Preparation and application of recombinant MANNase-GLP-1 and homolog of recombinant MANNase-GLP-1

A technology of GLP-1 and homologues, applied in the field of biomedicine, can solve problems such as the inability to realize the limitations of oral administration, and achieve the effects of good application value, high purity, and high protein expression

Active Publication Date: 2020-11-10
ANHUI NEW SIMON BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both albiglutide and dulaglutide are injected once a week, which cannot achieve the limitations of oral administration

Method used

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  • Preparation and application of recombinant MANNase-GLP-1 and homolog of recombinant MANNase-GLP-1
  • Preparation and application of recombinant MANNase-GLP-1 and homolog of recombinant MANNase-GLP-1
  • Preparation and application of recombinant MANNase-GLP-1 and homolog of recombinant MANNase-GLP-1

Examples

Experimental program
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Effect test

Embodiment 1

[0049] This embodiment provides a high-efficiency expression and purification of mannanase and GLP-1 recombinant fusion protein (MANNase-GLP-1), including the following steps:

[0050] (1) Use primer pairs to clone the target fragment encoding GLP-1 and mannanase, perform double enzyme digestion after PCR amplification, and then connect the obtained gene sequence encoding GLP-1 and mannanase to the pPICZαA plasmid , the recombinant expression vector pPICZαA-MANNase-31P was constructed;

[0051] The gene sequences encoding GLP-1 and mannanase are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; the sequence of the primer pair for amplifying the gene sequence encoding GLP-1 is shown in SEQ ID Shown in NO.3 and shown in SEQ ID NO.4; The sequence of the gene sequence primer pair for amplifying the gene sequence encoding mannanase is shown in SEQ ID NO.5 and shown in SEQ ID NO.6; the fusion The amino acid sequence of protein MANNase-31P is shown in SEQ ID NO.7;

[0052] (2) Tra...

Embodiment 2

[0062] This embodiment provides a high-efficiency expression and purification of mannanase and GLP-1 recombinant fusion protein, including the following steps:

[0063] (1) Use primer pairs to clone the target fragment encoding GLP-1 and mannanase, perform double enzyme digestion after PCR amplification, and then connect the obtained gene sequence encoding GLP-1 and mannanase to the pPICZαB plasmid , the recombinant expression vector pPICZαB-MANNase-31P was constructed;

[0064] The gene sequences encoding GLP-1 and mannanase are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; the sequence of the primer pair for amplifying the gene sequence encoding GLP-1 is shown in SEQ ID As shown in NO.3 and shown in SEQ ID NO.4, the sequence of the gene sequence primer pair for amplifying the gene sequence encoding mannanase is shown in SEQ ID NO.5 and shown in SEQ ID NO.6; the fusion The amino acid sequence of protein MANNase-31P is shown in SEQ ID NO.7;

[0065] (2) Transform the re...

Embodiment 3

[0075] This embodiment provides a high-efficiency expression and purification of mannanase and GLP-1 recombinant fusion protein, including the following steps:

[0076] (1) Use the primer pair to clone the target fragment encoding GLP-1 and mannanase, carry out double enzyme digestion after PCR amplification, and then connect the obtained gene sequence encoding GLP-1 and mannanase to the pPICZαC plasmid , the recombinant expression vector pPICZαC-MANNase-31P was constructed;

[0077]The gene sequences encoding GLP-1 and mannanase are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; the sequence of the primer pair for amplifying the gene sequence encoding GLP-1 is shown in SEQ ID Shown in NO.3 and shown in SEQ ID NO.4; The sequence of the gene sequence primer pair for amplifying the gene sequence encoding mannanase is shown in SEQ ID NO.5 and shown in SEQ ID NO.6; the fusion The amino acid sequence of protein MANNase-31P is shown in SEQ ID NO.7;

[0078] (2) Transform the r...

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Abstract

The invention relates to efficient expression purification and application of mannase and homolog thereof and GLP1 recombinant fusion protein. By carrying out high-density fermentation induced secretory expression on pichia pastoris by using a gene recombination technique, the mannase and homolog thereof and GLP-1 recombinant fusion protein are obtained; the high-yield mannase and homolog thereofand GLP-1 recombinant fusion protein are obtained by a filtering and concentrating separation and purification method, the fermentation expression quantity is high, the separation steps are simple, and the limitations of low drug yield, high cost, frequent injection and the like of the existing GLP-1 analogue are solved. the mannase and homolog thereof and GLP-1 recombinant fusion protein not onlyhave an injection hypoglycemic effect, but also can reduce blood sugar and reduce weight through oral administration, and have a good application value in patients with obesity and early diabetes; and meanwhile, a basis is provided for further researching the mechanism of reducing blood sugar and reducing weight of mannase and homolog thereof and GLP-1 recombinant fusion protein.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to the high-efficiency expression, purification and application of mannanase and its homologue and GLP-1 recombinant fusion protein. Background technique [0002] Diabetes is one of the important diseases that seriously threaten the life and health of human beings around the world in modern society. Diabetes mellitus is an endocrine-metabolic syndrome characterized by chronic hyperglycemia. According to the National Health and Family Planning Commission, 50.1% of adults are currently in pre-diabetes, and they may become new diabetics every day. More data shows that the prevalence of diabetes in China has grown rapidly in recent decades: from 0.67% in the 1980s to 11.6% in 2010, an increase of 17 times, and it is increasingly younger. [0003] In recent years, glucagon-like peptide-1 (GLP-1) not only has an excellent hypoglycemic effect, but also has the characterist...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/62C12N15/11C07K19/00A61K38/47A61K38/26A61K47/64A61K47/55A61P3/10A61P3/04
CPCC12N9/2488C12N15/815C07K14/605A61K38/26A61K38/47A61K47/64A61K47/55A61P3/10A61P3/04C12Y302/01025C07K2319/00C12R2001/84C12N1/165
Inventor 姜伟楼慧强王岩余卫雄徐冲
Owner ANHUI NEW SIMON BIOTECH CO LTD
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