CN1059836C - Production of inactivated vaccine for epidemic hemorrhagic fever using primary cells of kidney of golden yellow hamster - Google Patents
Production of inactivated vaccine for epidemic hemorrhagic fever using primary cells of kidney of golden yellow hamster Download PDFInfo
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- CN1059836C CN1059836C CN94115202A CN94115202A CN1059836C CN 1059836 C CN1059836 C CN 1059836C CN 94115202 A CN94115202 A CN 94115202A CN 94115202 A CN94115202 A CN 94115202A CN 1059836 C CN1059836 C CN 1059836C
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Abstract
The present invention relates to a method for preparing epidemic hemorrhagic fever vaccine I and II, and bivalent vaccine I and II by kidney cells of golden hamsters, and a concentration and purification method of the vaccines, and the kidney cells of golden hamsters are sensitive to epidemic hemorrhagic fever virus.
Description
The present invention relates to epidemic hemorrhagic fever vaccine, relate to particularly that to use golden hamster kidney cells be cellular matrix and be seed culture of viruses, produce the method for epizootic hemorrhagic heat-inactivated vaccine with the epidemic haemorrhagic fever virus strain that is adapted to golden hamster kidney cells.
Epidemic hemorrhagic fever has another name called hemorrhagic fever with renal syndrome (being called for short EHF or HFRS), is with the disease of natural focus of Mus as communication media, is a kind of viral acute infectious disease of serious harm people ' s health.In recent years, there are 150,000 cases in the whole world every year, and mortality rate reaches 3%-10%.Popular epidemic-stricken area is in continuous expansion, and serious symptom type epidemic-stricken area mainly occurs in the east in Asia and Europe, and light disease type is all over the world.In China, existing 26 provinces have the generation of primary disease and in various degree popular.110,000 cases once took place in 1986, and this all causes the people's health and the national economic development and has a strong impact on.For this reason, to the epidemic-stricken area high-risk group and will enter epidemic-stricken area work or the people of tourism carry out the specific immunity inoculation, be to reduce sickness rate, effectively control the fundamental way of the outbreak of epidemic of primary disease.
The eighties initial stage, Korea S Lee pick Wang reports that at first being separated to the epidemic hemorrhagic fever cause of disease from nature confirms as virus (Lee HW.et al.J infect Dis 146.638.1982), after this, domestic and international many researcheres all set about carrying out the epidemic hemorrhagic fever Inactivated Vaccine.Korea S LeeHW at first develop suckling mouse brain purification vaccine (Lee HW.etal.Arch Virol[Suppl.I], 35.1990), Korea's gold Lip river Ji etc. is developed white mouse and suslik suckling mouse brain purification vaccine (Jin Luoji etc., China's experiment and clinical virology magazine 5,487,1991).In China, the Lanzhou life grind the Mus brain purification vaccine of also developing (Sun Zhuchen etc., Chinese media biology and control magazine 7 phases of 3＜special issue; 203.1992), the Shanghai life grind institute and Zhejiang Province's epidemic prevention station develop long melon gerbil jird nephrocyte vaccine (Zhu Zhiyong etc., Chinese media biology and control magazine 7 phases of 3＜special issue; 279.1992), stream grinds the chick embryo cell vaccine of having developed (Yan Yuchen etc., Chinese media biology and control magazine 3; 166.1992) the calibrating Vero cell vaccine of having developed (Nie Zilin etc., the Chinese biological goods are learned magazine 4; 17.1991).
Main purpose of the present invention provides a kind of with the cellular matrix of golden hamster kidney cells as infective virus, prepares the method for epizootic hemorrhagic heat-inactivated vaccine.
Another main purpose of the present invention provides a kind of preparation method that obtains high titre, high antigen amount production seed culture of viruses, and this method is to comprise I type and the cell seed culture of viruses of II type seed culture of viruses after the eventually last diluted passage of golden hamster kidney cells adapts to.
A further object of the present invention provides a kind of popularity hemorrhagic fever vaccine semi-finished product and concentrates the method for purifying, this method be with semi-finished product vaccine liquid through centrifugal clarification, ultrafiltration dialysis concentrates, and purifies through the sulfate cellulose gel chromatography column again.
