CN1557491A - Specific antibody preparation for inhibiting decayed tooth bacteria - Google Patents
Specific antibody preparation for inhibiting decayed tooth bacteria Download PDFInfo
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- CN1557491A CN1557491A CNA2004100158613A CN200410015861A CN1557491A CN 1557491 A CN1557491 A CN 1557491A CN A2004100158613 A CNA2004100158613 A CN A2004100158613A CN 200410015861 A CN200410015861 A CN 200410015861A CN 1557491 A CN1557491 A CN 1557491A
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- streptococcus mutans
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Abstract
The present invention is method of inoculating health pregnant cow with vaccine containing variant streptococcus antigen, preparing specific antibody resisting variant streptococcus with the cow colostrums or milk secreted by the inoculated cow and preparing preparations of various forms for preventing and treating dental caries. The present invention features that multivalent antigen vaccine in freeze dried deactivated variant streptococcus is prepared by means of available bacterial vaccine technology and inoculated to health cow before or after parturition through intramuscular injection, and the collected cow colostrums or milk is prepared into the specific immune globulin resisting variant streptococcus for preventing and treating dental caries through defatting, filtering out casein, ultrafiltering, concentration and other steps.
Description
Affiliated technical field
The present invention relates to a kind of effective inhibition or reduce the Antivariant streptococcus specific antibody preparation that dental caries takes place, more particularly, the present invention relates to a kind of by healthy cow in calf inoculation is contained the antigenic vaccine of Streptococcus mutans, and from excretory colostrum of immunity inoculation milk cattle and milk the specific antibody of preparation Antivariant streptococcus, preparation various ways preparation is applied to prevention and treatment dental caries.
The invention technical background
Modern study confirms, the existence of antibacterial is the essential condition that dental caries takes place, a large amount of bacteria live is arranged in the oral cavity, the some of them antibacterial is Streptococcus mutans especially, ability with the chemical substance that produces harmful tooth, dental caries depend on the aperture bottom on the gingival surface or the identical slit of the tooth that growing is covered by some flora, if in flora breeding generation Streptococcus mutans (Streptococcus mutans, MS), will form the dental caries dental plaque.Streptococcus mutans (MS) positive patient dental caries is the process of reason more than, generally believe it mainly is that the glucide that contains in the food is swallowed through chewing, a part residues in the oral cavity, utilized by cariogenic bacteria, generation has corrosive material to tooth, thereby destroy dental hard tissue, and then cause the generation of dental caries.Experimental results show that, using specific antibody resists such flora (MS), can obviously suppress the growth of bacteria flora,, exist certain opposing factor usually although Streptococcus mutans is present in most of people's the oral cavity, limit its growth, in case exist in the oral cavity and be fit to vegetative environment, this bacteria flora will be grown fast, but in a single day it is white to use some specific immunity ball dawn, no matter be oral form or injecting pathway, all can play the effect of obvious its growth of inhibition.
People such as Michalek are verified a kind of to be infected by Streptococcus mutans by the isolating Antivariant streptococcus antibody capable inhibition of immune colostrum dental surface; in zoopery, confirm; add above-mentioned Antivariant streptococcus antibody in the living dental caries diet of white mice, can take care of one's teeth and not give birth to dental caries.Clinical trial confirms, compares with matched group, gargles sooner or later one minute with above-mentioned specific antibody solution, use fortnight continuously, significantly reduced Streptococcus mutans ratio in the youthful dental plaque, but this part research report does not provide specific embodiment, preparation technology and application technology.
In the technology that Chinese invention patent (application number 96199507) is announced, mention the immunity inoculation cow and prepare anti-otitis media pathogen specific antibody, although wherein mention treatment to dental caries, do not comprise Streptococcus mutans antibody, do not mention concrete grammar to the prevention of dental caries and treatment yet.
Technical problem to be solved by this invention is, prepare a kind of with low cost, contain the preparation of specificity to Antivariant streptococcus antibody, be used for suppressing or reducing generation, the development of dental caries.
Summary of the invention
The technical solution adopted for the present invention to solve the technical problems is, by known bacterial vaccine technology, the polyvalent antigen vaccine of preparation Streptococcus mutans deactivation antibacterial lyophilized powder, in the forward and backward gradation intramuscular injection of giving a birth of healthy cow, collect excretory colostrum of immune milk cattle and milk, white by the specific immunity ball dawn that degrease, filtering cheese dawn are white, the ultrafiltration and concentration supervisor prepares Antivariant streptococcus, be applied to prevention and treatment dental caries.Technical characterictic of the present invention is, the present invention is selected, being used for preparing vaccinated bacterial species is Streptococcus mutans (Streptocaccus mutans, MS), its bacterial species includes but not limited to Streptococcus mutans AHT (S.mutans), Streptococcus mutans BHT (S.mutans), Streptococcus mutans 10449 (S.mutans 10449), Streptococcus mutans 6715 (S.mutans 6715), these Streptococcus mutans are the main bacteria that cause dental caries.
