CN1235553C - Method for raising SPE hamster for preparing various biological products - Google Patents
Method for raising SPE hamster for preparing various biological products Download PDFInfo
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- CN1235553C CN1235553C CNB03157520XA CN03157520A CN1235553C CN 1235553 C CN1235553 C CN 1235553C CN B03157520X A CNB03157520X A CN B03157520XA CN 03157520 A CN03157520 A CN 03157520A CN 1235553 C CN1235553 C CN 1235553C
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Abstract
The present invention relates to a method of producing a SPF (specific pathogen free: SPF) hamster for the preparation of a variety of biological products. More particularly, the present invention relates to a method of producing the SPF hamster for the preparation of a variety of biological products, that is free of microbiological contaminants such as parasites, bacteria, and exogenous- and endogenous viruses, etc. with the potential to cause diseases. Since the SPF hamster produced according to the method of the present invention carries no contaminants such as parasites, bacteria, and exogenous- and endogenous viruses, etc., the primary cells originated from said SPF hamster and the biological products using the same do not induce any opportunistic infection by microbial contaminants that can be present in animal cells used for existing biological products such as vaccine, etc. Therefore, the SPF hamster produced according to the present invention can be safely used for preparing a variety of biological products that are applied to a human.
Description
Technical field
The present invention relates to a kind ofly be used to generate SPF (the specific pathogen that do not contain: the SPF) method of hamster, this hamster can be used for preparing various biological product.Particularly, the present invention relates to a kind of method that is used to generate the SPF hamster, with the various biological product of this hamster preparation be not subjected to parasite, antibacterial and endogenous-, exogenous virus etc. can potentially cause the infected by microbes of disease.
Background technology
Vaccine virus immunization is a kind ofly to make human host obtain protectiveness and initiative immunity and do not produce the method for any disease on the host, has utilized the antigen relevant with infectious disease in this method.For example; the antigen of disease association pathogen or pathogen itself all can suitably cause very special immunoreation; this immunoreation is regulated bone-marrow-derived lymphocyte or is regulated the T cell mediated by cellular immunization by humoral immunization; therefore, can make the titre of protection antibody be increased to identical or be comparable to the immune state in rehabilitation stage (or the lymphocytic persistent memory behavior of cytotoxin T) after host's natural infection.According to the antigenic dissimilar and different preparation methoies that adopted, vaccine can be divided into toxoid vaccine, inactivated vaccine, attenuated live vaccine and recombinant DNA vaccine etc.Up to the present, the vaccine majority of having developed is inactivated vaccine or attenuated live vaccine, and its preparation process comprises the following steps: live virus is inoculated on certain type the animal sources cell, it is cultivated and extract, then it is carried out purification and suitably processing.Because virus has the characteristic that can only breed in living cells, so must use the cell substrate of animal sources (comprising the mankind) cell as the preparation viral vaccine.The cell line that is used to prepare vaccine mainly is divided into germinal cell system, diploid cell line and continuous cell line.By being at a certain moment frozen cell, can prepare diploid cell line and continuous cell line, can or be controlled to be parent or the working cell storehouse system that is used to produce with its establishment then.When needs prepare a kind of vaccine, with the cell thawing of requirement, cultivate in the specific stage then, promptly can be used as the cell line of preparation vaccine.
Equally, germinal cell can be used for preparing poliomyelitis-, measles-, German measles-, Japanese encephalitis-or yellow fever live vaccine.For each batch vaccine product, all can use germinal cell to produce vaccine product, this germinal cell is directly separated from animals such as chicken, Canis familiaris L., monkey or hamster, cultivates then.Therefore, relevant with animal groups (as raw material sources) self-security quality management is extremely important.Through for many years to the PMK cell (
pRimary
mOnkey
kIdney cell, the original nephrocyte of monkey) research of safety, find to exist in the PMK cell 20 kinds of dissimilar potential virus, some of them may cause the mortality disease in human body, and during beginning vaccine the fifties from twentieth century, enormous function has been brought into play in the elimination of paralytic measles with the Measles Vaccine of this PMK cell preparation.For example, many batches of inactivated measles vaccines and live measles vaccine be inoculated into millions of children for many years after, find SV40 virus (a kind of in the simian virus), can cause the cancer of rodent.Experiment in vitro finds that also above-mentioned virus can change into the human normal cell cancerous cell (Hayflick L.Science, 276 (5311): 337-341,1997).Above-mentioned discovery has evoked the arguement to the possible carcinogenecity of relevant Measles Vaccine (based on the PMK cell) inevitably.Equally, measles-, parotitis-or yellow fever vaccine (is the cell substrate with the chick-embryo cell) wide-scale distribution and use after, find that this chick-embryo cell has been subjected to the pollution of a kind of live poultry retrovirus (AL's virus).Above-mentioned incident has caused much about the concern of vaccine to human body possibility carcinogenecity.More subsequently arguements to vaccine safety focus on the potential microbial contamination in the cell substrate (be used for preparing vaccine).Because above-mentioned reason, about as the animal of raw material sources with preparation biological product (as vaccine), in the world suggestion uses proved conclusively as SPF (
sPecific
pAthogen
fRee, specificity do not contain pathogen) animal groups.
To the roughly screening of SPF animal based on several experiments, as obduction, microscopy, culture experiment, serology experiment and histopathology experiment etc.But above-mentioned experiment only helps to detect the infection of parasite, antibacterial and some main viruses, does not also set up the clear standard that is used for screening the SPF animal.For SPF mouse and SPF mice, the existing internationally recognized standard that detects its microbial contamination in contrast to this, does not also have an international validation criteria that is used for screening the SPF hamster like this.The SPF hamster of present commercial use be a kind of VAF (
vIral
aNtigen
fRee, virus-free antigen) hamster, this VAF hamster is not polluted by several viral micro-organisms.Even by internationally recognized be the Charles River laboratory company or the Harlan Sprague Dawley company of the main source of supply of SPF animal, only by to 3-15 kind antibacterial, 2-3 kind parasite and 5 or the easy detection of 6 kind of virus screen the SPF animal.Often be found from one of them VAF hamster of above-mentioned two companies and be subjected to viral pollution.And, in some cases, because symptomless infection can't detect virus for a long time at all.Therefore, biological product (as vaccine etc.) are to serve as the basis preparation with commercial VAF hamster probably, and this VAF hamster has been subjected to contamination by micro that may be harmful.So the safety of above-mentioned biological product is not guaranteed so far.
The cell line for preparing vaccine with the SPF hamster is PHK cell (the original nephrocyte of hamster) normally.With the PHK cell serves as that the biological product that the basis prepares comprise following example: Japanese encephalitis's attenuated live vaccine, Japanese encephalitis's inactivated vaccine, rabies vaccine, dengue vaccine, hemorrhagic fever muroid vaccine and Ticks source property encephalomyelitis vaccine.Utilize each antigenic virus can be in the PHK cell effectively, the biological characteristics of propagation stably, prepared above-mentioned vaccine.So, set up SPF hamster as raw material sources, be even more important to be used for extracting PHK cell (as the cell substrate of different vaccine products).
Therefore, carried out many researchs with cultivate a kind of can be used safely in to prepare the hamster of the biological product that are used for human body after, SPF hamster group has been set up in wood invention, and this hamster group is not subjected to manyly can potentially causing parasite, antibacterial and the endogenous of disease, the infection of exogenous virus.Further, the PHK cell by confirming to take from described SPF hamster and with the safety of the biological product of its preparation has been finished the present invention.
Summary of the invention
Therefore, the purpose of this invention is to provide a kind of method of the SPF hamster of preparation biological product safely that to be used for that is used to generate.
In order to reach the foregoing invention purpose, the invention provides a kind of method that is used to generate the SPF hamster that is used for preparing biological product, its step comprises:
(a) at first, by microbial contamination experiment to parasite, antibacterial and hamster virus, choose the hamster of uninfection, and by the above-mentioned hamster of choosing of sib mating breeding, from the hamster that is bred, choose for the second time and be not subjected to the hamster that mentioned microorganism infects then;
(b) selected hamster is carried out copulation, got its uterus, an isolation room is introduced in the uterus of being got in 12-14 days after female hamster pregnancy, get its embryo by cesarean, in rank is lower than 10000 isolation area, utilizes generation to educate female Mus and cultivate the above-mentioned embryo who gets then, and
(c) breed the hamster of being cultivated in the above-mentioned steps (b) by sib mating, and it is carried out microbial contamination experiment of parasite, antibacterial, hamster virus, Murivirus, endogenous hamster retrovirus, hamster polyomavirus (HPyV), hamster lymphoma virus and unknown hamster virus, from the hamster of above-mentioned breeding, choose the hamster that not infected by mentioned microorganism then.
Among the present invention, term " biological product " (or " biological product ") refers to its objective is the diagnosis, immunity inoculation or the treatment that are used for human body or animal, or be used for correlational study by living tissue or the prepared vaccine of its product, culture and other preparation.
To describe the present invention in detail below.
The invention is characterized in that it provides the method for a kind of SPF of cultivation hamster, this hamster can be used to prepare safely the biological product that are used for human body and animal.In addition, the invention is characterized in, for stable and germinal cell is provided constantly, to be used for preparing above-mentioned biological product, set up an aseptic hamster group, this hamster group is not subjected to contamination by micro such as parasite, antibacterial and endogenous or exogenous virus, and standard and the method that is used for determining above-mentioned hamster safety is provided.
Hamster among the present invention comprises the various hamsters that can be used for preparing biological product.Preferred golden Syria hamster.Although Chinese Chengdu Inst. of Biological Products has used golden hamster, with its animal sources, but specially among the present invention be that selected golden hamster is used to the embryo that provides initial as vaccine product cell substrate, this embryo is transferred to purebred generation and educates female Mus, is cultivated by it.
