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A kind of human insulin analogue precursor and preparation method thereof

A technology for human insulin and analogues, applied in the fields of biochemistry and molecular biology, which can solve the problems of cumbersome process and low yield

Active Publication Date: 2021-11-12
JIANGSU HENGRUI MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Escherichia coli is expressed in the form of inclusion bodies, which require lysis and renaturation of inclusion bodies, which is a cumbersome process and low yield

Method used

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  • A kind of human insulin analogue precursor and preparation method thereof
  • A kind of human insulin analogue precursor and preparation method thereof
  • A kind of human insulin analogue precursor and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Construction of recombinant yeast expression systems analogue of human insulin precursor

[0052] For two steps as follows

[0053] (1), recombinant human insulin analogue precursor Expression Vector

[0054] Recombinant human insulin C-peptide analogs of the precursor with different spacer peptide corresponding to a different gene sequence.

[0055] Amino acid sequence

[0056] EEGEPKFVNQHLCGSHLVEALYLVCGERGFFYTDKERWRGIVEQCCTSICSLYQLENYCN (SEQ IDNO.8)

[0057] Corresponding to the following nucleotide sequence, they are in italics EcoRI and NotI restriction sites.

[0058] GAAGAAGGTGAACCAAAGTTCGTCAACCAGCACTTGTGTGGTTCCCATTTGGTTGAGGCTCTGTACTTGGTCTGTGGAGAAAGAGGTTTCTTTTACACCGATAAGGAAAGATGGAGAGGTATCGTTGAGCAATGTTGCACCTCTATTTGTTCCCTGTATCAGTTGGAAAACTACTGCAACTAAGCGGCCGC (SEQ ID NO.24)

[0059] Amino acid sequence EEGEPKFVNQHLCGSHLVEALYLVCGERGFFYTDKEKFRKGIVEQCCTSICSLYQLENYCN (SEQ ID NO.12)

[0060] Corresponding to the following nucleotide sequence, they are in italics E...

Embodiment 2

[0076] Example 2: High molecular weight determination and expression screening recombinant strains

[0077] (1) Yeastern blot expression screening

[0078] Each recombinant strain selected from different Zeocin concentrations of each monoclonal plate 80, a total of about 400, Yeastern blot for expression screening.

[0079] Yeastern blot can quickly detect the expression of large quantities of proinsulin colonies. To do is picked from the various concentrations of Zeocin plates containing the corresponding monoclonal streaked YPD Zeocin plates concentration, 30 ℃ culture incubator for 1-2 days until colonies full length. Cells by replica-plated to a method sterilized bacterial cellulose nitrate (Nitrocellulose, NC) membrane, the NC membrane printed with colonies attached BMMY placed on plates covered with methanol saturated placed WHATMAN NO. the cover plate 1 filter paper, at 30 deg.] C incubator for 2 days induced inversion. Detected by Western blot for each monoclonal proinsuli...

Embodiment 3

[0088] 5L insulin analogue precursor fermentation: Example 3

[0089] The Production Example 5L fermentation yeast Pichia as a host strain of the insulin analogue precursor EEGEPK-B (1-27) -DKE-RWER-A (1-21) (SEQ ID NO.13) as an example, the present invention the other insulin analogue precursor can be obtained by the fermentation method.

[0090] Minutes following four steps.

[0091] (1) to take 0.2ml glycerol was inoculated with 200ml YPG medium (yeast extract 10g / L, soy peptone 20g / L, glycerol, 20g / L) in 1L shake flasks at 30 ℃ 250rpm shaking for about 22h, strains were grown to an OD600 of about 12.

[0092] (2) good seed culture was inoculated into 5L fermenter for fermentation, using 2.5L BSM medium (which component: 85% phosphoric acid 26.7ml / L, CaSO 4 · 2h 2 O 1.2g / L, K 2 SO 4 18.2g / L, MgSO 4 7.3g / L, KOH 4.1g / L, Glycerol 40.0g / L, PTM1 4.4ml / L, 25% aqueous ammonia to adjust the pH 5.5. PTM1: CuSO 4 · 5h 2 O, 6.0g / L; KI, 0.08g / L; MnSO 4 · H 2 O, 3.0g...

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Abstract

The invention relates to a human insulin analog precursor and a preparation method thereof. Specifically, the present invention relates to a human insulin analog precursor containing a novel connecting peptide and a preparation method thereof. The novel human insulin analog precursor can be highly expressed in yeast and the downstream purification method is simple and easy to operate.

Description

Technical field [0001] The present invention relates to a human insulin analog precursor and a preparation method belongs to the field of biochemistry and molecular biology, molecular construct comprising a technical application information, cell selection, fermentation and the like. Background technique [0002] Diabetes is a defect in insulin secretion or insulin resistance characterized by hyperglycemia resulting from metabolic disorders, disease has now become a major disease after three hazardous cardiovascular diseases and cancer. Will be divided into four clinical diabetes, i.e. diabetes type Ⅰ, Ⅱ diabetes, type 2 diabetes and other special gestational diabetes, secondary diabetes, insulin (insulin, Ins) is applied to all the main and most type Ⅰ diabetes type Ⅱ therapeutic drug for diabetes. Insulin is secreted from pancreatic β cells polypeptide hormone consisting of two polypeptide chains, A and B, the two polypeptide chains are linked by disulfide bonds between the two...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/62C12N15/17C12N1/21C12N1/19C12R1/21C12R1/84C12R1/865
CPCC07K14/62
Inventor 王菲菲王宏伟曾翔赵亮亮牛宁宁王亚里田静
Owner JIANGSU HENGRUI MEDICINE CO LTD