Above-mentioned bacteria culture can be collected center (American Culture Collection) from U.S.'s antibacterial culturing and obtain, also can obtain from relevant university, the institute of having set up strain, the bacteria culture that is obtained method is in accordance with regulations cultivated, the purity of control flora, after definite institute obtains strain purity, can inoculate single Pseudomonas in the standard bacteria culture media of 500ml, method is according to the rules cultivated.
Technical characterictic of the present invention is, all Streptococcus mutans adopt static cultural method to cultivate, each culture vessel was cultivated 48 hours at 37 ℃, be heated to 56 ℃ then and continue two hours, to kill the antibacterial of cultivation, bacteria sample with deactivation is seeded on the bacteria culture media, under 37 ℃ of temperature, hatched 24 hours, whether bacterial detection complete inactivation, have only antibacterial to be inactivated fully and bacterium culture test when negative, just can carry out next step, the disinfectant culture is washed five times with distillation, and high speed centrifugation is collected culture on centrifuge then, and centrifugal speed is 4000 rev/mins, clean, the bacterial cultures of high-temperature inactivation be immersed in-90 ℃ the liquid nitrogen freezing after, send into freeze drying plant and prepare dry powder.
Technical characterictic of the present invention is, adopt the antibacterial dry powder of variety classes Streptococcus mutans preparation, weighed by equivalent, these different types of antibacterial dry powder blend, with a certain amount of normal saline solution dissolving, stir, mixing, prepare spissated Streptococcus mutans antigenic solution, dilute repeatedly with normal saline, reach following standard until concentration, adopt spectrophotometer measurement, under the wavelength of 660 dusts, the optical density counting is at 0.5～0.7% light absorption reading, final diluted concentration is stored down at 0 ℃ at 2.times.10.sup.8cells/ml, and above-mentioned preparation procedure should be finished before immunity inoculation.For example, prepare Streptococcus mutans AHT respectively, BHT, the antibacterial dry powder of 10449 and 6715 strains, every kind of strain antibacterial dry powder 1 gram of weighing, prepare antibacterial dry powder 4 grams altogether, dry powder blend with different strain, and be mixed with suspension with the 500ml sterile saline, in the suspension of 500ml, contain 4 gram Streptococcus mutans dry powder, every ml soln contains 8 milligrams antigen, from above-mentioned high-concentration suspension liquid, take out the 1ml sample and dilute repeatedly, until being that ultimate density is greatly about 2.times.10.sup.8 cells/ml on 0.5～0.7% the light absorption reading in optical density readings on the wavelength of spectrophotometer 660 dusts, the antigen suspension of preparation is contained in a plurality of sterile vials, and stores under 0 ℃ of low temperature.
Technical characterictic of the present invention is, the diluent of preparation ultimate density prepares before closing on immunity inoculation from concentrated solution, conventional program is the antigen that dissolve sufficient amount, prepare a plurality of bottles, with inoculation bull cow, in order to confirm the aseptic of antigenic solution, take out from refrigerated storage preceding 24 hours of immunity inoculation, antigen concentration is adjusted to ultimate density (2.times.10.sup.8cells/ml) with physiological saline solution, and branch is filled in the different disinfecting containers, the sample of getting 1ml was hatched 24 hours under 37 ℃ of temperature, with its deactivation degree of final inspection, have only the antigenic solution of complete inactivation to use, after inoculation, take advantage of surplus vaccine to be dropped in the bottle.
Technical characterictic of the present invention is, by method for preparing Streptococcus mutans vaccine of the present invention, the dosage of every milk cattle of immunity inoculation is 5ml Streptococcus mutans antigenic solution (under 2.times.10.sup.8 cell/ml concentration), the injection site is at the abundantest place of buttocks muscles, be injected at for the first time date of expected delivery 3～4 weeks a few days ago, later on once, in the whole phase duplicate injection all the time of giving milk every the injection of 2 weeks.
