CN1435260A - Yolk antirotavirus antibody preparation, method for preparing same and use thereof - Google Patents
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Abstract
An immune globulin composition containing chicken yolk resisting group-A rotaviruses, its preparing process, and its application in treating baby rotavirus diarrhea are disclosed. It is an enteric preparation which is prepared through immunizing hen, biochemical separation and purifying.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of novel immunoglobulin, more specifically, the present invention relates to a kind of anti-A group rotavirus Yolk immunoglobulin preparation and its production and use.Be suitable for intestinal tract diseases such as prevention and treatment infantile diarrhea.
Background technology
Infantile diarrhea is the infant commonly encountered diseases, in emergency case and inpatient, account for the 2nd (being only second to respiratory tract infection) of all disease incidences, estimation according to World Health Organization (WHO), infantile diarrhea case number below 5 years old surpasses 4,000 ten thousand, because the infant physiogenesis is incomplete, in case ill, many severe complications often appear, and mortality rate is higher.According to statistics, about every year is died from diarrheal infant case load more than 8,000,000 in the whole world, and particularly in developing country, because medical level is limited, the harm of diarrhoeal diseases is very huge.
Determined that rotavirus infection was the main pathogens that causes infantile diarrhea in 1973 first, rotavirus infection has now become the topmost cause of disease (Bishop in several diarrhoea of the infant that is found everywhere through the world, R.F.et al.Lancet1:149-151.1974) (Bishop, R.F.et al.Lancet2:1281-1283.1973).Developing country annual since due to the rotavirus mortality rate of infantile diarrhea estimate more than 1,000,000.Reports such as Schaller, be defined as the cacatory common cause of child in developed country's Human reoviruslike agent, in the serious diarrhoea of the child of developing country, second of Human reoviruslike agent (HRV) row (Schaller, J.P.et al.J.Infect.Dis.165:623-630.1992).Show that according to the up-to-date statistical result of the Center for Disease Control (CDC) the annual rotavirus case load that infects of the U.S. is more than 5,000,000.Be rotavirus diarrhea more than about 1/3-1/2 in the infant that acute gastroenteritis is in hospital, no matter be in developed country or developing country, the diarrhoea that the A group rotavirus causes all has higher sickness rate, be the infant morbidity and the lethal important cause of disease, in developing country, be lethal second cause of disease that is only second to respiratory tract infection of infant.
Rotavirus is a genus of Reoviridae, is to cause the important cause of disease of humans and animals diarrheal.Virion is gained the name because of be typical wheel shape under Electronic Speculum, thereby also be different from other reovirus, studies show that complete rotavirus granule has three layers of protein shell, inner shell was called virus nuclear in the past, be wrapped in it viral nucleic acid and virus replication must and the enzyme that do not have in the infection cell, inner shell is outward to be to penetrate shape subunit formation mesochite (being called inner shell in the past), diameter is about 60nm, the duovirus granule is coarse 20 bodies, outermost is thin and slick shell, complete virion is smooth wheel shape, the about 70nm of diameter.
The rotavirus granule contains 11 segmental cores of bifilar RNA by SI viral capsid and 1 to be formed, and the coat protein of rotavirus A group is made up of two kinds of structural protein: a kind of VP4 of being is coded by RNA fragment 4; A kind of is VP7, coded by one in last 7,8,9 three fragments of RNA.These two kinds of albumen are relevant with neutralization of virus, different according to A group rotavirus VP4 with the composition of VP7, this group rotavirus can be divided into different P type (determining) and G type (determining) according to different VP7 according to different VP4, the A group rotavirus can be divided into P1A, P1B, four serotypes of P3, P4 according to VP4 at present, according to VP7, identified 14 different types, caused that wherein the serotype of infantile diarrhea comprises G1, G2, G3, G4, G8, G9 and G12.
The clinical manifestation of rotavirus infection differs, and suffers from diarrhoea up to dehydration serious or even lethal from the slightest subclinical infection, laxativeness.Generally all show clinically diarrhoea, vomiting and low grade fever are arranged.The vomiting time that general rotavirus causes is grown (2.6 days), suffers from diarrhoea sustainable 1-9 days, and the sick child often has dehydration and compensated acidosis in various degree, and average time in hospital treatment time is more than 4 days.
