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Indirect ELISA method for detecting recombinant human interleukin-29 in fermentation liquor

A technology of interleukin and fermented liquid, applied in the field of biotechnology detection, can solve the problems of complicated operation, inability to reflect the activity of the expression product, and long time-consuming

Inactive Publication Date: 2015-03-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

IL-29 is a cytokine with a wide range of biological activities. Due to the complicated operation and long time-consuming of various animal experiment methods, it is not suitable for detecting the activity of the expression product rhIL-29 in the process of optimizing fermentation conditions. The expression level of protein in can not reflect the activity of the expression product rhIL-29

Method used

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  • Indirect ELISA method for detecting recombinant human interleukin-29 in fermentation liquor
  • Indirect ELISA method for detecting recombinant human interleukin-29 in fermentation liquor

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1 Detection of activity of rhIL-29 in fermentation broth during optimization of the amount of methanol added to induce the expression of rhIL-29 in Pichia pastoris engineered strains

[0021] 1. Take strains from the preserved Pichia pastoris engineering strain tube and inoculate them on the YPD plate. After activating the strains, pick a single colony from the YPD plate and inoculate it in the YPD liquid medium; culture at 30°C and 220rpm / min with shaking 24h, as the primary seed for fermentation optimization.

[0022] 2. According to the inoculum amount of 4%, inoculate the first-grade seeds of Pichia pastoris engineering bacteria into BMGY liquid medium, shake and cultivate to OD at 30°C and 220rpm / min 600 = 2-6 (16-18 hours) as secondary seeds optimized for fermentation.

[0023] 3. Leave the secondary seeds at room temperature for 1 hour, discard the supernatant, resuspend the cells with 25mL BMMY, and continue culturing at 30°C and 220rpm / min; at 0h, 24h,...

Embodiment 2

[0034] Example 2 Detection of the activity of rhIL-29 in the fermentation broth during the optimization of the culture temperature for inducing Pichia pastoris engineering strains to express rhIL-29

[0035] 1. Take strains from the preserved Pichia engineering strain tube and inoculate them on YPD plates. After activating the strains, pick a single colony from the YPD plates and inoculate them in YPD liquid medium; culture with shaking at 30°C and 220rpm / min 24h, as the primary seed for fermentation optimization.

[0036] 2. According to the inoculum amount of 4%, inoculate the first-grade seeds of Pichia pastoris engineering bacteria into BMGY liquid medium, shake and cultivate to OD at 30°C and 220rpm / min 600 = 2-6 (16-18 hours) as secondary seeds optimized for fermentation.

[0037] 3. Leave the secondary seeds at room temperature for 1 hour, discard the supernatant, resuspend the cells with 25mL BMMY, and continue culturing at 22°C, 24°C, 26°C, 28°C, and 30°C at 220rpm / m...

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Abstract

The invention provides an indirect ELISA (Enzyme Linked Immunosorbent Assay) method for detecting recombinant human interleukin-29 activity in fermentation liquor, which is used for simply, conveniently and quickly detecting the activity of an expression product rhIL-29 in the fermentation liquor during the fermentation condition optimization process of genetic engineering strains. The detecting method comprises the following steps: diluting the supernatant of the fermentation liquor of the engineered strain expressing rhIL-29 with a coating liquid, directly coating a microtiter plate, hatching for 3 hours at 37 DEG C, and staying overnight at 4 DEG C; washing the plate for three times, adding a confining liquid, and hatching for 3 hours at 37 DEG C; washing the plate for three times, adding goat anti-human IL-29 antibody, and then hatching for 30 minutes at 37 DEG C; washing the plate for three times, adding HRP-labeled rabbit anti-human IgG, and hatching for 30 minutes at 37 DEG C; washing the plate for three times, adding a TMB (Tetramethylbenzidine) substrate, and developing for 10 minutes at 37 DEG C; adding a stop solution to stop the reaction, and determining the OD (Optical Density) value at wavelength A450. The detecting method disclosed by the invention can be applied to detecting the activity of the expression product in the fermentation liquor during the fermentation condition optimization process of the genetic engineering strains, and can be used for determining the OD450 value of the fermentation liquor under different fermentation conditions, so as to determine the optimal fermentation condition of the genetic engineering strains.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and relates to an indirect ELISA method for detecting recombinant human interleukin-29 (rhIL-29) in fermentation broth. Background technique [0002] Human interleukin-29 (interleukin 29, IL-29) is a new cytokine newly discovered in 2003, its structure and function are similar to type I interferon (interferon, IFN), so it is also called IFN-λ1 . [0003] Human IL-29 is mainly synthesized by epithelial cells and hepatocytes and secreted extracellularly to act on target cells. Studies have found that the distribution of IL-29 receptors in different tissues and organs of the human body is significantly different, with high levels of expression in the gastrointestinal tract, respiratory system, heart and some gland cells, and the lowest expression level in the brain, indicating that the target of IL-29 Cells are more localized than type I IFN. The tissue and organ differences in the distribu...

Claims

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Application Information

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IPC IPC(8): G01N33/543
CPCG01N33/6869G01N2333/7155
Inventor 陈伟郑海军陆源李利云邬敏辰吴静
Owner JIANGNAN UNIV
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