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Recombinant anine interferon alpha standard substance, preparation method and potency determination method thereof

A technology of canine interferon and standard product, which is applied in the field of preparation of recombinant interferon standard product, can solve the problems of inconvenient research and production of canine interferon, inappropriate use of canine interferon and the like, and achieves low production cost, high potency, Reference accurate results

Inactive Publication Date: 2017-01-04
ANHUI JIUCHUAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, human interferon standard products are available, and pig interferon standard products have obtained invention patents. However, there is currently no standard product of recombinant canine interferon α at home and abroad. Because interferon is species specific, interference from humans or pigs Standard products of canine interferon are not suitable for canine interferon, which brings a lot of inconvenience to the research and production of canine interferon in the prevention and treatment of viral diseases

Method used

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  • Recombinant anine interferon alpha standard substance, preparation method and potency determination method thereof
  • Recombinant anine interferon alpha standard substance, preparation method and potency determination method thereof
  • Recombinant anine interferon alpha standard substance, preparation method and potency determination method thereof

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Experimental program
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Effect test

Embodiment 1

[0038] Preparation of recombinant canine interferon-α standard

[0039] 1. Preparation of Recombinant Canine Interferon α Protein Solution

[0040] 1.1 Inoculate engineering bacteria BL21 / pET-28α-rCaIFNα on solid LB medium containing ampicillin, culture at 37°C for 16-24 hours, inoculate positive clones into 3ml LB liquid medium containing ampicillin, shake at 37°C Shake culture for 18 hours to obtain the bacterial liquid;

[0041] 1.2 Inoculate the bacterial liquid obtained in step 1.1 into 50ml of culture medium, the volume ratio of the bacterial liquid to the medium is 1:10, culture it on a shaker at 37°C for 18 hours, and the rotation speed is 200r / min, as the fermentation seed bacterial liquid;

[0042]1.3 Add the prepared LB medium into the fermenter, connect the pH probe and the dissolved oxygen probe for in-situ sterilization, the sterilization temperature is 121°C, and the sterilization time is 20 minutes;

[0043] 1.4 Inoculate the fermented seed liquid into a ferm...

Embodiment 2

[0055] Detection of Recombinant Canine Interferon α Standard

[0056] 1. Determination of protein content

[0057] The protein concentration of the purified rCaIFN-α standard was detected by the Lowry method, and the protein content was 0.4 mg / ml.

[0058] 2. SDS-PAGE for purity identification

[0059] The purity of the rCaIFN-α standard substance was identified by SDS-PAGE, and its purity was 97.34%.

[0060] 3. HPLC purity identification

[0061] The rCaIFN-α standard was analyzed by μRPC C18ST 4.6 / 100 reversed-phase chromatography column and only one main absorption peak was obtained as figure 2 , and one peak is a miscellaneous peak. By calculating the peak area, the main peak area accounts for 99.79% of the total area ( figure 1 ).

[0062] 4. N-terminal amino acid sequence determination

[0063] 4.1 Digestion: Take 200 μg of purified rCaIFN-α protein, add 4 units of recombinant enterokinase, and digest with enzyme digestion buffer at 4°C for 16 hours.

[0064] 4...

Embodiment 3

[0070] Potency Determination of Recombinant Canine Interferon α Standard

[0071] This detection method is based on the principle that rCaIFN-α can protect bovine kidney cells (MDBK) from vesicular stomatitis virus (VSV), and inhibits the cytopathic effect (CPE) of the virus with interferon phenomenon as a method to detect its activity. That is, the reciprocal of the dilution that can still protect half of the cells (50%) from virus attack at the highest dilution per milliliter of interferon test product is defined as the interferon unit (or potency), often expressed in units (U), The results were calibrated with national standards. After the result was observed by staining the surviving MDBK cells with crystal violet dye, the potency of the determined interferon was calculated according to the Reed-Munch formula according to the depth of the staining.

[0072] 1. Experimental materials

[0073] Recombinant human interferon α standard: purchased from Beijing China Food and ...

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Abstract

The invention discloses a recombinant anine interferon alpha standard substance, a preparation method and potency determination method thereof. BL21 / pET-28 alpha-rCaIFN alpha recombinant bacteria of the expression recombinant anine interferon alpha is fermented and induced, and supernatant after bacterial cell disruption is collected directly to extract the total protein; thereafter, a pure product of the preparation is obtained by three-step purification, a freeze-drying protective additive is added in the pure product for vacuum freeze drying, then a standard substance is obtained, wherein the valence is 1.0*1.04 IU / mL, the purity is 97.34% by a SDS-PAGE detection, the purity is 99.79% by a HPLC detection, and the relative molecular weight is 33 KD. According to the preparation methd, the prepared pecombinant anine interferon alpha as a standard substance can be used for comparison of characteristic identification and potency determination of the recombinant anine interferon alpha, when the standard substance is taken as a standard, the determining results of the potency determination of different batches of the recombinant anine interferon alpha are more reliable and more dependable.

Description

technical field [0001] The invention relates to a preparation method of a recombinant interferon standard product, in particular to a recombinant canine interferon alpha standard product, its preparation method and titer determination method. Background technique [0002] With social progress and changes in people's lifestyles, dogs have become a part of people's lives as companion animals. Viral infectious diseases such as canine distemper and canine parvo seriously endanger the development of the dog industry. At present, there is no effective treatment drug. Vaccination is mainly used for prevention, but the immune effect is not good. Simultaneously, zoonoses such as rabies also cause people's attention, therefore, adopt a kind of medicine of safety and no side effect to prevent and treat canine virus disease just seem particularly important. [0003] Interferon is a kind of protein with broad-spectrum anti-virus, anti-tumor and immunomodulatory effects induced by viruse...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K14/56C07K1/22C07K1/18C07K1/16C12Q1/02G01N33/68C12R1/19
CPCC07K14/56C12N2503/02G01N33/5008G01N33/6866G01N2333/56G01N2500/10
Inventor 赵俊王明丽李树启赵雨戚仕梅符修乐赖鹏飞马腾飞王利利高耀辉汪良青周炜
Owner ANHUI JIUCHUAN BIOTECH
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