CN104164410A - Newcastle disease virus strain and application thereof in preparation of Newcastle disease vaccine - Google Patents
Newcastle disease virus strain and application thereof in preparation of Newcastle disease vaccine Download PDFInfo
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Abstract
The invention discloses a Newcastle disease virus strain and an application thereof in preparation of a Newcastle disease vaccine. A preparation method of the Newcastle disease virus strain comprises the following step: culturing recombinant plasmids pBrClone30-GM-CSF, pBL-N plasmids, pBL-P plasmids and pBL-L plasmid cotransfection mammalian cells together so as to obtain the Newcastle disease virus strain, wherein the recombinant plasmids pBrClone30-GM-CSF are of the plasmids of DNA molecules shown in No.2703 to No.18408 nucleotides from the 5' tail end of a sequence 3 with a sequence table. According to the Newcastle disease virus strain, molecular adjuvants GM-CSF are led into the genomes of the existing Newcastle disease vaccine, so that the immune effect is effectively improved and the immune blank period is greatly shortened. Meanwhile, the Newcastle disease virus strain can be used for resisting a maternal antibody so as to improve the immune protective rate. Thus, the Newcastle disease virus strain has an important value for preventing and treating Newcastle diseases.
Description
Technical field
The present invention relates to a kind of Newcastle disease poison strain and the application in preparing Newcastle disease vaccine thereof.
Background technology
Newcastle Disease (Newcastle Disease, ND), claim again philippine fowl disease, by Avian pneumo-encephalitis virus (Newcastle Disease Virus, NDV) a kind of bird height contact causing and the transmissible disease of acute septic are one of great deadly infectious diseases of serious harm world aviculture.NDV is a kind of RNA viruses, almost can infect all birds, and sick chicken is the main contagium of this disease, and after chicken infects, front 24h appears in clinical symptom, and its mouthful, nasal discharge and ight soil just have virus to discharge.Virus is present in all histoorgans, body fluid, secretory product and the movement of disease chicken.The band poison chicken of popular intermittent phase, it is also this sick contagium.Birds are also important circulators.Virus can, through digestive tube, respiratory tract, also can be invaded body through eye conjunctiva, injured skin and cloaca mucous membrane.This disease all can occur throughout the year, but more with spring and autumn.Once this disease occurs the chicken in chicken house, can in 4～5d, involve full group.
At present, the main method of prevention newcastle disease is vaccination immunity.Because attenuated vaccine is easy to use and can induce humoral immunization and local mucosa-immune, at home and abroad generally applied.But this type of vaccine usually need to could produce corresponding protection antibody at postvaccinal about 15 days first, has the Blank immunization phase.Once if newcastle disease broke out in the Blank immunization phase of vaccine inoculation, chicken group cannot obtain effective immunoprotection.Therefore, in the prevention work of newcastle disease, thereby produce fast in the urgent need to a kind of body that can stimulate the novel attenuated vaccine that antibody shortens the highly effective and safe of Blank immunization phase.In addition, the interference of maternal antibody is one of major reason of newcastle disease vaccine early immune failure, the newcastle disease vaccine that opposing maternal antibody disturbs, and in newcastle disease immunity practice, tool is of great significance.
Summary of the invention
The object of this invention is to provide a kind of Newcastle disease poison strain and the application in preparing Newcastle disease vaccine thereof.
Newcastle disease poison strain provided by the invention (called after rClone30-GM-CSF virus), its preparation method comprises the steps: recombinant plasmid pBrClone30-GM-CSF, pBL-N plasmid, pBL-P plasmid and pBL-L plasmid co-transfection mammalian cell and cultivates, obtains described Newcastle disease poison strain; Described recombinant plasmid pBrClone30-GM-CSF has the sequence 3 of sequence table from the plasmid of the DNA molecular shown in 5 ' end 2703-18408 position Nucleotide.The sequence 3 of sequence table is the genomic dna of rClone30-GM-CSF virus from the DNA molecular shown in 5 ' end 2703-18408 position Nucleotide.
Described recombinant plasmid pBrClone30-GM-CSF specifically can be the plasmid shown in the sequence 3 of sequence table.
Described mammalian cell specifically can be BHK-21 cell.
