CN106834240B

CN106834240B - Virus clone purified strain for producing Peste des petits ruminants live vaccine and application thereof

Info

Publication number: CN106834240B
Application number: CN201710092018.2A
Authority: CN
Inventors: 支海兵; 薛青红; 印春生
Original assignee: China Institute of Veterinary Drug Control
Current assignee: China Institute of Veterinary Drug Control
Priority date: 2017-02-21
Filing date: 2017-02-21
Publication date: 2020-03-10
Anticipated expiration: 2037-02-21
Also published as: CN106834240A

Abstract

The invention relates to a peste des petits ruminants virus clone purified strain and application thereof. The PPRV is a PPRV75/1 Clone9 strain obtained by cloning and purifying an introduced PPRV75/1 strain with weak toxicity of peste des petits ruminants virus, and the virus titer is improved by 10-100 times after cloning; the virus titer of the peste des petits ruminants live vaccine prepared by the method is reduced by no more than 0.3 titer in the process of storage and use; the safety and the immune efficacy are both higher than the OIE specified standard, the test result of immune attack and protection is shown in the specification, and the immunity is 10²TCID₅₀The protection rate is 96.7 percent; immunization 10³TCID₅₀The protection rate is 100%.

Description

Virus clone purified strain for producing Peste des petits ruminants live vaccine and application thereof

Technical Field

The invention relates to establishment and application of a virus clone purified strain for producing a Peste des petits ruminants live vaccine. Belongs to the field of biological products for animals.

Background

Peste des pests viruses (PPR), also known as Peste-des-bovis pestilence (Pseudo-rinderpest), are acute, virulent, and contact infectious diseases caused by Peste des-pests viruses (PPRV); OIE ranks the infectious diseases as A-class infectious diseases, and the morbidity of susceptible groups reaches 100 percent, and the mortality reaches 100 percent when the outbreak is serious. At present, no effective treatment method for the disease exists, and the disease is mainly controlled by early diagnosis and vaccine immunization. Since the first report in kortewa in the fortieth of the nineteenth century, the disease was prevalent in most african countries, the arabian peninsula, israel, syria, irak, jodan and turkish, the middle east region, and the indian peninsula of south asia. Since 2003, large-scale PPR epidemic has been outbreaked in Laos, India, Nepal, Russia, Pakistan, Burma and other countries around China. In 2007, PPR epidemic occurs in the Tibet part of China.

Peste des petits ruminants can cause massive morbidity and mortality in the Peste des petits. The peste des petits ruminants virus has an immunosuppressive effect, and the immunity of an animal body is reduced after infection, so that secondary infection of other pathogens is caused, and the epidemic situation is more complicated and difficult to deal with. Currently, approximately 62.5% of the ruminants worldwide face the threat of the disease, and therefore, the economic and social impact of the disease is enormous.

The production strain of live vaccine against Peste Des petits ruminants recommended by the world animal health organization (Office International Des epidemiology, OIE) is a vaccine strain obtained by separating PPRV75/1 strain from Nigeria in 1975, culturing through Vero cells, and weakening after continuous passage for 70 generations. Currently, OIE reference laboratories have been passaged to 120 generations, and the virus still maintains a certain immunoprotection. The strains were stored in lyophilized form at-20 ℃ after propagation on cells.

Disclosure of Invention

The applicant discovers that cytopathy is irregular, the occurrence time of lesions is more than 96 hours and is atypical, the virus content is low, and particularly the lesions are irregular when virus neutralization tests are carried out in the virus breeding process after the vaccine strain is introduced from a small ruminant animal plague reference laboratory. The invention aims to obtain a strain which has strong reproductive capacity, high virus titer, good safety and immunogenicity and is more suitable for vaccine production by introducing the Peste des petits ruminants 75/1 strain and carrying out cloning, purification and selection.

Technical scheme of the invention

1. A clone purified strain of Peste des pests virus (Peste des pests viruses) is characterized in that the virus strain is delivered to the general microorganism preservation center of China Committee for microorganism research institute, China academy of sciences, China national institute of microbiology, No. 3, West Lu No.1, Anthony-ward, Beijing, 12 months and 15 days in 2016, and the preservation numbers are respectively as follows: CGMCC No. 13388.

2. The clone-purified strain of Peste des petits ruminants virus of claim 1, wherein the strain is obtained by cloning and purifying introduced Peste des petits ruminants virus attenuated 75/1 strain.

