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Expression of human insulin and analogues thereof in dual-promoter of methanol yeast and preparation of human insulin and analogues thereof

A technology of human insulin and methanol yeast, applied in the biological field, can solve the problems of reduced curative effect, antibody production, fat atrophy, etc., and achieve the effect of reducing production cost and efficient preparation

Active Publication Date: 2011-05-11
CHONGQING PEG BIO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, animal insulin is a heterologous protein for humans, which has strong immunogenicity, and is prone to allergic reactions and lipoatrophy at the injection site. Long-term use may produce antibodies, resulting in reduced curative effect

Method used

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  • Expression of human insulin and analogues thereof in dual-promoter of methanol yeast and preparation of human insulin and analogues thereof
  • Expression of human insulin and analogues thereof in dual-promoter of methanol yeast and preparation of human insulin and analogues thereof
  • Expression of human insulin and analogues thereof in dual-promoter of methanol yeast and preparation of human insulin and analogues thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0046] Example 1, the efficient preparation of recombinant human insulin glargine (Glargine)

[0047] 1.1 Upstream build:

[0048] According to the cDNA sequence of SEQ ID NO: 1 Glargine:

[0049]

[0050]

[0051] Entrust Bao Biological Engineering (Dalian) Co., Ltd. to artificially synthesize the whole gene. And embedded in pPMD18-T vector and named pPMD18-T-Glargine. First use EcoR I and Not I to double digest the pPIC9K plasmid and pPMD18-T-Glargine plasmid, and recover the fragment of about 9300bp after digestion of pPIC9K by gel, and recover the fragment of about 200bp after digestion of pPMD18-T-Glargine, and use T4DNA Ligase, 16 ℃, 30min, connect the two target fragments, and transform into TOP10F', use LB-Amp plate to screen the recombinants containing exogenous genes, extract positive clone plasmids, identify by EcoR I and Not I double enzyme digestion, and send to Dalian Baobiology After correct sequencing, it was named pPIC9K-Glargine plasmid. The pPIC9K-...

example 2

[0058] Example 2, the efficient preparation of recombinant human insulin detemir (Detemir)

[0059] According to the cDNA sequence of SEQ ID NO: 2 Detemir:

[0060]

[0061] Detemir samples were obtained with reference to the upstream construction, expression and purification methods of Example 1.

example 3

[0062] Example 3, the efficient preparation of recombinant human insulin (Insulin)

[0063] cDNA sequence according to SEQ ID NO: 3 Insulin:

[0064]

[0065] Refer to the upstream construction and expression method of Example 1 to obtain a single-chain Insulin fermentation broth. The fermentation supernatant was taken, concentrated by ultrafiltration through an ultrafiltration column with a cut-off of 3000, and added with a final concentration of 0.5M (NH 4 ) 2 SO 4 And adjust the pH to 5.0, on the Butyl.F.F column rough purification. With 20mM acetic acid-sodium acetate, 2mM EDTA, 0.5M (NH 4 ) 2 SO 4 , equilibrate Butyl.F.F column with pH5.0 equilibrium solution, load the sample, then equilibrate with equilibrium solution, and then use eluent 1 (20mM acetic acid-sodium acetate, 2mM EDTA, pH5.0), eluent 2 (DDW) and Eluent 3 (50 mM Tris, 2 mM EDTA, pH 10.0) was eluted to obtain a single-chain Glargine sample with a purity greater than 95%. The single-chain Glargine ...

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Abstract

The invention discloses expression of human insulin and analogues thereof in a methanol yeast containing dual promoters and a high-efficient preparation method of the human insulin and the analogues thereof, and the method is characterized in that recombinant human insulin and the analogues thereof are prepared by constructing an expression vector containing the dual promoters and engineering cells in host cells of the methanol yeast, and performing the optimal fermentation technology and the high-efficient purification technology, so that the production cost of the recombinant human insulin and the analogues thereof can be effectively reduced.

Description

technical field [0001] The present invention relates to the field of biology, specifically, the expression and recombinant preparation of human insulin and its analogs in methanolic yeast containing dual promoters, including the construction of related expression vectors and engineered cells, and the production of recombinant human insulin and its analogs. expression and purification. Background technique [0002] Insulin is a protein hormone secreted by pancreatic β cells stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, and glucagon. The islet β cells first synthesize preproinsulin, which then becomes insulin through the action of specific proteases. The mature insulin molecule consists of two peptide chains, A and B. The insulin functions of animals of different races (human, cow, sheep, pig, etc.) are roughly the same, but the amino acid sequence is slightly different. The A chain of mature human insulin (Insulin Human) has 2...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12P21/02C12R1/88C12R1/78C12R1/84
Inventor 范开陈海容高剑坤陈勇张益张继兰
Owner CHONGQING PEG BIO BIOTECH CO LTD
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