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Improved gene construction method and property testing of fast-acting insulin precursor

A technology of insulin precursor and insulin, applied in the fields of biotechnology and pharmacy, can solve the problem of low expression of the precursor

Active Publication Date: 2012-10-10
广东南模生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although monomeric insulin has great advantages, the expression level of its precursor in yeast or prokaryotic expression system is very low

Method used

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  • Improved gene construction method and property testing of fast-acting insulin precursor
  • Improved gene construction method and property testing of fast-acting insulin precursor
  • Improved gene construction method and property testing of fast-acting insulin precursor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Construction of plasmid pPIC9K / GEMIP

[0094] Synthesize the insulin analogue gene fragment GEMIP (Monomeric insulinprecursor) by chemical synthesis, according to the codon preference of P. pastoris (strain GS115, purchased from Invitrogen), chemically synthesize the monomeric insulin precursor (GEMIP) gene fragment :

[0095] 5-gaattcaagttcgtcaaccaacacttgtgtggttcccacttggtcgaggctttgtacttggtctgtggtgaaa

[0096] gaggtttcttctacaagggtgaaagaggtttcttctacacccctgctgctaagggtatcgtcgaacaatgttgta

[0097] cctccatctgctccttgtaccaattggagaactactgtaactaggcggccgc-3 (SEQ ID NO: 2).

[0098] Appropriate prepro-leader helps the secretory expression of insulin precursor in P. pastoris. The chemically synthesized DNA fragment is cloned into the EcoRI and NotI sites of the pPIC9K plasmid to form the α-Mating factor leader-EAEAYVEFK-GEMIP expression framework. Among them, the spacer peptide rich in EA is beneficial to the secretion of insulin precursor. GEMIP is the precursor of DTI, and its structure ...

Embodiment 2

[0112] Electrotransformation of Methanol Yeast and Screening of High-Expression Clones

[0113] Plasmids pPIC9K / GEMIP, pPIC9K / MIP, and pPIC9K / PIP were respectively linearized by Bgl II digestion. The digestion reaction solution was extracted with phenol and chloroform, precipitated with ethanol, and dissolved in 1 mol / L sorbitol before being transformed. After the linearized plasmid enters the yeast cell, it is partially recircularized and inserted into the chromosome at a single point to obtain Mut + Phenotypic transformant, the other part is integrated into the chromosome by linear substitution to obtain Mut s Phenotypic transformants will spontaneously form multi-copy insertion integration during this process.

[0114] The pPIC9K / GEMIP plasmid carries an anti-G418 gene, and the copy number of the plasmid is proportional to the G418 (0~4mg / ml) resistance of the transformant. The inventors selected clones on the 4mg / ml G418 YPD plate for expression experiments.

Embodiment 3

[0116] Test tube expression comparison results of MIP, PIP, GEMIP expression

[0117] Inoculate the strains containing 3 kinds of target precursors into test tubes for culture, then receive 2ml YPD test tube culture medium, culture at 30℃ shaker 260r / min for 2 days, centrifuge at 4℃ 3000r / min for 5min, discard the supernatant and use fresh Resuspend the bacteria in 2ml YP medium (without glucose), transfer to a new test tube, add 20μl methanol every day for induction, induce 72h, centrifuge to take the supernatant and concentrate 10 times and load the sample for native electrophoresis identification, see figure 2 .

[0118] According to the electrophoresis diagram, the photoshop analysis software can roughly get the test tube expression levels MIP, PIP, GEMIP are 39μg / ml, 150μg / ml, 80μg / ml, see image 3 .

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Abstract

The invention discloses a small peptide for increasing the polymerization property of an insulin precursor. The carboxyl tail end of an insulin B chain is added with the small peptide and then is connected with an insulin A chain, the formed insulin precursor has good polymerization property, and the expression is obviously improved. The invention also discloses a method for preparing insulin from the insulin precursor.

Description

Technical field [0001] The present invention relates to the fields of biotechnology and pharmacy; more specifically, the present invention relates to a small peptide for increasing the polymerization properties of insulin precursor, insulin precursor containing the small peptide and its coding gene, and their use in insulin preparation application. Background technique [0002] In recent years, the incidence of diabetes has risen sharply, becoming a major disease that threatens human health after cardiovascular and cerebrovascular diseases and tumors. There are about 233 million people with diabetes in the world, and it is expected to reach 300 million in 2025, of which 75% are in developing countries such as India and China. [0003] Since insulin was used to treat diabetes in the 1920s, it has been an irreplaceable specific medicine. However, the secretion of insulin in normal humans is regulated by blood glucose concentration. Generally, the blood insulin concentration reaches...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K14/62C12N15/11C12N15/17C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12P21/04C07K1/14
Inventor 王一成费俭石嘉豪陈婷沈如凌
Owner 广东南模生物科技有限公司
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