Because the infant immune system still is in the stage of development; special rotavirus resisting immune is reflected at digestive system and plays a role and also have obstacle; a little less than the immunoreation to pathogenic microorganism; and lost protection because of the termination of age of sucking by body consumption from the adoptive immunity power (content with first Ruzhong is maximum) of breast milk; therefore in case after ill; the intravital rotavirus of machine is difficult for being removed by body, causes sick child's the course of disease long, more complication severe patient occurs and causes death.Lack a kind of effective Therapeutic Method clinically for rotavirus infection diarrhoea, many at present employing symptomatic treatments are main, comprise and adopt oral or intravenous injection fluid infusion correct dehydration etc.Producing the intravital virus of response to active immunization removing invasion by body fully recovers; aspect prevention; the use of Rotavirus Vaccine also is in the experimental stage; but infant for morbidity; adopt vaccine therapy must rely on competence exertion effect behind the antibody that body produces anti-rotavirus automatically; onset is slow, so adopt the passive immunity protection for the control rotavirus infection provides new method, its important clinical meaning is arranged.
Guarino etc. adopt 98 viral diarrhea children of oral rotavirus resisting immune globulin treatment, after the result shows disposable oral 300mg/kg, the average diarrhoea time of infant shortened to 76 hours by 131 hours, and the average toxin expelling time of intestinal shortened to 114 hours by 180 hours.Other clinical test results also confirms to adopt oral immune globulin treatment rotavirus diarrhoea all can obtain curative effect (Guarino, A.et al.Pediatrics93:12-16.1994) preferably.
But since in the world wide most HRV epidemic strains its distribute and very big-difference to be arranged because of time and geographical position, research according to the domestic epidemiologic data of China, it is P1AG1 type (accounting for 66.4%) and P1BG2 type (29.2%) (schaller that present China causes the serotype of the main rotavirus of infantile diarrhea, J.P.et al.J.Med.Virol.19:167-173.1986) (Liou, Y.L.Zhonghua Liu Xing.Bing.Xue.Za Zhi.3:225-226.1982).
The method of traditional mode of production antibody is to obtain specific antibody in the animal bloods such as the rabbit of adopting the specific antigen immunity, sheep.Owing to adopt oral antibody to carry out passive immunotherapy, need a large amount of antibody to bring into play effect, therefore adopt traditional immune animal to obtain a large amount of therapeutic antibodies because the source is limited, the production cost height, and need to sacrifice a large amount of laboratory animals, so the research of this aspect can't be carried out always.Adopt monoclonal antibody to obtain monoclonal antibody by cultivating hybridoma in addition in laboratory, production cost is higher, complex process, and also the antibody of producing is because at single antigen, protective effect is poor.
Along with the increase of laboratory and clinical research antagonist demand, studying extensive antibody production techniques becomes new research topic.The shortcoming that there are the cost height in traditional animal serum antibody and method for preparing monoclonal antibody, yield poorly, utilize the hyperimmune chicken specific antibody can be stablized the characteristic that is transported to yolk, adopt biotechnology from the Cold boiled chicken of laying eggs, to prepare specific polyclonal antibody, it is low to have production cost, the characteristics that output is high are expected to become the effective means of mass preparation antibody.Known, hen is subjected to cause pathogeny imcrobe infection or can produces specific antimicrobial antibody after accepting artificial immunity, and the antibody capable in these serum is transported to yolk by body on one's own initiative in a large number from blood, be called as Yolk immunoglobulin.
Main immunoglobulin is IgY in the chicken serum, and this immunoglobulin is made up of two heavy chains and two light chains, and its molecular weight is respectively 66Kd and 22Kd.IgY and mammal IgG are quite similar.Therefore after chicken was subjected to pathogenic infection or accepts artificial immunity, the IgY that produces in the serum optionally was transferred in the Ovum Gallus domesticus Flavus, IgY dense in yolk.
