CN102988974B

CN102988974B - Method for preparing porcine pseudorabies live vaccine by using passage CEF (chicken embryo fibroblast)

Info

Publication number: CN102988974B
Application number: CN201210541795.8A
Authority: CN
Inventors: 杨灵芝; 牛成明; 张静; 马延滨
Original assignee: SHANDONG BINZHOU WO HUA BIOTECH ENGINEERING Co Ltd
Current assignee: SHANDONG BINZHOU WO HUA BIOTECH ENGINEERING Co Ltd
Priority date: 2012-12-14
Filing date: 2012-12-14
Publication date: 2015-05-20
Anticipated expiration: 2032-12-14
Also published as: CN102988974A

Abstract

The invention discloses a method for preparing a porcine pseudorabies live vaccine by using the passage CEF (chicken embryo fibroblast), comprising the following steps: firstly, inoculating the primary CEF in the porcine PRV (pseudorabies virus) low virulent strain to obtain a low porcine PRV; then, preparing a DF-1 cell monolayer; next, inoculating the DF-1 cell monolayer in the prepared low porcine PRV; subsequently, putting the DF-1 cell monolayer in a constant-temperature incubator to incubate; and finally, observing the cytopathy, harvesting a virus solution at the right time, freezing and drying the harvested virus solution in vacuum to obtain the porcine pseudorabies live vaccine. According to the method, the passage CEF (DF-1) replaces the primary CEF to culture the PRV, and the process for culturing is optimized systematically, the PRV is easier to culture, the obtained PRV has higher viral titer, the vaccine has more stable quality, and the production cost is lower.

Description

A kind of method preparing pseudorabies live vaccine by the chick embryo fibroblast that goes down to posterity

Technical field

The present invention relates to and belong to veterinary biologics Cultivating techniques field, be specifically related to a kind of method that chick embryo fibroblast that goes down to posterity prepares pseudorabies Seedling alive.

Background technology

Pseudorabies is sick also known as AujeszkyShi, be the multiple domestic animal caused by pseudorabies virus (Pseudora-bies virus, PRV), the one of poultry and wild animal is the acute infectious disease of cardinal symptom with heating, miscarriage, very itch (except pig), encephalomyelitis.This virus ownership herpetoviridae (Herpesviridae) A type herpesviral subfamilies (Alphaherpesvirinae of alphavirinae), therefore also becomes herpesvirus suis type (Suid herpesvirus), infective bulbar bee paralysis virus or syndrome virus of very itching.The pattern of infection of this virus is extremely wide, and to show to have 40 kinds of animals can be infected, pig be most important reservoir and the carrier of this virus, thus plays an important role to the propagation of this virus.Because infected animal species is extremely many, virus can exist at nature iterative cycles, belongs to typical disease of natural focus, is also one of infectious disease of extremely difficult anti-system.

After pig generation pseudorabies, its clinical symptoms depends on the age of infected pigs, the virulence route of infection of Strain and dosage.Adult and sow main manifestations is respiratory symptom, many in transient, is the main source of band poison and toxin expelling.Farrowing sow miscarriage can be caused, produce stillborn fetus, mummy fetus and the boar comprehensive syndrome such as sterile.Piglet death rate of the onset within 15 ages in days is more up to 100%, and ablactational baby pig sickness rate can reach 40%, and dead pig is increasing.

Primary disease there is no specific treatment medicine at present, the immunity inoculation of vaccine is the essential measure of prevention and corntrol pseudorabies, the conventional attenuated vaccine of pseudorabies, inactivated vaccine and gene-deleted vaccine are developed at present both at home and abroad, these vaccines can alleviate or prevent and treat the clinical symptoms of pseudorabies to a certain extent, thus reduce the economic loss caused.

But the method for the weak poison of PRV is generally cultivated in the preparation method of current porcine pseudorabies virus live vaccine by primary chick embryo fibroblast (CEF), there is the supply that loaded down with trivial details, the malicious valency of operating process complexity is low, difference between batch is large, production cost is high, a large amount of production is limited by SPF egg in this method, and easily introduces exogenous virus or other polluter, is easy to defects such as causing vaccine quality unstable.

