CN101732709A - Method for preparing haemorrhagic fever vaccine by culturing primary cells in multilayer culture flask - Google Patents

Abstract

The invention relates to a method for preparing a haemorrhagic fever vaccine by culturing primary cells in a multilayer culture flask. The method comprises the following steps: anatomizing an animal to obtain the primary cells; inoculating half or one third cell of cell amount required by a spinner flask into the multilayer cell culture flask, adding proper culture solution in a hot house for culturing; and inoculating virus for culturing after the cell grows full of a single layer, obtaining virus culture solution, and preparing the vaccine after purification. The multilayer cell flask has the advantages of small amount of inoculated primary cells, large amount of the cultured cells, small occupied space, no need of a flask spinning machine and high yield.

Description

A kind of method of preparing haemorrhagic fever vaccine by culturing primary cells in multilayer culture flask

Affiliated technical field

The invention belongs to the cultivation and the virus breeding of primary cell, mainly is a kind of method of preparing haemorrhagic fever vaccine by culturing primary cells in multilayer culture flask.

Background technology

Cell culture (Cell culture) is an analog machine body physiological condition, cell is taken out from body, under artificial condition, make its existence, growth, breed and go down to posterity, carry out the research of problems such as cell life process, cell carcinogenesis, cell engineering and produce vaccine.By directly take out in human body or the animal body tissue or cell cultivate cry former be commissioned to train foster.The primary cultured cell isolated time is short, and character is interior similar to body.In general, the tissue of puerilism and cell, as: the embryo of animal, young baby's internal organs etc. are easier carry out former be commissioned to train foster.The method of cell culture is adopted in the traditional vaccine preparation mostly.(single or multiple lift, former generation of large-scale culture or passage cell are treated to prepare vaccine after cell covers with virus inoculation behind bottle wall, cultivation, results virus-culturing fluid, purification promptly to adopt the glass rolling bottle of different capabilities.The shortcoming that rolling bottle is cultivated is: the cell culture area is little, take that the hot house space is big, labor intensity is high and be easy to pollute.

Multilayer culture flask is a kind of novel cell culture vessel that development in recent years is got up, its advantage is that culture surface is through special handling, very suitable cell growth, and the cell culture area big, take up room little, do not need Rotary Machine, adopt the pipeline closed-loop operation, be difficult for polluting, the required cell concentration of unit culture area is few and expressing viral titre height, yield than the high 2-3 of rolling bottle doubly.

Summary of the invention

The objective of the invention is to overcome the deficiency of above-mentioned technology, and a kind of method of preparing haemorrhagic fever vaccine by culturing primary cells in multilayer culture flask is provided.

For solving the problems of the technologies described above, the present invention proposes following technical scheme to realize: the method for this preparing haemorrhagic fever vaccine by culturing primary cells in multilayer culture flask, and step is as follows:

(1), select the cleaning level gerbil jird of 10-20 age in days for use, smoked dead with ether, clean and sterilization Mus corpse; The aseptic kidney of getting shreds, through trypsinization, and with containing the calf serum MEM culture fluid cell dispersion of 3-5%, preparation cell suspension, packing multi-layer cellular culture bottle, every bottle of 8000ml puts 37 ± 1 ℃ and cultivates to change after 2-3 days and contain 1% calf serum MEM culture fluid;

(2), treat that 4-5 days cells cover with fine and close monolayer after, press the 0.2MOI virus inoculation, every 100cm ²Cell inoculation 1ml titre be the hemorrhagic fever virus work seed of 6.0lgCCID50/ml; Put 34.5 ± 1 ℃ and cultivated 22-26 hour, discard culture fluid in the bottle, every bottle of PBS flush away calf serum with pH7.2 adds the liquid of keeping that does not contain calf serum then and continues to cultivate;