IgY can be specifically combines with corresponding antigens on the pathogen, avoids the pathogen infringement by performance neutralizing antibody active protection body.Compare with monoclonal antibody, this antibody belongs to polyclonal antibody, can react with the multiple antigen on the pathogen, and is therefore stronger to the protective effect of body generation.
IgG has good heat stability.Under the room temperature condition, the biologic activity of IgY can keep more than 6 months.Can store the 6-7 activity for 4 ℃ only descends in 5%.In addition, IgY is more heat-resisting, alkaline-resisting, and is stable when PH=4.0-11.0, but active decline rapidly when PH=3.0 is following, active reduction during PH=12 is when temperature reaches 65 ℃, the IgY activity still can keep more than 24 hours, 70 ℃ of heating active obviously declines after 90 minutes.IgY solution under the 4000kg/cm2 high pressure and temperature handled 30 minutes when being lower than 60 ℃, still maintain activity, show that IgY has bigger anti-high-voltage performance.In addition, IgY can resist the digestion (Leslie, G.A.et al.Japanese quail.J.Immunol.105:1215-1222.1970) of trypsin and chymase in the intestinal.
After IgY enters intestinal, mainly be subjected to the effect of intestinal protease, these enzymes are based on trypsin family, its similar performance, the IgY of anti-HRV has certain resistance to the enzyme action of protease, and result of study is expressed, IgY after 8 hours, still has suitable neutralization activity through trypsinization.Therefore Ovum Gallus domesticus Flavus becomes an important source of the specific antibody that is used for the oral immunity therapy that is concerned by people.
Levine in 1981 has reported and has been used to cause that the E.Coli bacterial strain (ATCC23985) of people's diarrhoeal diseases carries out immunity to chicken, obtains the IgY of anti-this E.Coli of high titre, and it can make the E.Coli antibacterial assemble, thereby suppresses breeding and the growth of antibacterial.It is believed that this gathering specificity is very important; particularly, the E.Coli growth of enteral is suppressed, and can easily it be discharged from enteral at enteral; IgY made an addition to just can stop antibacterial in the food, make human body obtain the protectiveness passive immunity the infecting of food.HRV is the main pathogens, particularly baby that causes baby and child's intestinal tract disease, owing to autoimmune as yet not zoon be difficult to pathogen is produced active immunity.In recent years, the neonatal rat that many scholars infect with HRV is a model, has carried out the protectiveness experiment of anti-HRV chicken antibody (IgY).1993, the effect of Hatta etc. have detected 3 kinds of main digestive enzymes right-HRV IgY and anti--HRV IgY preserved 91% activity with pepsin and IgY, even have still had 63% activity behind the 10h infecting the protective effect of HRV behind pH4 incubation 1h.IgY respectively with trypsin and chymase incubation 8h, active preserve 39% and 41%, illustrate that IgY has resistant function within a certain period of time to the digestion of these two kinds of enzymes.Add this when IgY arrives digestive tract, it can be preserved enough activity and prevent infecting of pathogen in intestinal.Experiment shows if neonatal rat 1h before infecting HRV takes IgY, almost can prevent the diarrhoea that HRV causes fully, if take IgY in the 24h after infecting HRV, also can prevent the neonatal rat diarrhoea that HRV causes very effectively.The protection effect of IgY with infect HRV and take the interval of IgY relevant, weaken with the growth of blanking time.Even the pH value in the 5 monthly age baby stomaches after feeding newborn 2-3h also through being everlasting between 4～5.Therefore, IgY is unlikely serious inactivation in stomach, so add IgY in baby food, it is possible fully providing effective passive immunity to the baby.
Obtaining specific antibody IgY from Ovum Gallus domesticus Flavus is a high yield, approach of fine quality and good easily, very big potentiality and wide prospect are being arranged aspect the comprehensive development and utilization of biological product, and along with updating of IgY preparation and purification process, its application will further enlarge.And from the immunoglobulin that through the no cause of disease hen of Human reoviruslike agent immunity, extracts and the preparation method and the purposes of this immunoglobulin.The invention provides based on this immunoglobulin, be equipped with the enteric coated preparation of the prepared one-tenth of other medicinal adjuvant, also confirmed the therapeutical effect of enteric coated preparation experimental rotavirus diarrhea.
