Secretory expression for human insulin gene in methyl alcohol yeast

A technology of human insulin and methanol yeast, applied in the field of genetic engineering, can solve problems such as low expression level, and achieve the effects of increasing yield, simple process and shortening working hours

Inactive Publication Date: 2005-05-11
马延高 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] The technical problem to be solved by the present invention is: provide a kind of human insulin gene in methanol yeast (Pichia Pastoris) GS115 / pPICM # 101. Secretion expression in CCTCC NO.M204071 with the deposit number, which solves the defects of the low expression level of the expression gene obtained in the prior art and the need for extremely complicated processes such as transpeptide removal of the C chain

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  • Secretory expression for human insulin gene in methyl alcohol yeast
  • Secretory expression for human insulin gene in methyl alcohol yeast
  • Secretory expression for human insulin gene in methyl alcohol yeast

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Embodiment Construction

[0060] The present invention will be further described below in conjunction with embodiment, as shown in Figure 5, the present invention specifically comprises the following steps:

[0061] Step 1: Construction of expression plasmid pPIC9K(+B+A):

[0062] 1. Preparation of plasmid pPIC9K (B-C'-A) containing 2-peptide C-chain human proinsulin (B-C'-A).

[0063] The molecular biology operations involved in the present invention are all carried out according to conventional classical methods (Ausubel, F.M., et al., Short Protocols in Molecular Biology Second Edition, 1992).

[0064] a) Preparation of B-C'-A DNA fragment: using the yeast preferred codon human proinsulin (C') sequence (Figure 1). Synthesize the following two single-stranded DNA fragments of similar primers at Integrated DNA Technologies, Inc. in the United States:

[0065] 5'-USAp (100nt)

[0066] 5'- GCATTACGTA TTCGTTAACCAACACTTGTGTGGTTCTCACTTGGTTGAAGCTTTGTACTTGGTTTGTGGTGAAAGAGGTT TCTTTCTACACTCCAA AGACT

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Abstract

The invention was involved in expression of human insulin gene, especially for the expression of human insulin gene in Pichia Pastoris GS115 / pPICM#101 whose storage number wais CCTCC NO.M204071. It contained B and A strand synthesis, construction of express plasmid and engineering strain, transform of host cell, filter of transformant and over expression engineering strain, etc. The method left out the difficult procedure, reduced the process steps and shortened time, so the technique was simple. It overexpressed 10-100 folds than the others. It provided one new simple method for human insulin commercial process.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and it relates to the secretory expression of a human insulin gene, in particular to the expression of the human insulin gene in methanol yeast (Pichia Pastoris) GS115 / pPICM # 101. The secretory expression in the deposit number of CCTCC NO.M204071. Background technique [0002] Insulin is the only natural hormone secreted by B cells that lowers blood sugar. Its main function is to regulate the content of glucose in the blood and transport glucose to certain target cells in the body, and participate in the metabolism of the three major substances and energy in the body. Too high or too low blood sugar in the human body is a disease. No matter what the cause is, the decrease in insulin secretion, which is not needed for the regulation of blood sugar in the body, will cause the patient to develop insulin-dependent diabetes mellitus (IDDM). Insulin is necessary to treat these patients. Human in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/62C12N15/17C12N15/81C12P21/02
CPCC07K14/62C07K2319/50C12P21/02
Inventor 马延高马向东
Owner 马延高
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