(3), behind the 5-6 days, carry out the virus results first time, scrape the cell that takes a morsel before the results in culture bottle, with the viral infection rate and the viral type of specific monoclonal fluorescent antibody inspection cell, infection rate should be more than 95%; Each results back adds the fresh liquid of keeping continues to cultivate, and each 40 8000ml of confluent monolayer cells factory every 72 ± 4 hours results once, gathers in the crops 4-5 time;

(4), single virus results liquid is with the 15000r/min continuous flow centrifugation, flow velocity is 2500ml/min, collects supernatant, and supernatant is put 2-8 ℃ of deactivation in 3 days in 1: 4000 ratio adding beta-propiolactone, deactivation is to after date, and each inactivation of virus container is taken a sample immediately and carried out the inactivation of virus demonstration test respectively; Treat that the deactivation demonstration test merges into unit price virus results liquid after qualified, behind 30 times of the filter membrane ultrafiltration and concentration in 100KD aperture, carry out column chromatography, wash the collection first peak with the PBS of 0.02MpH7.2;

(5), the viral liquid behind the merging purification; film aseptic filtration through 0.22um; add the human albumin then as protective agent; human albumin's ultimate density is 0.3% in the semi-finished product; thimerosal content 50ug/ml; virus protein content 31.1-74.22ug/ml, antigenic content is diluted to 1: 128, adds the aluminum hydroxide adjuvant of 0.5mg/ml.

The invention has the advantages that: dissect animal and obtain primary cell, with 1/2 or 1/3 cell inoculation of the required cell concentration of rolling bottle in the multi-layer cellular culture bottle, add an amount of culture fluid and put into hot house and cultivate, wait to cover with behind the monolayer that virus inoculation is cultivated, prepare vaccine behind the results virus-culturing fluid, purification.Use the advantage of multi-layer cellular bottle to be: inoculation primary cell amount cell concentration few, that turn out is many, it is little to take up room, do not need Rotary Machine, yield height.

Description of drawings

Fig. 1 is the growth tendency sketch map of gerbil jird nephrocyte of the present invention in multi-layer cellular bottle and two kinds of different vessels of rolling bottle;

The antigenic content sketch map that Fig. 2 expresses in multi-layer cellular bottle and two kinds of different vessels of rolling bottle for HFRS virus of the present invention.

The specific embodiment

Below in conjunction with drawings and Examples invention is described further:

The method of preparing haemorrhagic fever vaccine by culturing primary cells in multilayer culture flask is as follows:

Select the cleaning level gerbil jird of 10-20 age in days for use, smoked dead with ether, clean and sterilization Mus corpse; The aseptic kidney of getting shreds, through trypsinization, and with containing the calf serum MEM culture fluid cell dispersion of 3-5%, preparation cell suspension, packing multi-layer cellular culture bottle, every bottle of 8000ml puts 37 ± 1 ℃ and cultivates to change after 2-3 days and contain 1% calf serum MEM culture fluid.

After treating that 4-5 days cells cover with fine and close monolayer, press 0.2MOI virus inoculation (every 100cm ²Cell inoculation 1ml titre be the hemorrhagic fever virus work seed of 6.0lgCCID50/ml), putting 34.5 ± 1 ℃ cultivated 22-26 hour, discard culture fluid in the bottle, every bottle of PBS flush away calf serum with pH7.2 adds the liquid of keeping that does not contain calf serum then and continues to cultivate.

After 5-6 days, carry out the virus results first time, scrape the cell that takes a morsel before the results in culture bottle, with the viral infection rate and the viral type of specific monoclonal fluorescent antibody inspection cell, infection rate should be more than 95%.Each results back adds the fresh liquid of keeping continues to cultivate, and each 40 8000ml of confluent monolayer cells factory every 72 ± 4 hours results once, gathers in the crops 4-5 time.