In one embodiment of the invention, above-mentioned golden hamster group is carried out the microbial contamination experiment of parasite, antibacterial and hamster virus, thereby first screening is not organized by the hamster of microbial contamination.The above-mentioned hamster of choosing of breeding in laboratory animal raising chamber, and carry out the mentioned microorganism experiment once more.Thereby programmed screening is not organized by the hamster of microbial contamination.For the mentioned microorganism experiment, disclosed any method all can be used in the relevant technologies.Particularly, utilize the method described in the reference example 1, the Experiment on Microbiology of being carried out among the present invention comprises 6 kinds of exogenous and endogenous parasites, 11 kinds of antibacterials and 7 kinds of hamster viruses (as listed in the following table 1).
Table 1 is for choosing the Experiment on Microbiology project that conceived female Mus carries out
| Microbial name | |
| Parasite | Hepaticola hepatica |
| Hymenolepis | |
| Encephalitozoon cuniculi | |
| Mus giardia lamblia stiles (Girdia muris) | |
| Mus six flagellates (Spironucleus muris) | |
| Syphacia | |
| Vermin | |
| Antibacterial | Bronchus deteriorated blood Bao Te bacterium |
| Corynebacterium kutscheri | |
| The spiral Pseudomonas | |
| Mycoplasma pneumoniae | |
| Salmonella | |
| Salmonella typhimurium | |
| Streptococcus pneumoniae | |
| Patina color pseudomonas | |
| Parent's pneumonia pasteurella | |
| Golden yellow Portugal coccus | |
| Hair shape clostridium | |
| Virus | SEND (celestial platform) |
| PVM (pneumonia of mice virus) | |
| MVM (Mus piconavirus) | |
| KRV (Mus Kilham virus) | |
| Reo-3 (3 type reovirus) | |
| LCMV (lymphatic choroid plexus encephalitis) | |
| H-1 (Toolans H-1 virus) |
In another embodiment of the present invention, for choosing conceived female Mus (conceived female hamster, afterwards its embryo was transferred to for educating female Mus), two female hamsters (at least once having once childbirth experience) are placed in the same cage with a male Mus (having the copulation experience).Then, choose the hamster that vaginal suppository occurred, it is handled,, and continue to raise until conceived first day.In addition, fetch VAF hamster, it is carried out mentioned microorganism pollute experiment from Charles River experiment company.After guaranteeing that this VAF hamster is not comtaminated, used as the generation educate female Mus (female hamster, it has accepted the embryo of conceived female Mus, and look after young Mus until the wean).
Fig. 1 shows the cultivation sketch of SPF hamster among the present invention.After female Mus pregnancy 12-14 days, get its uterus by cesarean, by soaking bath (being full of sterile solution), getting uterus is sent in the isolation room.According to the bulge degree of the female Mus abdominal part of pregnancy, the congested degree and the relax level of vaginal orifice, decide and carry out caesarean suitable opportunity.For above-mentioned sterile solution, can use peracetic acid soln, 2%Mikro-quat solution or iodine tincture liquation.Preferred peracetic acid soln.In addition, between the preferred 0.1-0.2% of the concentration of described peracetic acid soln.
After from the uterus, getting its embryo, educate female Mus by generation and cultivate.This cultivating process preferably carries out in rank is lower than 10000 SPF isolation area, and cultivating process is abideed by zoopery guide (by Korea S's Academy of Medical Sciences establishment) and carried out.Particularly, the cultivating process that the inventor carries out in the SPF isolation area is provided by the iaboratory animal medicine portion of Korea S Yonsei university medical college medical research center, and this cultivating process is adjusted under following condition and carries out: temperature 22-26 ℃, relative humidity 45-55%, ventilation frequency 8-12 time/hour, light intensity are lower than 10000 greater than 25 luxs, rank.Come breeding animals by sib mating.
In another embodiment of the present invention,, carried out microbial contamination experiment to various parasites, antibacterial and virus (shown in table 2, table 3, table 4) for from the animal of above-mentioned breeding, choosing a SPF hamster group.In infra tabulation 2, table 3 and the table 4, microbial contamination experimental project that the present invention carries out and the experimental project of VAF hamster (taking from Charles River experiment company or HarlanSprague Dawley company) have at present been compared when selecting the SPF hamster.
Organize the Experiment on Microbiology project of carrying out for choosing a SPF hamster among table 2 the present invention: antibacterial
| Sequence number | Antibacterial | The present invention | Charles River tests company | Harlan Sprague Dawley company |
| 1 | Bronchus deteriorated blood Bao Te bacterium | ● | ● | ● |
| 2 | Corynebacterium kutscheri | ● | ● | |
| 3 | The spiral Pseudomonas | ● | ● | |
| 4 | Mycoplasma pneumoniae | ● | ● | |
| 5 | Salmonella | ● | ● | ● |
| 6 | Salmonella typhimurium | ● | ||
| 7 | Streptococcus pneumoniae | ● | ● | ● |
| 8 | Rhodopseudomonas | ● | ● | |
| 9 | Patina color pseudomonas | ● | ● | ● |
| 10 | The crust phase moral Bordetella | ● | ● | |
| 11 | Parent's pneumonia pasteurella | ● | ● | |
| 12 | Golden yellow Portugal coccus | ● | ● | |
| 13 | Hair shape clostridium | ● | ● | |
| 14 | The klebsiella spp of hastening parturition | ● | ● | |
| 15 | Pasteurella multocida | ● | ● | |
| 16 | Klebsiella pneumoniae | ● | ● | |
| 17 | B group beta chain ball | ● | ● | ● |
| 18 | G group beta chain ball | ● | ● | |
| 19 | The beta chain Coccus | ● | ● |
Organize the Experiment on Microbiology project of carrying out for choosing a SPF hamster among table 3 the present invention: parasite
| Parasite | The present invention | Charles River tests company | Harlan Sprague Dawley company |
| Endoparasite | ● | ● | ● |
| Hepaticola hepatica | ● | ● | ● |
| Hymenolepis | ● | ● | ● |
| Encephalitozoon cuniculi | ● | ● | ● |
| Mus giardia lamblia stiles (Girdia muris) | ● | ● | ● |
| Mus six flagellates (Spironucleus muris) | ● | ● | ● |
| Syphacia | ● | ● | ● |
| Vermin | ● | ● | ● |
Organize the Experiment on Microbiology project of carrying out for choosing a SPF hamster among table 4 the present invention: virus
| Virus | The virus group | The present invention | Charles River tests company | Harlan Sprague Dawley company |
| Hamster specificity virus (10 kinds) | ||||
| SEND (celestial platform) | Parainfluenza virus | ● | ● | ● |
| PVM (pneumonia of mice virus) | Paramyxovirus | ● | ● | ● |
| MVM (Mus piconavirus) | Parvovirus | ● | ||
| KRV (Mus Kilham virus) | Parvovirus | ● | ||
| Reo-3 (3 type reovirus) | Reovirus | ● | ● | ● |
| LCMV (lymphatic choroid plexus encephalitis) | Arenavirus | ● | ||
| H-1 (Toolans H-1 virus) | Parvovirus | ● | ● | ● |
| GD-7 (Taylor murine encephalomyelitis virus) | Picornavirus | ● | ||
| SV5 (simian virus 5) | Parainfluenza virus | ● | ||
| HANT (hantaan virus) | This refined virus | ● | ||
| Mus specificity virus (16 kinds) | ||||
| LCMV (lymphatic choroid plexus encephalitis) | Arenavirus | ● | ||
| MHV (murine hepatitis virus) | Coronavirus | ● | ● | |
| PVM (pneumonia of mice virus) | Paramyxovirus | ● | ||
| MVM (Mus piconavirus) | Parvovirus | ● | ||
| SEND (celestial platform) | Paramyxovirus | ● | ||
| ECTRO (lacking the limb deformity) | Poxvirus | ● | ||
| EDIM (young Mus epizootic disease diarrhoea) | Rotavirus | ● | ||
| REO-3 (3 type reovirus) | Reovirus | ● | ||
| GDV II (Mus encephalomyelitis) | Picornavirus | ● | ||
| MAD (murine adenovirus) | Adenovirus | ● | ||
| POLY (polyoma virus) | Papovavirus | ● | ||
| HANT (hantaan virus) | This refined virus | ● | ||
| MTV (Mus thymus virus) | Herpesvirus | ● | ||
| MCMV (murine cytomegalovirus) | Herpesvirus | ● | ||
| K (pneumonia of mice virus) | Papovavirus | ● | ||
| LDV (lactate dehydrogenase-short virus) | Togavirus | ● | ||
| Endogenous hamster retrovirus | Retrovirus | ● | ||
| Hamster polyomavirus | Papovavirus | ● | ||
| Unknown hamster lymphoma virus | - | ● | ||
| Unknown hamster virus | - | ● | ||
At first, the present invention has carried out the microbial contamination experiment to antibacterial (table 2 is listed), parasite (table 3 is listed), 10 kinds of hamster specificity virus and 16 kinds of Mus specificity virus (table 4 is listed).As a result, select one and be proved the SPF hamster group (seeing Table 5-table 9) that is not subjected to microbial contamination.About antibacterial and parasitic microbial contamination experiment, answer the requirement of medical college medical research center iaboratory animal medicine portion of Korea S Yonsei university, about the microbial contamination experiment of hamster specificity virus and Mus specificity virus, answer the requirement of Glasgow, United Kingdom Q-one Biotech Co., Ltd.
About experiment and serology experiment,, carried out the hamster antibody of 10 species specificity exogenous viruses (listed as table 4) is generated test (HAP test) by isolating PHK cell from hamster to described hamster virus.
