Pichia yeast recombinant bacterial strain highly expressing Neuritin truncated protein, and application thereof

A technology of recombinant strains, Pichia pastoris, applied in the field of microorganisms, can solve the problems of lack of effective treatment and/or preventive drugs for nervous system diseases and nerve injuries, achieve stable passage and large-scale culture, facilitate large-scale culture, and promote regeneration and the effect of functional recovery

Inactive Publication Date: 2015-07-15
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Contemporary medicine still lacks effective treatment and / or preventive drugs for neurological diseases and nerve injuries

Method used

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  • Pichia yeast recombinant bacterial strain highly expressing Neuritin truncated protein, and application thereof
  • Pichia yeast recombinant bacterial strain highly expressing Neuritin truncated protein, and application thereof
  • Pichia yeast recombinant bacterial strain highly expressing Neuritin truncated protein, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Screening of Pichia pastoris recombinant strains highly expressing Neuritin truncated protein

[0032] The screening steps of the Pichia pastoris recombinant strain highly expressing Neuritin truncated protein in the present embodiment are as follows:

[0033] 1. Construction of recombinant Pichia pastoris

[0034] According to the method of Example 1 in Chinese invention patent CN200810227347-"Neuritin truncated protein active fragment and its expression system and special vector", the neuritin truncated body was used as a template to amplify the neuritin truncated body fragment, and the PCR product was used EcoRI, After NotⅠ restriction endonuclease digestion and purification, the digestion products were connected, transfected into DH5α competent cells, and the positive transformants identified as positive by PCR were electrotransformed with Pichia pastoris GS115 competent cells, and the bacterial solution was coated with Spread on the MD plate, cultivate...

Embodiment 2 Embodiment 1

[0052] Example 2 Purification of Pichia recombinant strains highly expressing Neuritin truncated protein obtained through screening in Example 1

[0053] The specific implementation steps are as follows:

[0054] (1) Utilize the highly expressed bacterial strain screened out in Example 1 to induce a large amount of expressed protein;

[0055] (2) After running the AKTA purification system, first wash the affinity chromatography column with 8mol / L urea, 1ml / min, 30min;

[0056] (3) 0.22um filtered ddH 2 O wash affinity chromatography column, 1ml / min, 20min;

[0057] (4) 0.1mol / L NiSO 4 , 1ml / min, 10min;

[0058] (5)ddH 2 O, 1ml / min, 10min;

[0059] (6) 1×start buffer equilibrium chromatography column, 1ml / min, 10min;

[0060] (7) Protein liquid collected, 0.7ml / min;

[0061] (8) 1×start buffer (20mM sodium phosphate, 0.5M NaCl, 10mM imidazole, pH7.4) equilibrates the chromatography column again, 1ml / min, until the protein absorption detection line is balanced;

[0062]...

Embodiment 3

[0068] Example 3 Identification of the Neuritin truncated protein purified in Example 2

[0069] 1. Western blot to identify the expressed protein, the specific implementation steps are as follows:

[0070] (1) Electrophoresis: stacking gel 80v, 30min; separating gel: 110v, 1h.

[0071] (2) Membrane transfer: Use a semi-dry transfer apparatus to transfer the protein from the gel to the PVDF membrane, place it in order from bottom to top: filter paper, membrane, glue, filter paper, and use a test tube to drive out the air bubbles between the layers, set the voltage and time : 21V, 42min.

[0072] (3) Blocking: Place the membrane in 5% skimmed milk powder (TBST dissolved), and block at room temperature for 2 hours on a shaker.

[0073] (4) Incubate the primary antibody: 6×His monoclonal antibody diluted 1:2000 in 5% skimmed milk powder-TBST solution, overnight at 4°C on a shaker.

[0074] (5) Washing: Put the membrane face up, add TBST solution, shake and wash once for 5 minu...

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Abstract

The invention discloses a pichia yeast recombinant bacterial strain highly expressing a Neuritin truncated protein, and an application thereof. The recombination bacterial strain is Pichia pastoris and has a preservation number of CGMCC NO:9912. Compared with Neuritin truncated proteins expressed by Escherichia coli and sold in the present market, the Neuritin truncated protein expressed by the pichia yeast recombinant bacterial strain has the following advantages: an exogenous protein expressed by eukaryotic cells has post-translational modification bioactivity, and exogenous gene is stably integrated in yeast chromosome, and can realize stable passage and easy large scale culture. A suitable concentration of Neuritin can promote the regeneration and function recovery of early stage sciatic nerves after injuries, provides a new method for treatment of peripheral neuropathy injury, and has good application prospect.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and more specifically, the invention relates to a Pichia recombinant strain capable of stably and highly expressing Neuritin truncated protein and its application. Background technique [0002] Various nerve injuries and degenerative diseases of the nervous system (such as Alzheimer's disease, Parkinson's disease, etc.) And other characteristics, have brought heavy burden to society and family. The key to treating various nervous system diseases and nerve injuries is to improve the repair ability of damaged nerves. Numerous experiments have shown that various neurotrophic factors can provide a good microenvironment for nerve regeneration, which can prevent the degeneration of nerve cells and promote nerve regeneration. Both play a very important role in the growth of protrusions. [0003] Neuritin (CPG15, NRN1) is a neurotrophic factor discovered in recent years, which plays an important...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19A61K38/18A61P25/02C12R1/84
CPCA61K38/18C12N1/165C12R2001/84A61K2300/00
Inventor 黄瑾杨磊朱井玲张云华崔丽娟单莉娅
Owner SHIHEZI UNIVERSITY
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