3. The use of a cloned and purified strain of Peste des petits ruminants virus according to claim 1, wherein the strain of virus is used for preparing a live Peste des petits ruminants vaccine.

4. The use of a Clone-purified strain of Peste des petits ruminants virus as claimed in claim 3, wherein the preparation method of the Peste des petits ruminants live vaccine is to use Peste des petits ruminants attenuated 75/1Clone9 strain (CGMCC No.13388) virus as a vaccine production strain, inoculate Vero cells, when the cytopathic effect of the cells reaches more than 70%, move the cell bottle to-20 ℃ for repeated freeze thawing for 3 times, and respectively harvest virus liquid; mixing, adding freeze-drying protective agent, and vacuum drying to obtain Peste des petits ruminants live vaccine.

5. The use of a cloned and purified strain of Peste des petits ruminants virus according to claim 4, wherein the preparation method of the Peste des petits ruminants live vaccine comprises the following steps: 0.75 percent of sucrose, 0.40 percent of dextran, 0.40 percent of sorbitol, 0.25 percent of fish gelatin and Na₂HPO₄0.051，NaH₂PO₄0.125, adding a small amount of double distilled water for dissolution, adjusting the pH to 7.2 by 1mol of KOH, supplementing the double distilled water to 100 percent, and carrying out autoclaving at 115 ℃ for 30 min.

Detailed description of the invention

1. Establishment of cloned and purified strain of peste des petits ruminants virus

(1) Virus propagation PPRV75/1 strain virus (introduced by OIE peste des petits ruminants reference laboratory, original strain 125 generations) was freeze-dried, hydrated, diluted 1:10 with serum-free MEM nutrient solution, inoculated with a Vero cell (introduced by American Type Culture Center (ATCC), F150 generation) monolayer, inoculated and then adsorbed at 37 ℃ for 1 hour, MEM containing 2% bovine serum was added, cultured at 37 ℃ and the cytopathic effect was observed daily. And when the cytopathic effect reaches more than 80%, placing the cell bottle at-20 ℃ for repeated freeze thawing for 3 times, harvesting the culture, uniformly mixing with a freeze-drying protective agent, and freeze-drying. Batch number 200201F126, as the original seed stock.

(2) Cloning of virusesDiluting the reproduced PPR virus solution to 10 times by serum-free MEM nutrient solution^-8Inoculating 3 pieces of 96-well cell culture plate, inoculating 24 wells per dilution, 0.1ml per well, simultaneously adding 0.1ml Vero cell suspension, and culturing at 37 deg.C and 5% CO₂Culturing under the condition. Observing cell lesions every day, selecting cell pore marks with only 1 plaque lesion, continuing culturing and observing until 120h, harvesting cultures with only 1 plaque lesion inoculation hole, and preparing PPRV monoclonal basic virus seeds by 4 generations of amplification culture according to the method (1). 13 PPRV clones are obtained from 3 cell culture plates, and a purified Clone strain Peste des petits ruminants virus (PPRV) 75/1Clone9 strain is obtained from the PPRV clones through a comparative test, and is delivered to China general microorganism collection center of microorganism collection institute of China academy of sciences, national institute of microbiology, No. 3, Naja, Beijing, Chajing, 12.15 days 2016, and the preservation numbers are respectively: CGMCC No. 13388.

2. Preparation of Peste des petits ruminants live vaccine

(1) The production seed virus PPRV75/1 Clone9 strain production basic seed virus is provided by Chinese veterinary medicine supervision.

(2) Vero cells are used for production, and preserved and provided by the supervision of Chinese veterinary medicaments.

(3) The preparation of the Peste des petits ruminants epidemic virus liquid comprises the steps of hydrating PPRV virus seeds with serum-free MEM, and synchronously inoculating Vero cell suspensions with different cell concentrations according to the proportion of 0.1%, 0.5%, 1% and 2%, wherein the cell suspension concentrations are respectively adjusted to be 20-30 ten thousand/ml and 50-60 ten thousand/ml. Mixing, subpackaging with cell bottles, culturing in 37 deg.C incubator, observing pathological changes of cells every day, recording the time of pathological changes of more than 70%, transferring the cell bottles to-20 deg.C, repeatedly freezing and thawing for 3 times when pathological changes reach more than 70%, and collecting virus solution.

(4) Virus fluid test virus fluid samples were tested for sterility, mycoplasma and virus content.