The preparation method of rClone30-GM-CSF virus is specific as follows:
(1) by described recombinant plasmid pBrClone30-GM-CSF, pBL-N plasmid, pBL-P plasmid and pBL-L plasmid co-transfection BHK-21 cell (every 1 * 10
6individual cell transfecting 1 μ g recombinant plasmid pBrClone30-GM-CSF, 0.5 μ g pBL-N plasmid, 0.25 μ g pBL-P plasmid and 0.1 μ g pBL-L plasmid), be placed in 5%CO
2, standing cultivation 72h in 37 ℃ of environment;
(2) get the transfectional cell that step (1) obtains, multigelation 3 times, the centrifugal cell conditioned medium liquid of collecting, is then inoculated in 9-11 age in days SPF chick embryo allantoic cavity, be placed in 37 ℃ of environment and cultivate 72h, collect chick embryo allantoic liquid (wherein containing rClone30-GM-CSF virus).
The preparation method of rClone30-GM-CSF virus is specific as follows:
(1) by described recombinant plasmid pBrClone30-GM-CSF, pBL-N plasmid, pBL-P plasmid and pBL-L plasmid co-transfection BHK-21 cell (every 1 * 10
6individual cell transfecting 1 μ g recombinant plasmid pBrClone30-GM-CSF, 0.5 μ g pBL-N plasmid, 0.25 μ g pBL-P plasmid and 0.1 μ g pBL-L plasmid), be placed in 5%CO
2, standing cultivation 72h in 37 ℃ of environment;
(2) get the transfectional cell that step (1) obtains, multigelation 3 times, the centrifugal cell conditioned medium liquid of collecting, is then inoculated in 9-11 age in days SPF chick embryo allantoic cavity, is placed in 37 ℃ of environment and cultivates 72h, collects chick embryo allantoic liquid;
(3) get the chick embryo allantoic liquid that step (2) obtains, be inoculated in new 9-11 age in days SPF chick embryo allantoic cavity, be placed in 37 ℃ of environment and cultivate 72h, collect chick embryo allantoic liquid;
(4) get the chick embryo allantoic liquid that step (3) obtains, be inoculated in new 9-11 age in days SPF chick embryo allantoic cavity, be placed in 37 ℃ of environment and cultivate 72h, collect chick embryo allantoic liquid;
(5) chick embryo allantoic liquid that the chick embryo allantoic liquid that chick embryo allantoic liquid step (2) being obtained, step (3) obtain and step (4) obtain mixes, and obtains mixed solution, is rClone30-GM-CSF virus liquid.
The present invention also protects a kind of Newcastle disease poison strain (called after rClone30-GM-CSF virus), and its genomic dna is if the sequence 3 of sequence table is from as shown in 5 ' end 2703-18408 position Nucleotide.
The present invention also protects the application of above arbitrary described Newcastle disease poison strain in preparing Newcastle disease vaccine.
The present invention also protects a kind of Newcastle disease vaccine, comprises above arbitrary described Newcastle disease poison strain.
The invention provides a kind of new Newcastle disease poison strain, called after rClone30-GM-CSF, is therefore called for short strain rClone30-GM-CSF.Strain rClone30-GM-CSF is inserted into Newcastle disease poison strain Clone30 by the encoding gene of chGM-CSF polypeptide (to be called for short strain Clone30, low virulent strain is existing Newcastle Disease living vaccine) genomic F gene and HN gene between the recombinant virus that obtains.After strain rClone30-GM-CSF vaccinated flock, can make chicken produce fast antibody, the Blank immunization phase was shortened to 7 days from 15 days, made up the shortcoming that existing newcastle disease living vaccine produces antibody overlong time, in the situation that maternal antibody exists, still can excite young chicken to produce antibody, solve the shortcoming that existing vaccine can not be resisted maternal antibody.
The recombinant Newcastle disease virus that the present invention builds can be considered the attenuated vaccine strain after genetic engineering modified, can be prepared as any conventional formulation, as freeze-dried powder, liquid vaccine preparation etc.
The present invention, by introduce molecule adjuvant GM-CSF in existing newcastle disease living vaccine genome, can effectively improve immune effect, greatly shortens the Blank immunization phase, can resist maternal antibody simultaneously, improves immune protective rate.The present invention has great value for the control of Newcastle Disease.
Accompanying drawing explanation
Fig. 1 is the element schematic diagram of recombinant plasmid pBrClone30-GM-CSF.
