CN103865888A

CN103865888A - Adapting method of rabies virus (RV) CTN-1 strain to primary chicken embryo fibroblast

Info

Publication number: CN103865888A
Application number: CN201410131925.XA
Authority: CN
Inventors: 郭采平; 王春华; 罗姗; 刘永娣; 容伟华; 李慧; 周维; 周兰贞; 田华; 张佩; 丁玉江; 黄伟荣; 吴开永; 张运佳; 王红冰; 王锦才; 吴恩应
Original assignee: Shenzhen Weiguang Biological Products Co Ltd
Current assignee: Shenzhen Weiguang Biological Products Co Ltd
Priority date: 2014-04-03
Filing date: 2014-04-03
Publication date: 2014-06-18
Anticipated expiration: 2034-04-03
Also published as: WO2015149387A1; CN103865888B

Abstract

The invention provides an adapting method of a rabies virus (RV) CTN-1 strain to a primary chicken embryo fibroblast. The adapting method comprises the following steps of carrying out 10 continuous passages on RV CTN-1V5 in a vero cell to obtain a CTN-1V15 strain; carrying out one passage on a virus seed of the CTN-1V15 strain in a chicken embryo to obtain a first-generation virus of the RV CTN chicken embryo; carrying out passage on the first-generation virus of the RV CTN chicken embryo in the chicken embryo fibroblast to enable the first-generation virus of the RV CTN chicken embryo to gradually adapt to the chicken embryo fibroblast. The invention provides the adapting method of the RV CTN-1 strain to CEC (chicken embryo cardiomyocytes); by using the method, the CTN strain can be rapidly and efficiently multiplied in the CEC; moreover, the obtained RV CTN chicken embryo cell adapting strain can be stably multiplied on the CEC, and has favorable stability and immune protection. The invention also provides an inactivated vaccine prepared by using the RV CTN chicken embryo cell adapting strain, and the inactivated vaccine has favorable immune protection and can be used for producing refined and purified RV for people.

Description

The adaptive method of rabies virus CTN-1 strain to primary chick embryo fibroblast

Technical field

The present invention relates to Rabies Vaccine field, be specifically related to rabies virus CTN-1 strain to be adapted to the method for primary chick embryo fibroblast.

Background technology

Rabies virus (Rabies virus, RV) be height neurotropic virus, for the sub-thread minus-stranded rna virus of non-segmented negative in Rhabdoviridae (Rhabdoviridae) lyssavirus (Lyssavirus genus), can cause the Pandemic infection disease-rabies of Zoonosis.It is reported that the whole world is every year because rabic death toll approximately has 5.5 ten thousand examples, actual death toll should be apparently higher than these statistical figure (http://www.worldrabiesday.org/).At present, except a few countries such as Japan, Britain, Hawaii and area do not have rabies generation, this disease is worldwide distribution.Asia and Africa are that the most serious area occurs human rabies, account for 99% of whole world total toll.In the rabic country of formal report, India has 30,000 people to die from above rabies every year, ranks first; The nearly death toll counting on every year for 20 years of China exceedes 3000 examples, occupies second (Yunpeng wang et al., 2012.Journal of Applied Virology.1:10-19).There is no at present legal medical expert and control rabies, vaccine inoculation immunity is antirabic unique effective way.

Rabies Vaccine is to be prepared by the RV of deactivation or attenuation.Because RV has a liking for nervosa virus, almost all mammiferous nervous tissues are all had to infectivity.RV infected rabbits spinal cord development for Rabies Vaccine on probation first in the world, with after through animal nerve tissue culture such as rabbit brain, mouse brain, sheep brains, prepare vaccine.But because such vaccine potency is low and have serious transformation reactions and (the .1992. rabies .203-221 such as Lin Fangtao that stopped using by WHO; Meslin F.X.et.al., 1996.Laboratory techniques in rabies.4 ^thed.WHO.223-313).Kissling in 1958 etc. RV street strain and fixed virus are gone down to posterity in primary hamster kidney cell (Kissing P.E.et al., 1958.Proc.Soc Exp Biol Med.98:223-225).After the sixties in 20th century, prepare vaccine with cell and had very great development, people start to utilize various cells to carry out scale operation Rabies Vaccine, particularly human diploid cell is cultivated the development (Wiktor etal., 1964.J Immunol.93:353-366) of vaccine (HDCV).Owing to there not being neuronal tissue, compared with cerebral tissue vaccine before, tissue culture vaccine is not only safer, and more effective.The several tissue culture vaccine of approved at present, it has the efficacy and saferry suitable with HDCV, for example purifying chick embryo cell vaccine (PCEC) (Barth et al., 1984.J BiolStand.12:29-64; Schgal et al., 1993.J.Commun.Dis.27:36-43) and purifying vero cell rabies (PVRV) (Suntharasamal et al., 1986.Lancet.2:129-131).

RV CTN-1 strain is that WHO and relevant department of China ratify the strain for production of vaccine, builds strain and preservation by National Institute for Food and Drugs Control.Li Hongling etc. cultivated RV CTN-1 strain and adapted to go down to posterity in vero cell the eighties in 20th century, obtained the CTN vero cell adapted strain of higher titre, can be used for production of vaccine .1989. biological products such as (learn magazine .2:22-25) Li Hongling.Dong Guanmu etc. further confirm that RV CTN-1 strain can adapt to fast in vero cell, and titre can arrive 7.0logLD ₅₀more than/ml, and can Multiple harvests virus-culturing fluid .1995. microbiology immunology progress .23:82-85 such as () Dong Guanmu.At present existing many production unit application RV CTN-1 strains produce vero cell vaccines and obtain production code and operation (Yu Yongxin .2008. rabies and Rabies Vaccine. the second edition: 192-208).But vero cell matrix is immortality continuous cell line, be therefore necessary to detect the cell residue DNA(Ref. European Pharmacopoeia .2004. Chinese Pharmacopoeia .2010 in finished product) because it may have the risk of transmitting latent virus and other materials.WHO also stipulates that people should be no more than 10ng(WHO Expert Committee on Biological Standardition.Recommendations inactivated rabies vaccine for human use produced in cell substrates andembryonated eggs.Genava.WHO.2005 with the residual DNA dosage of goods).

