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 fields of pharmacy, biochemistry, molecular biology, biotechnology and pharmacy, biological products, clinical pharmacy, can solve the problem of activity loss

Inactive Publication Date: 2010-05-05
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • 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

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  • 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 pAO815-2 (IGF-II-IGFBP-6)

[0071] Utilize the existing expression vector pAO815-2IGF-II and pAO815-2IGFBP-6 to construct the co-expression vector pAO815-2 (IGF-II-IGFBP-6), the method is as follows figure 1 As shown, pAO815-2IGFBP-6 was digested with BglII and BamHI to obtain two copies of the IGFBP-6 expression unit. After recovering the fragment, it was ligated with the expression vector pAO815-2IGF-II that had been linearized by BamHI and dephosphorylated by CIP .Pick ampicillin resistance-positive clones, prepare plasmids, and identify positive clones inserted with the target fragment according to the size of the plasmids, and then use BglII and BamHI double enzyme digestion to identify and screen two copies of IGFBP-6 expression units and connect them in the correct direction The recombinant expression vector pAO815-2(IGF-II-IGFBP-6). Prepare the plasmid, linearize it with SalI, and transform it into Pp strain GS115....

Embodiment 2

[0080] Example 2. Screening of positive co-expression transformants by sticking membrane method

[0081]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 2-3mm. 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 should...

Embodiment 3

[0083] Embodiment three, shaking flask expression

[0084] After SalI linearized pAO815-2 (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-term s...

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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 geneorder; 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

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Application Information

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Patent Type & Authority Patents(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
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