Fig. 2 is that the HA of mixed solution tires and the HI result of tiring.
Fig. 3 is that PCR identifies electrophorogram.
Fig. 4 is the result of embodiment 2.
Fig. 5 is the result of embodiment 3.
Fig. 6 is the result of embodiment 4.
Fig. 7 is the result of embodiment 5.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.
Mention the document of " pBrClone30 plasmid ": " Enhancement of anti-tumor activity of Newcastle disease virus by the synergistic effect of cytosine deaminase ", Zheng LV, Asian Pac J Cancer Ptev..
Mention the document of " pBL-N plasmid ", " pBL-P plasmid " and " pBL-L plasmid ": Genetically engineered Newcastle disease virus expressing interleukin 2 is a potential drug candidate for cancer immunotherapy, Fuliang Bai, Immunology letters..
NP gene, P gene, M gene, F gene, HN gene and the L gene in pBrClone30 plasmid with Avian pneumo-encephalitis virus, wherein have HpaI between F gene and HN gene and MluI enzyme is cut recognition site.By pBrClone30 plasmid, pBL-N plasmid, pBL-P plasmid and pBL-L plasmid co-transfection mammalian cell and cultivate that (pBL-N plasmid, pBL-P plasmid and pBL-L plasmid help out, pBrClone30 plasmid provides the full genome of virus), obtain strain Clone30.
Mention the document of " the strong malicious BJ strain of NDV ": " isolation identification of high virulent strains of Newcastle disease virus and Genetic Variation Analysis thereof ", school Hai Xia, Agricultural University Of Hebei's master thesis.
BHK-21 cell: ATCC is numbered CRL-13001.
DF-1 cell: ATCC is numbered CRL-12203.
SPF level white Leghorn: Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center.
The full name of chGM-CSF polypeptide is chicken granulocyte-macrophage colony stimutaing factor, and as shown in the sequence 2 of sequence table, the open reading frame of its encoding gene is if the sequence 1 of sequence table is from as shown in the 16th to 450 Nucleotide of 5 ' end.
DMEM substratum is the DMEM substratum that contains 5% foetal calf serum completely.
The concrete grammar of chicken hemaglutination (HA) test is as follows: (1) respectively adds 50 μ L physiological saline in the 1-12 hole of blood-coagulation-board first row; (2) in the 1st hole, add 50 μ L virus liquid to be measured, mix rear suction 50 μ L to the 2 holes, so doubling dilution is until the 10th hole, and the 10th hole mixes rear reject 50 μ L; (3) in 1-12 hole, respectively add 50 μ L1% chicken erythrocytes; (4) the standing 20-30min of room temperature after concussion, observations.
The concrete grammar of chicken hemaglutination inhibition (HI) test is as follows: (1) respectively adds 25 μ L physiological saline in the 1-12 hole of blood-coagulation-board; (2) the 1st holes add serum 25 μ L to be checked, mix rear suction 25 μ L to the 2 holes, and doubling dilution is until the 10th hole, and the 10th hole mixes rear reject 25 μ L; (3) in 1-12 hole, respectively add the rClone30 virus liquid (4 HAUs) of 25 μ L embodiment 1 preparations, the standing 30min of room temperature; (4) in 1-12 hole, respectively add 25 μ L1% chicken erythrocytes; (5) the standing 30-40min of room temperature after concussion, observations.
The preparation of embodiment 1, rClone30-GM-CSF virus liquid
One, the structure of recombinant plasmid
1, the double chain DNA molecule shown in the sequence 1 of composition sequence table.
In the sequence 1 of sequence table, from the 1st to 6 Nucleotide of 5 ' end, it is the recognition sequence of restriction enzyme HpaI, 10-15 position Nucleotide is Kozac sequence, the 16th to 450 Nucleotide are the encoding gene of chGM-CSF polypeptide, and the 451st to 456 Nucleotide are the recognition sequence of restriction enzyme Mlu I.
2, the double chain DNA molecule obtaining by restriction enzyme HpaI and MluI double digestion step 1, reclaims enzyme and cuts product.
3,, with restriction enzyme HpaI and MluI double digestion pBrClone30 plasmid, reclaim the carrier framework of about 16000bp.