The people (US4115195) such as Rudolph Barth have described the process of producing Rabies Vaccine, wherein multiple RV strain all can utilize chick embryo fibroblast (Chicken embryo fibroblast cells, CEC) prepare Rabies Vaccine, as VP11 strain, Pasteur strain, PM strain, Flury LEP and Flury HEP.They specifically provide the example that utilizes RV to fix strain VP11, Flury LEP and Flury HEP infection CEC in patent.PATEL Pradip Maganlal and PATEL PankajRamanbhai(PCT/IN2008/000262) describe Pitman moore strain (Wistar strain PM-HDCS 1503-3M) and be adapted to CEC, the RV strain output that obtains is high and the production time is short, is easy to produce on a large scale.1984 the end of the year China Chen road people and Lin Fangtao select with Flury(LEP) strain 68 generation chicken embryo fixed virus (living vaccine strain for animals) adapts to CEC and cultivates Antirabic Vaccine strain, tentatively obtain a strain and can use CEC to there are again rabies chick-embryo cell adapted strain-Wuhan (Wuhan) 34 strains similar with a G strain (3) the immunogenicity ill magazine .4:28-30 altogether of .1988. China Life Insurance such as () Chen Daomin.But not seeing relevant this adaptation strain further reports.The people such as Wang Yuanzheng also attempt RV aG strain on CEC, to carry out adaptation of virus, obtain titre and can reach 7.0lgLD ₅₀the strain of/ml, but whether this strain is adapted to still uncertain (the .2012. Products in China such as Wang Yuanzheng is learned magazine .25:669-671) of CEC completely.Up to now, also not having document to mention makes RV CTN-1 strain be adapted to CEC.

Summary of the invention

The technical problem to be solved in the present invention is to provide and a kind ofly makes rabies virus CTN-1 strain be adapted to the method for primary chick embryo fibroblast and utilize CTNCEC25 strain that method the obtains purposes in preparation rabies virus inactivated vaccine.

The invention provides the adaptive method of rabies virus CTN-1 strain to primary chick embryo fibroblast, comprise the steps:

Step 1: RV CTN-1V5 is passed continuously in vero cell to 10 generations, obtain CTN-1V15 strain.

Step 2: CTN-1V15 strain seed culture of viruses is passed to 1 generation in chicken embryo, the RV CTN chicken embryo generation virus of acquisition.

Step 3: RV CTN chicken embryo generation virus is gone down to posterity in chick embryo fibroblast, make it adapt to gradually chick embryo fibroblast.

In described step 1, be, with the PBS of pH7.4, RV CTN-1V5 is done to 10 times of serial dilutions, then, according to the ratio inoculation vero monolayer cell of 1:100～1:1000,37 ℃ of absorption were added cell maintenance medium after 60 minutes, were placed in 37 ℃, 5%CO ₂in incubator, leave standstill and cultivate, cultivate and within 4～6 days, gather in the crops viral supernatant liquor, passed so continuously for 10 generations, obtain CTN-1V15 strain.

In described step 2, be by CTN-1V15 strain seed culture of viruses through the PBS of pH7.4 after 1:10～1:1000 dilution, get the SPF chicken embryo of viral dilution liquid through yolk sac inoculation 6～7 ages in days, every embryo 0.5ml, be placed in 37～39 ℃, in the full-automatic hatching box of relative humidity 40～80%, hatch, hatch to 72～144 hours, collect and be at death's door but not dead embryo, remove after the head of chicken embryo, trunk is ground, and add virus protection liquid to be prepared into 10% viral suspension according to chicken embryo weight, then 2000rpm, 4 ℃ centrifugal 10 minutes, so pass a generation and obtain CTNCE01.

In described step 3, be with 10 by the PBS of CTNCE01 pH7.4 ⁰～10 ^-4after dilution, according to inoculative proportion 1:10～1:5 × 10 ⁵mix with CEC suspension, be distributed in Tissue Culture Flask, be placed in 35～37 ℃, 5%CO ₂cultivate in incubator, be cultured to cell and occur pathology results virus liquid, continue virus to go down to posterity according to above-mentioned culture condition on CEC suspension, adapt to and stablize after obtain CTNCEC25 strain.

The used substratum that goes down to posterity in step 3 is take 199 substratum as basis, supplement bovine serum, HEPES and human serum albumin, the whole content that makes bovine serum is 3～10%, the whole content of HEPES is 20mmol/L, the whole content of human serum albumin is 0.3～3%, and with 7.5% sodium hydrogen carbonate solution by pH regulator to 7.4～7.8.

The used temperature that goes down to posterity in step 3 is 33～36 ℃.

The best MOI of virus inoculation of going down to posterity in step 3 is 0.001～0.05FFU/ cell, the Best Times of results virus liquid for inoculation after 72～96 hours.

The virus titer of every generation seed culture of viruses all adopts the experiment of cell fluorescence kitchen range conversion unit to carry out.