Single virus results liquid is with the 15000r/min continuous flow centrifugation, and flow velocity is 2500ml/min, collects supernatant.Supernatant is put 2-8 ℃ of deactivation in 3 days in 1: 4000 ratio adding beta-propiolactone.Deactivation is to after date, and each inactivation of virus container is taken a sample immediately and carried out the inactivation of virus demonstration test respectively.Treat that the deactivation demonstration test merges into unit price virus results liquid after qualified, behind 30 times of the filter membrane ultrafiltration and concentration in 100KD aperture, carry out column chromatography, wash the collection first peak with the PBS of 0.02MpH7.2.

Viral liquid behind the merging purification through the film aseptic filtration of 0.22um, adds the human albumin then as protective agent.Human albumin's ultimate density is 0.3% in the semi-finished product, thimerosal content 50ug/ml, and virus protein content 31.1-74.22ug/ml, antigenic content is diluted to 1: 128, adds the aluminum hydroxide adjuvant of 0.5mg/ml.

Through many batches of trial productions, to test with traditional method, the every test rating of the hemorrhagic fever vaccine of multilayer culture flask production all meets national examination criteria, and cell is relatively seen appendix figure A and figure B at the stand density and the results liquid antigenic content of different time.

This container is equally applicable to former generation hamster kidney cell and produces the various vaccines with the primary cell preparation such as Japanese encephalitis purified vaccine.

The above description of this invention does not have restricted, if those of ordinary skill in the art is enlightened by it, in the situation of the protection that does not break away from claim of the present invention, makes other malformation of the present invention and embodiment, all belongs to protection scope of the present invention.

Claims (1)

1. the method for a preparing haemorrhagic fever vaccine by culturing primary cells in multilayer culture flask, it is characterized in that: step is as follows:

(1), select the cleaning level gerbil jird of 10-20 age in days for use, smoked dead with ether, clean and sterilization Mus corpse; The aseptic kidney of getting shreds, through trypsinization, and with containing the calf serum MEM culture fluid cell dispersion of 3-5%, preparation cell suspension, packing multi-layer cellular culture bottle, every bottle of 8000ml puts 37 ± 1 ℃ and cultivates to change after 2-3 days and contain 1% calf serum MEM culture fluid;

(2), treat that 4-5 days cells cover with fine and close monolayer after, press the 0.2MOI virus inoculation, every 100cm ²Cell inoculation 1ml titre be the hemorrhagic fever virus work seed of 6.0lgCCID50/ml; Put 34.5 ± 1 ℃ and cultivated 22-26 hour, discard culture fluid in the bottle, every bottle of PBS flush away calf serum with pH7.2 adds the liquid of keeping that does not contain calf serum then and continues to cultivate;

(3), behind the 5-6 days, carry out the virus results first time, scrape the cell that takes a morsel before the results in culture bottle, with the viral infection rate and the viral type of specific monoclonal fluorescent antibody inspection cell, infection rate should be more than 95%; Each results back adds the fresh liquid of keeping continues to cultivate, and each 40 8000ml of confluent monolayer cells factory every 72 ± 4 hours results once, gathers in the crops 4-5 time;

(4), single virus results liquid is with the 15000r/min continuous flow centrifugation, flow velocity is 2500ml/min, collects supernatant, and supernatant is put 2-8 ℃ of deactivation in 3 days in 1: 4000 ratio adding beta-propiolactone, deactivation is to after date, and each inactivation of virus container is taken a sample immediately and carried out the inactivation of virus demonstration test respectively; Treat that the deactivation demonstration test merges into unit price virus results liquid after qualified, behind 30 times of the filter membrane ultrafiltration and concentration in 100KD aperture, carry out column chromatography, wash the collection first peak with the PBS of 0.02MpH7.2;

(5), the viral liquid behind the merging purification; film aseptic filtration through 0.22um; add the human albumin then as protective agent; human albumin's ultimate density is 0.3% in the semi-finished product; thimerosal content 50ug/ml; virus protein content 31.1-74.22ug/ml, antigenic content is diluted to 1: 128, adds the aluminum hydroxide adjuvant of 0.5mg/ml.

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