Technical characterictic of the present invention is, select the milk cattle of Streptococcus mutans inoculation, must be healthy through the veterinary's inspection and the proof of various routines, the cow in calf of no disease, tuberculin test and other known infectious disease detect should be normal, any have colostrum and the milk of the milk cattle of disease probability should not be used to prepare preparation of the present invention, and, after cow in calf is by immunity inoculation, should regularly detect its serum sample, after in its serum, the Streptococcus mutans antibody of high titre occurring, could collect its colostrum and milk raw material as preparation preparation of the present invention, its Streptococcus mutans antibody titer assay method can adopt the method for any known mensuration pathogen antibody titer, ELISA method etc. for example, for its Streptococcus mutans antibody horizontal of more accurate mensuration, in case of necessity can be in contrast with the serum that does not carry out the immunity inoculation calver.
Technical characterictic of the present invention is, prepared white method of specific immunity ball dawn immunity inoculation milk cattle colostrum and the milk and be from above-mentioned, colostrum or milk are diluted in 1: 3 ratio with distilled water, from solution, remove fat with centrifugation, mode coagulation and precipitation cheese dawn by acidify, heating diluent are white, it is white to remove the bag agglutinative cheese dawn with centrifugal or filter type, concentrate with supermicro filtration membrane then, liquid preparation or powdered preparation are made in aseptic filtration, lyophilization or spray drying.Another kind of flexible method can prepare the specific antibody of similar purity too, promptly splash into sour agent and make its acidify post-heating and coarse filtration at skimmed colostrum or defat Niu Chuzhong, white to remove the cheese dawn, dilute with sodium chloride solution subsequently, collect supernatant, the preliminary filtration concentrates then, neutralizes, and carries out aseptic filtration or lyophilization or spray drying at last and obtains similar purity product.
Technical characterictic of the present invention is, adopt the Antivariant streptococcus specific antibody of the inventive method preparation, can be prepared into the pharmaceutical preparation of various ways, nutritional preparation or condensed food form, be applied to prevention and treatment dental caries, these preparations or food include but not limited to: be mixed with collutory with suitable solution, adjuvant and adjuvant with sugar-free are modulated into chewing gum, confection, ice fine jade pouring etc. comprises the food of above-mentioned specific antibody, be prepared into the oral cavity cleaning powder or the tooth film of canonical form, or be prepared into milk powder, tea, coffee or other form beverages, and other any type of preparation that above-mentioned specific antibody can be applied to dental surface.
Technical characterictic of the present invention is, the Antivariant streptococcus specific antibody preparation of the inventive method preparation, both can use separately, also can with other drug composition, nutrition use in conjunction, to reach the purpose of prevention and treatment dental caries, these compositions include but not limited to the composition of xylitol, fluoride and other dental caries, when use in conjunction, can be prepared into mix preparation with the composition of uniting use.
The invention has the beneficial effects as follows, compared with prior art, the Antivariant streptococcus immunoglobulin of applied immunology of the present invention and the preparation of biotechnology method, can effectively resist Streptococcus mutans in the oral cavity, suppress or reduce the generation and the development of dental caries, method is simple, effect is remarkable, with low cost, promotional value is arranged.
Specific embodiments
The invention will be further described below in conjunction with embodiment:
Embodiment 1
Preparation Streptococcus mutans vaccine
Collect Streptococcus mutans AHT, BHT, 10449 and 6715 strains, be inoculated in respectively in the standard bacteria culture media, under 37 ℃ of temperature, cultivated 38 hours, use centrifuge with the 4000 rev/mins of centrifugal collection of speed antibacterials, clean five times with DDW, being diluted to ultimate density with the phosphate buffer of 0.1m is 2.times.10.sup.8 cells/ml, continues 30 minutes deactivation antibacterials with 60 ℃, is placed on 4 ℃ of refrigerator and cooled and hides.Measure different strain (Streptococcus mutans AHT, BHT, 10449 and 6715) the deactivation bacterial solution of 4 parts of equivalent, be mixed with complex antigen solution.
Embodiment 2
Immunity inoculation Streptococcus mutans vaccine
Select healthy cow in calf, carry out the total health inspection by known veterinary method, adopt the vaccine of embodiment 1 preparation, in predetermined childbirth day first three week, in cow in calf intragluteal injection 5ml vaccine, duplicate injection is once weekly later on, per injection position, injected dose are identical, begin to detect the Antivariant streptococcus antibody titer next day from inoculating cows serum after the injection, when antibody titer obviously raises than matched group antibody titer, just determines to use its colostrum and milk as raw material.