The present invention also provides the purposes of CTNCEC25 strain in preparation rabies virus inactivated vaccine.

The present invention has following beneficial effect:

First aspect, the invention provides the adaptive method of RV CTN-1 strain to CEC, has obtained strain RVCTN chick-embryo cell adapted strain-RV CTNCEC25 strain by the method, and this strain can be stablized propagation on CEC, and has satisfactory stability and immune protective.

Second aspect, the invention provides a kind of suitable culture process, can make CTN strain breed fast and efficiently in CEC.

The third aspect; the invention provides one and utilize RV chick-embryo cell adapted strain to set up three grades of viral seed banks, the inactivated vaccine of preparation, has good immune protective; its concentrated and purified front stoste is tired and can be arrived 5IU/ml, can be used for producing refining purifying Antirabic Vaccine.

Accompanying drawing explanation

The history that goes down to posterity of Fig. 1 RV CTNCEC25 strain.

The early lesion phenomenon of Fig. 2 RV CTN-1 strain on CEC.

The later stage pathology phenomenon of Fig. 3 RV CTN-1 strain on CEC.

Virus multiplication trend in Fig. 4 RV CTN-1 strain domestication process.

The CP of Fig. 5 RV CTNCEC25 strain study on the stability generation.

Embodiment

The invention provides the adaptive method of rabies virus CTN-1 strain to primary chick embryo fibroblast, as shown in Figure 1, comprise the following steps:

Step 3: RV CTN chicken embryo generation virus is gone down to posterity in chick embryo fibroblast (CEC), make it adapt to gradually CEC.

The present invention relates to adapt to RV CTN-1 strain on CEC, what adopt is that the white Leghorn kind of SPF level egg (is called for short SPF level kind egg, source: emerging great Hua Nong birds, beasts and eggs company limited, address: SPF field, mountain after the general headquarters of Lan Gen Wen Shi group of Le Zhu town, Xinxing County of Chinese Guangdong province, lower same), the RV CTN-1 strain that domestication adopts derives from National Institute for Food and Drugs Control, is CTN-1 strain vero cell 5 generation seed culture of viruses, i.e. RV CTN-1V5 strain.SPF kind egg and RV CTN-1 strain are matrix and the virus strain of WHO approval for the manufacture of human rabies vaccine.

The preparation process of CEC suspension involved in the present invention is as follows:

Select output one week with interior, form normal, eggshell thickness uniformity, flawless, the dense thick white Leghorn kind of the SPF level egg of albumen (source: emerging great Hua Nong birds, beasts and eggs company limited, address: SPF field, mountain after the general headquarters of Lan Gen Wen Shi group of Le Zhu town, Xinxing County of Chinese Guangdong province, lower same), whether put into 37～39 ℃, the incubator of relative humidity 40～80% and hatch, observing with ovoscopy lamp is zygote and vigor thereof.Select 9～11 ages in days, normal, the visible blood vessel clearly of chick embryo development and movable chicken embryo.With 0.2%(m/v) bromogeramine solution soaks after 5 minutes and pulls out, air chamber is upwards placed in egg holder, then uses 2%(m/v) tincture of iodine and 75%(v/v) move in super clean bench after alcohol disinfecting.With aseptic nipper taking-up chicken embryo, put into the plate that fills 1 × Hanks solution.Remove head, the internal organ of chicken embryo, put into sterilizing wide-necked bottle, cut into 1～3mm by sterile scissors ³tissue block, add according to the amount of every piece of chicken embryo 5～8ml the 0.1%(m/v that is preheated to 37 ℃) pancreatin solution, be placed in 37 ℃ of water baths digestion 15～30 minutes.Digest completely, add the bovine serum of 50～250ml cell growth medium (take 199 substratum as basis, interpolations final concentration is 3～5%(v/v), 199 substratum can be purchased from the clear large day Science and Technology Ltd. in Beijing, and model is M199MD505, lower same.For difference substratum below, this substratum final solution is labeled as M0), use without mycetocyte piping and druming pipe piping and druming cell dispersion and obtain cell suspension.Get 1.0ml cell suspension through phosphate buffered saline buffer (PBS, pH7.4, lower with) after 10 times of dilutions with equal-volume 0.4%(m/v) after trypan blue mixes, carry out cell counting with blood counting chamber, according to count results, adjusting cell density is 0.8～1.4 × 10 ⁶the suspension of individual cell/ml, for subsequent use.

PBS(pH7.4 involved in the present invention) preparation method as follows:

Take 8.0g sodium-chlor, 0.2g Repone K, 0.27g dipotassium hydrogen phosphate, 1.42g SODIUM PHOSPHATE, MONOBASIC, adds the abundant stirring and evenly mixing of 800ml water for injection, then adds 38% concentrated hydrochloric acid tune pH to 7.4, is finally settled to 1000ml.After sterilizing in 121 ℃, 15 minutes, room temperature preservation is for subsequent use.

Below will be elaborated by specific embodiment:

Embodiment 1

Take CTN-1V5 strain as female strain, by the relatively screening of many go down to posterity approach and techniques, find by following operational path go down to posterity obtain strain can adapt to gradually the growth in chick-embryo cell, this rabies virus chick-embryo cell adapted strain is named as CTNCEC25 strain.

Step 1: CTN-1V5 strain is gone down to posterity on vero cell, to improve virus titer.