1) The sterility test and the mycoplasma test were carried out according to the current "national animal pharmacopoeia" two O-five year edition (three division) Chinese agricultural publishing agency, 2016 (edited by Committee of Chinese veterinary pharmacopoeia), hereinafter referred to as "Chinese veterinary pharmacopoeia" in the present invention.

2) Virus content determination harvested virus fluid samples were serially diluted 10-fold with serum-free MEM, 10 samples were taken^-1～10^－ ⁸Inoculating 96-well cell culture plates with 8 dilutions of virus solution, inoculating 0.1ml of each well in 5-well dilution, and adding 0.1ml of goat kidney cell suspension in each well after inoculation. Placing at 37 ℃ with 5% CO₂Culturing in an incubator for 5-7 days, and calculating TCID according to cytopathic hole number by a Reed-Muench method₅₀。

(5) The PPR virus liquid and the protective agent which are qualified in inspection are mixed according to the ratio of (V/V)1:1, then are fully and uniformly mixed, are subpackaged, are boxed, are freeze-dried according to the freeze-drying curve designed by the invention, and are subjected to vacuum freeze-drying to obtain the PPR virus liquid.

Drawings

FIG. 1 PPRV Clone9 infected cells caused a in the pathogram: PPRV75/1 Clone9 infected cells 60h pathogram, B: PPRV75/1 Clone9 infected cells 72h pathogram, C: PPRV75/1 Clone9 infected cells 84h pathogram, D: PPRV Clone9 infected cells 96h pathogram, E: PPRV75/1 Clone9 infected cells 108h pathogram, F: PPRV75/1 Clone9 infected cells 120h pathogram.

The invention relates to biomaterial resource information

Peste Des petits ruminants virus (PPRV) 75/1 strain (see DIALLO A, TAYLOR W P, LEFEVRE P C, et al. patent of a strain of pest Des ruminants virus: spatial homology living vaccine. Revued' animal medicine vector genes patent, 1989,429(3): 311) was derived from the world animal health organization (Office International Des epidemics, OIE), Peste Des ruminants virus (PPRV) 75/1 strain (PPRV 1339 strain), which has been deposited in North American society for microbiological culture Collection of microorganisms, national culture Collection of Japan, Inc. 2016, 15, Japan, No. 1. Microbiol. Council., Japan, No. 2016, No. 88, No.1, CGM. 7, CGM. 3, strain, CGM. 3, Clinton, Clintoe, CGM. 7, strain, Min, A. Vero cells: generation F168, which was maintained and provided by the china veterinary medicine institute (the china veterinary medicine institute, the china veterinary microbial culture preservation management center, the china veterinary bacterial species catalog (second edition), the china agricultural science and technology press, 2002, p 171-172).

Positive significance of the invention

The invention relates to a peste des petits ruminants virus clone purified strain and application thereof. The virus strain is a PPRV75/1 Clone9 strain obtained by cloning and purifying an introduced PPRV75/1 strain with weak toxicity of peste des petits ruminants virus, and the virus titer is improved by 10-100 times after cloning; the virus titer of the peste des petits ruminants live vaccine prepared by the method is reduced by no more than 0.3 titer in the process of storage and use; the safety and the immune efficacy are both higher than the OIE specified standard, the test result of immune attack and protection is shown in the specification, and the immunity is 10²TCID₅₀The protection rate is 96.7 percent; immunization 10³TCID₅₀The protection rate is 100%.

Examples

The following examples are intended to further illustrate the present invention and are not intended to limit the invention.

Example 1

Cloning and purification of Peste des petits ruminants live vaccine production seed Virus

The applicant discovers in the breeding process of virus seeds that the cell pathological changes are irregular after the vaccine strain is introduced from a small ruminant epidemic reference laboratory, the occurrence time of the pathological changes is more than 96 hours and is atypical, the virus content is low, particularly the pathological changes are irregular when virus neutralization test is carried out, in order to select the strain which has strong reproductive capacity on Vero cells, high virus titer and is suitable for vaccine production, the introduced strain is cloned and purified, and the cloned strain obtained by cloning and purifying is named Pester des viruses (PPRV) 75/1Clone9 strain, and the strain is delivered to the general microorganism preservation center of China Committee for culture Collection of microorganisms, China institute of microbiology, institute of Microbiol Collection, No. 3, North West institute of western medicine, No.1, Beijing rising south Asian province, 12 months and 15 days in 2016 years, wherein the preservation numbers are respectively: CGMCC No. 13388.