Embodiment 3
Preparation Antivariant streptococcus antibody
Get give a birth 13 liters of freezing cattle colostrumss in back 10 hours of immune milk cattle, under 5 ℃～10 ℃, thaw, with centrifuge with the defat in centrifugal 20 minutes of 3000 rev/mins of speed, with 700ml 1N HCL acidify colostrum, regulate PH to 4.8, in 40 ℃ of temperature heating 30 minutes, leaving standstill storage in 4 ℃ spends the night, draw up clear liquid with pump and enter hold-up vessel by the nylon cloth filter, the filtering cheese dawn is white, is diluted to 26 liters with 0.9% sodium chloride solution.Filter by hollow fiber column with the dilution supernatant, the hollow fiber column aperture is 0.4 μ m, and surface area is 3m
2Fibre diameter 1.2mm, sodium chloride solution with 100 liter of 0.9% concentration filters, working pressure surpasses atmospheric pressure 0.2～0.6 crust, carrying out the coarse filtration while first time, the filtrate of generation is carried out the coarse filtration second time by the system that is made up of three hollow fiber columns again, and the separation extreme of each post is 10,000D, surface area are 1.4M
2, in this system, for the first time the filtrate of coarse filtration obtains tentatively concentratedly, uses the sodium chloride solution diafiltration of 100 liter of 0.9% concentration then, last reconcentration to 2.3 liter.Resultant white content of specific immunity ball dawn is 6%, uses the known method filter-sterilized, prepares dry powder with freeze-drying method.
Embodiment 4
Measure the Antivariant streptococcus antibody titer
Get the Antivariant streptococcus antibody dry powder of embodiment 3 preparations, measure the Streptococcus mutans antibody titer with the ELISA method, with the biestings of immunity inoculation not in contrast, the result shows that the titre of Antivariant streptococcus antibody is higher than nearly 100 times of matched group in the Antivariant streptococcus preparation of the inventive method preparation.
Claims (10)
1. one kind by containing the antigenic vaccine of Streptococcus mutans to the inoculation of healthy cow in calf, and from excretory colostrum of immunity inoculation milk cattle and milk preparation Antivariant streptococcus specific antibody, preparation various ways preparation, be applied to prevent and treat the method for dental caries, it is characterized in that, by known bacterial vaccine technology, the polyvalent antigen vaccine of preparation Streptococcus mutans deactivation antibacterial lyophilized powder, before healthy cow childbirth, back gradation intramuscular injection, collect excretory colostrum of immune milk cattle and milk, pass through degrease, the filtering cheese dawn is white, it is white that the ultrafiltration and concentration supervisor prepares specific immunity ball dawn of Antivariant streptococcus, is applied to prevention and treatment dental caries.
2. in accordance with the method for claim 1, technical characterictic of the present invention is, the present invention is selected, be used for preparing vaccinated bacterial species and be Streptococcus mutans (Streptocaccus mutans, MS), its bacterial species includes but not limited to Streptococcus mutans AHT (S.mutans), Streptococcus mutans BHT (S.mutans), Streptococcus mutans 10449 (S.mutans10449), Streptococcus mutans 6715 (S.mutans 6715), these Streptococcus mutans are the main bacteria that cause dental caries.
3. in accordance with the method for claim 1, technical characterictic of the present invention is, all Streptococcus mutans adopt static cultural method to cultivate, each culture vessel was cultivated 48 hours at 37 ℃, be heated to 56 ℃ then and continue two hours, to kill the antibacterial of cultivation, bacteria sample with deactivation is seeded on the bacteria culture media, under 37 ℃ of temperature, hatched 24 hours, whether bacterial detection complete inactivation, have only antibacterial to be inactivated fully and bacterium culture test when negative, just can carry out next step, the disinfectant culture is with distillation washing five times, high speed centrifugation is collected culture on centrifuge then, centrifugal speed is 4000 rev/mins, cleans, the bacterial cultures of high-temperature inactivation be immersed in-90 ℃ the liquid nitrogen freezing after, send into freeze drying plant and prepare dry powder.
4. in accordance with the method for claim 1, technical characterictic of the present invention is, adopt the antibacterial dry powder of variety classes Streptococcus mutans preparation, weighed by equivalent, these different types of antibacterial dry powder blend, with a certain amount of normal saline solution dissolving, stir, mixing, prepare spissated Streptococcus mutans antigenic solution, dilute repeatedly, reach following standard until concentration with normal saline, adopt spectrophotometer measurement, under the wavelength of 660 dusts, optical density is counted at 0.5～0.7% light absorption reading, and final diluted concentration is at 2.times.10.sup.8 cells/ml, store down at 0 ℃, above-mentioned preparation procedure should be finished before immunity inoculation.
