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Engineered bacteria Pichia pastoris for high yield of insulinotropic hormone and its construction method

A Pichia pastoris, construction method technology, applied in the directions of microorganism-based methods, hormone peptides, biochemical equipment and methods, etc., can solve the problems that the product is not easy to purify, easy to produce immunogenicity, etc., and achieve the effect of easy purification

Inactive Publication Date: 2009-06-10
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention uses the Pichia pastoris eukaryotic expression system to produce GLP-1, and its expression product protein will not produce high antigenicity, the expression amount is very high and can be secreted to the outside of the cell. When the expression system produces GLP-1, the product obtained is not easy to purify and is prone to immunogenicity. The product produced by the strain of the present invention is particularly suitable for medical treatment purposes

Method used

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  • Engineered bacteria Pichia pastoris for high yield of insulinotropic hormone and its construction method
  • Engineered bacteria Pichia pastoris for high yield of insulinotropic hormone and its construction method
  • Engineered bacteria Pichia pastoris for high yield of insulinotropic hormone and its construction method

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Experimental program
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Effect test

Embodiment 1

[0030] 1) Construction of Pichia pastoris (P. pastoris) expression vector pPIC9GLP-1:

[0031] According to the sequence characteristics of the multiple cloning site of the pPIC9 plasmid, select the upstream primer cccgggcaaa agacattctg agggaac, and the downstream primer gcggccgctta acggccatct acg; directly connect the cloned GLP-1 into T-vector to obtain pMDGLP-1, and digest it with Smal I and Not I GLP-1 gene fragment; Plasmid pPIC9 was digested with XhoI, then the sticky end was blunted, and then digested with NotI, and the large fragment was recovered; the GLP-1 gene fragment and the large pPIC9 fragment were connected by T4DNA ligase, and positive transformants were screened.

[0032] 20μL PCR reaction system

[0033] 10×Buffer 2.0

[0034] Upstream primer 0.5

[0035] Downstream primer 0.5

[0036] dNTP 0.4

[0037] Taq DNA polymerase 0.7

[0038] template 0.3

[0039] Sterile double distilled water 15.6

[0040] 20 μL total

[0041] Reaction ...

Embodiment 2

[0052] 1) Fermentation culture of Pichia pastoris

[0053] Each transformant Pichia cell was inoculated in 5 ml of BMGY liquid medium, cultured with shaking at 30° C. for 40 to 48 hours, the supernatant was discarded by centrifugation under sterile conditions, and replaced with 10 ml of BMMY liquid medium. During the induction process, an appropriate amount of methanol was added every 24 hours to maintain a final concentration of about 0.75%. After culturing for 60 hours, the supernatant was collected by centrifugation, and induced without adding methanol was used as a blank control.

[0054] Recipe for BMGY and BMMY medium:

[0055] 1% yeast extract

[0056] 2% peptone

[0057] 100 mM potassium phosphate, pH6.0

[0058] 1.34% YNB

[0059] 4x10 -5 % biotin

[0060] 1% glycerol or 0.5% methanol

[0061] 2) Collection and precipitation of the target protein and electrophoresis detection

[0062] The fermentation broth was centrifuged at 12000X fo...

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Abstract

The invention discloses a Pichia pastoris engineering bacterium and building method of high-production insulinotropic hormone, which is characterized by the following: building pPIC9GLP-1 as P.pastoris expressing carrier firstly; transmitting the lineated P.pastoris expressing carrier into GS115 to produce the product; keeping long time activity for Pichia pastoris.

Description

technical field [0001] The invention relates to a high-production insulin-stimulating hormone Pichia pastoris engineering bacterium (Pichia pastoris) and a construction method thereof, belonging to the field of molecular biology. Background technique [0002] The chemical name of insulin-stimulating hormone (GLP-1) is glucagon-like peptide-1 (glucagon-like peptide-1), which is a kind of insulin induced by glucose by interacting with specific receptors on the surface of pancreatic beta cells. Secretion of significantly enhanced substances. Studies have confirmed that GLP-1 can stimulate insulin secretion, and the higher the blood sugar, the stronger the effect of GLP-1; but when the blood sugar concentration returns to normal, GLP-1 will no longer continue to act, so hypoglycemia will not occur; In addition, GLP-1 also reduces islet cell damage, improves islet cell structure, repairs islet cell function, improves islet cell viability and prolongs its lifespan, inhibits the r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/66C12N15/10C07K14/575C07H21/00C12R1/84C12P21/02
Inventor 李明刚侯建华方园园薄涛王翠艳杨少辉武志强闫瑞香
Owner NANKAI UNIV
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