Compared with the original PPRV75/1 virus seed introduced in 2002, the PPRV75/1 Clone9 strain has the advantage that the virus content of the cloned and purified PPRV75/1 Clone9 strain is increased by 2.5 times and can reach 10^5.6TCID₅₀0.1ml, safety and immunogenicity after virus clone purificationGood and is more suitable for vaccine production.

1. Cloning of viruses

The PPRV75/1 strain virus (introduced by OIE Peste des petits ruminants reference laboratory, original strain is 125 generations) is frozen, hydrated, diluted by serum-free MEM nutrient solution at a ratio of 1:10, inoculated with Vero cell (introduced by American center for culture (ATCC), F150 generation) monolayer, adsorbed at 37 ℃ for 1 hour after inoculation, added with MEM containing 2% bovine serum, cultured at 37 ℃, and observed for cytopathia every day. And when the cytopathic effect reaches more than 80%, placing the cell bottle at-20 ℃ for repeated freeze thawing for 3 times, harvesting the culture, uniformly mixing with a freeze-drying protective agent, and freeze-drying. Batch number 200201F126, as the original seed stock.

Serial 10-fold dilution of the propagated PPRV virus liquid to 10 times with serum-free MEM nutrient solution^-8Inoculating 3 pieces of 96-well cell culture plate, inoculating 24 wells per dilution, 0.1ml per well, simultaneously adding 0.1ml Vero cell suspension, and culturing at 37 deg.C and 5% CO₂Culturing under the condition. Observing cell lesions every day, selecting cell pore marks with only 1 plaque lesion, continuing culturing and observing until 120h, harvesting cultures with only 1 plaque lesion inoculation hole, and carrying out 4-generation amplification culture on the cultures to prepare PPRV monoclonal basic virus seeds.

13 PPRV75/1 strain clones are obtained from 3 cell culture plates and distributed at 10^-6And 10^-7And (4) dilution degree. Inoculating Vero cells with 13 clone virus liquid for 3 generations of expansion culture, and harvesting about 300ml of virus liquid for each clone virus culture.

2. Identification of virus species

(1) And (3) carrying out purity test on virus seeds of peste des petits ruminants:

1) the sterility and mycoplasma inspection is carried out according to the appendix of the Chinese veterinary pharmacopoeia;

2) exogenous virus test different clone virus seed virus liquid is added with PPRV positive serum with the same volume, neutralized for 1 hour at 37 ℃, respectively inoculated with Vero, PK15, BHK21 and bovine testis cells, cultured at 37 ℃, and subjected to exogenous virus test according to the appendix of the current Chinese veterinary pharmacopoeia (three parts).

The results of the original and cloned PPRV75/1 clones are shown in Table 1.

TABLE 1 Peste des petits ruminants virus seed purity test results

Remarking: "-" indicates that the test result was negative, and "+" indicates that the test result was positive; "ND" means no test was performed.

(2) Virus content determination different clone PPRV75/1 virus seeds were serially diluted 10-fold with serum-free MEM cell culture medium, 10 were taken^-1～10^-6Inoculating 96-well cell plate with six dilutions, inoculating 5-well cell plate with each dilution, each well containing 0.1ml Vero cell suspension, and adding 5% CO at 37 deg.C₂Cultured under the condition for 6 days, and the cytopathic effect is observed. According to the number of cytopathic wells appearing at each dilution, the TCID contained in each 0.1ml of virus solution was calculated by the Reed-Muench method₅₀。

And (3) measuring the virus content of the original virus seeds and virus liquid of each PPRV75/1 clone virus seed in 200201F126 batches, and recording the first appearance time and 100% lesion time of fused cell spots of each clone virus seed during daily observation of cytopathic process. The results are shown in Table 2.

The picture of the cytopathic characteristics and the time of the appearance of the lesions of the PPRV Clone9 strain is shown in FIG. 1.

TABLE 2PPRV 75/1clone virus seed lesion time and virus content determination results

60-72 h is selected to generate fusion plaque for the first time, the pathological changes reach 100% in 120h, and the virus toxic value reaches 10%^5.6TCID₅₀0.1ml of the cloned viruses Clone9, Clone11, Clone12 as preselected clonal strains of the base seed virus were subjected to safety and immunogenicity assays to determine the base seed virus clonal strains.