5. in accordance with the method for claim 1, technical characterictic of the present invention is, the diluent of preparation ultimate density prepares before closing on immunity inoculation from concentrated solution, conventional program is the antigen that dissolve sufficient amount, prepare a plurality of bottles, with inoculation bull cow, in order to confirm the aseptic of antigenic solution, take out from refrigerated storage preceding 24 hours of immunity inoculation, antigen concentration is adjusted to ultimate density (2.times.10.sup.8 cells/ml) with physiological saline solution, and branch is filled in the different disinfecting containers, the sample of getting 1ml was hatched 24 hours under 37 ℃ of temperature, with its deactivation degree of final inspection, have only the antigenic solution of complete inactivation to use, remaining vaccine should be dropped in the bottle after inoculation.
6. in accordance with the method for claim 1, technical characterictic of the present invention is, by method for preparing Streptococcus mutans vaccine of the present invention, the dosage of every milk cattle of immunity inoculation is 5ml Streptococcus mutans antigenic solution (under 2.times.10.sup.8cell/ml concentration), the injection site is at the abundantest place of buttocks muscles, be injected at for the first time date of expected delivery 3～4 weeks a few days ago, later on once every the injection of 2 weeks, in the whole phase duplicate injection all the time of giving milk.
7. in accordance with the method for claim 1, technical characterictic of the present invention is, select the milk cattle of Streptococcus mutans inoculation, must be healthy through the veterinary's inspection and the proof of various routines, the cow in calf of no disease, tuberculin test and other known infectious disease detect should be normal, any have colostrum and the milk of the milk cattle of disease probability should not be used to prepare preparation of the present invention, and, after cow in calf is by immunity inoculation, should regularly detect its serum sample, after in its serum, the Streptococcus mutans antibody of high titre occurring, could collect its colostrum and milk raw material as preparation preparation of the present invention, its Streptococcus mutans antibody titer assay method can adopt the method for any known mensuration pathogen antibody titer, ELISA method etc. for example, for its Streptococcus mutans antibody horizontal of more accurate mensuration, in case of necessity can be in contrast with the serum that does not carry out the immunity inoculation calver.
8. in accordance with the method for claim 1, technical characterictic of the present invention is, prepared white method of specific immunity ball dawn immunity inoculation milk cattle colostrum and the milk and be from above-mentioned, colostrum or milk are diluted in 1: 3 ratio with distilled water, from solution, remove fat with centrifugation, by acidify, the mode coagulation and the precipitation cheese dawn of heating diluent are white, it is white to remove the agglutinative cheese dawn with centrifugal or filter type, concentrate with supermicro filtration membrane then, aseptic filtration, lyophilization or spray drying are made liquid preparation or powdered preparation, another kind of flexible method can prepare the specific antibody of similar purity too, promptly splash into sour agent and make its acidify post-heating and coarse filtration at skimmed colostrum or defat Niu Chuzhong, white to remove the cheese dawn, dilute with sodium chloride solution subsequently, collect supernatant, the preliminary filtration concentrates then, neutralization is carried out aseptic filtration or lyophilization or spray drying at last and is obtained similar purity product.
9. in accordance with the method for claim 1, technical characterictic of the present invention is, adopt the Antivariant streptococcus specific antibody of the inventive method preparation, can be prepared into the pharmaceutical preparation of various ways, nutritional preparation or condensed food form, be applied to prevention and treatment dental caries, these preparations or food include but not limited to: be mixed with collutory with suitable solution, adjuvant and adjuvant with sugar-free are modulated into chewing gum, confection, ice fine jade pouring etc. comprises the food of above-mentioned specific antibody, be prepared into the oral cavity cleaning powder or the tooth film of canonical form, or be prepared into milk powder, tea, coffee or other form beverages, and other any type of preparation that above-mentioned specific antibody can be applied to dental surface.
10. in accordance with the method for claim 1, the Antivariant streptococcus specific antibody preparation of the inventive method preparation, both can use separately, also can with other drug composition, nutrition use in conjunction, to reach the purpose of prevention and treatment dental caries, these compositions include but not limited to the composition of xylitol, fluoride and other dental caries, when use in conjunction, can be prepared into mix preparation with the composition of uniting use.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2004100158613A CN1557491A (en) | 2004-01-15 | 2004-01-15 | Specific antibody preparation for inhibiting decayed tooth bacteria |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2004100158613A CN1557491A (en) | 2004-01-15 | 2004-01-15 | Specific antibody preparation for inhibiting decayed tooth bacteria |
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| CN1557491A true CN1557491A (en) | 2004-12-29 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1309420C (en) * | 2004-12-31 | 2007-04-11 | 长沙德文生物科技开发有限公司 | Anticarious preparation of antibody |
-
2004
- 2004-01-15 CN CNA2004100158613A patent/CN1557491A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1309420C (en) * | 2004-12-31 | 2007-04-11 | 长沙德文生物科技开发有限公司 | Anticarious preparation of antibody |
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