Preparation method and application of mutain of human insulin-like growth factor binding protein 7 (IGFBP7)

A technology of growth factor and human insulin, applied in the field of biomedical materials

Active Publication Date: 2011-08-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no literature reports on the key amino acid sites of IGFBP7 binding to insulin

Method used

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  • Preparation method and application of mutain of human insulin-like growth factor binding protein 7 (IGFBP7)
  • Preparation method and application of mutain of human insulin-like growth factor binding protein 7 (IGFBP7)
  • Preparation method and application of mutain of human insulin-like growth factor binding protein 7 (IGFBP7)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The introduction of a certain point mutation in the embodiment

[0021] 1. Design of mutant primers: Design the mutant amino acid codons into the primer sequence according to the corresponding primer design principles, namely Arg(AGG)→Ile(GAG), His(CAC)→Phe(TCC). The primer sequences are: upstream, SEQ ID NO. 3cctgtcctcatctggaacaaggtaaaaATAggtTTCtatggagttcaaaggac; downstream, SEQ ID NO. 4gtcctttgaactccataGAAaccTATttttaccttgttccagatgaggacagg.

[0022] 2. Introduce mutant PCR reaction: Dissolve the above primers to 10uM, dilute the wild-type IGFBP7-pET28a plasmid to 10ng / ul, use a DNA polymerase with high fidelity performance, and use a 50ul amplification system as follows: 5*buffer 10ul, dNTP 4ul , upstream primer 1ul, downstream primer 1ul, DNA template 1ul, polymerase 1ul, double distilled water 32ul. PCR reaction conditions: One-step annealing and extension method, that is, denaturation at 98°C for 1min, 10sec at 98°C, 6min30sec at 68°C, and the reaction ends after 3...

Embodiment 2

[0027] Expression and purification of target protein

[0028] 1. Transform the expression strain BL21 and select the positive expression strain: transform the expression vector IGFBP7-pET28a successfully sequenced into the E. coli expression strain BL21, take 2ul of the plasmid (100ng / ul) and add it to 200 μl of prepared competent E. coli BL21, ice After bathing for 30 minutes, heat shock at 42°C for 90 seconds, then ice-bath for 3 minutes; add 800 μl LB liquid medium, shake the bacteria at 37°C, 150 rpm for 1 hour, and then take 200 μl of bacterial liquid and spread evenly on kanamycin-positive ( 50 μg / ml) on LB agar plates. Invert the plate and place in a 37°C incubator for 12-16h. Pick 3-5 clones and shake them overnight for culture, and re-inoculate each clone into two tubes of 4ml kanamycin-resistant liquid LB medium to divide them into (+) (-) controls. After shaking at 300rpm for 2 hours, the bacterial concentration reaches When the OD value is 0.6-1, add the inducer ...

Embodiment 3

[0036] Example 3 Western blot verification of the binding ability of the mutant protein to insulin

[0037] 1. Membrane spotting: Take 2.5ul each of wild-type protein (W) and two mutant proteins (M2) at the same concentration and spot on nitrocellulose membrane (NC membrane). After natural drying, use 5% bovine serum at room temperature Albumin (BSA) blocked for 2 hours.

[0038] 2. Quantification: The membrane was incubated with a monoclonal antibody to IGFBP7 (mouse source, TBST milk dilution ratio 1:5000), incubated at room temperature for half an hour and then placed at 4°C overnight. The next day, they were incubated at room temperature for 20 minutes, and washed three times with Tris-HCl buffer (TBST) containing Tween-20 for 5 minutes each time. Then incubate with a fluorescently labeled secondary antibody (goat anti-mouse, dilution ratio 1:5000) at room temperature for 45 minutes, wash with TBST as above, and scan the membrane with Odyssey after washing to quantify the...

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Abstract

The invention provides mutain of a human insulin-like growth factor conjugated protein 7 (IGFBP7). The mutain contains mutation of Arg198Ile/His200Phe, and has an amino acid sequence of SEQ ID NO.1. An igfbp7 gene coding sequence is selected from an SW480cDNA library of a human colorectal cancer cell line and is constructed into pET28a plasmids of a prokaryotic expression vector, so that the IGFBP7 protein can be expressed in a colon bacillus strain BL21. On the basis of the expression vector, mutain of the IGFBP7 is constructed, BL21 is converted, the mutain is expressed and purified, and a key site where the IGFBP7 is bonded with insulin is pointed out. The mutain provided by the invention has simple steps in a preparation method and high success rate, and the bonding capacity of the protein with insulin is remarkably reduced, so that insulin resistance can be suppressed and the mutain can be applied to the preparation of a medicament for treating type 2 diabetes.

Description

technical field [0001] The invention belongs to the technical field of biomedical materials, and relates to a mutant protein preparation method of insulin growth factor binding protein 7 (IGFBP7) and its application. Background technique [0002] Insulin growth factor binding proteins (IGFBPs) are a class of secreted proteins with high affinity to insulin-like growth factors (IGF), including IGFBP1-15. It can regulate the bioavailability of IGF through reversible binding in vivo, thereby regulating embryonic development, cell proliferation and other processes. Studies in recent years have found that this protein family not only participates in regulating the function of IGF, but also has a function independent of IGF molecular network (IGF-independent). Among them, the insulin-like growth factor binding protein 7 (insulin-like growth factor binding protein 7, IGFBP7) gene is a subtractive cDNA library from colon adenocarcinoma-normal mucosa by suppression subtractive hybrid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/70C12N15/10A61K38/18A61P3/10
Inventor 来茂德李有朝阮文静
Owner ZHEJIANG UNIV
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