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Fermentation medium and fermentation method of human recombinant neutrophil inhibitory factor and hirulog hybrid

A technology of inhibitory factor and fermentation method, applied in the field of microbial fermentation, can solve the problems of low TNHH fermentation density, high acetic acid production, and high plasmid loss rate, and achieve good industrial application prospects, low acetic acid production, and low plasmid loss rate. Effect

Active Publication Date: 2021-02-23
LUNAN PHARMA GROUP CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] In order to solve the problems of low TNHH fermentation density, high acetic acid production, high plasmid loss rate, and low fermentation yield, the present invention provides a fermentation medium and fermentation method for recombinant leukocyte inhibitory factor and leech peptide chimeric protein

Method used

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  • Fermentation medium and fermentation method of human recombinant neutrophil inhibitory factor and hirulog hybrid
  • Fermentation medium and fermentation method of human recombinant neutrophil inhibitory factor and hirulog hybrid
  • Fermentation medium and fermentation method of human recombinant neutrophil inhibitory factor and hirulog hybrid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067](1) Preparation of seed fluid:

[0068]Solid seed medium: yeast powder 5 g / l, protein 胨 10g / L, sodium chloride 10g / L, agarose 20g / L.

[0069]Liquid seed medium: yeast powder 5 g / l, protein 胨 10g / L, sodium chloride 10g / L.

[0070]A, Screening of strains: Take the bacteria PET-TNHH / BL21 (DE3) PLYSS, to LB flat line activation, 37 ° C culture overnight, picking up single collapse from plate to 8 mLB liquid test tube, 37 ° C, 180R / MIN Culture to OD600It was 1.0-1.5, and IPTG was added to a concentration of 0.2 mmol / L. After 4 hours, centrifugation was collected, and the SDS-PAGE electrophoresis was analyzed, and the TnHH single colonies were screened, and the higheststade of the row line plate was selected from 37 ° C to cultivate overnight. Save at 4 ° C after completion;

[0071]B, first-level seed liquid culture: from the retracted trained plate to pick up the single collaboration in a liquid test tube, 37 ° C, 150R / min culture for 15 h to OD600It is 0.5-1.0, as a see...

Embodiment 2

[0083](1) Preparation of seed fluid:

[0084]Solid seed medium: yeast powder 5 g / l, protein 胨 10g / L, sodium chloride 10g / L, agarose 20g / L.

[0085]Liquid seed medium: yeast powder 5 g / l, protein 胨 10g / L, sodium chloride 10g / L.

[0086]A, Screening of strains: Take the bacteria PET-TNHH / BL21 (DE3) PLYSS, to LB flat line activation, 37 ° C culture overnight, picking up single collapse from plate to 8 mLB liquid test tube, 37 ° C, 180R / MIN Culture to OD600It was 1.0-1.5, and IPTG was added to a concentration of 0.2 mmol / L. After 4 hours, centrifugation was collected, and the SDS-PAGE electrophoresis was analyzed, and the TnHH single colonies were screened, and the higheststade of the row line plate was selected from 37 ° C to cultivate overnight. Save at 4 ° C after completion;

[0087]B, first-level seed fluid culture: from the retracted trained flattening of single collapse into a liquid test tube, 33 ° C, 150R / min culture to OD loading with 8 ml seed medium600It is 0.5-1.0...

Embodiment 3

[0098](1) Preparation of seed fluid:

[0099]Solid seed medium: yeast powder 5 g / l, protein 胨 10g / L, sodium chloride 10g / L, agarose 20g / L.

[0100]Liquid seed medium: yeast powder 5 g / l, protein 胨 10g / L, sodium chloride 10g / L.

[0101]A, Screening of strains: Take the bacteria PET-TNHH / BL21 (DE3) PLYSS, to LB flat line activation, 37 ° C culture overnight, picking up single collapse from plate to 8 mLB liquid test tube, 37 ° C, 180R / MIN Culture to OD600It was 1.0-1.5, and IPTG was added to a concentration of 0.2 mmol / L. After 4 hours, centrifugation was collected, and the SDS-PAGE electrophoresis was analyzed, and the TnHH single colonies were screened, and the higheststade of the row line plate was selected from 37 ° C to cultivate overnight. Save at 4 ° C after completion;

[0102]B, primary seed fluid culture: from the retracted culture of flattened single collapse into a liquid test tube equipped with 8 ml seed medium, 35 ° C, 150R / min culture 12 h to OD600It is 0.5-1....

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Abstract

The invention belongs to the technical field of microbiologic fermentation, and particularly relates to a fermentation medium and a fermentation method of a human recombinant neutrophil inhibitory factor and hirulog hybrid (TNHH). According to the invention, a basal culture medium and a feeding culture medium are optimized with the fermentation culture method changed; more specifically, carbon source is not added for culture at the initial stage of the fermentation, but fed after culture for a certain period of time, so that thallus growth is controlled. Thus, high-density fermentation of theTNHH can be stably realized. Compared with low-density fermentation, the technical scheme disclosed by the invention improves bacterium density by 3-4 times, increases yield of fermentation inclusionbody to as high as 23.8 g / L (namely by more than 5 times,), and ensures low plasmid loss rate and low acetic acid yield. Therefore, the fermentation medium and the fermentation method are capable of improving production efficiency of the TNHH, and has a very good industrial application prospect.

Description

Technical field[0001]The present invention belongs to the field of microbial fermentation techniques, and more particularly to fermentation medium and fermentation methods of recombinant white blood cell inhibitory factor and hydrotide chimeric protein.Background technique[0002]Cerebrovascular disease is one of the three causes of human diseases, with high residual and mortality characteristics, seriously endangering human health, among which ischemia cein vascular disease (ICVD) is more common. About all cerebrovascular patients are 60% to 70%, which can be divided into two categories of whole cerebral ischemia and focal cerebral ischemic. The pathogenesis is not fully understood. Studies have shown that the inflammatory response of cerebral edema and transition is the main cause of cerebral ischemia and post-ischemic reperfusion injury. After the cerebral ischemia, the corresponding receptor binding to the cell membrane after local large amount of thrombin is one of the main cause...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12N1/20C12R1/19
CPCC12P21/02C12N1/20Y02A50/30
Inventor 张贵民冀成法邵明学
Owner LUNAN PHARMA GROUP CORPORATION
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