4, the carrier framework of the enzyme of step 2 being cut to product and step 3 is connected, and obtains recombinant plasmid pBrClone30-GM-CSF.The nucleotide sequence of recombinant plasmid pBrClone30-GM-CSF is (in the sequence 3 of sequence table, being the genome of rClone30-GM-CSF virus from 5 ' end 2703-18408 position Nucleotide) as shown in the sequence 3 of sequence table.Fig. 1 is shown in by the element schematic diagram of recombinant plasmid pBrClone30-GM-CSF.
Two, the preparation of recombinant virus
1, by recombinant plasmid pBrClone30-GM-CSF, pBL-N plasmid, pBL-P plasmid and pBL-L plasmid co-transfection BHK-21 cell (every 1 * 10
6the about transfection 1 μ g recombinant plasmid pBrClone30-GM-CSF of individual cell, 0.5 μ g pBL-N plasmid, 0.25 μ g pBL-P plasmid and 0.1 μ g pBL-L plasmid), be placed in 5%CO
2, standing cultivation 72h in 37 ℃ of environment.
2, get the transfectional cell that step 1 obtains, multigelation 3 times, the centrifugal cell conditioned medium liquid of collecting, then be inoculated in 9-11 age in days SPF chick embryo allantoic cavity, be placed in 37 ℃ of environment and cultivate 72h, collect chick embryo allantoic liquid (HA of this chick embryo allantoic liquid tires be that 4096, HI tires be 256).
3, get the chick embryo allantoic liquid that step 2 obtains, be inoculated in new 9-11 age in days SPF chick embryo allantoic cavity, be placed in 37 ℃ of environment and cultivate 72h, collect chick embryo allantoic liquid (HA of this chick embryo allantoic liquid tires be that 2048, HI tires be 1024).
4, get the chick embryo allantoic liquid that step 3 obtains, be inoculated in new 9-11 age in days SPF chick embryo allantoic cavity, be placed in 37 ℃ of environment and cultivate 72h, collect chick embryo allantoic liquid (HA of this chick embryo allantoic liquid tires be that 1024, HI tires be 512).
5, chick embryo allantoic liquid that the chick embryo allantoic liquid that chick embryo allantoic liquid step 2 being obtained, step 3 obtain and step 4 obtain mixes, and obtains mixed solution (HA of this mixed solution tires be that 2048, HI tires be 512, see Fig. 2).
6, get the mixed solution that step 5 obtains, extracting total RNA reverse transcription is cDNA, and the primer pair that adopts F1 and R1 to form carries out PCR evaluation (F1:5 ˊ-GTTAACGCCGCCACCATGCTGGCACAGCTGACTATTCTGC-3 ˊ; R1:5 ˊ-ACGCGTTTAGATGCAGTCTTTCTCCTCTGGC-3 ˊ), the results are shown in Figure 3.Pcr amplified fragment size is about 450bp, conforms to expection.
Result shows, has successfully prepared the NDV virus that possesses infective expression chGM-CSF polypeptide, the mixed solution called after rClone30-GM-CSF virus liquid that step 5 is obtained.
Three, contrast viral preparation
With pBrClone30 plasmid, replace recombinant plasmid pBrClone30-GM-CSF to carry out 1 to 5 of step 2, the mixed solution called after rClone30 virus liquid obtaining.
The expression amount of chGM-CSF in embodiment 2, detection virus liquid
1, the DF-1 cell of logarithmic phase is inoculated in to six orifice plates, with the dosage of 0.1MOI, infect the rClone30-GM-CSF virus liquid (rClone30-GM-CSF virus liquid dilutes with complete DMEM substratum) of embodiment 1 preparation, 37 ℃ of standing 1h of hatching, then with complete DMEM substratum washing three times, then add containing the tryptic complete DMEM substratum of 1 μ g/mL and be placed in 5%CO
2, standing cultivation 48h in 37 ℃ of environment, then multigelation is 3 times, gets supernatant liquor.
2, with the rClone30 virus liquid of embodiment 1 preparation, replace rClone30-GM-CSF virus liquid, other is with step 1.
3, adopt GM-CSF test kit (CSB-E17363C, Cusabio) by specification to operate, the concentration of the chGM-CSF in the supernatant liquor that the supernatant liquor that detecting step 1 obtains respectively and step 2 obtain.
The results are shown in Figure 4.Result shows, rClone30-GM-CSF virus can stably express chGM-CSF, and rClone30 virus is not expressed chGM-CSF.
