Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Recombinant targeted infuse protein for promoting thrombocytopoiesis and its preparation method

A fusion protein and platelet technology, which is applied to medical preparations containing active ingredients, drug combinations, peptide/protein components, etc., can solve the problems of increasing the cost of use and increasing the amount of use, and achieve enhanced effects, reduced dosage, The effect of improving bioavailability

Active Publication Date: 2006-02-22
ARMY MEDICAL UNIV +1
View PDF0 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to hGH's broad-spectrum cell growth-promoting effect, when it is applied in the body, it will widely affect tissue cells throughout the body. In order to make it have a prominent effect on a specific tissue or cell, such as improving The ability of megakaryocyte proliferation and platelet production must be increased
In this way, it will not only increase its use cost, but also bring other adverse reactions to the human body.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Recombinant targeted infuse protein for promoting thrombocytopoiesis and its preparation method
  • Recombinant targeted infuse protein for promoting thrombocytopoiesis and its preparation method
  • Recombinant targeted infuse protein for promoting thrombocytopoiesis and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Cloning of hGH

[0067] The cDNA sequence of hGH gene was obtained by PCR from the fetal liver cDNA library, and the PCR upstream and downstream primers used for amplification were GH1 and GH2, respectively. The base sequences of GH1 and GH2 are as follows:

[0068] GH-1: 5′-ATG TTC CCA ACT ATT CCA CTG-3′

[0069] GH-2: 5′-TTA GAA GCC ACA CGA CCC TTC-3′

[0070] The PCR reaction system is 100ul, which contains 100pmol of primers GH-1 and GH-2, 20nmol of dNTP, 1ul of fetal liver cDNA library, 10ul of 10×PCR reaction buffer, and 5U of high-fidelity Pyrobest DNA polymerase. The reaction conditions of PCR were as follows: the first stage of pre-denaturation at 95°C for 5 minutes; the second stage of denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, extension at 72°C for 1 minute, and 32 cycles; the third stage of extension at 72°C for 5 minutes, 10°C for 10 minutes. The obtained PCR product is detected by 1% agarose electrophoresis, and an el...

Embodiment 2

[0071] Example 2: Construction and Identification of TMP and hGH Fusion Protein Expression Plasmid

[0072] In order to enable TMP to form a fusion protein with hGH, the following two PCR primers were first designed and synthesized:

[0073] MH-1: 5′-ACT GAT ATC GAA GGT CCG ACT CTG CGT CAG TGG CTG GCT GCACGT GCT GGT TCT GGT TCT GGT TTC CCA ACT ATT CCA CTG AG-3′

[0074] MH-2: 5′-AGT AAG CTT TTA GAA GCC ACA CGA CCC TTC-3′

[0075] In MH-1, the underlined part is the recognition site of the endonuclease EcoRV, the bold part is the nucleotide sequence encoding TMP (SEQ ID NO1 of the sequence table), and the italic part is the encoding bridge peptide Gly-Ser-Gly-Ser - the nucleotide sequence of Gly. The underlined part in MH-2 is the recognition site of endonuclease Hind III.

[0076] Such as figure 1 As shown, the pMD-hGH plasmid DNA was used as a template to amplify the hGH gene by PCR, and the PCR amplification conditions were the same as above. The obtained PCR produ...

Embodiment 3

[0093] Embodiment 3: the fermentation of engineered yeast and the purification of TMP-hGH fusion protein

[0094] Inoculate pPIC9K / TMP-hGH engineered yeast strains containing pPIC9K / TMP-hGH stored at -70°C on a YPD plate for activation, pick a single colony with good appearance and inoculate it in BMGY medium, and culture in a constant temperature shaker at 280rpm at 28°C-30°C for 16-18 hours to OD 600 ≈4, the bacterial liquid is used as the seed liquid. Then the seed solution was inoculated in a 15L fermenter (B. Braun company, Germany) equipped with 2.5L BMMY medium for fermentation. The fermentation temperature is controlled at 30°C, the dissolved oxygen is greater than 20% saturation, the pH value is around 5.0, and cultured to OD 600 When ≈150, methanol was added for induction, so that the final concentration of methanol was always maintained at about 1%. After induction culture for 72 hours, the supernatant was obtained by centrifugation, 20% ammonium sulfate was adde...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a fusion protein produced through restructuring targeted thrombopoietin (TPO), which comprises a TPO simulated peptide region and a human growth hormone polypeptide region, bridge joint peptides are arranged between the two regions. The invention also provides the preparing method and use of the fusion protein.

Description

technical field [0001] The invention belongs to the field of bioengineering and pharmacy, and specifically relates to the preparation and application of a fusion protein of TPO mimic peptide and human growth hormone with the effect of targeting megakaryocyte proliferation-thrombocytosis. Background technique [0002] Primary and secondary thrombocytopenia caused by hematopoietic dysfunction are relatively common clinically, and it is an important scientific problem to effectively protect and promote platelet recovery. Among the many platelet-increasing schemes, the hematopoietic growth factor has the best effect, among which the most significant platelet-increasing effects are thrombopoietin (TPO) and interleukin-11 (IL-11). TPO is also called megakaryocyte growth and development factor (MGDF) or platelet growth factor (TSF). Human TPO consists of 332 amino acid residues and has a molecular weight of 38KD. TPO stimulates the proliferation and differentiation of megakaryotic...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K38/27A61P7/00
Inventor 王军平粟永萍艾国平刘晓宏程天民
Owner ARMY MEDICAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com