The preparation of I, epidemic hemorrhagic fever vaccine seed culture of viruses
[be the JR strain, draw separating the nephridial tissue specimen of prolonging the geographic morbidity in limit rabbit from Jilin Province from " Chinese public health journal " 9 (2); 74.1990] [be the L99 strain, draw from " Jiangxi Medical College's journal " (3) with the lung tissue sample that separates from the Jiangxi Province Rattus losea; 1.1984] prior to 2-4 age in days neonatal rat encephalocoele inoculation adaptation of virus, when viral infection neonatal rat encephalocoele LD50 reached 8.0Log, transferred species was in hamster kidney cell, and pass more than 10 generations continuously with last diluted passage method eventually.Proceed to when going down to posterity for the last time, treat the hamster kidney cell 7-9 of virus strain infection days after, check intracellular virus antigen with exempting from fluorescence method (IFA), IFA reaches when infection cell ++ +～++ ++ the time gather in the crops viral liquid, add the packing of 10-20% Ox blood serum after, put-60 ℃ of preservations.Seed culture of viruses carries out infectious titration at hamster kidney cell, titre 〉=8.5LogTCID50/ml, and mycoplasma inspection and sterility test are all negative.Using monoclonal antibody typing reagent is determined the specificity and the interior no exogenous factor of proof seed culture of viruses of the antigenicity and the serotype of seed culture of viruses simultaneously, with the seed culture of viruses of this seed culture of viruses as the production epidemic hemorrhagic fever vaccine.
II, production of vaccine
1, preparation hamster kidney cell
Select the Golden Hamster of 10-14 age in days health for use, take out nephridial tissue with aseptic formality, shred the back and add 0.125% pancreatin, put 4-8 ℃ 18-20 hour, discard trypsin solution, use the nutritional solution cell dispersion that contains 0.2% lactalbumin hydrolysate Earle ' s liquid and be added with 8% calf serum, make cell suspension, plant in the 10 liter culture bottles, every bottle of kind is gone into cell suspension 1500ml.Tissue Culture Flask is put 37 ℃ of thermostatic chambers, rotating and culturing 2-3 day, can form even, fine and close cell monolayer.
2, virus inoculation and vaccine semi-finished product results
Select cell monolayer well-grown Tissue Culture Flask for infective virus.Need infect the JR seed culture of viruses if produce I type vaccine; If produce the II vaccine then need infect the L99 seed culture of viruses.Infective virus dosage is 3-4Log TCID
50/ 1.0ml.Infecting with liquid is 2% Ox blood serum, 0.2% lactalbumin hydrolysate Earle ' s liquid.Place 32-35 ℃ of thermostatic chamber internal rotation to cultivate 1-3 days the Tissue Culture Flask of infective virus, use Earle ' s liquid flushing cell, and then add the immersion of 0.1% human albumin, 0.07 ‰ cysteine Earle ' s liquid after about 20 hours, reuse with the method flushing once, and then change to 0.2% human albumin, 199 comprehensive culture fluid, put 32-35 ℃ of thermostatic chamber and continue to cultivate.7-9 days results behind the infective virus are collected earlier culture fluid, cell are hit down again, mix with culture fluid after putting-60 ℃ of freeze thawing again, by the silk filtration, sample and do sterility test and titration of virus.Culture fluid and Cell sap mixed liquor after filtering are added 1/4000 formalin (inactivator), put 33-37 ℃ earlier after 24 hours, transposition 2-8 ℃ deactivation, this is the semi-finished product of inactivated vaccine, gets an amount of mixing of I type and II type vaccine semi-finished product to can be made into the epidemic hemorrhagic fever bivalent vaccine.
The semi-finished product vaccine liquid that so makes by the requirement of biological product determination rules, carries out stock solution virus titer mensuration, antigen quantitative determination, remaining Ox blood serum content, and deactivation safety test and potency test etc. are after the assay was approved a series of, can packing carry out finished product inspection.The finished product calibrating comprises sterility test, and pH value should be 7.0-8.0, aluminium hydroxide content 0.4-0.7mg/ml, and free formaldehyde content is inaccurate to surpass 0.025 ‰, and thimerosal content is inaccurate to surpass 0.01%, and 4 of mouse toxicity tests should be good for and be deposited.