With PBS(pH7.4) RV CTN-1V5(is derived to National Institute for Food and Drugs Control, for CTN-1 strain vero cell 5 generation seed culture of viruses) do 10 times of serial dilutions, then (derive from National Institute for Food and Drugs Control according to the ratio inoculation vero monolayer cell of 1:100～1:1000,121 generations), 37 ℃ of absorption are added cell maintenance medium (take 199 substratum as basis after 60 minutes, add final concentration 10%(v/v) bovine serum, pH7.2～8.0), be placed in 37 ℃, 5%CO ₂in incubator, leave standstill and cultivate, cultivate and within 4～6 days, gather in the crops viral supernatant liquor, passed so continuously for 10 generations, obtain virus titer and can reach 7.5lgLD ₅₀cTN-1V15 strain more than/ml.

Subsequently by the CTN-1V15 strain seed culture of viruses obtaining through PBS(pH7.4) after 1:10～1:1000 dilution, get the SPF chicken embryo of viral dilution liquid through yolk sac inoculation 6～7 ages in days, every embryo 0.5ml, be placed in 37～39 ℃, in the full-automatic hatching box of relative humidity 40～80%, hatch, day by day observe (in 24 hours, dead inoculation embryo is designated as non-specific death), hatch to 72～144 hours, collect and be at death's door but not dead embryo, remove after the head of chicken embryo, trunk is ground, and add virus protection liquid (take 199 substratum as basis according to chicken embryo weight, add final concentration 20%(v/v) bovine serum, pH7.2～8.0) be prepared into 10%(m/v) viral suspension, then 2000rpm(Revolutions Per Minute, rpm), 4 ℃ centrifugal 10 minutes, getting supernatant props up and point is filled to special cryopreservation tube according to 1.0ml/, virus titer mensuration is carried out in sampling, so pass a generation, obtaining RV CTN chicken embryo generation virus is CTNCE01, detecting its titre is 5.2lgLD ₅₀/ ml.

The PBS(pH7.4 for RV CTN chicken embryo generation virus (CTNCE01) that step 2 is obtained) after suitable dilution (10 ⁰～10 ^-4), according to different inoculative proportion (1:10～1:5 × 10 ⁵) (M0, cell density is 0.8～1.4 × 10 with the aforementioned CEC suspension preparing ⁶individual cell/ml) mix, be distributed in Tissue Culture Flask, be placed in 35～37 ℃, 5%CO ₂in incubator, cultivate.Be cultured to cell occur pathology (main manifestations is that sick cell is assembled, circle contracting, faster aging with come off, see Fig. 2) results virus liquid, continue virus to go down to posterity according to above-mentioned culture condition on CEC.

In the process that goes down to posterity, adopt inverted microscope to observe the metamorphosis of the cell after virus inoculation, found that the increase along with passage number, the pathology of virus on cell strengthens gradually; the phenomenon (see figure 3) that the cell of pathology engenders that loose, cavity, kytoplasm are concentrated, organoid considerable damage even dissolves in endochylema, is different from that initial stage sick cell is just assembled, the phenomenon of circle contracting.

Adopt cell fluorescence kitchen range to transform unit experiment (concrete operations are as follows for Fluorescence Focus Units Assay, FFU) to the equal sampling and measuring virus titer of every generation seed culture of viruses simultaneously.Result shows, RV CTN starts well to copy, and passes four

After generation, virus titer is down to minimum (4.5lgFFU/ml), but virus titer starts rising subsequently, reaches after 20 generations,

Titre raises and stablizes to 6.0lgFFU/ml(the results detailed in Fig. 4).

Cell fluorescence kitchen range transforms unit experiment (Fluorescence Focus Units Assay, FFU) and measures virus titer:

1. viral sample to be measured is first carried out to 10 times of serial dilutions with PBS, then carry out 3 times of serial dilutions.Then getting virus liquid after 50 μ l dilutions, to inoculate 50 μ l cell densities be 1 × 10 ⁶the bsr cell (deriving from CDC virus disease prevention and control institute, 25 generations) of individual cell/ml.Mix and be placed on 37 ℃, 5%CO ₂cultivate 24 hours.

2. acetone is fixed and is dyeed

A. above-mentioned cultivation, after 24 hours, is abandoned supernatant liquor, with PBS(pH7.4) wash one time.

B. every hole adds 50 μ l80%(v/v) cold acetone, be placed in-20 ℃ and fix 30 minutes.

C. every hole adds the Antibody agaianst rabies virus (Millipore company, Cat.NO.5100) of 50 μ l FITC marks, is placed in 37 ℃ and hatches 30 minutes, removes antibody-solutions.

D. with PBS washing three times, dry, every hole adds 50 μ l80%(v/v) glycerine, be directly placed in the each dilution fluorescence kitchen range number of fluorescence microscopy Microscopic observation.

E. get the adjacent two dilution data in the hole calculation result according to the following formula of 10 of > and 10 fluorescence kitchen ranges of <.

The low dilution fluorescence mean number of fluorescence mean number × 3+ of testing sample titre (lg FFU/ml)=lg{[(high dilution)]/2 × low extension rate × 1000/50}

Table 1

Remarks: form blank space represents that fluorescence kitchen range number is too much or very few, without keeping in mind.

