Method for producing alpha interferon and dedicated bacteria therefor

A technology of recombinant bacteria and host bacteria, applied in the field of production of alpha interferon, can solve the problems of no immunotherapy reagents, poor efficacy, excessive animal food and drug residues, etc., and achieve improved immunity, high specific activity and stable production performance. Effect

Active Publication Date: 2009-09-16
中科拜克(天津)生物药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no effective immunotherapeutic agents
The traditional treatment is to inject "virinin" or "ribavirin". These chemical drugs not only have poor curative effect but also easily lead to excessive drug residues in animal food

Method used

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  • Method for producing alpha interferon and dedicated bacteria therefor
  • Method for producing alpha interferon and dedicated bacteria therefor
  • Method for producing alpha interferon and dedicated bacteria therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, the construction of engineering bacteria

[0051] 1. Construction of engineering bacteria

[0052] 1. Acquisition of porcine interferon alpha gene

[0053] The whole genome DNA of the pig liver was extracted, and the genome DNA was used as a template to carry out PCR amplification with the following primers:

[0054] Upstream: 5'-GCA GAATTC ATGTGTGACCTGCCTCAGACCC-3' (SEQ ID NO: 2), downstream: 5'-CC GGATCC TTA CTCCTTCTTCCTGAGTCTGTCT-3' (sequence 3) (primers were synthesized by Beijing Saibaisheng Company);

[0055] PCR reaction system: template 1μg, upstream and downstream primers 50pmol / L, total volume 50μL; reaction conditions are 98°C, 8min, 94°C, 1min, 54°C, 1min, 72°C, 1min, 30 cycles, and finally at 72°C , 10min.

[0056] The PCR product was electrophoresed on a 1.2% agarose gel, and the results were as follows: figure 1 As shown (M is the molecular weight standard; 1 is the PCR sample; 2 is the negative control), indicating that the size of ...

Embodiment 2

[0086] Embodiment 2, the production technology that prepares alpha interferon with engineering bacterium

[0087] 1. The production process of interferon-alpha prepared by engineering bacteria

[0088] (1) Fermentation production

[0089] Composition of LB medium for fermentation: 10 g / L trypton, 5 g / L yeast extract, 10 g / L NaCl, 30 g / L glycerol, pH 7.2, prepared with distilled water.

[0090] 1. Preparation of seed solution

[0091] Streak inoculate strain DH5α / pBV220 / Sw IFN-αCGMCC NO.2845 on LB agar plate, activate overnight, and select a single colony that grows well; inoculate into a test tube containing LB medium, and cultivate overnight at 30°C; then transfer to In a 1000ml Erlenmeyer flask, add ampicillin (100mg / L) at a ratio of 1:1000, and expand culture at 30°C and 200r / min for 10h.

[0092] 2. Fermentation

[0093] (1) Empty sterilization: Empty cans are sterilized before each fermentation. After the tank body is washed with water, connect the pipelines, tighten...

Embodiment 3

[0123] Embodiment 3, porcine alpha interferon stability (storage period) detection that the present invention obtains

[0124] Dilute the IFN-α stock solution prepared above, add a stabilizer, and then perform the following detection. Take 3 batches for testing.

[0125] 1. The influence of strong light irradiation on the stability of IFN-α: place the IFN-α test product in a light box equipped with fluorescent tubes for 10 days under the condition of illuminance of 4500lx±500lx, and on the 5th and 10th days Three groups were selected for biological specific activity and purity analysis.

[0126] 2. Accelerated test at 25°C: Place the test product of porcine α-interferon in a thermostat for 6 months at 25°C and a relative humidity of 60%±10%, and take samples for analysis at the end of the 1st, 3rd, and 6th months. Test sterility, biological potency, and purity indicators.

[0127] 3. Long-term stability test at 2-8°C: put the porcine interferon-alpha test product in the ref...

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Abstract

The invention discloses a method for producing alpha interferon and a dedicated bacterial strain therefor. The bacterial strain is obtained by introducing the following genes into a host strain: (1) DNA molecules with the nucleotide sequence as shown in the sequence one in the sequence table; (2) DNA molecules whose functional protein is hybridized with and provided with the same coding with the DNA sequence as prescribed in (1) under strict conditions; and (3) DNA molecules whose functional protein has over 90% of homology with and has the same coding with the DNA sequence as prescribed in (1). The method for preparing the alpha interferon comprises the following step of fermenting the recombined strains to obtain the alpha interferon. The Escherichia coli DH5a/pBV220/Sw IFN-aCCMCC NO.2845 of the invention has good genetic stability, stable production performance, and the fermentation expressive recombined alpha interferon has high specific activity, high yield and high safety. The method of the invention is used for preparing the alpha interferon, thus the operation is simple, the cost is low and the efficiency is high. Therefore, the method for preparing alpha interferon of the invention has wide application prospect.

Description

technical field [0001] The invention relates to a method for producing alpha interferon and special bacteria. Background technique [0002] Pig farming is the largest industry in my country's rural areas except planting. Its income accounts for one-third of farmers' total annual income and is the main source of farmers' cash income. In the pig industry, the mortality rate of PRRS, pig "high fever", piglet viral diarrhea and pig infectious pleurisy is the greatest threat to the pig industry, and it is a difficult problem for the veterinary field to solve. Especially during the summer and autumn of 2006, the pig "high fever" epidemic occurred in some areas of my country, which caused great losses to the pig industry. In March 2007, the Ministry of Agriculture made the highly pathogenic porcine blue-ear disease the focus of porcine disease prevention and control to prevent the spread of the disease during the high-incidence season of porcine diseases. However, effective immun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/00C12N1/21C12P21/02C12R1/19
Inventor 毕清贵毕清华
Owner 中科拜克(天津)生物药业有限公司
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