3, the purification of vaccine concentrates
Select qualified semi-finished product vaccine liquid, remove most of cell debris through high speed centrifugation (7000rpm40 minute), reuse G2 filter bulb filters once, afterwards through ultrafiltration dialyse concentrated (dialysis solution is pH7.2 0.2MPB), with pH7.2 0.2MPB balance sulfate cellulose gel chromatography column, the vaccine upper prop that dialysis equilibrium is good, wash post with same buffer afterwards and do not flow out (A280nm monitoring) to there being albumen, change pH8.5 0.2MPB eluting virus protein, collect the eluting peak part, the back adding preservative agent (1: 20000 thimerosal) that keeps sample is put 4 ℃ of preservations.The purification sample detection comprises, remaining Ox blood serum assay (＜50ng/ml) render a service calibrating (〉=1: 20, positive rate of rotation 〉=3/4) above-mentioned detection be all qualified after, the purification vaccine carries out aseptic filtration, adds Al (OH)
3To 0.5mg/ml, then, do sterility test and pyrogen testing, carry out the packing lyophilizing after qualified, be finished product.The finished product calibrating comprises sterility test, toxicity test, sterility test, antiseptic test, formaldehyde determination, Al (OH)
3Mensuration, pH mensuration, freeze dried vaccine determination of moisture etc., quality index is as the criterion with inactivated vaccine.
III, crowd's clinic trial
Ministry of Public Health approval II type vaccine carried out clinical observation first in 1989, and the human vaccination does not have obvious adverse reaction, and serum neutralizing antibody all sun changes.Nineteen ninety Ministry of Public Health approval bivalent vaccine carries out human clinical's observation, and we are to volunteer's intramuscular injection 2 pins (0-28) of 206 preimmune serum antibody response feminine genders, intramuscular injection 3 pin (0-28-42; 0-7-28), 56 days inspection serum I FAT antibody male rotary rates are 95.9% after the first pin vaccination; Check that with the Elisa method antibody male rotary rate is 93.5%.Checking that with enzyme plaque reduction neutralization test (EFRNT) to II type virucidin sun rate of rotation be 100%, is that 88.2%, 1992 year three batches of Ministry of Public Health approval II type unit price carries out pilot scale 1500 people II phase clinical observations to I type virus.Injecting 2 pins (0-28) MCPENT method detection neutralizing antibody sun rate of rotation is 96%, and injecting the positive rate of rotation of 3 pins (0-28-42) is 99%.I type vaccine carries out the 3 batches of clinical observations of II phase in approval in 1993, observes 500 people for every batch, and injecting 2 pins (0-28) neutralizing antibody sun rate of rotation is 92%, and injecting 3 pins is 93%.People more than 4000 is inoculated in I type and three batches of clinical observations of II type vaccine pilot scale altogether, inject first pin occur in strong responder be 8 people, inject second pin and the 3rd pin is 22 people.In strong response rate＜1%.Proof epidemic hemorrhagic fever I type, II type and two valency type vaccine are all safe and effective.
Embodiment:
Time: 8 ,-July 19,94 on April
Get the Golden Hamster of 12-14 age in days, put to death the back and sterilize with 0.3% bromo geramine.The aseptic kidney of getting shreds, and cleans blood cell, adds 0.125% the trypsin solution of PH8.0, puts 8 ℃ and carries out cold digestion, takes out after 18-19 hour.Discard trypsin solution, use the washing of Earle ' s liquid, add a small amount of bead jog 5-6 time, collect each time suspension, with 2 layers of filtered through gauze, make cell be suspended from (growth-promoting media is 8% Ox blood serum lactalbumin liquid) in the growth-promoting media, shake up the back branch and install in the 15 liter bottles, 1500 milliliters every bottle, put 37 ℃ of rotating and culturing, after three days, cell grows up to fine and close monolayer.