The method of calculation of carrying out titre according to result in table 1 are as follows:

1# titre (lgFFU/ml)=lg{[(8+9)/2] × 3+ (17+12)/2}/2 × 10 ²× 9 × 1000/50}=5.56

2# titre (lgFFU/ml)=lg{[(8+6)/2] × 3+ (25+29)/2}/2 × 10 ³× 27 × 1000/50}=7.11

Embodiment 2

Condition according to embodiment 1 step 3 reached for 28 generations continuously, obtained RV CTNCEC25 strain 28 generation seed culture of viruses.If continue to go down to posterity according to above-mentioned condition, find that virus titer does not continue rising (titre is hovered in 6.0～6.5lgFFU/ml left and right) along with the increase of the generation that goes down to posterity, therefore for further improving the titre of the strain obtaining, the present invention carries out process optimization from aspects such as culture medium prescription, culture temperature, MOI, pH values, more than the technique after optimization can be increased to 7.0lgFFU/ml by the titre of strain.Processing parameter after optimization is as follows:

Determine culture medium prescription: take 199 substratum as basis, supplement appropriate HEPES(4-hydroxyethyl piperazine ethanesulfonic acid), bovine serum and human serum albumin, make HEPES(4-hydroxyethyl piperazine ethanesulfonic acid) whole content be 20mmol/L, the whole content of bovine serum is 3～10%(v/v), the whole content of human serum albumin is 0.3～3%(m/v), and with 7.5%(m/v) sodium hydrogen carbonate solution is pH regulator to 7.4～7.8.

Virus inoculation MOI is 0.001～0.05FFU/ cell.

Virus culture temperature is 33～36 ℃.

The Best Times of results virus liquid is latter 72～96 hours of inoculation.

Concrete operations are as follows:

(1) the selection of culture medium prescription

A. substratum combination 1(M1): take 199 substratum as basis, supplement appropriate bovine serum, HEPES, the whole content that makes bovine serum is 3%(v/v), the whole content of HEPES is 20mmol/L, and with 7.5%(m/v) sodium hydrogen carbonate solution is pH regulator to 7.4～7.8.

B. substratum combination 2(M2): take 199 substratum as basis, supplement appropriate HEPES, bovine serum, human serum albumin, the whole content that makes HEPES is 20mmol/L, the whole content of bovine serum is 3%(v/v), the whole content of human serum albumin is 0.3%(m/v), and with 7.5%(m/v) sodium hydrogen carbonate solution is pH regulator to 7.4～7.8.

C. substratum combination 3(M3): take 199 substratum as basis, supplement appropriate HEPES, bovine serum, human serum albumin, making the whole content of HEPES is 20mmol/L, the whole content of bovine serum is 10%(v/v), the whole content of human serum albumin is 3%(m/v), and with 7.5%(m/v) sodium hydrogen carbonate solution is pH regulator to 7.4～7.8.

The RV CTNCEC25 strain 28 generation seed culture of viruses (M0 culture medium culturing) that test selects continuous passage to obtain is viral as going down to posterity strain, (except adopting different culture media, prepare CEC suspension according to method as previously mentioned, cell quantity is adjusted into 0.8～1.4 × 10 all to adopt the inoculative proportion of 1:5000 and the CEC suspension preparing ⁶individual cell/ml) mix, then will infect viral cell suspension inoculation in Tissue Culture Flask or cell factory, be placed in 33～35 ℃, 5%CO ₂in environment, cultivate.There is pathology results virus liquid in the cell being cultured to more than 80%, obtain the first-generation of this substratum, continue to go down to posterity by equal conditions with this venom, the s-generation that the virus liquid of results is this substratum, in three kinds of all 3 generations of parallel continuous passage like this of substratum combination, experiment repeats 2 times.

Each generation virus liquid all utilizes FFU method to measure virus titer, the virus culture effect of three kinds of substratum of assessment.Result is as shown in table 2:

Table 2

From table, the result of titer determination can be found out, compared with M1, adopts M2 and M3 to cultivate RV, passes continuously after three generations, and virus titer obviously improves and stablizes to more than 7.0lgFFU/ml.

Carry out T-check analysis simultaneously, see the otherness that three kinds of substratum are cultivated for RV.In showing, in data input Excel table, the p value that calculates M1 and M2 is that 0.001, t value is 5.96.P < 0.05, t > t _0.05(6)(t _0.05(6)=2.447), prove that M1 and M2 also have significant difference.The p value of M1 and M3 is that 0.002, t value is 5.15.P < 0.05, t > t _0.05 ₍₆₎, prove that M1 and M3 also have significant difference.The p value of M2 and M3 is that 0.88, t value is 0.16.P > 0.05, t < t _0.05(6), prove that M2 and M3 do not have notable difference.Therefore take 199 substratum as basis, supplement bovine serum, HEPES and human serum albumin, the whole content that makes bovine serum is 3～10%, the whole content of HEPES is 20mmol/L, the whole content of human serum albumin is 0.3～3%, and with 7.5% sodium hydrogen carbonate solution be the substratum after optimizing by the substratum of pH regulator to 7.4～7.8.

(2) suitable culture temperature

Temperature 1(Tm1): cultivate 2 days for 37 ℃, turn 32 ℃ of cells that continue to be cultured to more than 80% and occur pathology results virus liquids (4th～5 days) results.

Temperature 2(Tm2): there are pathology results virus liquids (4th～5 days) results in 33 ℃ of cells that are cultured to more than 80%.

Temperature 3(Tm3): there are pathology results virus liquids (4th～5 days) results in 36 ℃ of cells that are cultured to more than 80%.

Prepare CEC cell according to above-mentioned steps, the RV CTNCEC25 strain 28 generation strain obtaining with embodiment 1 is the seed culture of viruses that goes down to posterity, and the preparation of CEC suspension adopts M2 substratum, puts the 5%CO of differing temps ₂in incubator, cultivate, there is pathology results virus liquids (4th～5 days) results virus liquid in the cell being cultured to more than 80%, is the first-generation under this temperature condition, then take this virus liquid as kind, continuous passage three generations.Experiment repeats 2 times.

