Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Preparation method of activated insulin-like growth factor-II mediated by insulin-like growth factor binding protein-6

A growth factor, insulin-like technology, applied in the preparation of proteins or polypeptides, controlling the co-expression of the number of molecules according to a certain ratio, co-expression of proteins or polypeptides and their molecular partners, can solve the problem of loss of activity

Inactive Publication Date: 2007-02-28
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
View PDF0 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, in the in vitro expression of IGF-II, there is a problem of forming aggregates, which inactivates its activity
And there are reasons to think that IGF-II used alone as a drug in the body has the same problems as IGF-I, and will produce a variety of side effects

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation method of activated insulin-like growth factor-II mediated by insulin-like growth factor binding protein-6
  • Preparation method of activated insulin-like growth factor-II mediated by insulin-like growth factor binding protein-6
  • Preparation method of activated insulin-like growth factor-II mediated by insulin-like growth factor binding protein-6

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1. Construction of co-expression vector pA0815-2 (IGF-II-IGFBP-6)

[0071] Utilize the existing expression vectors pA0815-2IGF-II and pA0815-2IGFBP-6 to construct the co-expression vector pA0815-2 (IGF-II-IGFBP-6), the method is shown in Figure 1, BglII and BamHI double digestion pA0815-2IGFBP -6. Obtain two copies of the IGFBP-6 expression unit, recover the fragment, and connect it with the expression vector pA0815-2IGF-II that has been linearized by BamH I and dephosphorylated by CIP. Pick ampicillin-resistant positive clones, prepare plasmids, identify positive clones inserted with the target fragment according to the size of the plasmid, and then use BglII and BamHI double enzyme digestion to identify and screen two copies of the IGFBP-6 expression unit connected in the correct direction. Recombinant expression vector pA0815-2 (IGF-II-IGFBP-6). The plasmid was prepared, linearized by SalI, and transformed into Pp strain GS115.

[0072] The cDNA sequence of...

Embodiment 2

[0104] Example 2. Screening of positive co-expression transformants by sticking membrane method

[0105] Inoculate the positive transformants grown on the MD plate onto a new MD plate in sequence, and about 30-40 clones can be inoculated on a plate with a diameter of 9 cm. After culturing at 28-30°C for 3-4 days, the diameter of the clones can reach about 2-3 mm. Stick a round sterile cellulose acetate membrane with a diameter of 6 cm on the flat plate to make the colonies adhere to the cellulose acetate membrane. On a freshly prepared BMMY plate, place a piece of sterile nitrocellulose membrane of the same size, put the side of the cellulose acetate membrane with the colony up on the nitrocellulose membrane, so that the two membranes overlap, Try to eliminate air bubbles between the two membranes and the plates. Induce the expression for 24 hours at 28-30°C, and remove the nitrocellulose membrane for antibody hybridization. For each experiment, two identical membranes shou...

Embodiment 3

[0107] Embodiment three, shaking flask expression

[0108] After the SalI linearized pA0815-2 (IGF-II-IGFBP-6) was transformed into GS115, the HIS4 gene on the vector and the his4 gene on the yeast chromosome underwent homologous recombination and integrated into the yeast chromosome without affecting the yeast AOX1 promoter pair The utilization of methanol, the obtained positive transformants should all be Mut + Phenotype. Increase the volume of culture when inducing expression. Scrape the positive clones on the MD plate, inoculate them in 25ml YPD liquid medium, shake and culture at 28~30℃, 200~230rpm for 20h, then transfer an appropriate amount of bacteria solution to 100ml BMG and continue culturing overnight to make the OD 600 ≈10, centrifuge at room temperature at 1500×g, collect the bacteria, resuspend in 500ml BMMY, induce culture with 1% methanol for 72 hours, collect the supernatant by centrifugation at 8000×g at 4°C, store at 4°C for short-term storage, if long-te...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
diameteraaaaaaaaaa
Login to View More

Abstract

The invention discloses the method for producing polypeptides and protein, comprising the following steps: 1 obtaining the gene order of polypeptides and protein, and obtaining the mate molecule gene order; 2 using the order to build expression carrier; 3 transferring host cell, making the expression carrier express the polypeptides and protein or their mate molecule; 4 collecting the composites which express products. The invention builds the expression carrier, transfers the host cell, makes their composites, and the insulin-like growth factors -II is real folding active molecule. The composites can be used to make drugs.

Description

technical field [0001] The present invention relates to the fields of molecular biology, biochemistry, biotechnology and pharmacy, in particular to the preparation of protein or polypeptide, especially the preparation of protein or polypeptide with pharmaceutical activity. The present invention also relates to the use of insulin-like growth factor binding protein-6, a molecular chaperone of insulin-like growth factor-II. The present invention also relates to the co-expression of a protein or polypeptide and its molecular partner, in particular, the co-expression of controlling the number of its molecules according to a certain ratio. The invention provides a method for preparing active insulin-like growth factor-II, comprising constructing an expression vector containing the insulin-like growth factor-II gene and the insulin-like growth factor-binding protein-6 gene, transforming the host, and screening out transformants ; Culture the transformed host, co-express insulin-like...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/12C12N15/17C12N15/63C07K19/00
Inventor 陈照丽陈虹黄秉仁
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com