Described HAP test generates on the basis of testing (MAP test) in murine antibody to be revised, and carries out (Rowe WP et al, J.Exp.Med., 109:379-391,1959 by Rowe and colleague thereof at first; AndRowe WP et al., Virology 11:645-649,1960).It is a kind of sensitivity, specific and comprehensive method, is used to detect the virus that can infect the hamster tissue.This experiment on the quick hamster host of height (not being subjected to the infection of 10 species specificity exogenous viruses), is carried out virus inoculation then responsive, specific determination of serology and is detected antiviral antibody.
For determining the main chance of any adventitious viruses infected animal, 3 kinds of inoculation routes have been used.Inoculum has brought and can enter gastral enterovirus in mouthful.The intranasal vaccination thing has brought the Respirovirus that can enter respiratory system.The virus that the intraperitoneal inoculum brings may be passed the digestive tract mucosa and be entered intracorporeal organ.
About experiment, carried out the murine antibody of 16 species specificity viruses (listed as table 4) is generated test (MAP test) to described Murivirus.(Rowe WP.，et al，J.Exp.Med.，109：379-391，1959；Rowe WP.，et al.，In，The Problems of Laboratory Animal Disease.pp 132-141.ed.RC Harris.Academic Press Inc.，New York，1962；Smith AL.，In，Viral andMycoplasmal Infections of Laboratory Rodents.pp 731-750 eds PN Bhatt，ROJacoby，MC Morse and AE New.Academic Press Inc.，New York，1986)
As the main method that detects the external Muridae virus in cell and/or the tumor system, the MAP test was extensive use of more than 20 years.This experiment be by being stored in any Muridae virus inoculation in the experiment material in mouse body, detect by the inoculation intravital antibody product of mouse again and carry out.
For determining the maximum likelihood of any adventitious viruses infected animal, 4 kinds of inoculation routes have been used.Inoculum has brought and can enter gastral enterovirus (MHV﹠amp in mouthful; GDVII).The intranasal vaccination thing has brought the Respirovirus (PVM﹠amp that can enter respiratory system; SEND).The virus that the intraperitoneal inoculum brings may be passed the digestive tract mucosa and be entered intracorporeal organ.The site of puncture has also served as a kind of admission passage, can cause ectromely.Inoculum has brought and can directly enter big cerebromeningeal LCMV virus in the brain.
In the LCM excitation experiment, excite mouse with the fatal strain of a kind of known virus, observe the M ﹠ M of every workday in the time of 12 days.If in experimental project, there is the avirulent strain of LCMV, the immunity of above-mentioned mouse is excited, mouse will survive down.If do not contain described avirulent strain in the experimental project, mouse will die from booster dose in the time of 4-12 days.
Whether laboratory animal has been subjected to infection, and whether contains virus in the experiment material, and the antibacterial possible to difference determined by different technology.The serological technique that uses among the present invention comprises that direct fluorescent antibody (IFA), enzyme-linked immunoassay (ELISA), complement are in conjunction with (CFA), immunoelectron microscope (IEM) and hemagglutination inhibition reaction (HAI).
Then, in order to detect in the selected SPF hamster group whether have the latent virus that may cause opportunistic infection, carried out the infection experiment of hamster polyomavirus (hereinafter, referring to ' HPyV ').Syria hamster is very responsive to the infection that HPyV virus causes, this virus can cause its follicular epithelium cancer.Viral genome comprises the double-stranded DNA that is approximately the 5.3kb size, and the formation of its open reading frame is typical polyoma virus shape (Delmas V, et al., EMBOJournal, 4:1279-1286,1985).
The copy number that uses PCR to detect the nucleotide target molecule in real time carries out quantitative assay.PCR detects in real time the 5 '-nucleotide 5 prime excision enzyme activity that utilizes the Taq polymerase, the Taq Man internal probe of fluorescence indicator that come a kind of labelling of hydrolysis, and cooling dyeing (Lee LG, et al., Nucleic Acids Research21:3761-3766,1993).In ABI 7700 sequencing systems, to the calculating of copy number based on the amplification that when detects target molecule at first.7700 systems are detected to be threshold cycle number (C
T), this period is the minority period that fluorescence can pass on fixed threshold value baseline.By unknown sample and standard curve are compared, can determine its absolute quantity, this standard curve generates (PE Applied Biosystems by the target molecule of dose known amounts, 1997, Relative quantitation of gene expression, UserBulletin#2.ABI PRISM 7700 Sequence detection system).Sample C by experiment
TNumber and dose known amounts recombiant plasmid molecule C
TThe contrast of number can be determined the quantity of the HPyV genomic DNA that exists in the experiment sample to comprise HPyV DNA in the described recombiant plasmid molecule, to be used to generate a standard curve.Under the conventional determining condition, measure the genome of 100 pH-PyV.
The result in any tissue of SPF hamster, does not detect HPyV-specificity virus (seeing Table 10) in the present invention.
The cell line that is used to prepare biological product can produce retrovirus in the host range widely.So, should detect the infection whether SPF hamster among the present invention has been subjected to endogenous hamster retrovirus (endogenous HaLV).Be subjected to infection if confirmed the SPF hamster, also answered the human body cell whether to be subjected to the retroviral infection of SPF hamster.Whether have retrovirus (can infect the mankind or germinal cell) in the experimental project cell line, available following detection cell line is measured: human diploid cell line MRC-5, Burkitt ' s lymphoma cell line Raji, African titi kidney cell line Vero, baby hamster kidney cell are BHK etc.MRC-5 and Raji cell are used for detection and whether have the somatic retrovirus of energy infected person.Reproducible C-type retrovirus among the MRC-5, and also reproducible D-type retrovirus (comprising squirrel monkey retrovirus) among the Raji.Show with the co-cultivation result of Vero cell, the reproducible retrovirus that can infect primates in this cell, and bhk cell has been represented a kind of cell line in the hamster cell system.Use the reason of multiple detection cell like this to be, the co-cultivation of project cell and different host cells by experiment, the expression chance of unknown virus (may hide in the experimental project cell) may increase.
When carrying out above-mentioned experiment, with the experimental project cell with survey the cell co-cultivation, and going down to posterity for 1 stage, remove the experimental project cell.For the lower virus of the infection level that increases, at least the detection cell is carried out 5 times and go down to posterity.After going down to posterity for 5 times (, perhaps more going down to posterity), from culture, collect supernatant, and promote sexual inversion record enzyme (PERT) algoscopy, detect whether there is retrovirus by product one if suitable.For the active mensuration of reverse transcriptase (HA), it is a kind of highstrung assay method that PERT measures, and reports that the sensitivity of this method is 10 of general RT algoscopy
6Doubly above (Silver J, et al., Nucleic Acids Research, 21:3593-3594,1993; And Pyra H, et al., PNAS, U.S.A., 91:1544-1548,1994).Have endogenous retrovirus in the bhk cell, the result is carrying out positive findings having occurred when PERT measures, and based on the above-mentioned fact, uses the RT algoscopy to detect (to take from the BHK culture) in the supernatant whether to have retrovirus.
Particularly, in the present invention, with PHK cell (from SPF hamster of the present invention, separating) respectively with MRC-5 and Raji cell co-cultivation.As a result, with the human cell line of described PHK cell co-cultivation in, do not find that the activity of reverse transcriptase (RT), the activity of this reverse transcriptase are the active indicant of retrovirus (see Table 11 and table 12).
In the 3rd experiment, detected the infection whether SPF hamster of the present invention has been subjected to the unknown virus pollutant.When carrying out this experiment, will survey cell and PHK cell (from SPF hamster of the present invention, separating) co-cultivation, and in whole culture period, with CPE whether having occurred in this detection cell of microscopic examination.In addition, be used to erythrocyte (rbcs), make above-mentioned detection cell carry out the erythrocyte adsorption from different animals such as the inferior pig of guinea, chicken and human body etc.Particularly, with PHK cell (from SPF hamster of the present invention, separating) respectively with MRC-5, Raji cell and Vero cell co-cultivation.Utilize the inferior swine erythrocyte of guinea, chicken red blood cell and the erythrocytic mixture of human body ' O ' type, make above-mentioned detection cell carry out the erythrocyte adsorption equally.As a result, in the various detection cells of the PHK cell co-cultivation in above-mentioned and the present invention, do not observe cytopathy reaction (CPE) or erythrocyte adsorption.
In last experiment, utilize a kind of immunosuppressive drug-ciclosporin, detected whether there is the unknown infection that causes lymphoma virus in the SPF hamster of the present invention.In the 100 day time after the immunologic function that has suppressed hamster, the symptom of tumor or other similar tumor does not take place in the SPF hamster among the present invention.
As mentioned above, can prove in the SPF hamster in the present invention, and in the PHK cell that from this SPF hamster, extracts, there are not bacterial infection, parasite, 10 species specificity hamster viruses, 16 species specificity Muriviruses, hamster polyomavirus, endogenous retrovirus (HaLV), unknown hamster lymphoma virus (can potentially cause malignant lymphoma) and other unknown hamster pathogen.And, in the VAF series hamster of cultivating by Charles River experiment company or Harlan Sprague Dawley company, only detected and whether had several antibacterials, parasite and 4-5 species specificity hamster virus, not as the present invention, carry out an affirmation of safety widely (see Table 2, table 3 and table 4).Therefore, in safety, the SPF hamster among the present invention is better than present VAF or other SPF hamster.So clearly, from SPF hamster of the present invention, extract germinal cell (preferred PHK cell), and be very favourable as the cell substrate to be used to preparing biological product it.