Utilize inverted microscope to evaluate adhering to and growing of cell, find that culture temperature is high in earlier stage, the adherent speed of CEC, spreading rate and growth velocity are all better than the cell under low temperature cultivation, show that temperature usury is in Growth of Cells in earlier stage.

Adopt FFU method to detect virus titer, the virus culture effect under assessment differing temps.Result is as shown in table 3:

Table 3

From table, the result of titer determination can be found out, compared with Tm1, the more favourable RV of Tm2 and Tm3 breeds on primary CEC, and it passes three generations continuously, and the virus titer of every generation is all more than 7.0lgFFU/ml.

Carry out T-check analysis simultaneously, see the otherness that different culture temperature are cultivated for RV.In showing, in data input Excel table, the p value that calculates Tm1 and Tm2 is that 0.002, t value is 5.04.P < 0.05, t > t _0.05(6)(t _0.05(6)=2.447), prove that Tm1 and Tm2 have significant difference.The p value of Tm1 and Tm3 is that 0.001, t value is 3.80.P < 0.05, t > t _0.05(6), prove that Tm1 and Tm3 also have significant difference.The p value of Tm2 and Tm3 is that 0.66, t value is 2.15.P > 0.05, t < t _0.05(6), prove that Tm2 and Tm3 do not have significant difference, that is to say and adopt 33～36 ℃ of cultivation RV all can obtain satisfied virus titer.

(3) best MOI and best harvest time

The RV CTNCEC25 strain 28 generation strain obtaining with embodiment 1 is the seed culture of viruses that goes down to posterity, and (except substratum difference, according to preparing as previously mentioned CEC suspension, its cell quantity is adjusted into 0.8 × 10 to infect CEC suspension with the RV of different quantities ₆individual cell/ml), virus inoculation MOI(multiple of infection, MOI) be respectively 0.1,0.05,0.01,0.001,0.0001 and 0.00001FFU/ cell.The preparation of all test group CEC suspensions all adopts substratum M2, and is placed in 33～36 ℃, 5%CO ₂cultivate.Experiment repeats 2 times, adopts the virus culture effect of FFU method assessment different vaccination amount and different incubation times.Result is as shown in table 4, and it has shown the impact of viral initial inoculum on viral ultimate capacity.

Table 4

Remarks: in table, "/" represents that cell comes off completely, does not have sample.In table, data are the mean values of 2 parallel test results.

As can be seen from the table, adopt MOI=0.001～0.1FFU/ cell that RV is inoculated in to CEC and infect, all can obtain virus harvest liquid more than titre 7.0lgFFU/ml at 96～120h.But from the replicative cycle result of RV CTNCEC virus in early stage on CEC, MOI is larger in inoculation, early stage, residual virus was just more in supernatant liquor, and therefore the best MOI of virus inoculation is 0.001～0.05FFU/ cell, the Best Times of results virus liquid for inoculation after 72～96 hours.

Embodiment 3

The present embodiment provides the preparation that utilizes CTNCEC25 strain to carry out primordial seed to criticize seed culture of viruses, main seed lot seed culture of viruses and work seed lot seed culture of viruses.

Primordial seed is criticized the preparation of seed culture of viruses: the CTNCEC25 strain 28 generation seed culture of viruses that embodiment 1 is obtained is initial, and the processing parameter that utilizes embodiment 2 to optimize adopts substratum M2, cell quantity 0.8～1.4 × 10 ⁶individual cell/ml and the inoculation of MOI=0.01FFU/ cell, be placed in 33～36 ℃, 5%CO ₂in incubator, cultivate, cultivate 72～96 hours (cytopathy 80% left and right) and receive liquid, passed continuously for 5 generations to 33 generations of CTNCEC25 strain, cleer and peaceful bottom cell in virus is gathered in the crops after being mixed together, add bovine serum (final concentration 20%, v/v), human serum albumin (final concentration 1～2%, m/v), sucrose (final concentration 3～5%, m/v), gelatin (final concentration 0.5～1.0%, m/v), after the amount packing of propping up according to 1.0ml/ after mixing, be primordial seed through frozen drying and criticize seed culture of viruses.

The preparation of main seed lot seed culture of viruses: get primordial seed and criticize after seed culture of viruses PBS solution (pH7.4) redissolution, passed continuously for 3 generations by above-mentioned condition, cleer and peaceful bottom cell in CTNCEC25 strain 36 generation virus is gathered in the crops after being mixed together, the virus liquid of results adds bovine serum (final concentration 20%, v/v), human serum albumin (final concentration 1～2%, m/v), sucrose (final concentration 3～5%, m/v), gelatin (final concentration 0.5～1.0%, m/v), after the amount packing of propping up according to 1.0ml/ after mixing, be the seed culture of viruses of main seed lot through frozen drying.

The preparation of work seed lot seed culture of viruses: get after main seed lot seed culture of viruses PBS solution (pH7.4) redissolution, passed continuously for 4 generations by above-mentioned condition, cleer and peaceful bottom cell in CTNCEC25 strain 40 generation virus is gathered in the crops after being mixed together, results virus liquid according to final concentration 20%(v/v) ratio add bovine serum, be mixed evenly the seed culture of viruses that is work seed lot.

Adopt FFU method to measure virus infection titer.Result shows that primordial seed prepared by the present invention criticizes the infection titer of seed culture of viruses, main seed lot and work seed lot seed culture of viruses and be respectively 6.97lgFFU/ml, 6.80lgFFU/ml and 7.59lgFFU/ml.

