Porcine alpha interferon mature-protein recombinant adenovirus, construction method and application thereof

A recombinant adenovirus, alpha interferon technology, applied in the application, antiviral agent, virus/phage and other directions, can solve the problem of low titer of recombinant adenovirus, inability to use, etc., achieve inhibition of virus proliferation, stable expression, titer and stable effect

Inactive Publication Date: 2012-11-21
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The recombinant adenovirus obtained by this method has a low titer and has a GFP tag, so it cannot be applied clinically

Method used

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  • Porcine alpha interferon mature-protein recombinant adenovirus, construction method and application thereof
  • Porcine alpha interferon mature-protein recombinant adenovirus, construction method and application thereof
  • Porcine alpha interferon mature-protein recombinant adenovirus, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The construction method of pig interferon alpha mature protein recombinant adenovirus comprises the following steps:

[0038] 1. Construction of porcine alpha interferon mature protein adenoviral plasmid (pAd-mIFNα)

[0039] 1. Extraction of total cellular RNA

[0040] The peripheral blood mononuclear lymphocytes of pigs were separated with lymphocyte separation medium (purchased from Shanghai Sangong Company), and the cells were diluted to 5×10 with RPMI-1640 medium (purchased from HyClone Company). 4 / mL, planted in 24-well cell plate, 1mL per well. Add ConA (purchased from Sigma) to 5 μg / mL in each well, place at 37°C, 5% CO 2 cultured in an incubator. After 24 hours, the supernatant was discarded, and 1 mL TRIzol (purchased from Invitrogen) was added to extract the total cellular RNA. The RNA extraction steps were all performed according to the steps in the instructions of the RNA extraction kit produced by Invitrogen.

[0041] 2. Primers

[0042] Primers were ...

Embodiment 2

[0077] The method for constructing recombinant adenovirus of porcine alpha interferon mature protein as described in Example 1, the difference is that the liposome is 293Fectin, and the transfected cells are AD293 cells.

Embodiment 3

[0079] Mass Preparation of Porcine mIFNα Recombinant Adenovirus

[0080]The recombinant adenovirus rAd-mIFNα purified in Example 1 and Example 2 was continuously passaged on 293 cells for 50 generations, and the expression of porcine mIFNα was detected, which proved that the expression of porcine mIFNα was stable, and the titer of the recombinant virus was stable at 10 8.67 TCID 50 / mL.

[0081] During the expanded culture, the purified porcine mIFNα recombinant adenovirus was used for 10 4 TCID 50 293 cells grown as monolayers were seeded. The virus was harvested when the cytopathy reached 80-90%, and the porcine mIFNα recombinant adenovirus was obtained after freezing and thawing, which were labeled as sample 1 and sample 2, respectively.

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Abstract

The invention relates to a porcine alpha interferon mature-protein recombinant adenovirus, a construction method and an application of the porcine alpha interferon mature-protein recombinant adenovirus, wherein porcine alpha interferon expression genes are inserted into a shuttle vector pShuttle-CMV to obtain pShuttle-CMV-mIFN alpha; the pShuttle-CMV-mIFN alpha occurs homologous recombination with an adenovirus skeleton vector pAdEasy-1 to obtain recombinant adenovirus plasmid pAd-mIFN alpha; and then the porcine alpha interferon mature-protein recombinant adenovirus rAd-mIFN alpha is obtained through liposome-mediated cell transfection and packaging. The porcine alpha interferon mature-protein recombinant adenovirus constructed by the method is characterized by stable foreign protein expression and high and stable titer; the titer of the recombinant adenovirus is stable at 108.67TCID50 / mL; and compared with the existing interferon, the titer of the recombinant adenovirus constructed by the method is increased by 10000 times.

Description

technical field [0001] The invention relates to a recombinant adenovirus of porcine alpha interferon mature protein (mIFNα) and its construction method and application, belonging to the technical field of biotechnology. Background technique [0002] Porcine α-interferon has broad-spectrum, high-efficiency anti-virus and anti-tumor effects, and plays a key role in regulating the immune system. Porcine α-interferon has a non-specific inhibitory function on invading viruses and is effective against many major infectious diseases. ground defense function. Compared with blood-derived interferon, porcine alpha interferon prepared by genetic engineering has the advantages of no pollution, high safety, high purity, high specific activity, low cost, and definite curative effect. [0003] Chinese patent document CN101845441A (application number: 201010125057.6) discloses a compound porcine alpha interferon gene and its recombinant vector, belonging to the technical field of genetic e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/21C12N15/861A61K48/00A61K38/21A61P31/12C12R1/93
Inventor 孙文博王金宝黄保华吴家强宋恩亮杜以军李俊丛晓燕时建立任素芳吕伟郭立辉
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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