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Insulin mimetic peptide fusion protein, mutant and applications thereof

A fusion protein and mutant technology, applied in the direction of peptide/protein components, peptides, hybrid peptides, etc., can solve the problems of low efficacy and achieve high biological activity, avoid insulin resistance, and low ability to stimulate cell division.

Active Publication Date: 2012-06-27
XUANZHU BIOPHARMACEUTICAL CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However in vitro studies of the fusion protein showed low efficacy (Kim et al., Diabetes. 2003; 52(3):751-759)

Method used

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  • Insulin mimetic peptide fusion protein, mutant and applications thereof
  • Insulin mimetic peptide fusion protein, mutant and applications thereof
  • Insulin mimetic peptide fusion protein, mutant and applications thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0069] The pharmaceutical compositions of the present invention can be prepared by known methods for preparing pharmaceutically acceptable carriers that can be administered to patients, so that an effective amount of nucleotide or polypeptide molecule is combined with a pharmaceutically acceptable carrier to form a mixture. Such suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, Pa., USA). On this basis, the pharmaceutical composition may comprise the active compound in association with one or more pharmaceutically acceptable carriers or diluents in a buffer solution having an appropriate pH value and isotonic with physiological fluids or Substances such as complex nucleic acid molecules, polypeptide molecules or fusion eggs. Methods of combining active molecules and carriers or both with diluents are well known to those skilled in the art. The composition may include a targeting agent for delivering the acti...

Embodiment 1

[0089] Embodiment 1: the construction of carrier

[0090] The vectors encoding the fusion protein containing IMP and IgG1-Fc, IMP / IgG-Fc (QL) and IMP / IgG-Fc (QA) vectors were constructed by overlapping PCR (overlap PCR). The IgG4-Fc region contains the IgG4 constant heavy chain (hinge-CH2-CH3 part). The murine IgK secretion leader sequence was fused to the IMP sequence to direct the secretion of the synthetic fusion protein into the culture medium. The cDNA encoding the IMP / hIgG-Fc fusion protein was chemically synthesized and ligated to the PCR-amplified product encoding human IgG4-Fc (hinge, CH2, and CH3), then inserted into the EcoRV and BamHI of the pcDNA3.1 vector The IMP / hIgG-Fc / pcDNA3.1 vector was constructed between the sites. Secretable IMP / hIgG-Fc and its mutant fusion proteins contain an IgG4 constant heavy chain (hinge-CH2-CH3). The mouse IgK secretion leader peptide sequence fused to the IMP sequence directs the secretion of the synthesized protein into the cel...

Embodiment 2

[0096] Example 2: CHO cell expression of IMP / IgG-Fc, IMP / IgG-Fc(QL) and IMP / IgG-Fc(QA) fusion proteins

[0097] In order to evaluate the ability of the vector to express and secrete IMP / IgG-Fc fusion protein, the constructed fusion protein vector was transfected into CHO-S cells for transient expression. 48h after transfection, total RNA was extracted from the transfected cells and the expression effect was evaluated by RT-PCR. Application of gene-specific primers detected the expression of the IMP / IgG-Fc fusion protein gene at the transcriptional level and the expression of the IgG-Fc control gene ( figure 2 A). No transcript level expression was detected in the control samples.

[0098] Transfected CHO-S cell lysates and cell culture fluid were detected by Western blot and anti-mouse antibody to confirm the expression of the fusion protein at the translational level. Such as figure 2 As shown in B, Fc fusion proteins were detected in both culture medium and cell lysate...

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PUM

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Abstract

The invention relates to an amino acid sequence of a fusion protein for treating human type I and type II diabetes, and a production method and an application thereof. The invention relates to a noninsulin diabetes treatment method which can be used for avoiding insulin resistance. The fusion protein related to the invention is formed by fusing an insulin mimetic peptide and an IgG-Fc or IgG-Fc mutant through a connecting peptide, so that the in-vivo half-life of the insulin mimetic peptide can be prolonged remarkably while the blood sugar lowering activity of the insulin mimetic peptide is kept. The mutant of IgG-Fc can be used for further prolonging the in-vivo half-life of the fusion protein.

Description

field of invention [0001] The present invention relates to the prevention and treatment medicine of human diabetes. Specifically, the present invention provides a fusion protein for detecting and treating human type I and type II diabetes and its preparation method and application. The fusion protein contains insulin mimetic peptide (IMP) and human IgG-Fc (hinge-CH2-CH3) protein and / or IgG-Fc mutants (T250Q / M428L; QL and T307Q / N434A; QA), which can effectively reduce The blood sugar level of diabetic patients increases the half-life of insulin-mimetic peptides in vivo, especially the mutant of IgG-Fc (QL / QA), which can further increase the metabolic half-life of insulin-mimetic peptides in vivo and reduce the frequency of drug injection. Background technique [0002] With the improvement of people's living standards, the aging population and the increase in the incidence of obesity, the incidence of diabetes is increasing year by year. Diabetes is one of the main causes of...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/21A61K38/28A61K47/48A61K48/00A61P3/10
Inventor 张海涛
Owner XUANZHU BIOPHARMACEUTICAL CO LTD
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