Summary of the invention
The purpose of this invention is to provide a kind of anti-A group rotavirus Yolk immunoglobulin preparation that contains of novel Gong oral application, this immunoglobulin has neutralization to the main Causative virus type of Chinese Infant and Children rotavirus diarrheal.
Another object of the present invention provides the method for preparing the alleged rotavirus resisting immune globulin preparation of the present invention.
Another object of the present invention provides the purposes of the alleged immunoglobulin preparation of the present invention aspect prevention, treatment infantile rotavirus enteritis.
Another object of the present invention provides wheel virus antigen material that can be used for the no cause of disease hen of immunity and preparation method thereof.
This realization above-mentioned purpose, the present invention adopts following technical scheme:
1. the invention provides a kind of separation, specific amplification rotavirus strain, and it is prepared into and can reaches the multivalent immunogen prepared product that biologically uses for medical science.
In the invention of this part, at first according to the epidemic research data, filter out Causative virus serotype common in the Chinese Infant and Children, adopt corresponding Strain to carry out amplification in vitro; The serotype that is used for the bacterium strain that optimizes is G1P1A, G2P1B.
The amplification of virus can be carried out in suitable cell culture.Said cell culture comprises MA104 cell, PK-15 cell, Vero cell etc., preferably Vero cell in the invention of this part.
2. of the present inventionly also provide a kind of preferred animal immunization method, after rotavirus deactivation with preparation, add a certain amount of adjuvant, preferred scheme be will preparation 1ml contain the rotavirus extract (2 * 105TCID) mix with the Freund's complete adjuvant of equivalent and carry out initial immunity after afterwards fully emulsified, and 2-4 days at interval, be used for the bird inlay of the no cause of disease of immunity.And in the booster immunization process, use incomplete Freund to mix, immunizing potency is better.
The present invention also provide a kind of from yolk the method for efficiently purifying immunoglobulin.Particularly, by yolk is placed/4 ℃, in the 20mM NaAc buffer under the pH=5.0, adopt then add 40% saturated ammonium sulphate after, adopt the Phenyl-Sepharose column chromatography purification, after ultrafiltration, can obtain specificity rotavirus resisting immune globulin active component.Purification scheme technology provided by the invention is simple and practical, and with active high, final purity can reach more than 95% in the immunoglobulin behind the purification.
The present invention also provide at last a kind of preparation contain height tire rotavirus resisting immune globulin, be not subjected to gastric acid and the destructive enteric coated preparation of pepsin.The height rotavirus resisting immune globulin preparation of tiring that contains provided by the invention comprises enteric coated granule, and enteric-coated microcapsule tablet.Preferred preparation is an enteric coated granule, and said preparation is easy to infant taking, can obviously improve the therapeutic effect of specificity rotavirus resisting immune globulin.
Description of drawings
Fig. 1 rolls behind the flask culture different time titre of rotavirus in the culture.
Fig. 2 is the content of specificity anti-rotavirus antibody in the different time yolk of immunity back.
Fig. 3 is the hydrophobic chromatography figure of specificity anti-rotavirus antibody.
Fig. 4 is that variable concentrations anti-rotavirus antibody is to the protective effect of the experimental rotavirus diarrheal of neonatal rat.
Specific embodiments
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
Embodiment 1: the separation of rotavirus
The separation of the pathogenic rotavirus of people
Get infant diarrhoea excrement juice 10ml, the PBS do dilution in 1: 5 with 0.15M pH=7.6 adds in the aseptic centrifuge tube, fully vibrates 30 minutes, and adding final concentration is the trypsin of 10 μ g/ml, and the solution of gained is hatched 20min at 37 ℃.Draw suspension, high speed centrifugation 10000rpm/min.After the filter of 0.22um filtered, cultivate and to annotate the MEM culture medium of 2 μ g/ml trypsin solutions (interpolation know clearly) with 10 times of filtrate serial dilutions, the solution of generation was used for next step amplification and evaluation with virus multiplication with supernatant.