Above-mentioned biological product comprise all viral vaccines, and this virus refers to that all can be with germinal cell (separating) as host cell (cell substrate) from the SPF hamster, thus the virus of propagation therein.Specific example comprises: the Japanese encephalitis's attenuated live vaccine for preparing in invention (Tsai et al., Japaneseencephalitis vaccine.3rd ed.Vaccines, ed.S.A.Plotkin and W.Orenstein., 1999, Philadelphia:WB Saunders, 672-710), Japanese encephalitis's inactivated vaccine (Lu et al., Japaneseencephalitis vaccine.1st ed.Medical Biological Products.1995, Beijing:Peoples Medical Publishing House, 528), rabies vaccine (Plotkin SA, et al., Rabiesvaccine, In Plotkin SA, Orenstein WA, eds. (1999) Vaccines (3rd ed.), Philadelphia, PA:WB Saunders company, 743-766; Lu et al., Rabies vaccine.1st ed.Medical Biological Products.1995, Beijing:Peoples Medical Publishing House, 552), dengue vaccine, hemorrahgic fever with renal syndrome vaccine (Lu et al., Hemorrhagic fever vaccine.1st ed.Medical Biological Products.1995, Beijing:Peoples Medical PublishingHouse, 652), and Ticks source property encephalomyelitis vaccine (Lu et al., Tick-borne encephalitis vaccine.1st ed.Medical Biological Products.1995, Beijing:Peoples Medical PublishingHouse, 540) etc.
The accompanying drawing summary
Fig. 1 illustration produce the process sketch of a SPF hamster among the present invention.
The preferred embodiments of the present invention
To be described in further detail the present invention by different examples below.But it should be noted that the present invention not only is confined to these examples or is subjected to the restriction of these examples.
＜reference example 1 〉
The microbial contamination experiment
1-1) to parasitic detection
After getting specimen with adhesive tape, whether there is any exogenous parasite with light microscopic examination.Simultaneously, whether there is any endogenous parasite, laboratory animal put to death, and dissect, be ready to stomach, small intestinal, large intestine tissue specimen then for checking.Getting specimen is dyeed with h and E, under light microscope, check then.
1-2) to the detection of antibacterial
Based on following three kinds of methods, check whether there is antibacterial.
A. culture method-to all antibacterials except that spiral Pseudomonas, mycoplasma pneumoniae, hair shape clostridium.
Get trachea, nasal sinuses and intestinal material with disinfecting cotton swab, and with its drawout on culture plate, in order to carrying out Bacteria Detection.Then, abideing by SOP (Standard Operating Procedure, S.O.P.) handles.
The b.PCR method-to the spiral Pseudomonas
With SEQ ID NO.1 is preceding primer, serves as that contrary primer carries out PCR (J.Clin.Microbiol., 35 (6): 1620-1623,1997 with SEQ ID NO.2; J.Clin.Microbiol., 39 (11): 3920-3926,2001).PCR is reflected under the following operating condition and carries out: degeneration (94 ℃, 30 seconds); Annealing (55 ℃, 30 seconds); Extend (72 ℃, 2 minutes), carry out 30 circulation amplifications altogether, final step was carried out under 72 ℃ 5 minutes.By the 1.5%NuSieve agarose gel electrophoresis, differentiate above-mentioned gained the PCR product (FMC BioProducts, Rockland, Maine).
The c.ELISA method-to mycoplasma pneumoniae and hair shape clostridium
Abide by the requirement of manufacturer's workbook, utilize Monilisa test kit (ICLAS, laboratory animal ultimate survey center, Japan) and Murin antigenic reagent box (Intracel, the U.S.) to detect.
1-3) to the detection of virus
With the ELISA method is that the virus detection is carried out on the basis.Abide by the requirement of manufacturer's workbook, utilize Monilisa test kit (ICLAS, laboratory animal ultimate survey center, Japan) and Murin antigenic reagent box (Intracel, the U.S.) to carry out ELISA.Particularly, detection to 7 kinds of listed in the above-mentioned table 1 hamster viruses, abide by Smith method (Smith AL. (1986) Serologic tests for detection of antibody torodent viruses.In, Viral and Mycoplasmal Infections of Laboratory Rodents.pp731-750.eds.PN Bhatt, RO Jacoby, MC Morse and AE New.Academic Press Inc., Orlando) and Lewis method (Lewis AM, Rowe WP, Turner HC and Heubner RJ. (1965) Lymphocytic choriomeningitis virus in hamster tumor:spread to hamsters andhumans.Science, 150,363-364) grade is carried out.Except that hamster virus,, in corresponding example, have been described in detail the detection of other virus.
＜reference example 2 〉
The preparation of operation isolation room
(Daehan Biolink Co.Ltd., inside Korea) keeps its malleation with movable air-flow then with ethanol and the sterilization of gluconic acid chlorhexidine with an operation isolation room.To be full of 0.2% peracetic acid soln in the immersion bath.Operation tool and other necessarys are put in this isolation room,, prepared a blood agar plate (SIGMA) in addition, in order to detecting the microbial contamination that may be present in the specimen (just having taken from the embryo) immediately in order to surgical operation usefulness.
＜example 1 〉
Screening is used to produce the hamster of SPF hamster
1-1) the first screening of hamster group
Abide by the method described in the reference example 1, the golden Syria hamster that will take from Chinese Chengdu Inst. of Biological Products carries out microbial contamination experiment (as table 1).As a result, filtered out for the first time and not organized by the hamster of microbial contamination.
1-2) breed the hamster group that filters out first, and carry out programmed screening
In laboratory animal raising chamber, by sib mating, with above-mentioned routine 1-1) in the hamster group selected carry out the above breeding of 3 generations.Carry out above-mentioned routine 1-1 once more) in microbial contamination experiment, thereby programmed screening goes out unpolluted animal.Then, the animal of selecting is assigned in the new group, then breeds.
＜example 2 〉
Generation of SPF hamster and breeding
2-1) conceived female Mus and the generation educate female Mus screening
For selecting conceived female Mus that the embryo can be provided by cesarean, from routine 1-2) the hamster that the filters out group, choose two female Mus that have once above childbirth experience, and it is had the male Mus that copulation experiences and be placed in the same cage with one.Second day, select the female Mus that has generated vaginal suppository, it is handled, until conceived first day, and continue to raise.In addition, to test the golden Syria hamster of VAF (Harlan USA) carries out mentioned microorganism and pollutes as described in the experiment (as above-mentioned routine 1-1)) of company from Charles River, and use and above-mentioned same method, to guarantee that unpolluted hamster carries out copulation, to obtain conceived female Mus.Obtained thus for educating female Mus.In generation, educated female Mus than the Zao copulation of conceived female Mus one day.Select animal, and it is educated female Mus as generation, to be used for cultivating the embryo of the female Mus of described pregnancy in production the previous day expected date of confinement of the female Mus of pregnancy.
2-2) obtain the embryo by cesarean
Based on the bulge degree of the female Mus abdominal part of pregnancy, the congested degree and the relax level of vaginal orifice, determine caesarean suitable opportunity.Cesarean is carried out in after pregnancy the 13.5th day.At first, injection CO
2To put to death conceived female Mus, use disinfectant soap (hands iodine soap, U.S. QUIP tests company) to clean the corpse appearance then, and carry out disinfection with alcohol gauze.The reuse iodine solution is further sterilized.When carrying out disinfection, vagina open zone of conceived female Mus and genital area need especially to note, should thoroughly clean.Open the abdominal part of dead animal, take out the uterus.With the zone of each ovary both sides of pliers ligation, and cut off with operating scissors.Neck region, reuse pliers ligation uterus, and cut off with operating scissors.With iodine solution incision is carried out disinfection, and make cornua uteri down, the immersion bath of isolation room is passed in the uterus, thereby introduce the inside (see figure 1) of isolation room, described isolation room is got ready in above-mentioned reference example 2.Then, with antiseptic gauze and the fast as far as possible disinfectant solution of removing the surface, uterus of cotton balls, then cut the uterus to take out the embryo.The above-mentioned embryo who obtains is moved on the operating-table, and this operating-table is got ready in advance and has been carried out warming.Note not allowing the embryo contact chemical solution such as disinfectant solution etc.Approximately after half an hour, check out embryo's situation, select the embryo of energy autonomous respiration.Then, will be coated onto on the embryo, and distribute to each generation and educate 8 embryos of female Mus for the Excreta of educating female Mus.Utilization isolating Placenta Hominis etc. in operation carries out the microbial contamination experiment to the embryo.
＜example 3 〉
In gnotobasis, raise and the breeding embryo
In the SPF isolation area, raise the embryo who obtains in the above-mentioned example 2, described SPF isolation area is provided by the iaboratory animal medicine portion of Korea S Yonsei university medical college medical research center.Described isolation area is adjusted to following condition: temperature 22-26 ℃, relative humidity 45-55%, ventilation frequency 8-12 time/hour, light intensity is lower than 10000 greater than 25 luxs, rank.About drinking water, supply with chloride concentration and be lower than 2% ionized water.About the raising raw material, and supply process roentgenization disinfectant rodent common food (LabDiet Co., USA).About hamster offspring's composition, the hamster of raising is divided into groups, thereby make the breeding group that has in each generation more than 8.In order to carry out inbreeding, adopt the sib mating method, in the method, copulation is carried out in brood.
＜example 4 〉
The screening of SPF hamster
4-1) antibacterial and parasite pollute experiment.
Each hamster that to select in the breeding group of example 3 carries out the microbial contamination experiment of antibacterial and parasite (listed as table 2, table 3).Method described in the above-mentioned reference example 1 of experimental basis is carried out.As shown in table 5 below, the result has confirmed not detect parasite and antibacterial in the above-mentioned hamster group of selecting.