Embodiment 3, rClone30-GM-CSF virus and the power growth curve of rClone30 virus in host cell
1, the DF-1 cell of logarithmic phase is inoculated in to six orifice plates, dosage with 0.1MOI is inoculated in cell monolayer by the rClone30-GM-CSF virus liquid (complete DMEM substratum dilution for rClone30-GM-CSF virus liquid) of embodiment 1 preparation, adopt containing the tryptic complete DMEM substratum of 1 μ g/mL, be placed in 5%CO
2, standing cultivation 96h in 37 ℃ of environment, every 24h, get supernatant liquor and measure TCID
50.
2, with the rClone30 virus liquid of embodiment 1 preparation, replace rClone30-GM-CSF virus liquid, other is with step 1.
The results are shown in Figure 5.RClone30-GM-CSF virus and rClone30 virus are keeping consistent breeding titre.Result shows, the insertion of the encoding gene of chGM-CSF does not affect the multiplication capacity of rClone30 virus.
HI antibody titer after embodiment 4, rClone30-GM-CSF virus or rClone30 virus immunity chicken
With reference to Ministry of Agriculture's standard, it is qualified during antibody titer >=5log2 of young chicken, to be judged to.Particularly before and after 30 ages in days, antibody horizontal, lower than this standard, if there is during this period strong poison to invade, just likely breaks out newcastle disease.If chicken group energy reaches this standard during this period, the threat of Avian pneumo-encephalitis virus will be subject to.
The SPF level white Leghorn of getting 30 ages in days, is divided into 3 groups, 10 every group at random.Process respectively following (being single immunization):
First group: (rClone30-GM-CSF virus liquid dilutes with complete DMEM substratum the rClone30-GM-CSF virus liquid of every mode immunity 200 μ L embodiment 1 preparations by collunarium eye droppings, for the concentration of immune virus liquid, is 10
5tCID
50/ 0.1ml);
Second group: (rClone30 virus liquid dilutes with complete DMEM substratum the rClone30 virus liquid of every mode immunity 200 μ L embodiment 1 preparations by collunarium eye droppings, for the concentration of immune virus liquid, is 10
5tCID
50/ 0.1ml);
The 3rd group: every by collunarium eyedripping way immunity 200 μ L0.9% physiological saline.
Respectively at 7d, 14d, 21d wing venous blood collection separation of serum after immunity, detect HI and tire.
The results are shown in Figure 6.7d after immunity; the HI of first group of serum tires higher than 5log2; second group tire lower than 3log2; be that rClone30-GM-CSF virus liquid can be realized early protection effect; make chicken avoid the threat of Avian pneumo-encephalitis virus, and rClone30 virus liquid cannot be after immunity 7d realize the threat that effective protection chicken avoids Avian pneumo-encephalitis virus.
Embodiment 5, recombinant virus rClone30-GM-CSF or rClone30 immunity are containing the HI antibody titer after the chicken of maternal antibody
In poultry is produced, a chicken group's maternal antibody level is often uneven, the chick that some of them maternal antibody level is lower is malicious at the wild poison of its young age grade section easy infection and band, when other chick maternal antibody level declines, they are also infected successively, make the propagation of wild poison in chicken group without stop, leave over very large potential threat.According to related documents, after laying hen hatching, in different days, measure its maternal antibody against newcastle disease level, result shows: 1 age in days maternal antibody is 6.6log2; During 7 age in days, antibody horizontal drops to 4.7log2; During to 10 age in days, antibody horizontal drops to below 4log2, if take antibody titer 4log2 as critical protection value, 1 Japanese instar chickling maternal antibody protection ratio is that 95%, 7 age in days maternal antibody protection ratio is 80%, and during to 10 age in days, maternal antibody protection ratio only has 50%.
The white Leghorn containing maternal antibody of getting 1 age in days, is divided into 3 groups, 10 every group at random.Process respectively following (being single immunization):
First group: (rClone30-GM-CSF virus liquid dilutes with complete DMEM substratum the rClone30-GM-CSF virus liquid of every mode immunity 200 μ L embodiment 1 preparations by collunarium eye droppings, for the concentration of immune virus liquid, is 10
5tCID
50/ 0.1ml);
Second group: (rClone30 virus liquid dilutes with complete DMEM substratum the rClone30 virus liquid of every mode immunity 200 μ L embodiment 1 preparations by collunarium eye droppings, for the concentration of immune virus liquid, is 10
5tCID
50/ 0.1ml);
The 3rd group: every by collunarium eyedripping way immunity 200 μ L0.9% physiological saline.