According to version " Chinese Pharmacopoeia " related request in 2010, three grades of viral seeds are carried out to quality examination, result meets pharmacopeia and requires (in table 5).

Table 5

Remarks: * represents selectable calibrating project.

Embodiment 4

" the prevention vaccine preclinical study technical director principle " issuing according to version " Chinese Pharmacopoeia " in 2010 and State Food and Drug Administration must be investigated stability and the genetic stability of its biological property in the process going down to posterity for the seed culture of viruses of production of vaccine, for the restriction number of times that goes down to posterity of seed culture of viruses provides foundation.

Getting work seed lot seed culture of viruses (being CTNCEC25 strain 40 generation seed culture of viruses) continues to reach continuously for 60 generations by above-described embodiment 3 methods.

The cellular form that adopts inverted microscope to observe after virus inoculation changes, and original with it batch of strain compares.Found that the work seed lot seed culture of viruses virus inoculation CEC obtaining that continues to go down to posterity, be cultured to 72 hours, original batch of strain proterties of CP and its consistent (Fig. 5).

Detect its virus infection titer by FFU method, found that and reached continuously for 60 generations, RV CTNCEC25 strain still can be stabilized in 7.0lgFFU/ml(at the infection titer of CEC and see Fig. 4).

The seed culture of viruses of mitotic stability being investigated to generation with reference to 2010 editions " Chinese Pharmacopoeias " carries out immunogenicity test.Result is as shown in table 6:

Table 6

As can be seen from the above table; RV CTNCEC25 strain work seed lot seed culture of viruses continue to go down to posterity on CEC the virus immunity protective index that obtains all can reach 2010 version " Chinese Pharmacopoeia " the regulation of " protective index is not less than 100 ", show that the strain after 40 generations also has good immune protective.

Adopt molecular biological method to investigate the genetic stability of its major antigen albumen (G albumen).The sequence of the RV CTN-1 strain of announcing according to NCBI website Genbank, (primer sequence is CTN-MGL-F1Sequence (5 ' to3 '): CGTACTCTAGTGACTCGTAA to design RV CTNCEC25 strain G protein-specific sequencing primer; CTN-MGL-R (5 ' to3 '): ATCTTGCGTAGAAAGTTCAT), synthesized by Beijing Liuhe Huada Genomics Technology Co., Ltd.

Utilize the increase G protein gene sequence of this strain of the method for RT-PCR, carry out sequencing and splicing (being completed by Beijing Liuhe Huada Genomics Technology Co., Ltd), use the MegAlign instrument in DNAStar software to analyze the each generation of RVCTNCEC25 strain and RV CTN-1 strain G gene order, result is as shown in table 7:

Table 7

Can find out that from the result of upper table RV CTNCEC25 strain reaches continuously 60 generations its G albumen and do not make a variation completely on CEC, criticize seed culture of viruses with its primordial seed and be consistent, show that this strain has good genetic stability.

Show based on the above results CTNCEC25 strain on CEC, reach continuously 60 generations obtain strain all there is stable biological characteristics and major antigen protein gene heredity; and immune protective is good, show that three grades of viral seed banks of CTNCEC25 strain that we set up are suitable.

Embodiment 5

1. with predetermined three batches of former vaccines of work seed lot seed culture of viruses continuous production.

With the inoculum size of MOI=0.01FFU/ cell, RV CTNCEC25 strain work seed lot virus (being CTNCEC25 strain 40 generation seed culture of viruses) is inoculated in to CEC suspension and (prepares CEC suspension according to described in embodiment 1, substratum is replaced by M2 or M3, and its cell quantity is adjusted into 0.8～1.4 × 10 ⁶individual cell/ml) in, after mixing, be sub-packed in cell bottle or cell factory, be then placed in 33～36 ℃, 5%CO ₂cultivate.

Treat that more than 80% cell occurs that pathology can gather in the crops viral supernatant liquor, the viral supernatant liquor of results is through pin strainer (Ф 0.60 μ m, Millipore company) filter after clarification, add beta-propiolactone, make the final ratio of itself and viral suspension reach 1:4000(v/v), fully, after stirring and evenly mixing, be placed in 2～8 ℃ and hatch at least 24 hours, do not lose viral antigenicity to guarantee the infectivity of abundant inactivation of viruses.After 24 hours, the virus liquid of fully deactivation is placed in to 37 ℃ and places 2 hours, be fully hydrolyzed beta-propiolactone remaining in virus liquid, obtain vaccine to be measured, be stored in 2～8 ℃, detect in order to vaccine valence and antibody level of serum.

2. adopt NIH method to detect tiring of vaccine to be measured, to assess its immune protective.

According to rabies laboratory technique (Laboratory Technologies for Rabies, WHO, 1996) and 2010 version " Chinese Pharmacopoeia " carry out.Attack required experimental vaccine dosage and give identical protection required corresponding to vaccine (National Institute for Food and Drugs Control by relatively protecting mouse to avoid rabies virus in lethal quantity brain; lot number 250009-201108,6.6IU/ agent) determine tiring of the former vaccine of rabies.

After heavily molten with reference to vaccine use 1.0ml sterile water for injection, it is carried out to 5 times of gradient dilutions with PBS, finally getting dilution range is that 1:25～1:3125(is above-mentioned 4 kinds of extent of dilution, refers to respectively 1:25,1:125,1:625,1:3125, lower with) carry out mouse immune.

The vaccine to be measured preparing also carries out 5 times of gradient dilutions with PBS, and finally getting dilution range is that 1:25～1:3125 carries out mouse immune.