Table 5 antibacterial and parasite pollute experimental result
| Microorganism | Testing result | |
| Antibacterial | Bronchus deteriorated blood Bao Te bacterium | *- |
| Corynebacterium kutscheri | - | |
| The spiral Pseudomonas | - | |
| Mycoplasma pneumoniae | - | |
| Salmonella | - | |
| Salmonella typhimurium | - | |
| Streptococcus pneumoniae | - | |
| Rhodopseudomonas | - | |
| Patina color pseudomonas | - | |
| Pasteurella | - | |
| Parent's pneumonia pasteurella | - | |
| Golden yellow Portugal coccus | - | |
| Hair shape clostridium | - | |
| The klebsiella spp of hastening parturition | - | |
| Pasteurella multocida | - | |
| Klebsiella pneumoniae | - | |
| B group beta chain ball | - | |
| G group beta chain ball | - | |
| The beta chain Coccus | - | |
| Parasite | Hepaticola hepatica | - |
| Hymenolepis | - | |
| Encephalitozoon cuniculi | - | |
| Mus giardia lamblia stiles (Girdia muris) | - |
| Mus six flagellates (Spironucleus muris) | - | |
| Back of the body shape Turbatrix | - | |
| Vermin | - |
*-: do not detect
4-2) viral pollution experiment
Will be from routine 4-1) the hamster selected carry out the microbial contamination experiment of variety classes virus.The requirement of institute of Glasgow, United Kingdom Q-One Biotech company is answered in all virus experiments.
4-2-1) to the experiment of 10 kinds of hamster specificity virus
A serology experiment
From each routine 4-1) the experimental group that filters out of lining, the serum of getting 5 laboratory animals carries out elisa assay (Smith AL., Viral and Mycoplasmal Infections of Laboratory Rodents.731-750,1986; Lewis AM et al., Science, 150:363-364,1965; Baum SG et al., N.Eng.Med., 274:934-936,1966).At first, the antibody with 10 species specificity hamster viruses in the table 4 is overlying on the microtitration flat board in advance.The serum specimen of getting ready is added in each hole, and with flat board incubation 1 hour at room temperature.With the negative contrast of serum of VAF LVG Syria hamster, the age of this VAF LVG Syria hamster is 3-4 week, and is female, raises in isolation area.The positive contrast of serum with the hamster that once was exposed to each virus antigen.Use the PBS/0.05% polysorbas20 then, flat board is cleaned 3 times, and the xenogenesis lgG that will combine peroxidase is added in each dull and stereotyped hole.With flat board incubation 1 hour at room temperature, then it is cleaned 3 times with the PBS/0.05% polysorbas20 again.Thereafter, with o-phenylenediamine and carbamide/H
2O
2Substrate is added in each dull and stereotyped hole.Then under room temperature lucifuge condition, with dull and stereotyped incubation 20 minutes.After the incubation, read out in the absorptance under the 490nm, whether have specificity hamster virus thereby detect.As a result, filtered out the hamster group that is not subjected to any hamster viral infection fully.The experimental result of having summarized the above-mentioned hamster that filters out in the following table 6.
Table 6 hamster serum virus is learned experimental result
| Sample | Hamster virus | |||||||||
| SEND | PVM | KRV | MVM | REO-3 | GD-7 | LCMV | SV5 | H-1 | HANT | |
| 1 | *- | - | - | - | - | - | - | - | - | - |
| 2 | - | - | - | - | - | - | - | - | - | - |
| 3 | - | - | - | - | - | - | - | - | - | - |
| 4 | - | - | - | - | - | - | - | - | - | - |
| 5 | - | - | - | - | - | - | - | - | - | - |
| *NC | - | - | - | - | - | - | - | - | - | - |
| *PC | *+ | + | + | + | + | + | + | + | + | + |
*NC: negative control
*PC: positive control
*-: do not detect
*+: detect
B. hamster antibody generates test (HAP test)
Whether be present in the hamster group (in above-mentioned steps a, filtering out) in order to detect above-mentioned 10 species specificity viruses (the known hamster tissue that infected) more accurately, carried out the HAP test.The used HAP test of the present invention has been made slight modifications on the basis of MAP test, this MAP carries out (Rowe WP et al., J.Exp.Med., 109:379-391,1959 by Rowe and colleague thereof at first; And Rowe WP etal., Virology 11:645-649,1960).Isolate the PHK cell among above-mentioned steps a, and it is seeded in the female VAF LVG Syria golden hamster from hamster (filtering out), this gold Syria hamster is female, age 3-4 week.Experimental session places the hamster that is inoculated in autoclaved little isolated location, and straw mattress, water and food are arranged in this isolated location, thereby avoids the viral infection outside the Virus Pollution of PHK cell.At postvaccinal the 28 day, from all animals, get serum sample.Utilize the determination of serology method, in all serum samples, measure 10 kinds of different antiviral antibodies.According to methods such as Hartley (Hartley JW et al., Virology, 11:645-649,1960), determine HANT antibody with IFA method (direct immunization fluorescent mensuration).Determine all the other 9 kinds of antiviral antibodies with the ELISA algoscopy.As shown in following table 7, confirmed not produce any to being tried the antibody of hamster virus.This result is consistent with result among the above-mentioned steps a.
Table 7 HAP result of the test
| Hamster virus | Detection method | The PHK cell inoculation | MEM culture medium inoculated (contrast) |
| SEND | ELISA | *- | - |
| SV-5 | ELISA | - | - |
| PVM | ELISA | - | - |
| MVM | ELISA | - | - |
| KRV | ELISA | - | - |
| H-1 | ELISA | - | - |
| GD-7 | ELISA | - | - |
| REO-3 | ELISA | - | - |
| LCMV | ELISA | - | - |
| HANT | IFA | - | - |
*-: do not detect
4-2-2) to the detection of 16 species specificity Muriviruses
In order to confirm whether hamster group (filtering out) has infected Murivirus, utilizes isolated PHK cell from described hamster, has carried out MAP test (Rowe WP., et al, J.Exp.Med., 109:379-391,1959 in above-mentioned routine 4-2-1; Rowe WP, et al., In, The Problems of LaboratoryAnimal Disease.pp 132-141.ed.RC Harris.Academic Press Inc., New York, 1962; Smith AL., In, Viral and Mycoplasmal Infections of Laboratory Rodents.pp731-750 eds PN Bhatt, RO Jacoby, MC Morse and AE New.Academic Press Inc., New York, 1986).
At first, go up inoculation PHK cell 20 VAF mouse (Harlan UK company is taken from CD 1 strain), described PHK cell is taken from the hamster among the above-mentioned steps a.At postvaccinal the 4th day, put to death 4 mouse, isolate its blood plasma.Utilize the spectrophotometric determination method, detect the activity level of LDH (lactic acid dehydrogenase), thereby determine whether to exist LDV (lactic acid dehydrogenase one challenge virus).The result confirms, inoculating in the present invention in the mouse of PHK cell the active LDH activity identical (seeing the following form 8) with negative control of its LDH.The above results shows, above-mentioned 4-2-1) in the hamster that filters out not infected by LDV.
Table 8 LDV infection experiment result
| Inoculum | LDH activity (unit) |
| DMEM culture medium (negative control) | ＜160 |
| PHK cell among the present invention | ＜160 |
| LDV (Riley strain) is (positive control) | 2560 |
At postvaccinal the 23rd day, excite 4 mouse with a kind of known LCMV killer strain (lymphocytic choriomeningitis virus, Armstrong strain), and observe the M ﹠ M of each working day in the time of 12 days.If above-mentioned PHK cell does not infect LCMV, animal will be dead in 4-12 days.In 12 days behind inoculation LCMV, inoculated all death of 4 mouse of hamster PHK cell in the present invention.This result shows that the hamster that produces among the present invention does not infect LCMV.
At postvaccinal the 31st day, from remaining mouse, extract serum specimen, and, measure the antibody that whether has mouse virus by the serology experiment.Described in following table 9,, do not detect 15 kinds of dissimilar Muriviruses having inoculated in the present invention in the serum of the mouse of PHK cell.
Table 9 MAP result of the test
| Mouse virus | Detection method | The PHK cell inoculation | MEM culture medium inoculated (contrast) |
| LCMV | ELISA | *- | - |
| MHV | ELISA | - | - |
| PVM | ELISA | - | - |
| MVM | ELISA | - | - |
| SEND | ELISA | - | - |
| ECTRO | ELISA | - | - |
| EDIM | ELISA | - | - |
| REO3 | ELISA | - | - |
| GDVII | ELISA | - | - |
| MAD | ELISA | - | - |
| POLY | ELISA | - | - |
| HANT | IFA | - | - |
| MTV | IFA | - | - |
| MCMV | ELISA | - | - |
| K | ELISA | - | - |
*-: do not detect
According to above-mentioned experiment, confirmed that the SPF hamster group of being set up among the present invention does not infect any microorganism, comprise 19 kinds of antibacterials, 6 kinds of parasites and 10 kinds of viral and 16 kinds of Muriviruses of hamster.
＜example 5 〉
Detect the additional experiment of dissimilar viral infection
Among the present invention, the SPF hamster that filters out in the example 4 has been carried out further experiment, whether had endogenous or ectogenic viral infection to detect.