(3) Differential test PPRV clones were diluted to 200TCID₅₀After neutralizing with PPRV positive serum, inoculating Vero cells to culture for 7 days, and testing results of 13 clone virus seedsTypical lesions appeared in the virus control group, while no cytopathic effect appeared in both the virus neutralization group and the cell control group.

(4) Safety test PPRV75/1 strain clone virus seed is diluted to 10 by sterilized normal saline⁵TCID₅₀And/ml, injecting healthy and susceptible goats 10-15 months old (the serum neutralizing antibody is lower than 4 times) subcutaneously at the neck, and each goat is 1 ml. No body temperature increase and other clinical symptoms occurred 28 days after inoculation. Wherein the observation record of the body temperature of the virus liquid of the PPRV75/1 Clone9 strain is shown in Table 3

TABLE 3PPRV 75/1Clone9 Strain inoculation goat temperature recording chart

(5) Immunogenicity assay

PPRV75/1 clone virus seed immune sheep neutralizing antibody determination, the detected serum is inactivated at 56 deg.C for 30min, diluted with serum-free MEM at 1:4, and then diluted to 1:1024 in 96-hole cell culture plate, each dilution is inoculated with 5 holes, each hole is inoculated with 0.1ml, each hole is added with about 30 ten thousand/ml Vero cell suspension 0.1 ml. At the same time, PPRV75/1 control (virus content 100 TCID) was set up₅₀/0.1ml、10TCID₅₀/0.1ml、1TCID₅₀/0.1ml、0.1TCID₅₀/0.1ml)，37℃5％CO₂Culturing in an incubator, observing cytopathic effect every day until 120h, recording the number of cytopathic effect holes of each dilution, and taking the highest dilution of serum without cytopathic effect in 5 inoculation holes as the titer of serum antibody. Three PPRV75/1 Clone seeds of Clone9, Clone11 and Clone12 were diluted to 10 with serum-free MEM³TCID₅₀The method is characterized in that 5 susceptible goats (the serum neutralizing antibody is lower than 4 times) are inoculated subcutaneously in 1ml, the susceptible goats and 3 control goats are bred in mixed groups, abnormal clinical manifestations of all the inoculated goats are observed for 21 days, and the body temperature and the ingestion are normal. The blood sampling determination is carried out on the neutralizing antibodies of the inoculated goat serum, wherein the neutralizing antibodies are all larger than 16 times, and the neutralizing antibodies of the control goat serum are all smaller than 4 times. The results are detailed in Table 4.

TABLE 4PPRV 75/1clone virus seed immunogenicity assay results

(6) Freeze drying of cloned virus seed

According to the virus content, the cytopathic generation time, the cytopathic typical degree of 13 PPRV75/1 Clone strains and the immunogenicity determination results of 3 preselected Clone strains, the Clone9 strain is selected as the basic virus strain for producing the live vaccine of Peste des petits ruminants, virus liquid is unfrozen, and then freeze-drying protective agent is added according to the ratio of 1:1 for freeze-drying. Because Clone purification of Clone9 strain is carried out for 4 times, the freeze-dried virus strain is equivalent to 130 generations of original virus strain, the mark lot number is 200601F130 as the basic virus strain for producing the live vaccine of Peste des petits ruminants, the freeze-dried virus strain is stored at-70 ℃, the virus strain is named Pestedes Petits Ruminants Virus (PPRV) 75/1Clone9 strain, which is called PPRV Clone9 strain for short, the virus strain is delivered to China Committee for general microbiological culture Collection of China institute of microbiology, Ministry of microbiology, No. 3, Beijing Korean district, 12 months and 15 days in 2016 years, and the preservation numbers are respectively: CGMCC No. 13388.

The Peste des petits ruminants vaccine strain 75/1 is attenuated by 70 generations of Vero cells, and the established original basic virus strain is 120 generations, has good immunogenicity, and is a vaccine strain recommended by OIE for preventing Peste des petits ruminants. However, the vaccine strain introduced from the Peste des petits ruminants reference laboratory is found in the virus seed breeding process that the cytopathy is irregular, the occurrence time of the pathological changes is more than 96 hours and is atypical, the virus content is low, particularly the pathological changes are irregular when virus neutralization test is carried out, in order to select the virus strain which has strong breeding capability on Vero cells, high virus titer and is suitable for vaccine production, the introduced virus strain is cloned and purified, and compared with the PPRV original virus seed introduced in 2002, the result shows that the virus content of the PPRV75/1 Clone9 virus strain after cloning and purification is increased by 2.5 times and can reach 10^5.6TCID₅₀0.1ml, safety after virus clone purification andthe immunogenicity is still good, and the vaccine is more suitable for vaccine production.