In addition, DF-1 cell line is improved in 1998 by Univ Minnesota-Twin Cities USA Department of Animal Science Douglas doctor Foster the earliest, the ELL-0(originating from 10 ages in days navigates) chick embryo fibroblast (CEF), be spontaneous immortality, can Immortalization.It has fibroblastic typical cellular morphology.DF-1 cell reversal record enzyme is negative, does not have the endogenous gene sequence of Avian Sarcoma and leucovirus, so be non-tumorigenic.DF-1 cell line is the global commerce cell line of authorizing through FDA (FDA), has been widely used in the production of the propagation of avian viral, the expression of recombiant protein, the research of oncovirus and animals and humans vaccine at present.DF-1 cell is to infections chicken cloacal bursa virus, reovirus, avian leukosis virus, rous sarcoma virus, the equal susceptible of herpes turkey virus, the pathological changes of avian viral on DF-1 cell is more violent, more obvious, can copy on DF-1 cell, and obvious cytopathy can be formed.DF-1 cell has good multiplication potentiality, compares with chick embryo fibroblast, and the density of DF-1 cell can than large more than 4 times.Therefore, be a current main direction of studying with DF-1 cells produce pig PRV live vaccine.

Summary of the invention

The object of the invention is, a kind of method preparing pseudorabies live vaccine by the chick embryo fibroblast that goes down to posterity is proposed for the deficiencies in the prior art, the method utilizes the chick embryo fibroblast (DF-1) that goes down to posterity to replace primary chick embryo fibroblast CEF to cultivate the weak poison of PRV, and culture process is carried out to the optimization of system, to overcome complicated loaded down with trivial details, high cost, and easily cause the defect of vaccine quality instability.

Technical scheme of the present invention realizes as follows:

(1) primary chick embryo fibroblast (CEF) is first prepared, 20-28h after the preparation of CEF cell, PRV (Pseudorabies virus) (PRV) low virulent strain is inoculated primary chick embryo fibroblast (CEF), obtain the weak seed culture of viruses poison of PRV (Pseudorabies virus), the virus liquid of the weak seed culture of viruses poison of PRV (Pseudorabies virus) of results, surveys the median infective dose (TCID of virus according to a conventional method after multigelation 3 times ₅₀) should 10 be reached ^7.0tCID ₅₀/ more than ml.

(2) prepare chick embryo fibroblast (DF-1) cell monolayer that goes down to posterity, the DMEM culture medium of DF-1 cell containing 8% serum is tamed, is positioned over 37 DEG C, CO ₂content is cultivate in the constant incubator of 5%.In wherein said DMEM culture medium containing 1.5g sodium bicarbonate/liter, PH is transferred to 7.0-7.2.

(3) as about 40 ~ 48h after DF-1 passage, when cell density is 90-100% monolayer, by the PRV weak seed culture of viruses poison of preparation by 0.8% ~ 1%(v/v) dosage of inoculation be inoculated into DF-1 cell monolayer, add the DMEM culture medium containing 2% serum, continue to be positioned over 37 DEG C, CO ₂content is continue in the constant incubator of 5% to cultivate.In wherein said DMEM culture medium containing 1.5g sodium bicarbonate/liter, PH is transferred to 7.0-7.2.

(4) after virus inoculation, DF-1 cell can produce cytopathy, observation of cell pathological changes, when cytopathy reaches 85%, and results virus liquid.

(5) by results virus liquid mix with freeze drying protectant in the ratio of 1:1, through vacuum freezing lyophilizing, pseudorabies live vaccine.In wherein said freeze drying protectant, every 100ml contains the defatted milk powder of 5g sucrose and 10g.