The Vero cell is cultured to monolayer in 6 well culture plates after, discard the culture medium that is used to cultivate, every kind of viral parting material of above-mentioned 1ml is joined in each culture hole.Virus in the parting material is adsorbed on the cell, discards inoculation solution then.Every hole adds 5ml virus multiplication culture medium, when waiting cell to be generated to 80% pathological changes, collects supernatant.Behind high speed centrifugation, virus is carried out the serotype typing by ELISA or fluorescence immunoassay method.
After the ELISA detection kit detects, by above method, the present invention be separated to a strain rotavirus serotype be G2P1B.
Embodiment 2: the In vitro culture of rotavirus and amplification
With isolated rotavirus culture and rotavirus Strain is after the human-like Wa Strain of standard is mixed, and carries out external the cultivation altogether and amplification.
With the Vero cell (ATCC CCL81) of exponential phase in the DMEM-F12 that has added 10%FCS (Gibco company) cultivates to form cell monolayer, the culture area of culture bottle is 150cm2.Prepare single cell suspension after 0.25% trypsinization, be inoculated into and roll in the bottle, the cell initial concentration is 1 * 10
6/ ml treats cell culture when approaching monolayer (80%), and changing culture fluid is in the DMEM-F12 culture fluid of 2 μ g/ml for not containing 10%FCS, having added trypsin to final concentration.Behind hybrid virus strain prepared product usefulness trypsin 20 μ g/ml processing 20min, inoculation goes into to roll in the bottle, and the virus inoculation amount is 2 * 10
5The virus of TCID is adsorbed and is changed liquid once after 1 hour.Add the tryptic DMEM-F12 culture medium of 10%FCS and 2 μ g/ml and continue to cultivate, the virus titer in different time employing MA104 cytopathy political reform observation culture.Reach 80% o'clock results virus-culturing fluid in order to being further purified and being prepared into the wheel virus antigen prepared product that can be used for animal immune to cytopathy.See Fig. 1: roll behind the flask culture different time titre of rotavirus in the culture.
Embodiment 3: the preparation of wheel virus antigen
1. Bing Du purification
With the viral suspension 8L that collects behind the amplification in vitro.Adopt Millipore Pellicon ultrafiltration apparatus 10 times of 4 ℃ of following ultrafiltration and concentration, molecular cut off is 30KD.Adopt 50mMTris/HCl then, the conversion buffered liquid of pH=8.0.
To using 50mM Tris/HCl in advance, (2.6 * 10cm), fully eluting adopts 0-0.5M NaCl linear gradient elution not in conjunction with after the component to the DEAE-Sepharose FF post after the pH=8.0 balance with sample on the above-mentioned prepared product.It is standby to contain virus component merging back.
2. Bing Du deactivation
Through concentrating the rotavirus prepared product after reaching purification, use after can using inactivator deactivations such as joy Malin, phenol, glutaraldehyde, β-Bing Chunsuanneizhi, if use formalin, addition are between the 0.01-0.5%, the optimum concentration of using among the present invention is 0.1%.The temperature of deactivation can be at 2-38 ℃, and the optimum temperature that uses among the present invention is 25-28 ℃.Inactivation time is 5-10 days, and the suitableeest time is 7 days.
3. the selection of immunological adjuvant
Through the wheel virus antigen prepared product after the deactivation can with medically acceptable immunological adjuvant compatibility after act on, share such as incomplete Freund, Freund's complete adjuvant, both neutralizing antibodies in inducing hen serum produce and yolk in have different usefulness in the generation of neutralizing antibody amount.
Use 15ml PBS (pH=7.4) dilution rotavirus prepared product, making protein concentration is 50 μ g/ml.Add isopyknic Freund's complete adjuvant or Freund, be prepared into the prepared product that contains wheel virus antigen that can be used for immune hen.See Fig. 2: the content of specificity anti-rotavirus antibody in the different time yolk of immunity back.
Embodiment 4: animal immune
1. animal is selected
Select the purebred Lay aircraft carrier of 6 monthly age healthy high-yield eggs chicken, in standard I I level cleaning level Animal House, raise a week after.