5-1) to hamster polyomavirus (HPyV) INFECTION IN DETECTION
Get a laboratory animal in the SPF hamster that from example 4, filters out,, separate its liver, spleen, lymph node, lung and nephridial tissue according to known method.Then, utilize ABI 7700 sequence detection systems (PE AppliedBiosystems, 1997, Relative quantitation of gene expression, User Bulletin #2), detect in the above-mentioned isolated tissue whether have the HPyV-specific sequence.In the single hole of 96 hole reaction plate, carry out the PCR reaction.With isolated DNA from above-mentioned tissue is template.TaqMan PCR reactant mixture (is comprised TaqMan buffer A, Mg
2+, dNTPs, AmpErase UNG, AmpliTaqGold archaeal dna polymerase, TaqMan HPyV-Auele Specific Primer and fluorescent probe) add in the reaction.Reaction plate is put in ABI 7700 reactors, in this reactor, carried out activation and the amplified reaction of AmpErase and AmpliTaq.Under 50 ℃, carry out described AmpErase reaction 2 minutes.Under 95 ℃, carried out AmpliTaq Gold priming reaction 10 minutes then.In addition, setting the PCR reaction condition is: carry out 40 circulations altogether, be included in 95 ℃ of following degeneration 15 seconds, annealing/extension is 1 minute under 60 ℃.For matched group, in negative control group, do not use template to carry out PCR; And in positive controls, be that template is carried out PCR with the reorganization phaPV that contains the HPyV target gene.The result is as shown in following table 10.So, the institute that has confirmed SPF hamster in the present invention in a organized way in, do not detect HPyV one specific sequence.
Table 10 hamster polyomavirus experimental result
| The experiment grouping | The HPyV-specific sequence |
| Negative control | *- |
| The hepatic tissue of hamster among the present invention | - |
| The spleen tissue of hamster among the present invention | - |
| The lymph node tissue of hamster among the present invention | - |
| The lung tissue of hamster among the present invention | - |
| The nephridial tissue of hamster among the present invention | - |
| Positive control | *+ |
*-: do not detect
*+: detect
5-2) to the detection of retroviral infection
From the SPF hamster that example 4 filters out, separate the PHK cell, and with this PHK cell respectively with human diploid cell line MRC-5, human Burkitts lymphoma cell line Raji co-cultivation, detect retroviral generation then.At first, with PHK cell (from the present invention SPF hamster separate) with survey cell (MRC-5 or Raji cell) co-cultivation 4-5 days.Described detection cell line is from American type culture collection (ATCC).Going down to posterity for 1 stage, removing described PHK cell, and will survey cell culture more than 5 generations.For wrong positive signal is minimized, added CTNEAT (calf thymus activated dna), described wrong positive signal is caused by class-reverse transcriptase (RT) activity of archaeal dna polymerase, and this CTNEAT can disturb the class-RT activity of archaeal dna polymerase.In positive control, added based fine particles virus (VLPs).Then, collect to survey be commissioned to train supernatant after supporting of cell 5.According to disclosing known method, utilize PERT algoscopy (product strengthens sexual inversion record enzymatic determination) to detect whether have retrovirus (Lugert R, et al., Biotechniques, 20 (2): 210-217,1996; Pyra H, etal., PNAS.U.S.A., 91:1544-1548,1994).The bottom of cell inoculation at compound anti-hole flat board will be surveyed.As a result, in the culture of having inoculated the PHK cell, do not detect RT activity (related content see Table 11, table 12).The above results shows that the PHK cell of SPF hamster is not subjected to the pollution of retrovirus (possible infected person) among the present invention.
Table 11 retrovirus: the experimental result of MRC-5 cell line
| Specimen | *C TValue | The result | |
| Average | |||
| The cell of handling through CT NEAT | 40 | 40 | *- |
| Dilute the cell that CT NEAT handled through 1/10 | 40 | 40 | - |
| Cell without CT NEAT processing | 37.3 | 39.3 | - |
| Cell without 1/10 dilution CT NEAT processing | 39.8 | 39.9 | - |
| Through 10 5The cell of VLPs (1/10) peaking | 20.8 | 23.0 | *+ |
| Through 10 3The cell of VLPs (1/10) peaking | 28.6 | 26.2 | + |
*C
T: threshold cycle
*+: the positive
*-: feminine gender
Table 12 retrovirus: the experimental result of Raji cell line
| Specimen | *C TValue | The result | |
| Average | |||
| The cell of handling through CT NEAT | 40 | 39.7 | *- |
| Dilute the cell that CT NEAT handled through 1/10 | 40 | 40 | - |
| Cell without CT NEAT processing | 30.8 | 31.6 | - |
| Cell without 1/10 dilution CT NEAT processing | 32.9 | 36.4 | - |
| Through 10 5The cell of VLPs (1/10) peaking | 24.7 | 21.7 | *+ |
| Through 10 3The cell of VLPs (1/10) peaking | 31.6 | 30.6 | + |
*C
T: threshold cycle
*+: the positive
*-: feminine gender
5-3) to the detection of unknown hamster viral infection
In SPF hamster of the present invention, also detected whether there is unknown hamster viral pollution, this virus might cause the opportunistic infection to various detection cells.About surveying cell, adopted human diploid cell line MRC-5, human Burkitt lymphoma cell line Raji, African titi kidney cell line Vero, all detection cells are from ATCC.To survey cell (MRC-5, Raji or Vero cell) and PHK cell (from SPF hamster of the present invention, separating) co-cultivation 4-5 days.Going down to posterity for 1 stage, removing the PHK cell, will survey cell culture more than 5 generations.Then, in whole culture period, survey in the cell CPE whether occurs with microscopic examination.As a result, in the whole culture period, in the culture of inoculation, do not observe CPF.
In addition, utilize the mixture of guinea pig erythrocyte, chicken red blood cell and the mankind ' O ' type erythrocyte, checked and surveyed the erythrocyte absorbability of cell it.Under 4 ℃, make PHK cell (taking from the SPF hamster of the present invention) carry out the erythrocyte adsorption to the mixture of guinea pig erythrocyte, chicken red blood cell and the mankind ' O ' type erythrocyte, carried out at least 30 minutes.To have inoculated the negative matched group of detection cell of culture medium, to have inoculated the positive matched group of MRC-5 cell of influenza A virus.As a result, in negative control group with inoculated in the culture (5 times go down to posterity back) of PHK cell, do not observe human ' O ' type erythrocyte, chicken red blood cell and the erythrocytic erythrocyte adsorption of guinea pig.
Because with PHK cell and various detection cell co-cultivation for a long time after, in described PHK cell, do not observe CPF and erythrocyte adsorption yet, so we can say, in the PHK of SPF hamster of the present invention cell, not exist unknown virus to infect from hamster.
5-4) to unknown hamster lymphoma virus INFECTION IN DETECTION
Get 20 new lives' SPF young hamster, it is used immunosuppressive drug-cyclosporin, to suppress its immunocompetence.After guaranteeing to have reduced its immunocompetence, be experimental group with described animal.The experimental group hamster was raised more than 100 days, and whether monitoring malignant lymphoma or other tumors have taken place weekly.In positive controls, prepare 20 hamsters that inoculate HeLa tumor cell (ATCC).In negative control group, prepare 10 healthy hamsters of not using above-mentioned immunosuppressive drug.After 100 days, will determine not take place the laboratory animal execution of malignant lymphoma or other tumor, and perform an autopsy on sb.In each sacrificed animal, detect the tumor vestige in lymph node, spleen, pancreas, kidney, the lung regulating liver-QI etc.It is unusual to detect other simultaneously.The result shows, in the 100 day time after suppressing its immunity, does not find the infringement of tumor or other similar tumor in the SPF hamster of the present invention.The above results shows, in SPF hamster of the present invention, and the unknown hamster virus of not hiding.
By The above results, can prove in the SPF hamster that the present invention filtered out in example 4, do not infect any dissimilar parasite, antibacterial, virus, hamster virus, Murivirus and other known/unknown pathogen.And, can also prove that the PHK cell that extracts is applicable to preparation biological product (as vaccine etc.) from SPF hamster of the present invention.
＜example 6 〉
From SPF hamster of the present invention, separate the PHK cell, and with the Japanese hepatitis attenuation of this PHF cell preparation
Live vaccine
SPF hamster among the present invention (age is 10-14 days) is used CO
2Gas is put to death.After the sterilization, it is moved in the laboratory animal room, rank is lower than 10000.Get the kidney of described animal under the aseptic condition.The kidney of collected young hamster is cleaned with the EMEM medium, add trypsin solution then.To be immersed in kidney in this trypsin solution at 37 ℃ of following incubation 1-3 hours.Behind the incubation, the kidney suspension behind the trypsin acting is cleaned with medium (containing serum), and obtain single cell suspending liquid by bead.With the medium that contains serum above-mentioned cell suspending liquid directly is diluted to suitable volume, and it is transferred in the T-flask.Obtain the cell of requested number by breeding after, clean this cell with the medium that does not contain serum, and in the EMEM medium with about 1 hour of above-mentioned cell culture, contain Japanese hepatitis virus attenuated strain SA in this EMEM medium
14-14-2 (KCTC 0316 BP).After removing the culture medium that contains virus,, thereby breed described virus again with above-mentioned cell culture 3-4 days.Observe 75% or more CPE after, collect the suspension that contains virus, purify and aseptic filtration after, store down at 2-8 ℃, in order to the QC detection.Then, carry out last prescription (adding stabilizing agent), filling and vacuum lyophilization.
＜experimental example 1 〉
Safety test to Japanese hepatitis attenuated live vaccine among the present invention
For confirming of the safety of Japanese hepatitis attenuated live vaccine to human body, carried out safety test, described Japanese hepatitis attenuated live vaccine is prepared with the PHK cell of SPF hamster (example 5) among the present invention.
1-1) to the detection of HPyV
For detecting in Japanese hepatitis attenuated live vaccine of the present invention, whether have HPyV, utilize ABI 7700 sequence detection systems, determined whether there is the HPyV specific sequence, method is as routine 5-1) as described in.The result is summarized in the following table 13.