Wherein the serum described in above step 2,3 is domestic serum, elects at least one in Hangzhou Ilex purpurea Hassk.[I.chinensis Sims hyclone, the sharp hyclone in Wuhan three, Lanzhou Min Hai top grade new-born calf serum and Jinan strength cattle new-born calf serum as.

Pseudorabies low virulent strain wherein described in the present invention is Bartha K61 strain, purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture.

A kind of method preparing pseudorabies live vaccine by the chick embryo fibroblast that goes down to posterity of the present invention, the utilization chick embryo fibroblast (DF-1) that goes down to posterity replaces CEF to cultivate PRV weak poison, and culture process is optimized, such as 40 ~ 48h after DF-1 passage, cell is 90% to virus inoculation during just confluent monolayers, the virus liquid poison valency obtained is higher, when the malicious valency of kind of poison is 10 ^7.0tCID ₅₀/ ml ~ 10 ^7.5tCID ₅₀time between/ml, dosage of inoculation is 0.8% ~ 1%(v/v) time, the malicious valency obtained is more high.The advantages such as make the method have the operation of cultivating PRV virus easier, the virus titer obtained is higher, and vaccine quality is more stable, production cost is lower.

Detailed description of the invention

In order to make technical scheme of the present invention clearly understand, below in conjunction with specific embodiment, the present invention is further illustrated.

Embodiment:

Pseudorabies low virulent strain used in the present embodiment is Bartha K61 strain, purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture.The chick embryo fibroblast that goes down to posterity (DF-1) is purchased from Shanghai Inst. of Life Science, CAS cellular resources center.

When DF-1 cell is tamed with domestic serum and culture medium, have selected the serum of domestic several producer when cultivating and tame DF-1 cell, be respectively Hangzhou Ilex purpurea Hassk.[I.chinensis Sims hyclone, the sharp hyclone in Wuhan three, Lanzhou Min Hai top grade new-born calf serum and Jinan strength cattle new-born calf serum.Well can not adapt to domestic serum and domestic culture medium at domestication initial stage DF-1 cell, increase and occur that cell produces the phenomenon of a large amount of cavity.A certain amount of import serum is all added by these four kinds of domestic serum, carry out continuous passage, DF-1 cell is finally made to adapt to domestic serum gradually, the DF-1 cell of four kinds of serum free culture system domestications is carried out the contrast of cell state, comprise cellular morphology, the speed of growth, adherent speed, the factors such as edge index of refraction judge, find that 4 kinds of serum free culture system DF-1 cell state difference are little, because the new-born calf serum cost of Jinan strength cattle is lower, be applicable to the requirement reduced costs in large-scale production, therefore, by the new-born calf serum of Jinan strength cattle when the program is finally determined to cultivate DF-1 cell.

The DMEM culture medium that have selected domestic clear large sky one when cultivating and tame DF-1 cell replaces.Use the sodium bicarbonate of 13.78 grams, large clearly sky one DMEM culture medium powder and 1.5 grams to be dissolved in 1L water, adjust between PH to 7.0 to 7.2, the DF-1 cell forward direction of cultivation often rises in culture medium the Jinan strength cattle new-born calf serum adding 80ml.

Concrete implementation step is as follows:

1, prepare PRV weak seed culture of viruses poison, with the SPF Embryo Gallus domesticus of 9 ~ 11 ages in days decaptitate, extremity and internal organs, prepare chick embryo fibroblast.PRV (Pseudorabies virus) (PRV) low virulent strain is inoculated primary chick embryo fibroblast (CEF), cultivates about 72h, results virus liquid when cytopathy reaches 80%, by the virus liquid multigelation 3 times of results, obtain the weak seed culture of viruses poison of PRV (Pseudorabies virus).The TCID of the weak seed culture of viruses poison of Simultaneously test PRV ₅₀, the malicious valency of the PRV kind poison of mensuration is 10 ^7.4tCID ₅₀/ ml.

