Classical swine fever virus (CSFV) gene recombinant adenovirus and production method thereof

A technology of genetic recombination and swine fever virus, which is applied in the direction of biochemical equipment and methods, viruses, antiviral agents, etc., can solve problems such as heavy workload of personnel, cumbersome production process of tissue virus, and restrictions on the development of trade of live pigs and pork products

Inactive Publication Date: 2016-02-24
YEBIO BIOENG OF QINGDAO
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it is interfered by maternal antibodies, leading to the phenomenon of immunization failure, which is reported from time to time. The antibodies produced by inoculation with this attenuated vaccine cannot be distinguished from the antibodies produced by wild virus infection. The challenge of restricting the trade development of my country's live pigs and pork products in non-immune countries and regions
[0003] The currently widely used swine fever vaccines (spleen-associated swine fever vaccine, swine fever milk rabbit vaccine) all need to be produced with live animals. The tissue poisoning production process is cumbersome and the workload is heavy. Can the vaccine manufacturer strictly implement the use of vaccines that have never been vaccinated? It is doubtful that the rabbits used for the rabbit plague vaccine will be used as vaccines

Method used

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  • Classical swine fever virus (CSFV) gene recombinant adenovirus and production method thereof
  • Classical swine fever virus (CSFV) gene recombinant adenovirus and production method thereof
  • Classical swine fever virus (CSFV) gene recombinant adenovirus and production method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0018] Embodiment 1: the preparation of recombinant adenovirus of classical swine fever virus E0-E2 gene

[0019] 1.1 By analyzing the genome sequence of CSFV, an antigenic nucleotide fragment was obtained, and its sequence was modified. The modified nucleotide sequence is SEQ ID NO: 1, encoded The amino acid sequence is SEQ ID NO:2. Compared with the vaccines that have been reported, the vaccines made from the above fragments have better immune performance. The transformed gene was cloned into the PET32a(+) polyclonal restriction site, transformed into DH5a Escherichia coli, coated with ampicillin-resistant plates, single clones were picked, and sequenced for identification. Select positive colonies with correct sequencing, pick single clones, and sequence them for identification.

[0020] 1.2 The target gene was directional cloned into the shuttle plasmid vector pAdTrack-CMV, and the "two-step transformation method" was used for homologous recombination in bacteria to cons...

Embodiment 2

[0024] Example 2: Growth process of HEK293 cell adherent culture in different media

[0025] 2.1 HEK293 cell culture Take out 3 tubes of the same batch of HEK293 frozen cells from the liquid nitrogen tank, quickly put them in a 37°C water bath, and shake them gently to make them melt quickly. Aspirate the cryopreservation solution with lysed cells, add them to centrifuge tubes previously filled with culture solution (preheated at 37°C, 6-8mL), gently blow and beat to mix, centrifuge at 1000g / min for 5min, discard the supernatant, and add 10mL containing 20% serum (WeikeshengFBS, GibcoFBS, GibcoNCS) in DMEM medium, gently blown into cell suspension, respectively in 10mL culture flasks, 37 ° C, 5% CO 2 Cultivated under the incubator.

[0026] 2.2 Exploration of different culture media Change the medium once after resuscitating the cells for 48 hours. When the confluence of the cells is about 90%, wash once with PBS, add a little trypsin to cover the cell surface, and digest for...

Embodiment 3

[0028] Embodiment 3: the technique of different storage conditions of recombinant adenovirus of classical swine fever virus

[0029] 3.1 Operation methods under different storage conditions After the cells have been subcultured for 24 hours, when the adherent cells have reached 70%-80% monolayer cells, replace 10% of the medium with 1% of the medium. Recombinant adenoviruses were used to infect HEK293 cells at a ratio of 1% and 3%, respectively, and the virus was harvested in due course according to the disease conditions.

[0030] 3.2 IFU results under different storage conditions

[0031] Recombinant adenovirus was used to infect HEK293 cells at a ratio of 1% and 3%, respectively, and the results showed ( figure 2 ), the IFU of cells infected with freeze-dried virus is greater than that of cells infected with wet poison in the same proportion; at the same time, whether it is wet poison or freeze-dried virus, the IFU of 3% inoculation ratio is greater than the IFU of 1% ino...

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Abstract

The invention aims to provide a classical swine fever virus (CSFV) gene recombinant adenovirus and a production method thereof. An amino acid sequence of a gene segment used for preparing the CSFV gene recombinant adenovirus is SEQ ID NO:2 and a coded nucleotide sequence is SEQ ID NO:1. The CSFV gene recombinant adenovirus is prepared through homologous recombination of the gene segment to a multiple cloning site of a human type 5 replication-defective adenoviral genome. An optimal process for producing the CSFV E0-E2 gene recombinant adenovirus through adherent culture of HEK 293 cells is characterized by thawing the HEK 293 cells with 20% W-DMEM, after thawing for 48 hours, carrying out enlarged culture and subculture on the cells with 10% W-DMEM, after carrying out culture for 24 hours, inoculating the virus according to 3% until IFU is maximum, and along with repeated virus rejuvenation, infecting the cells for 48 hours according to the virus inoculation proportion of 1% and harvesting the virus, thus harvesting the virus with high virus titer. Therefore, the harvested virus solution is timely freeze-dried and different generations of freeze-dried virus seed batches are built, thus providing reliable virus seeds for preparing viruses with high virus titer levels in quantity for suspension culture.

Description

technical field [0001] The invention belongs to the technical field of virus vaccine preparation, and in particular relates to a recombinant adenovirus of swine fever virus gene and a production method thereof. Background technique [0002] Classical swine fever (CSF) is a highly contagious disease of pigs caused by Classical swine fever virus (CSFV), which has a high mortality rate and has caused huge economic losses to the world's pig industry. Swine fever has been listed as a class A infectious disease of livestock by the World Organization for Animal Health (OIE), and it is also listed as a class I animal disease in my country. Clinically, it is characterized by persistent high fever and a large number of bleeding spots on the skin and mucous membranes. Years of practical experience have proved that vaccination is the main means of preventing and controlling animal diseases. In the 1960s, my country developed the attenuated strain of classical swine fever (also known a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N7/01A61K39/187A61P31/14
CPCC07K14/1816A61K39/187C12N7/00C12N2710/10034
Inventor 张小苗张恒范根成孙永科
Owner YEBIO BIOENG OF QINGDAO
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