2. immunization method
Every hen under chicken wing, breast, abdominal part multi-point injection implement the prepared products that contain wheel virus antigen of 2 preparations.At interval 2 all booster immunizations once regularly detect tiring of anti-rotavirus neutralizing antibody in serum and the yolk.The antigen preparation thing that adds Freund's complete adjuvant is adopted in preceding twice immunity, and booster immunization subsequently all adopts the antigen preparation thing that adds Freund.
Embodiment 5: the isolation and purification of immunoglobulin
Adopt acid solution extracting, hydrophobic chromatography and anion exchange methods, preparation purity is greater than the anti-rotavirus antibody that can supply human body to use more than 90%.
1. acid solution extracting
Contain water 48% in the Ovum Gallus domesticus Flavus, protein 17.8% and fat 30.5%, wherein nearly all fat all forms lipoprotein with protein bound.Immunoglobulin is a water-solubility protein in the yolk, and the pollution of removing lipoprotein during separation and purification is very necessary to follow-up purification.
Get 100 in the egg that hen produced after the hyperimmune.Yolk is separated with Ovum Gallus domesticus album, with refiner with its homogenize after, add the 20mM NaAc buffer of 8 times of volumes, 4 ℃ leave standstill 4 hours after, centrifugal 9000rpm/min abandons precipitation.
2. ammonium sulfate precipitation
Under 4 ℃, add solid ammonium sulfate at supernatant, every ml adds ammonium sulfate 19.4g, and the limit edged stirs and dissolves fully up to ammonium sulfate.4 ℃ leave standstill 4 hours after.Centrifugal 9000rpm/min 30min, collecting precipitation, the resolution of precipitate of every g weight in wet base are in 10ml, and 50mM PB is in the pH=7.0 buffer.
Add 1.5g by every ml in the supernatant, adding solid ammonium sulfate is about 1.5M to final concentration, and centrifugal 9000rpm/min * 30min removes precipitation, and it is standby to get supernatant.
3.Phenyl-Sepharose FF (Low sub) column chromatography purification
Chromatographic column adopts Phenyl-Sepharose FF (Low sub) 2.6 * 10cm post
After chromatographic column adopts the abundant balance of buffer of 50mM PB+1.5M ammonium sulfate of 10 column volumes, press on the 40mg/ml glue behind the sample, fully eluting makes UV280 to baseline values.Adopt 50mM PB gradient elution, collect required component, adopting the conversion buffered liquid of Millipore ultrafiltration apparatus is 50mM PBS, pH=7.4.See Fig. 3: the hydrophobic chromatography figure of specificity anti-rotavirus antibody.
4. the preparation of anti-rotavirus lyophilized powder
At the last anti-rotavirus antibody that goes on foot the purification of preparation gained, behind adding protective agent mannitol and the albumin, vacuum lyophilization is stand-by.
Embodiment 6: the preparation of rotavirus antibody enteric coated particles
1. fill a prescription and consumption
Rotavirus antibody lyophilized powder 300g
Lactose 3000g
Gelatin 250g
Arabic gum 250g
Hydroxypropyl emthylcellulose 500g
Phthalic acid diethyl ester 10g
Pulvis Talci 100g
5% polyacrylic resin II 800g
2. preparation process
Take by weighing the mixing of each adjuvant → mistake 120 mesh sieves → fully by prescription, be made into soft material with an amount of binder solution, → sieve → 42 ℃ of bakings 8 hours → sieve with 14 mesh sieves granulate → mistake 30 mesh sieves and remove fine powder.
The enteric-coating material polyacrylic resin is dissolved in the ethanol for II number, add phthalic acid diethyl ester and Pulvis Talci after, adopt the air suspension coating coating, get final product anti-rotavirus antibody enteric coated granule.
Embodiment 7: the preparation of rotavirus antibody enteric-coated microcapsule tablet
Become capsule formula: rotavirus antiviral antibody concentrated solution 200ml
10% gelatin solution 200ml
Stearic acid 15g
95% ethanol is an amount of
The microencapsule tablet prescription:
Antibody microcapsule 30g
Microcrystalline Cellulose 6g
10% ethyl cellulose alcohol liquid 12ml
95% ethanol is an amount of
Pulvis Talci is an amount of
2. preparation process
Description of the process:
This tablet name is called the enteric-coated microcapsule sheet.