2, after DF-1 cell covers with monolayer, passage is carried out.First wash twice with PBS, add appropriate tryptic digestive juice, keep flat about 10s, observation of cell digestion situation, if it is smudgy to find that cellular layer occurs, then discard pancreatin, add appropriate culture fluid (DMEM containing 8% serum), with glass pipette blow and beat gently to cell dispersal be individual cells.Seed cell bottle number be vaccinated cell bottle number and can go down to posterity in the ratio of 1:2.DF-1 cell after going down to posterity puts the 5%CO of 37 DEG C ₂constant incubator in cultivate;

3, DF-1 cell cultivates about 48h in incubator, takes out cell observation cell state secret degree.The DF-1 cell state gone down to posterity is good, and cell density is about 90% ~ 100%.Now DF-1 cell is discarded culture fluid, washs 2 times with PBS, then by the PRV weak seed culture of viruses poison of preparation with 0.8% concentration inoculation DF-1 cell monolayer, put 37 DEG C containing 5%CO ₂constant incubator in adsorb 1h, discard virus liquid, add cell maintenance medium, put 37 DEG C containing 5%CO ₂constant incubator in continue cultivate.When cultivating about 96h, observation of cell pathological changes about reaches 85% results virus, and cells show is cell rounding, comes off.

4, results virus liquid through steriling test, viral level measure, after safety verification is qualified, add the sucrose skimmed milk of 5% and appropriate antibiotic quantitative separating in the ratio of 1:1, through vacuum freezing lyophilizing, pseudorabies live vaccine.

Claims

1. prepare a method for pseudorabies live vaccine by the chick embryo fibroblast that goes down to posterity, it is characterized in that cultural method and step as follows:

(1) primary chick embryo fibroblast (CEF) is first prepared, 20-28h after the preparation of CEF cell, PRV (Pseudorabies virus) (PRV) low virulent strain Bartha K61 strain is inoculated primary chick embryo fibroblast (CEF), obtain PRV (Pseudorabies virus) weak seed culture of viruses poison, after results multigelation 3 times, survey the median infective dose (TCID of virus according to a conventional method ₅₀) reach 10 ^7.0tCID ₅₀the virus liquid of the weak seed culture of viruses poison of PRV (Pseudorabies virus) of/more than ml;

(2) prepare chick embryo fibroblast (DF-1) cell monolayer that goes down to posterity, the DMEM culture medium of DF-1 cell with 8% serum is tamed, is positioned over 37 DEG C, CO ₂content is cultivate in the constant incubator of 5%;

(3) as 40 ~ 48h after DF-1 passage, when cell density is 90-100% monolayer, by the PRV weak seed culture of viruses poison of preparation by 0.8% ~ 1%(v/v) dosage of inoculation be inoculated into DF-1 cell monolayer, add the DMEM culture medium containing 2% serum, be positioned over 37 DEG C, CO ₂content is continue in the constant incubator of 5% to cultivate;

(4) after virus inoculation, DF-1 cell can produce cytopathy, observation of cell pathological changes, when cytopathy reaches 85%, and results virus liquid;

(5) by results virus liquid mix with freeze drying protectant in the ratio of 1:1, through vacuum freezing lyophilizing, pseudorabies live vaccine, in described freeze drying protectant, every 100ml contains the defatted milk powder of 5g sucrose and 10g.

2. a kind of method preparing pseudorabies live vaccine by the chick embryo fibroblast that goes down to posterity according to claim 1, it is characterized in that: described DMEM culture medium is the DMEM culture medium in domestic clear large sky one, wherein containing 1.5g sodium bicarbonate/liter, DMEM cultivates keynote PH to 7.0-7.2.

3. according to a kind of method preparing pseudorabies live vaccine by the chick embryo fibroblast that goes down to posterity described in claim 1, it is characterized in that: wherein the serum described in step (3) is domestic serum, elect at least one in Hangzhou Ilex purpurea Hassk.[I.chinensis Sims hyclone, the sharp hyclone in Wuhan three, Lanzhou Min Hai top grade new-born calf serum and Jinan strength cattle new-born calf serum as.