Example 2

Preparation of seed lot for producing seed of virus by Peste des petits ruminants live vaccine

An effective means of preventing peste des petits ruminants is to vaccinate the peste des petits ruminants vaccine. In order to develop the Peste des petits ruminants live vaccine, 2 batches of basic seeds for producing the Peste des petits ruminants live vaccine and 1 batch of production seeds are specially prepared, and systematic verification is carried out on the production seeds. The results of the study are now reported as follows:

1. viral propagation

Hydrating the PPR 75/1Clone9 freeze-dried virus seeds with serum-free MEM, inoculating a Vero cell monolayer according to 1% of a cell maintenance solution, culturing at 37 ℃ for about 120h after inoculation, freezing and thawing the cell bottles at-20 ℃ for 3 times when the cytopathic effect reaches more than 70%, harvesting virus solution, and passaging to 145 generations; adding the 141 and 145 generation virus solution into a freeze-drying protective agent according to the proportion of 1:1, subpackaging with 1ml of each ampoule, and freeze-drying after subpackaging. The basic virus seed numbers are 200601F130, 200701F141 and 200801F145 respectively. After lyophilization, the cells were stored in a refrigerator at-70 ℃ until use (see Table 5).

TABLE 5PPRV 75/1 strain original and basic seed and seed lot preparation details of seed

Replacement of virus seeds	Substitute cell and substitute	Date of lyophilization	Number of lyophilized fractions	Storage conditions
					200201F126	VeroF160	2002.10	300 pieces	-70 ℃ refrigerator
200601F130	VeroF161	2006.9	400 pieces	-70 ℃ refrigerator
					200701F141	VeroF163	2007.9	400 pieces	-70 ℃ refrigerator
200801F145	VeroF169	2008.4	300 pieces	-70 ℃ refrigerator

2. Virus seed detection

(1) The sterility and mycoplasma inspection is carried out according to the appendix of the Chinese veterinary pharmacopoeia;

exogenous virus test A virus seed is hydrated by serum-free MEM nutrient solution, then added with PPR positive serum with the same volume, neutralized at 37 ℃ for 1 hour, respectively inoculated with Vero, PK15, BHK21 and bovine testis cells, cultured at 37 ℃, and subjected to exogenous virus test according to the appendix of Chinese veterinary pharmacopoeia. The results of the test are shown in Table 6.

TABLE 6 Peste des petits ruminants virus purity test results

Note: "-" indicates that the test result was negative, and "+" indicates that the test result was positive; "ND" means no test was performed.

The identification test dilutes 200201F126, 200601F130, 200701F141, 200801F145 batches of poison seeds into 200TCID by serum-free MEM nutrient solution₅₀0.1ml, adding equal volume of PPRV positive serum, neutralizing at 37 deg.C for 1 hr, inoculating Vero cell monolayer, adsorbing at 37 deg.C for 1 hr, adding MEM containing 2% bovine serum,

after being cultured at 37 ℃ for 7 days, the virus control group has typical lesions caused by PPRV, and the virus neutralization group and the cell control group have no cytopathic effect.

(2) Measuring virus content by serial diluting lyophilized PPR basic virus seed with serum-free MEM nutrient solution by 10 times, and taking 10^-1～10^-6Diluting the virus solution, inoculating 96-well cell plate, inoculating 5 wells per dilution, each well is 0.1ml, adding 0.1ml Vero cell suspension, inoculating at 37 deg.C and 5% CO₂Culturing in an incubator for 6 days under the condition, observing cytopathic effect, and calculating the virus content according to the cytopathic hole number by a Reed-Muench method. The results of the determination of the virus content of the batches 200201F126, 200601F130, 200701F141 and 200801F145 are shown in the table 7.

TABLE 7 determination of the Virus content of the seed Virus

(3) Safety test the PPR lyophilized seeds of 200201F126, 200601F130, 200701F141 and 200801F145 are respectively diluted into 10 by serum-free MEM nutrient solution⁵TCID₅₀And/ml, injecting 5 healthy susceptible goats of 10-15 months old (the serum neutralizing antibody is lower than 4 times) into the neck part subcutaneously, and each goat is 1 ml. After inoculation, the body temperature is observed for 28 days, the body temperature is continuously measured for 15 days, all the inoculated goats have no abnormal clinical manifestations, and the body temperature and the ingestion are normal.