Take pharmaceutical pack to go into microcapsule and tablet is carried out enteric coated its stability of (under the sour environment) under one's belt of protecting.One step of spray drying method can reach and make the principal agent drying and make two step of microcapsule purpose.
Gelatin also is a protein, and it is less to influence main active probability in theory, after principal cartridge is gone into microcapsule, can further avoid further effect taking place with other adjuvants, and also better mobile.
Embodiment 8: the safety testing of anti-rotavirus antibody preparation
Acute toxicity test in mice
1. experimental animal
Strain: Kunming mouse
Body weight: 20 ± 2g
Sex: male and female half and half
Number of animals: totally 40
2. test method
Grouping: each 20 of administration group negative control group, every group of male and female half and half
Dosage is provided with: 12mg/g (be equivalent to effective dose 80 times)
Every animals administer amount: 1ml
Route of administration: irritate stomach
Method: in normal saline, negative control group waits the capacity normal saline solution with the medicine dissolution of effective dose, observes 7 days continuously after administration, and all surviving animals finish the back with taking off the execution of cervical vertebra method in the observation period, dissect inspection.
3. observation index
Death condition
The ordinary circumstance of toxic reaction comprises that mice is by hair, general behavior, appetite, feces etc.
The pathological change of perusal main organs.
4. result of the test
The reaction of animals end is seen and is seen death at the viewing duration end unusually after the administration, and mice is dense and glossy by hair, and appetite and feces are all normal, and the animal mental status is good, activity freely, toxic reaction appears in the end, dissects main organs end, back and sees ANOMALOUS VARIATIONS.
Embodiment 9: anti-rotavirus antibody enteric coated particles is to the protective effect of neonatal rat infection model
1. experimental animal
Strain: Balb/C kind 5 age in days neonatal rats
Number of animals: totally 40
2. test method
Grouping: each 20 of administration group negative control group, every group of male and female half and half
Dosage is provided with: 0.05,0.1,0.2,0.5, and five various dose groups of 1mg/g
Route of administration: irritate stomach
Method:
With after virus to be infected is mixed, 37 ℃ of effects 1 hour are carried out the enterovirus attack through irritating stomach to neonatal rat then with the rotavirus resisting immune globulin preparation of various dose.Observe whether neonatal rat diarrhoea, diarrhoea persistent period, weight increase situation occur in 96 hours.
3. experimental result
Experimental result shows that newborn neonatal rat gives the rotavirus resisting immune globulin of 0.2mg/g and can protect infection neonatal rat 100% to be immune to diarrhoea, compares with matched group and differs remarkable.See Fig. 4: variable concentrations anti-rotavirus antibody is to the protective effect of the experimental rotavirus diarrheal of neonatal rat.
Accompanying drawing of the present invention is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (18)
1. a yolk anti-rotavirus antibody preparation is characterized in that containing anti-A group rotavirus Yolk immunoglobulin, gelatin and pharmaceutic adjuvant etc.
2. yolk anti-rotavirus antibody preparation according to claim 1 is characterized in that said preparation is the enteric dosage form.
3. enteric dosage form according to claim 2 is characterized in that this dosage form is enteric coated particles or enteric-coated microcapsule tablet.
4. yolk anti-rotavirus antibody preparation according to claim 1 is characterized in that the immunoglobulin content in the said preparation is 5-10%.
5. enteric-coated microcapsule tablet according to claim 3 is characterized in that adopting in its enteric microcapsule gelatin as the capsule material.
6. enteric coated particles according to claim 3 is characterized in that containing hydroxypropyl emthylcellulose in its enteric coated particles.
7. method for preparing the described yolk anti-rotavirus of claim 1 antibody preparation is characterized in that adopting the following step:
(1) from the diarrhoea infant, separate and at cultured and amplified in vitro rotavirus granule, concentrate and purification after be prepared into can be for the medical science and the multivalent immunogen prepared product of use biologically;
(2) with the multivalence rotavirus immunity prepared product that is prepared into, after the inactivator deactivation, add a certain amount of medically acceptable immunological adjuvant, the bird inlay of the no cause of disease of immunity;
(3) high efficiency separation and purification immunoglobulin from yolk, through the acidic buffer defat, saltout, the hydrophobic chromatography method purifies, and obtains specificity rotavirus resisting immune globulin active component;
(4) add pharmaceutic adjuvant and make preparation.