Get reference vaccine and vaccine to be measured after dilution, by abdominal channels, mouse is carried out to initial immunity, at least 16 mouse of each extent of dilution, every mouse 0.5ml vaccine diluent.Just exempt from latter 7 days, according to the step of initial immunization and amount, mouse is carried out to second immunisation.After initial immunity 14 days, with containing 5～100LD ₅₀cVS viral suspension (derive from National Institute for Food and Drugs Control, be initially CVS-8, passed for 2 generations in mouse brain, i.e. CVS-10) mouse after immunity is carried out attacking in brain.After attacking in brain, observe day by day 14 days, in 3 days, dead mouse is designated as non-specific death.Calculate and tire by Reed-Muench method.Find:

Described vaccine to be measured and with reference to the actual ED of vaccine ₅₀value is all between the highest and lowest dose level that gives laboratory animal.

The challenging dose of described CVS strain virus suspension is 10LD ₅₀/ 0.03ml, all there is death in all laboratory animal of accepting this challenging dose viral suspension.

Tiring of test vaccine calculated according to the following formula:

Test vaccine relative effectivenes P=(T/S) × d _t/ d _s× D

Wherein in above-mentioned formula, P is vaccine valence to be measured, IU/ml

T is vaccine ED to be measured ₅₀inverse;

S is with reference to vaccine ED ₅₀inverse;

DT is 1 people's dosage of vaccine to be measured, ml;

DS is 1 people's dosage with reference to vaccine, ml;

D is with reference to the tiring of vaccine, IU/ml.

Net result is as shown in table 8:

Table 8

Can learn from above-mentioned experiment, on average tiring of vaccine to be measured is 5.39IU/ml, and each CVS strain virus challenge dose of applying in detecting is all at 5～100LD ₅₀between.The actual ED of the reference vaccine of application in detecting each time ₅₀all between the highest and minimum extent of dilution.Therefore can conclude, this vaccine has immune protective, and NIH tires higher than the quality standard 2.5IU/ agent of WHO and China's current version pharmacopeia.

Claims

1. the adaptive method of rabies virus CTN-1 strain to primary chick embryo fibroblast, is characterized in that, comprises the steps:

2. the adaptive method of rabies virus CTN-1 according to claim 1 strain to primary chick embryo fibroblast, it is characterized in that, in described step 1,, with the PBS of pH7.4, RV CTN-1V5 is done to 10 times of serial dilutions, then according to the ratio inoculation vero monolayer cell of 1:100～1:1000,37 ℃ of absorption were added cell maintenance medium after 60 minutes, were placed in 37 ℃, 5%CO ₂in incubator, leave standstill and cultivate, cultivate and within 4～6 days, gather in the crops viral supernatant liquor, passed so continuously for 10 generations, obtain CTN-1V15 strain.

3. the adaptive method of rabies virus CTN-1 according to claim 2 strain to primary chick embryo fibroblast, it is characterized in that, in described step 2, be by CTN-1V15 strain seed culture of viruses through the PBS of pH7.4 after 1:10～1:1000 dilution, get the SPF chicken embryo of viral dilution liquid through yolk sac inoculation 6～7 ages in days, every embryo 0.5ml, be placed in 37～39 ℃, in the full-automatic hatching box of relative humidity 40～80%, hatch, hatch to 72～144 hours, collect and be at death's door but not dead embryo, remove after the head of chicken embryo, trunk is ground, and add virus protection liquid to be prepared into 10% viral suspension according to chicken embryo weight, then 2000rpm, 4 ℃ centrifugal 10 minutes, so pass a generation and obtain CTNCE01.

4. the adaptive method of rabies virus CTN-1 according to claim 3 strain to primary chick embryo fibroblast, is characterized in that, in described step 3, is with 10 by the PBS of CTNCE01 pH7.4 ⁰～10 ^-4after dilution, according to inoculative proportion 1:10～1:5 × 10 ⁵mix with CEC suspension, be distributed in Tissue Culture Flask, be placed in 35～37 ℃, 5%CO ₂cultivate in incubator, be cultured to cell and occur pathology results virus liquid, continue virus to go down to posterity according to above-mentioned culture condition on CEC suspension, adapt to and stablize after obtain CTNCEC25 strain.

5. the adaptive method of rabies virus CTN-1 according to claim 1 strain to primary chick embryo fibroblast, it is characterized in that, the substratum that middle the used CEC suspension that goes down to posterity in step 3 adopts in preparing is take 199 substratum as basis, supplement bovine serum, HEPES and human serum albumin, the whole content that makes bovine serum is 3～10%, the whole content of HEPES is 20mmol/L, and the whole content of human serum albumin is 0.3～3%, and with 7.5% sodium hydrogen carbonate solution by pH regulator to 7.4～7.8.

6. the adaptive method of rabies virus CTN-1 according to claim 1 strain to primary chick embryo fibroblast, is characterized in that, the used temperature that goes down to posterity in step 3 is 33～36 ℃.

7. the adaptive method of rabies virus CTN-1 according to claim 1 strain to primary chick embryo fibroblast, it is characterized in that, the best MOI of virus inoculation of going down to posterity in step 3 is 0.001～0.05FFU/ cell, the Best Times of results virus liquid for inoculation after 72～96 hours.

8. the adaptive method of rabies virus CTN-1 according to claim 1 strain to primary chick embryo fibroblast, is characterized in that, the virus titer of every generation seed culture of viruses all adopts the experiment of cell fluorescence kitchen range conversion unit to carry out.

9. the purposes of the CTNCEC25 strain obtaining according to claim 4 in preparation rabies virus inactivated vaccine.