(4) High generation secondary virus immunogenicity determination 200701F141, 200801F145 PPR freeze-dried virus is diluted into 10 with sterilized normal saline³TCID₅₀Per ml, neckThe susceptible goat was injected subcutaneously with 5 (less than 4-fold neutralizing antibody in serum) 1ml each. 21 days after inoculation, blood is collected and serum is separated, the serum is inactivated in 56 ℃ water bath for 30 minutes, diluted 1:4 on a 96-well cell culture plate and then diluted to 1:1024, 5 wells are inoculated for each dilution, each well is 0.1ml, and 100TCID is added into each well₅₀0.1ml PPR virus solution, acting for 1h at 37 ℃, and adding 0.1ml Vero cell suspension into each hole; PPRV control (virus content 100 TCID) is set up at the same time₅₀/0.1ml、10TCID₅₀/0.1ml、1TCID₅₀/0.1ml、0.1TCID₅₀/0.1ml)，37℃5％CO₂The incubator is cultured for 6 days, the number of cytopathic holes per dilution is recorded, and the highest dilution of serum without cytopathic effect in all 5 test holes is used as the titer of the serum neutralizing antibody.

The results of the immunogenicity test of the poison seeds in batches of 200701F141 and 200801F145 for the high generation are shown in Table 8.

TABLE 8 immunogenicity test results of high-generation 200701F141, 200801F145 batches of PPR basic poison

(5) Shelf life test of the virus seeds original virus seeds introduced and propagated in 2002 are stored in 200201F126 batches, 200601F130 batches, 200701F141 batches and 200801F145 batches of Clone9 virus seeds at-70 ℃ and are subjected to comprehensive test once every 12-24 months.

1) PPRV basic virus content and immunogenicity at different storage times

The original seed of PPR of 200201F126 batches, which was prepared and identified in 2002, is stored in a refrigerator at-70 ℃ for 10 months in 2013. And respectively storing the basic poison seeds of 200601F130 batches, 200701F141 batches and 200801F145 batches in a refrigerator at the temperature of-70 ℃ for 4-6 years. The virus content and immunogenicity of the virus seeds were measured every 1-2 years, and the results are shown in Table 9.

TABLE 9 determination of immunogenicity and virus content after different generations of PPRV basic virus seeds are preserved for different times

Before the test, healthy susceptible goats are screened by adopting a neutralization test method, PPR (propylene glycol fatty acid) neutralizing antibodies of the goats used in the test are less than 4 times, 3 control goats are set in each immunogenicity determination test, the control goats and the goats inoculated with PPRV virus seeds are raised under the same condition, and blood is collected to determine the PPR neutralizing antibodies. The test result shows that PPRV neutralizing antibodies of all control sheep are less than 4 times, and the control is established.

2) Shelf life test of PPRV basic virus species

The results of the examination of the original and basic and production strains of PPRV of different generations after being stored for different periods are shown in Table 10. Test results show that the freeze-dried virus seeds in the 200201F126 batches can reach the regulation of the virus seed standard after being stored in a refrigerator at the temperature of-70 ℃ for 11 years, the freeze-dried virus seeds in the 200601F130 batches and the 200701F141 batches are stored in the refrigerator at the temperature of-70 ℃ for 6 to 7 years, and the freeze-dried virus seeds in the 200801F145 batches can reach the regulation of the virus seed standard after being stored in the refrigerator at the temperature of-70 ℃ for 5 years.

TABLE 10 shelf life results for PPRV virus seeds

And (4) conclusion:

the Peste des petits ruminants vaccine strain 75/1 is attenuated by 70 generations of Vero cells, and the Peste des petits ruminants vaccine strain taking 120 generations as a basic strain has good immunogenicity and is the only vaccine strain recommended by OIE for preventing Peste des petits ruminants.