8. preparation method according to claim 7, wherein the cultured cell line that uses in the amplification in vitro process is the Vero cell.
9. preparation method according to claim 7 wherein can be used for containing the human-like rotavirus strain Wa of standard strain (serotype is G1P1A) in the immune prepared product that contains wheel virus antigen and serotype is the rotavirus strain of G2P1B.
10. preparation method according to claim 7, wherein the serotype of rotavirus strain is G2P1B.
11. preparation method according to claim 7 wherein can be used for containing Freund's complete adjuvant or Freund in the multivalence rotavirus deactivation prepared product of animal immune.
12. preparation method according to claim 7, the immunologic process that wherein adopts multivalence rotavirus deactivation prepared product immune hen and produce Yolk immunoglobulin adopts the virus antigen be contained in Freund's complete adjuvant to carry out twice initial immunity.
13. immunization method according to claim 12 wherein adopts the prepared product that contains incomplete Freund to carry out booster immunization.
14. preparation method according to claim 7, use therein acidic buffer pH=5.0 when being used for defat, the buffering liquid of use is the 20mM sodium acetate.
15. preparation method according to claim 7, wherein the dewatering filling of hydrophobic chromatography method employing is phenyl-Speharose FF (low sub).
16. preparation method according to claim 7, wherein the concentration of the ammonium sulfate of salt analysis method use is 40% saturation.
17. according to the purposes of the described yolk anti-rotavirus of claim 1-3 antibody preparation, it is characterized in that the main Causative virus type of infant rotavirus diarrheal is had neutralization, indication is an infant colyliform characteristic of disease diarrhoea.
18. want the purposes of 17 arts as right, it is characterized in that route of administration is oral.
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| CN 02110671 CN1435260A (en) | 2002-01-28 | 2002-01-28 | Yolk antirotavirus antibody preparation, method for preparing same and use thereof |
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| CN 02110671 CN1435260A (en) | 2002-01-28 | 2002-01-28 | Yolk antirotavirus antibody preparation, method for preparing same and use thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102018955A (en) * | 2010-12-27 | 2011-04-20 | 吉林亚泰生物药业股份有限公司 | Purification method for producing viral vaccine in large scale |
| CN104502608A (en) * | 2014-12-23 | 2015-04-08 | 北京出入境检验检疫局检验检疫技术中心 | Group A rotavirus chromatography test paper strip based on low-noise excitation fluorescent label |
| CN104497138A (en) * | 2014-12-08 | 2015-04-08 | 广东京豪医药科技开发有限公司 | Preparation method and application of hand-foot-and-mouth disease resistant chicken egg-yolk antibody |
| CN107789647A (en) * | 2016-09-05 | 2018-03-13 | 上海赛伦生物技术股份有限公司 | It is a kind of to inactivate method viral in animal blood serum or blood plasma |
-
2002
- 2002-01-28 CN CN 02110671 patent/CN1435260A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102018955A (en) * | 2010-12-27 | 2011-04-20 | 吉林亚泰生物药业股份有限公司 | Purification method for producing viral vaccine in large scale |
| CN104497138A (en) * | 2014-12-08 | 2015-04-08 | 广东京豪医药科技开发有限公司 | Preparation method and application of hand-foot-and-mouth disease resistant chicken egg-yolk antibody |
| CN104502608A (en) * | 2014-12-23 | 2015-04-08 | 北京出入境检验检疫局检验检疫技术中心 | Group A rotavirus chromatography test paper strip based on low-noise excitation fluorescent label |
| CN107789647A (en) * | 2016-09-05 | 2018-03-13 | 上海赛伦生物技术股份有限公司 | It is a kind of to inactivate method viral in animal blood serum or blood plasma |
| CN107789647B (en) * | 2016-09-05 | 2021-02-02 | 上海赛伦生物技术股份有限公司 | Method for inactivating virus in animal serum or plasma |
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