The testing result of table 13 pair hamster polyomavirus
| Experimental group | The HPyV-specific sequence |
| Negative control group | *- |
| Japanese hepatitis attenuated live vaccine of the present invention | - |
| Positive controls | *+ |
*-: do not detect
*+: detect
1-2) HAP test
In Japanese hepatitis attenuated live vaccine of the present invention, whether have hamster virus for detecting, to utilize routine 4-2-1) method described in the step b, carried out the HAP test.The result is summarized in the following table 14, and is very obvious, in Japanese hepatitis attenuated live vaccine of the present invention, do not detect any hamster virus.
Table 14 HAP result of the test
| Virus | Detection method | Japanese hepatitis attenuated live vaccine of the present invention | MEM culture medium inoculated (contrast) |
| SEND | ELISA | *- | - |
| SV-5 | ELISA | - | - |
| PVM | ELISA | - | - |
| MVM | ELISA | - | - |
| KRV | ELISA | - | - |
| H-1 | ELISA | - | - |
| GD-7 | ELISA | - | - |
| REO-3 | ELISA | - | - |
| LCMV | ELISA | - | - |
| HANT | IFA | - | - |
*-: do not detect
1-3) the MAP test that excites with LCM
In Japanese hepatitis attenuated live vaccine of the present invention, whether have Murivirus for detecting, to utilize routine 4-2-2) described in method, carried out the MAP test.Japanese hepatitis attenuated live vaccine in the example 6 is resuspended in the DMEM medium of 1.5ml/ bottle.With the DMEM medium will in and antiserum with 1: 10 dilution proportion.Then, with in described vaccine and the above-mentioned dilution and antiserum with 1: 1 mixed, and cultivated 1 hour down, thereby generated experiment sample at 37 ℃.Go up this sample of inoculation 20 SPF mouse (CD1 strain, Harlan UK company).At postvaccinal the 13rd day, put to death 4 mouse, by the spectrophotometric determination method, measure the LDH activity level in the blood plasma of dead mouse.Described in following table 15, inoculating among the present invention on the mouse of PHK cell the active little LDH activity that is lower than negative control group of its LDH.
Table 15 LDV infection experiment result
| Inoculum | LDH activity (unit) |
| DMEM medium (negative control) | 449 |
| Japanese hepatitis attenuated live vaccine of the present invention | 419 |
| LDV (Riley strain) is (positive control 1) | 1005 |
At postvaccinal the 14th day, excite 4 mouse with LCM virus (Amstrong strain), observe the M ﹠ M of every workday in the time of 12 days.After 7 days, all mouse death.The above results can confirm, with in the Japanese hepatitis attenuated live vaccine of PHK cell preparation of the present invention, does not carry LCM virus.
In addition,, from remaining mouse, get blood, and its serum is carried out the serology experiment at postvaccinal the 34th day.As shown in following table 16, do not detect any in 15 kinds of Muriviruses.
Table 16 MAP result of the test
| Murivirus | Detection method | Japanese hepatitis attenuated live vaccine of the present invention | MEM culture medium (contrast) |
| LCM | ELISA | *- | - |
| MHV | ELISA | - | - |
| PVM | ELISA | - | - |
| MVM | ELISA | - | - |
| SEND | ELISA | - | - |
| ECTRO | ELISA | - | - |
| EDIM | ELISA | - | - |
| REO3 | ELISA | - | - |
| GDVII | ELISA | - | - |
| MAD | ELISA | - | - |
| POLY | ELISA | - | - |
| HANT | IFA | - | - |
| MTV | IFA | - | - |
| MCMV | ELISA | - | - |
| K | ELISA | - | - |
*-: do not detect
1-4) PERT measures
In Japanese hepatitis attenuated live vaccine of the present invention, to whether existing retrovirus to detect.The present invention has carried out PERT mensuration, and described PERT algoscopy is used to detect the reverse transcriptase microgranule with enzymatic activity, and this method has very high sensitivity.At first, get in the example 6 the live vaccine 3ml of preparation, with the rotating speed of about 11000g centrifugal 10 minutes, thus it is purified, then make its sterilizing filter, thereby remove remaining fragment through 0.45 μ m.Then, with the vaccine after the above-mentioned processing super centrifugal 60 minutes, make it be the ball shape with the rotating speed of 100000g.Each piller is resuspended in the 100 μ l lysis buffers, to discharge the activity of RT.Utilize the method (Pyra H, et al., Proc.Nat.Acad.Sci., 91:1544-1548,1994) of announcements such as Pyra to carry out PERT mensuration then.The result confirms: in Japanese hepatitis attenuated live vaccine of the present invention, the RT activity is negative.The above results shows, does not have retrovirus in Japanese hepatitis attenuated live vaccine of the present invention.
1-5) the external test that unknown virus is polluted
By with the Japanese hepatitis attenuated live vaccine among the present invention with survey the cell line co-cultivation, the present invention has determined whether viral infection is arranged in this vaccine.With human body near-triploid cell line H9 (ATCC) serve as the detection cell.At first, with the inoculation strain and the H9 cell line co-cultivation of the present invention Japan hepatitis attenuated live vaccine.Going down to posterity for 1 stage, removing the inoculation strain.To survey cell continues to go down to posterity more than 5 times.At whole culture period, survey in the cell CPE whether occurs with microscopic examination.In above-mentioned H9 culture, do not observe CPE.
After going down to posterity for 5 times, from the H9 culture, collect supernatant, and detect whether there is retrovirus (Pyra H, et al., Proc.Natl.Acad.Sci USA, 9:1544-1548,1994) by the PERT algoscopy.In the H9 culture of having inoculated the vaccination strain, do not detect the RT activity.
In addition, under 4 ℃,, make H9 cell (having inoculated Japanese hepatitis attenuated live vaccine of the present invention) carry out the erythrocyte adsorption, carried out at least 30 minutes by the inferior swine erythrocyte of guinea, chicken red blood cell and the erythrocytic mixture of the mankind ' O ' type.As a result, in this H9 cell, do not observe the erythrocyte adsorption.Because do not detect CPF, RT activity and erythrocyte adsorption in H9 cell (having inoculated Japanese hepatitis attenuated live vaccine of the present invention), so we can say, Japanese hepatitis attenuated live vaccine of the present invention does not carry viral pollution.
1-6) measure in the body to the unknown virus pollution
Requirement (Committee for Proprietary Medicinal Products:AdHoc Working Party on Biotechnology/Pharmacy.J.Biol.Stand., 17:213-222,1989 according to CPMP and FDA; And Points to consider in characterization of cell lines used to producebiologicals, 1993.Center for Biologics Evaluation and Research.Food and DrugAdministration, Gethesda MD 20205, USA), for detecting in the Japanese hepatitis attenuated live vaccine of the present invention whether have adscititious viral agent, by the inoculation to embryo, age of sucking mice, adult rats and the inferior pig of guinea, the present invention has carried out measuring in the following body.
(1) sample preparation
Japanese hepatitis attenuated live vaccine among the present invention (preparation in example 6) is resuspended among the DMEM of 1.5ml/ bottle.With the DMEM medium will in and antiserum with 1: 10 dilution proportion, with in above-mentioned vaccine and this dilution and antiserum with 1: 1 mixed, and 37 ℃ of cultivations 1 hour down.
(2) zoopery
A.
Experiment to mice age of sucking
At first, determine the safety of the vaccine for preparing among the present invention to mice age of sucking.By (0.01ml) injection path in the intramuscular (0.01ml), intraperitoneal (0.1ml), brain, the sample of preparation in the above-mentioned steps (1) is inoculated into the age of sucking mice goes up (inoculation for the first time), this, mice was at least 20 age of sucking, from 2 nests or more than 2 nests.By same path, the DMEM medium is inoculated on other one group of age of sucking mice, and as negative control group.Observe mice whether occur as weak, tremble, any disease reactions such as paralysis or death, continue to observe 14 days.After 14 days, put to death mice.Isolate the organ of mice, dissolve and stir evenly.With with inoculate for the first time same method, this organ suspension is seeded in other one group of new mice age of sucking goes up (inoculation for the second time).This group mice of same observation 14 days.In inoculating for the first time, behind 24 hours of inoculation, survived in 20 mices 19 (survival rate is 95%).After mice age of sucking of death performed an autopsy on sb, do not observe viral infection.In inoculating for the second time, behind 24 hours of inoculation, all mice survivals (survival rate is 100%).
B.
Experiment to adult rats
By (0.031ml) injection path in intramuscular (0.1ml), intraperitoneal (0.5ml), the brain, above-mentioned sample is inoculated into (SPF, CD 1 strain) on 10 adult rats.With same path, the DMEM medium is inoculated on the negative control group mouse.Then, observe mice and any disease reaction whether occurs, continue to observe 28 days.As a result, behind 24 hours of inoculation, all mouse survivals (survival rate is 100%).
C.
Experiment to guinea pig
In last experiment,, above-mentioned sample is inoculated on 5 adult guinea pigs (SPF, Duncan Hartley strain) by intramuscular (0.1ml) injection path.In negative control group, the DMEM medium is inoculated on 5 guinea pigs with same path.Observe above-mentioned guinea pig and any disease reaction whether occurs, continue to observe 28 days.Found that, behind 24 hours of inoculation, all guinea pig survivals (survival rate is 100%).