Choose the Tissue Culture Flask that grows up to fine and close monolayer, discard the growth-promoting media in the former bottle, seed culture of viruses is diluted in the lactalbumin liquid of 2% Ox blood serum, be sub-packed in the culture bottle 1500 milliliters every bottle after the jolting evenly.Put 33-35 ℃ of rotating and culturing 2 days, and discarded viral liquid, use Earle ' s liquid hydro-peening, add soak then, 1500 milliliters every bottle.Soak is 0.07 ‰ cysteine Earle ' s liquid, contains 0.1% human albumin.Soak after 20 hours, discard soak, with the same method again hydro-peening once change to 1500 milliliters of cell maintenance mediums then.Keeping liquid is that 199 comprehensive culture fluid contain 0.2% human albumin.The cell bottle is put 33-35 ℃ of rotating and culturing.Gather in the crops after 8 days, receive earlier 1200 milliliters of each flask culture liquid, retains 300 milliliters, the cell bottle is put-60 ℃ freeze taking-up after 5-6 hour.Jolt cell and make its fragmentation, then with culture fluid and Cell sap hybrid filtering.Add formalin to 1 by liquid measure: 4000,1: 20000 shake well of thimerosal rearmounted 37 ℃ 24 hours, 4 ℃ on transposition is 14 days again, in the meantime, jolting every day vaccine 2 times.The qualified back end hydrogenation aluminum adjuvant of safety test is to 0.5mg/ml.
The above-mentioned vaccine of gathering in the crops is semi-finished product, through sterility test, after the assay approvals such as titration of virus, antigen quantitative determination, remaining Ox blood serum content, inactivation of virus safety test, potency test, carries out packing, 1.2 milliliters every.Carry out the finished product calibrating after the packing, the finished product calibrating comprises sterility test, visual examination, pH value, aluminium hydroxide content, and free formaldehyde content, thimerosal content, toxicity test etc. are the epizootic hemorrhagic heat-inactivated vaccine behind the finished product assay approval.
Claims (3)
1, a kind of method for preparing the epizootic hemorrhagic heat-inactivated vaccine.This method comprises the cellular matrix of the golden hamster kidney cells of application popularity hemorrhagic fever virus sensitivity as infective virus, after the golden hamster kidney cells adaptation is gone down to posterity, the JR strain and the L99 strain virus cell adapted strain that improve the virulence titre are seed culture of viruses, prepare I type, II type and two valency epizootic hemorrhagic heat-inactivated vaccine, and vaccine is concentrated purification.
2, according to after the process of claim 1 wherein that said golden hamster kidney cells is meant that 10-14 age in days Golden Hamster nephridial tissue is with trypsinization, make cell suspension, cultivate the cell monolayer of preparation at the Tissue Culture Flask internal rotation.
3, according to the process of claim 1 wherein said vaccine is concentrated to purify be meant with semi-finished product vaccine liquid that through high speed centrifugation it is concentrated to carry out ultrafiltration dialysis after the G2 filter bulb filters, and purifies through the sulfate cellulose gel chromatography column again.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94115202A CN1059836C (en) | 1994-09-21 | 1994-09-21 | Production of inactivated vaccine for epidemic hemorrhagic fever using primary cells of kidney of golden yellow hamster |
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| Application Number | Priority Date | Filing Date | Title |
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| CN94115202A CN1059836C (en) | 1994-09-21 | 1994-09-21 | Production of inactivated vaccine for epidemic hemorrhagic fever using primary cells of kidney of golden yellow hamster |
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| CN1109782A CN1109782A (en) | 1995-10-11 |
| CN1059836C true CN1059836C (en) | 2000-12-27 |
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| CN94115202A Expired - Fee Related CN1059836C (en) | 1994-09-21 | 1994-09-21 | Production of inactivated vaccine for epidemic hemorrhagic fever using primary cells of kidney of golden yellow hamster |
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| CN103160477B (en) * | 2013-02-25 | 2015-06-03 | 湖南中岸生物药业有限公司 | Method for improving Japanese encephalitis virus titer |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6456620A (en) * | 1987-08-26 | 1989-03-03 | Univ Osaka | Production of hemorrhagic fever with renal syndrome virus antigen and vaccine and diagnostic agent using said antigen |
| US5252410A (en) * | 1991-09-13 | 1993-10-12 | Ballard Power Systems Inc. | Lightweight fuel cell membrane electrode assembly with integral reactant flow passages |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6456620A (en) * | 1987-08-26 | 1989-03-03 | Univ Osaka | Production of hemorrhagic fever with renal syndrome virus antigen and vaccine and diagnostic agent using said antigen |
| US5252410A (en) * | 1991-09-13 | 1993-10-12 | Ballard Power Systems Inc. | Lightweight fuel cell membrane electrode assembly with integral reactant flow passages |
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