparation of reagents for inhibiting RBE cell invasion

[0177] 1 Materials and methods

[0178] 1.1 Materials

[0179] Reagents: Lipofectamine 2000 (Invitrogen), 24-well Transwell Permeable Support (Corning, NY), Matrigelmatrix (BD, Franklin Lakes, NJ)

[0180] Instrument: CO 2 Incubator (Sanyo), 3K-18 refrigerated centrifuge (Sigma, Germany), OLYMPUS IX71microscope (OLYMPUS, Japan)

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Abstract

An insulin-like growth factor binding protein 7 eukaryotic expression plasmid is disclosed. The sequence of the insulin-like growth factor binding protein 7 eukaryotic expression plasmid is shown as SEQ ID NO.2 and is named as a pIRES2-ZsGreen1-IGFBP7 eukaryotic expression vector. The insulin-like growth factor binding protein 7 eukaryotic expression plasmid can be used for preparation of cell proliferation inhibiting agents, and can be used for preparation of cell invasion inhibiting agents.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a eukaryotic expression plasmid for insulin-like growth factor binding protein 7 (IGFBP7), a construction method and application thereof. Background technique [0002] Insulin-like growth factor-binding protein 7 (IGFBP7) is a member of the IGFBP family. It is a secreted protein whose expression can be detected in various tissues and organs in the human body. IGFBPs can regulate the bioavailability of insulin growth factors, thereby affecting cell growth, differentiation and proliferation. IGFBP7 can affect the activation of various human proto-oncogenes, thereby inhibiting the proliferation of fibroblasts and melanocytes and promoting cellular senescence. A large number of studies have shown that IGFBP7 is abnormally expressed in various tumors such as liver cancer, breast cancer, colon cancer and thyroid cancer, and may be a new potential tumor suppressor. In addition...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/12A61K48/00A61K38/17A61P35/00
Inventor 彭健岳春燕胥洪鹃廖明媚杨满意
Owner XIANGYA HOSPITAL CENT SOUTH UNIV
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