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Preparation method of standard product for recombinant ovine interferon Tau biological activity detection

A biological activity and interferon technology, which is applied in the field of preparation of a standard substance for the detection of biological activity of recombinant sheep interferon τ

Inactive Publication Date: 2017-02-01
ANHUI JIUCHUAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no standard product of rShIFN-τ at home and abroad, which brings a lot of inconvenience to the actual research work.

Method used

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  • Preparation method of standard product for recombinant ovine interferon Tau biological activity detection
  • Preparation method of standard product for recombinant ovine interferon Tau biological activity detection
  • Preparation method of standard product for recombinant ovine interferon Tau biological activity detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Preparation of standard substance for detection of biological activity of recombinant sheep interferon τ

[0044] 1. Raw liquid manufacturing

[0045] Fermentation and protein extraction of recombinant E. coli BL21 (pET-32a-rShIFNtau):

[0046] The engineered bacteria were fermented in a Bioengineering NLF22 30L fermentor, and the process was as follows:

[0047] 1. Inoculate the engineered bacteria on solid LB medium containing ampicillin and culture at 37°C overnight.

[0048] 2. Pick positive clones and inoculate them into 3 ml of LB medium containing ampicillin, and culture with shaking at 37°C for 18 hours at a speed of 220 r / min.

[0049] 3. Then inoculate the obtained bacterial liquid into 200ml LB medium at 1:100 (volume ratio), set it on a shaker at 37°C with a rotating speed of 220r / min, and shake it for 18h to serve as fermentation seeds.

[0050] 4. Autoclave the intake filter, sampler, NaOH feed bottle, defoamer feed bottle, etc.

[0051] 5. ...

Embodiment 2

[0071] Example 2 Identification of standard products

[0072] 1. Determination of protein content

[0073] The protein concentration of the purified recombinant sheep interferon τ sample was detected by Lowry method, and the protein content was 1.5 mg / ml.

[0074] 2. SDS-PAGE for purity identification

[0075] SDS-PAGE was used to identify the purity and molecular weight of protein samples obtained from protein expression conditions optimization and engineering bacteria fermentation.

[0076] 2.1 Prepare 5% stacking gel and 12% separating gel according to the molecular cloning test guidelines.

[0077] 2.2 Glue pouring Install the vertical plate SDS polypropylene phthalamide gel electrophoresis device according to the instruction manual of the instrument, quickly pour the separating gel between the two layers of glass plates, and cover the glue surface with a small amount of distilled water; after the separating gel is completely polymerized, The distilled water on the surf...

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Abstract

The invention relates to a preparation method of a standard product for recombinant ovine interferon Tau biological activity detection. The method comprises the following steps of after the fermentation of recombinant escherichia coli BL21 / pET-32a-rShIFNtau, extracting total protein; performing purification by a three-step method of dialysis renaturation, affinity chromatography and dialysis buffer liquid replacement; performing freeze drying; and obtaining the standard product. The recombinant ovine interferon biological activity standard product prepared by the method has the advantages that the work valence is 1.0*10<6>U / mL; through SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) electrophoresis and HPLC (High Performance Liquid Chromatography) detection, the purity is higher than 95 percent; the relative molecular weight is 36KD; and the N-terminal amino acid sequence is CYLSQRLMLDARENL from the tenth amino acid.

Description

technical field [0001] The present invention provides a method for preparing a standard product for detecting the biological activity of recombinant sheep interferon τ, which is a biological activity reference standard used for the identification of recombinant sheep interferon τ and the determination of titer. Background technique [0002] As an important industry in my country, animal husbandry has become an important pillar of the national economy, and it is closely related to people's lives. With the process of reform and opening up, the development of human society and living standards are constantly improving, and animal husbandry has also developed by leaps and bounds. Sheep farming is one of the important pillar industries in my country's animal husbandry and one of the advantageous industries in modern agriculture. Mutton is a necessities of life for Muslims in my country and an important vegetable basket product for urban and rural residents. Raw sheep farming has ...

Claims

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Application Information

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IPC IPC(8): C12N15/20C12N15/70C12N1/21C12R1/19
CPCC07K14/555
Inventor 赵俊王明丽李树启赵雨符修乐戚仕梅高耀辉赖鹏飞马腾飞王利利汪良青周炜
Owner ANHUI JIUCHUAN BIOTECH
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