According to the requirement of vaccine development, 4 batches of seed batches of virus seeds are prepared in the laboratory, and the virus seeds are pure and free from exogenous virus pollution through inspection, and the virus content of 4 batches is not lower than 10^4.7TCID₅₀0.1ml, 200201F126 batches, 200701F141 batches and 200801F145 batches with 10 poison seeds³TCID₅₀The neutralizing antibody of the immunized goat reaches over 1:10 in 21 days, and the immunogenicity of the virus is proved to be good. The goat is inoculated with the strains of 200701F141 and 200801F145 according to 100 immunization doses, and the goat is inoculated without any adverse reaction, thereby proving that the strainsThe safety of the seeds is good; the generation range of the basic virus seeds is limited to F130-F141, and the requirements for vaccine production can be met.

After the 200201F126 virus seeds are stored at the temperature of minus 70 ℃ for 11 years, the requirements of the virus seeds for producing the peste des petits ruminants vaccine can still be met, and after the 200601F130, 200701F141 and 200801F145 batches of virus seeds are stored at the temperature of minus 70 ℃ for 5 to 7 years, the virus content and the immunogenicity are not obviously reduced; can meet the requirements of the virus seeds for producing the peste des petits ruminants vaccine, and the storage life of the virus seeds is determined to be 10 years after the virus seeds are freeze-dried and stored at the temperature of-70 ℃.

Example 3

Preparation of Peste des petits ruminants live vaccine

(1) The production seed virus PPRV75/1 Clone9 strain production basic seed virus is prepared and provided by Chinese veterinary medicine supervision.

(2) Production cell Vero cell: the Chinese veterinary medicine inspection institute stores and provides.

1) The sterility test and mycoplasma test were performed according to the current pharmacopoeia of Chinese beasts.

2) Virus content determination harvested virus fluid samples were serially diluted 10-fold with serum-free MEM, 10 samples were taken^-1～10^－8Inoculating 96-well cell culture plates with 8 dilutions of virus solution, inoculating 0.1ml of each well in 5-well dilution, and adding 0.1ml of goat kidney cell suspension in each well after inoculation. Placing at 37 ℃ with 5% CO₂Culturing in an incubator for 5-7 days, and calculating TCID according to cytopathic hole number by a Reed-Muench method₅₀。

The experimental materials involved in the invention are:

(1) the Peste des petits ruminants virus attenuated 75/1 strain is introduced by OIE Peste des petits ruminants reference laboratory, the original strain is 125 generations, the Peste des petits ruminants vaccine strain 75/1 virus strain is attenuated by 70 generations of Vero cells, the established original basic virus strain is 120 generations, has good immunogenicity, and is the vaccine strain recommended by OIE for preventing Peste des petits ruminants.

(2) Vero cells, introduced by the American Type Culture Collection (ATCC), were preserved and supplied by the Chinese veterinary medicine inspection.

(3) Peste des petits ruminants positive serum was introduced by OIE Peste des petits ruminants reference laboratories.

(4) 30 healthy susceptible goats aged 10-15 months are purchased from southern mountains of Wulu wood city, Xinjiang. Neutralizing antibodies are measured by blood sampling before immunization, and sheep with the antibody titer of less than 4 times are selected for the test.

(5) Other conventional reagents are provided by the supervision of Chinese veterinary drugs.

Claims

1. A Clone purified strain of Peste des petits ruminants virus is characterized in that the virus is named Peste des petits ruminants virus 75/1Clone9 strain, and is delivered to the general microorganism collection center of China Committee for microorganism Collection, China institute of microbiology, No.1 institute of China academy of sciences, 3, the rising area of Beijing city, on 2016, 12 months and 15 days, with the preservation number: CGMCC No. 13388.

2. A Peste des petits ruminants live vaccine, characterized by that the preparation method of said Peste des petits ruminants live vaccine is to regard Peste des petits ruminants attenuated CGMCC No.13388 strain virus as the vaccine production strain, inoculate Vero cell, when the cell appears cytopathic change and reaches more than 70%, move the cell bottle to-20 duC and freeze-thaw repeatedly 3 times, harvest the virus liquid separately; mixing, and freeze-drying for protectionThe preparation is prepared into peste des petits ruminants live vaccine through vacuum drying; the formula and the preparation method of the freeze-drying protective agent are as follows: 0.75 percent of sucrose, 0.40 percent of dextran, 0.40 percent of sorbitol, 0.25 percent of fish gelatin and Na₂HPO₄0.051，NaH₂PO₄0.125, adding a small amount of double distilled water for dissolution, adjusting the pH to 7.2 by 1mol of KOH, supplementing the double distilled water to 100 percent, and carrying out autoclaving at 115 ℃ for 30 min.