High-adaptability HEK293 clonal cell strains for recombinant adenovirus vaccine strains and application of high-adaptability HEK293clonal cell strains

A technology of recombinant adenovirus and cell strain, which is applied in the direction of embryonic cells, animal cells, viruses/phages, etc., can solve the problems of recessive infection, immune failure, congenital infection, etc., and achieve the improvement of virus titer and virus titer , the effect of high application value

Active Publication Date: 2015-12-16
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI +1
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Problems solved by technology

However, in recent years, the incidence of swine fever in China has shown an upward trend again, and its epidemic characteristics have also undergone major changes, mainly caused by mild swine fever and persistent infection of sows, abortion, stillbirth and death of newborn piglets. The pig industry has caused serious economic losses
There are some deficiencies in the attenuated swine fever rabbit vaccine, which brings many difficulties to the purification and eradication of the disease
First of all, there are many problems in the production process of the current attenuated swine fever vaccine. The calf testicular cells and calf serum used to produce the vaccine are often contaminated with BVDV or its antibodies, which seriously affects the potency and quality of the attenuated swine fever vaccine; secondly, the attenuated vaccine Maternal antibodies to CSF ​​interfere with vaccination. If the immunization procedure is improper, it is easy to cause immune failure, congenital infection, recessive infection or immune tolerance

Method used

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  • High-adaptability HEK293 clonal cell strains for recombinant adenovirus vaccine strains and application of high-adaptability HEK293clonal cell strains
  • High-adaptability HEK293 clonal cell strains for recombinant adenovirus vaccine strains and application of high-adaptability HEK293clonal cell strains
  • High-adaptability HEK293 clonal cell strains for recombinant adenovirus vaccine strains and application of high-adaptability HEK293clonal cell strains

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Experimental program
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Effect test

Embodiment 1H

[0036] Cloning and Identification of Embodiment 1 HEK293 Clone Cell Strain

[0037] 1 Experimental method

[0038] 1.1 Cloning of HEK293 clone cell line

[0039] (1) Select the kind of cell HEK293;

[0040] (2) Cell subculture: first recover the seed cells selected in step (1) into a T25 (Corning) cell flask, culture for about 36 hours, and subculture according to the ratio of 1:4, and continuously subculture 10 times before cloning;

[0041] (3) HEK293 single cell cloning was performed in a 96-well plate by the limiting dilution method, the digested HEK293 cells were counted and diluted to 1 cell / mL, and the diluted cells were spread on a 96-well plate, 0.1 mL / well, 37 ℃5%CO 2 Cultivate in the incubator and observe for 10 days;

[0042] (4) Select a single clone for further subcloning. After subcloning twice, select a single clone for expansion culture in DMEM (containing 10% FBS), and further identify the cloned cells.

[0043] 1.2 Identification of HEK293 clone cell li...

experiment example 1

[0065] Experimental Example 1 Optimizing Test of Propagation of Recombinant Adenovirus rAdV-SFV-E2 Strain Conditions Using HEK293 Clone Cell Line HEK293-D9

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Abstract

The invention discloses high-adaptability HEK293 clonal cell strains for recombinant adenovirus vaccine strains and application of the high-adaptability HEK293 clonal cell strains, which belongs to the field of HEK293 clonal cell strains. The high-adaptability HEK293 clonal cell strains and the application thereof are characterized in that a limiting dilution method is adopted for cloning HEK293 single cells; and two monoclonal cell strains with excellent performance are finally screened by subcloning the cloned HEK293 single cells, with microbial preservation numbers of CGMCC (China General Microbiological Culture Collection Center) No. 11096 and CGMCC No. 11097. Compared with a female cell, the two HEK293 monoclonal cell strains provided by the invention have the advantages that the growth speed is remarkably improved, recombinant adenovirus rAdV-SFV-E2 strains can be better replicated, the replication level of viruses is obviously improved, and the better stability is obtained; the two HEK293 monoclonal cell strains are used for replicating the recombinant adenovirus rAdV-SFV-E2 strains, so that virus bulk with higher titer can be obtained, and the virus titer is significantly improved.

Description

technical field [0001] The present invention relates to cloning cell strains, in particular to HEK293 cloning cell strains. The invention also relates to the application of the HEK293 cloning cell strains in cultivating recombinant adenovirus rAdV-SFV-E2 strains, belonging to the field of cloning and application of HEK293 cells. Background technique [0002] Classical swine fever (CSF) is one of the most serious infectious diseases that harm the pig industry. It is listed in the OIE Animal Disease List by the World Organization for Animal Health (OIE), and China lists it as a first-class infectious disease. Since the late 1950s when the attenuated CSF vaccine was popularized and used, it has effectively controlled the large-scale outbreak and prevalence of CSF in China. However, in recent years, the incidence of swine fever in China has shown an upward trend again, and its epidemic characteristics have also undergone major changes, mainly caused by mild swine fever and persi...

Claims

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Application Information

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IPC IPC(8): C12N5/073C12N7/00C12R1/93
Inventor 仇华吉李彦伟孙元王克雄王牟平赵飞赵宪成
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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