(3) embryo's experiment
A. intraamniotic injection
When carrying out intraamniotic injection, choose 10 the biggest SPF embryos of 10-11, the above-mentioned sample of 0.1ml is inoculated in its amniotic cavity.Choose 10 the biggest SPF embryos of 10-11 in addition, the 0.1mlDMEM medium is inoculated in its amniotic cavity, and as negative control group.Choose the 3rd group of the biggest SPF embryo of 10 10-11, the 0.1ml influenza A virus is inoculated in its amniotic cavity, and as positive controls.After the inoculation, the embryo was cultivated 5 days down at 35-36 ℃.Collect amniotic fluid and mixing.Mixed amniotic fluid is carried out hemagglutination reaction (HA) determination of activity.By continuous 2 times of dilutions, on the microtitration flat board, carry out HA and measure amniotic fluid.After cleaning chicken and guinea pig, get its erythrocyte, with in this erythrocyte respectively the form with 0.5% suspension add in the above-mentioned flat board, and respectively 2-8 ℃ cultivate down 1-2 hour, cultivated 1-2 hour down at 17-20 ℃, observe the HA activity on this compound flat board then.The result shows that in being tried embryo's amniotic fluid, its HA is active close with negative control group.In addition, according to the back 24 hours existence test of inoculation, 9 are tried among the embryo, and 8 survivals (survival rate is 89%) are arranged.
B. injection in the allantois
When carrying out injecting in the allantois, choose 10 the biggest SPF embryos of 10-11, the above-mentioned sample of 0.1ml is inoculated in its allantoic cavity (goes down to posterity for the first time).Choose 10 the biggest SPF embryos of 10-11 in addition, the 0.1mlDMEM medium is inoculated in its allantoic cavity, and as negative control group.Choose the 3rd group of the biggest SPF embryo of 10 10-11, (ATCC VR-547) is inoculated in its allantoic cavity with the 0.1ml influenza A virus, and as positive controls.After the inoculation, the embryo was cultivated 3 days down at 35-36 ℃.Collect its allantoic fluid and mixing.Get the portion that mixes in the allantoic fluid of back, freezing and store in the time of-70 ℃ or below-70 ℃, until it being carried out the HA determination of activity or with till its subinoculation is in one group of new embryo.
Passed through allantoic inoculation the embryo of above-mentioned sample from initial, got the mixed allantoic fluid of its 0.2ml, it has been inoculated on 7 the biggest embryos of 10-11 one by one, thereby this allantoic fluid has been carried out going down to posterity (second pass generation).From the initial embryo who inoculates the DMEM medium, get its allantoic fluid, and by same approach, this allantoic fluid is gone down to posterity on one group 7 new the biggest embryos of 10-11.From the initial embryo who inoculates influenza A virus, get its allantoic fluid, and by same approach, this allantoic fluid is gone down to posterity on one group 7 new the biggest embryos of 10-11.After 3 days, collect allantoic fluid, mix and measure its HA activity.Utilize and the same method of the interior test of above-mentioned amniotic membrane, carry out HA and measure.
Measurement result shows that in being tried embryo's allantoic fluid, its HA is active close with negative control group.In addition, according to the back 24 hours existence of inoculation test, in going down to posterity for the first time, all embryos keep survival (survival rate is 100%), in second pass generation, have 5 to keep survival (survival rate is 71%) among 7 embryos.
C. yolk intracapsular injection
When carrying out the yolk intracapsular injection, choose 10 the biggest SPF embryos of 6-7, the above-mentioned sample of 0.1ml is inoculated in its yolk sac cavity (goes down to posterity for the first time).Choose 10 the biggest SPF embryos of 6-7 in addition, the 0.1mlDMEM medium is inoculated in its yolk sac, and as negative control group.Above-mentioned embryo was cultivated 9 days down at 35-36 ℃.Then, detect this embryo's survival rate.Collect above-mentioned yolk sac, clean and mixing.Then it is prepared into 10% suspension, and subinoculation (second pass generation) to one group 7 new the biggest embryos of 6-7.From the embryo who has injected DMEM, get its yolk sac, be prepared into 10% suspension equally, and with its subinoculation to one group 7 new the biggest embryos of 6-7, with as negative control group.After 9 days, detect all embryos' survival ability.
The above results shows, in going down to posterity for the first time, in postvaccinal 24 hours, 10 are tried the embryo and all keep survival (survival rate is 100%), and in second pass generation, 7 animal subjects all keep survival (survival rate is 100%).
Comprehensive above-mentioned experimental result confirms not to be subjected to any viral infection in the Japanese hepatitis attenuated live vaccine of the PHK cell preparation of using SPF hamster of the present invention.
Commercial Application
As mentioned above, the method in according to the present invention and the SPF hamster that generates is not carried any parasite, bacterium or other various endogenous or exogenous virus pollutant. Therefore, the initial cell that from this SPF hamster, extracts and the opportunistic infect that can not cause microorgranic contaminant with the biological products of this cell preparation, and in the zooblast for the preparation of present biological products (such as vaccine etc.), might there be this microorgranic contaminant. So in SPF hamster of the present invention, the hamster tissue that extracts and the supernatant of culture thereof can be used safely in to prepare various biological products, use in order to human body.
Claims (12)
1. thereby one kind is used to generate the method that the specific hamster that does not contain pathogen is used for preparing biological product, and its step comprises:
(a) at first, by microbial contamination experiment to parasite, antibacterial and hamster virus, choose the hamster of uninfection, and by the above-mentioned hamster of choosing of sib mating breeding, from the hamster that is bred, choose for the second time and be not subjected to the hamster that mentioned microorganism infects then;
(b) selected hamster is carried out copulation, got its uterus, an isolation room is introduced in the uterus of being got in 12-14 days after female hamster pregnancy, get its embryo by cesarean, in rank is lower than 10000 isolation area, utilizes generation to educate female Mus and cultivate the above-mentioned embryo who gets then, and
(c) breed the hamster of being cultivated in the above-mentioned steps (b) by sib mating, and it is carried out microbial contamination experiment of parasite, antibacterial, hamster virus, Murivirus, endogenous hamster retrovirus, hamster polyomavirus, hamster lymphoma virus and unknown hamster virus, from the hamster of above-mentioned breeding, choose the hamster that not infected by mentioned microorganism then.
2. method according to claim 1, the wherein said SPF hamster that is used to prepare biological product is to be used to extract the original nephrocyte of hamster.
3. method according to claim 1, wherein the hamster in the above-mentioned steps (a) is golden Syria hamster.
4. method according to claim 1, wherein above-mentioned steps (a) or (c) in parasite comprise endoparasite and vermin.
5. method according to claim 4, wherein said endoparasite comprise Hepaticola hepatica, Hymenolepis, encephalitozoon cuniculi, Mus giardia lamblia stiles, Mus six flagellates and Syphacia.
6. method according to claim 1, wherein the antibacterial in the above-mentioned steps (a) comprises bronchus deteriorated blood Bao Te bacterium, corynebacterium kutscheri, spiral Pseudomonas, mycoplasma pneumoniae, Salmonella, Salmonella typhimurium, streptococcus pneumoniae, patina color pseudomonas, close pneumonia pasteurella, golden yellow Portugal coccus and hair shape clostridium.
7. method according to claim 1, wherein the storehouse in the above-mentioned steps (a) belongs to virus and comprises celestial platform, pneumonia of mice virus, Mus piconavirus, Mus Kilham virus, 3 type reoviruss, lymphatic choroid plexus encephalitis, Toolans H-1 virus.
8. method according to claim 1, wherein to educate female Mus be a kind of hamster that does not contain virus antigen the generation in the above-mentioned steps (b).
9. method according to claim 1, wherein the antibacterial in the above-mentioned steps (c) also comprise Rhodopseudomonas, pasteurella, the klebsiella spp of hastening parturition, Pasteurella multocida, klebsiella pneumoniae, B group beta chain ball, G group beta chain ball, beta chain Coccus, and the antibacterial in the above-mentioned steps (a).
10. method according to claim 1, wherein the hamster virus in the step (c) also comprises Taylor murine encephalomyelitis virus, simian virus 5, hantaan virus, and the hamster virus in the above-mentioned steps (a).
11. method according to claim 1, wherein the Murivirus in the above-mentioned steps (c) comprises lymphatic choroid plexus encephalitis, murine hepatitis virus, pneumonia of mice virus, Mus piconavirus, celestial platform, lacks limb deformity virus, young Mus epizootic disease diarrhea virus, 3 type reoviruss, murine encephalomyelitis virus, murine adenovirus, polyoma virus, hantaan virus, Mus thymus virus, murine cytomegalovirus, pneumonia of mice virus, lactate dehydrogenase-short virus.
12. method according to claim 1, wherein said biological product are selected from one group of vaccine, comprising: Japanese encephalitis's attenuated live vaccine, Japanese encephalitis's inactivated vaccine, rabies vaccine, dengue vaccine, hemorrahgic fever with renal syndrome vaccine and Ticks source property encephalomyelitis vaccine.
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| KR10-2003-0001698A KR100482644B1 (en) | 2003-01-10 | 2003-01-10 | Method for producing specific pathogen free hamsters which can be used for production of a variety of biological products |
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| CN101971788A (en) * | 2010-11-18 | 2011-02-16 | 青岛康大食品有限公司 | Artificial propagation technology of SPF experimental hare |
| CN110122415B (en) * | 2019-05-28 | 2021-06-08 | 上海市农业科学院 | Breeding method of small pig closed group for high inbred experiment |
| CN110736654B (en) * | 2019-11-04 | 2021-11-19 | 上海青赛生物科技有限公司 | Mumps virus rat neurovirulence evaluation model |
| CN116769946A (en) * | 2022-12-22 | 2023-09-19 | 斯贝福（北京）生物技术有限公司 | Primer probe composition for detecting mouse six-wire flagellate and application |
| CN115804362A (en) * | 2023-02-08 | 2023-03-17 | 中国医学科学院医学生物学研究所 | IFN-alpha/beta R -/- Injection for enhancing infection of mice by antibody dependency and preparation method thereof |
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