Respectively at 0d, 6d wing venous blood collection separation of serum after immunity, detect HI and tire.
The results are shown in Figure 7.
Embodiment 6, challenge test
30 age in days SPF level white Leghorns are divided into 3 groups, 30 every group at random.Process respectively following (being single immunization):
First group: test the 1st day, (rClone30-GM-CSF virus liquid dilutes with complete DMEM substratum, for the concentration of immune virus liquid, is 10 for every rClone30-GM-CSF virus liquid by collunarium eyedripping way immunity 200 μ L embodiment 1 preparations
5tCID
50/ 0.1ml);
Second group: test the 1st day, (rClone30 virus liquid dilutes with complete DMEM substratum, for the concentration of immune virus liquid, is 10 for every rClone30 virus liquid by collunarium eyedripping way immunity 200 μ L embodiment 1 preparations
5tCID
50/ 0.1ml);
The 3rd group: test the 1st day, every by collunarium eyedripping way immunity 200 μ L0.9% physiological saline.
Test the 8th day, (every chicken adopts 10 to adopt the strong malicious BJ strain of NDV to attack poison
4eLD
50dosage through intramuscular injection path, attack poison, attacking malicious volume is 0.1ml), test the 18th day, statistics each group morbidity and death condition.The results are shown in Table 1.
The morbidity of each group of table 1 and death condition statistics (mean values of three tests)
That morbidity characterizes is following (meet 1.-in 4. one is judged as morbidity above): 1. the upper respiratory tract is secreted a large amount of mucus, from mouth and nose outflow, is sometimes hung on mouth end, often shakes the head and wants to get rid of; Expiratory dyspnea, during breathing, throat sends stridulate sound or the birdie of " chuckleing "; 2. due to expiratory dyspnea, in blood, oxygen is not enough, and carbonic acid gas increases, and makes wattle become livid purple look, front and after death particularly evident on one's deathbed; 3. diarrhea, ight soil is green, yellow-white, is sometimes mixed with a small amount of blood; 4. part chicken stomach is accumulated the smelly liquid of a large amount of acid, and while holding chicken, liquid flows out rapidly in mouth, carries kettle pouring general.
Result shows: rClone30-GM-CSF immunity can be in early days prior to produce antibody and strengthen humoral immunity level in rClone30 vaccine strain stimulation chicken body simultaneously, and poison protection efficiency is attacked in raising.
Claims (6)
1. a Newcastle disease poison strain, its preparation method comprises the steps: recombinant plasmid pBrClone30-GM-CSF, pBL-N plasmid, pBL-P plasmid and pBL-L plasmid co-transfection mammalian cell and cultivates, obtains described Newcastle disease poison strain; Described recombinant plasmid pBrClone30-GM-CSF has the sequence 3 of sequence table from the plasmid of the DNA molecular shown in 5 ' end 2703-18408 position Nucleotide.
2. Newcastle disease poison strain as claimed in claim 1, is characterized in that: the plasmid shown in the sequence 3 that described recombinant plasmid pBrClone30-GM-CSF is sequence table.
3. Newcastle disease poison strain as claimed in claim 1 or 2, is characterized in that: described mammalian cell is BHK-21 cell.
4. a Newcastle disease poison strain, its genomic dna is if the sequence 3 of sequence table is from as shown in 5 ' end 2703-18408 position Nucleotide.
5. the application of arbitrary described Newcastle disease poison strain in preparing Newcastle disease vaccine in claim 1 to 4.
6. a Newcastle disease vaccine, comprises arbitrary described Newcastle disease poison strain in claim 1 to 4.
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Effective date of registration: 20181224 Address after: 222001 No. 58 Haichang South Road, Haizhou District, Lianyungang City, Jiangsu Province Patentee after: Jiangsu Kangyuan Ruiao Biomedical Technology Co., Ltd. Address before: 150001 College of Life Sciences, Northeast Agricultural University, 59 Wood Street, Xiangfang District, Harbin, Heilongjiang Province Patentee before: Harbin Boao Bio-Medical Technology Development Company |