CN105646726A - Preparation method of type B haemophilus influenzae capsular polysaccharide and combined vaccine - Google Patents
Preparation method of type B haemophilus influenzae capsular polysaccharide and combined vaccine Download PDFInfo
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Abstract
The invention discloses a preparation method of a type B haemophilus influenzae capsular polysaccharide. The preparation method comprises the steps of fermentation culture, sterilization and precipitation, polysaccharide extraction and purification, polysaccharide derivatization and polysaccharide protein conjugate preparation. The invention further discloses a multivalent combined vaccine which is prepared through the steps that an acellular pertussis stock solution, refined diphtheria toxoid and refined tetanus toxoid are added into aluminum hydroxide to be adsorbed and then added into a type B haemophilus influenzae capsular polysaccharide stock solution, and the mixed solution is diluted with normal saline. According to the preparation method, the prior art is optimized, and the high-purity low-impurity-content type B haemophilus influenzae capsular polysaccharide can be obtained; meanwhile, the type B haemophilus influenzae capsular polysaccharide is combined with multiple component vaccines to form the multivalent vaccine, the multivalent vaccine can be immune to multiple diseases simultaneously, the inoculating times are decreased, the medical risk is reduced, and the better immune effect can be obtained through one-time inoculating.
Description
Technical field
The present invention relates to, be specifically related to preparation method and the combined vaccine of a kind of Type B hemophilus influenza capsular polysaccharide.
Background technology
Hemophilus influenza is common pathogenic bacterium, only to human disease, divides band Capsular strains and without Capsular strains. Capsular strains divides 6 serotypes (a-f) according to the specific antigen difference of capsular polysaccharide, and wherein b type antigen virulence is maximum, is the Main Pathogenic Bacteria of sepsis in children, meningitis and pneumonia. Hib pod membrane is made up of lipopolysaccharide and capsular polysaccharide, and main component is ribosyl alcohol acid phosphate phosphate (polyriboylribitolphosphate, PRP), and its structure includes a ribose, a ribitol and a phosphoric acid alkali.
Hib passes through droplet transmission, less than 5 years old child of main infection. Neonate transmits serum bactericidal antibody by Placenta Hominis, obtains from parent. The combined vaccine inoculation child being carrier with protein, the antibody produced is more much higher than independent capsular polysaccharide vaccine and can maintain the long period, the basis of these vaccines is to be combined with covalent bond with there being immunogenic protein carrier by PRP polysaccharide, hapten is made to become complete antigen, so that non-thymus-dependent antigen (Ti-Ag) is transformed into thymus-dependent antigen (Td-Ag), promote the T cell effect to B cell. Ti-Ag is only capable of exciting humoral immunization; and Td-Ag can not only excite humoral immunization; and cellular immunization can also be caused; produce immunological memory simultaneously; by the transformation of antigen; not only make little age child produce booster immunization reaction, and also can produce booster immunization reaction to again inoculating, which thereby enhance the protective value of vaccine.
Being incorporated into by PRP or derivatives thereof on a kind of protein carrier (such as diphtheria toxoid albumen, with the diphtheria toxoid PROTEIN C PM197 of attenuation, tetanus toxoid, meningitis how Salmonella outer membrane protein composite etc.) for these people makes it be transformed into T cell dependency polysaccharide antigen to strengthen response, and combined vaccine has good immunogenicity.
In recent years, abuse due to antibiotics, Type B hemophilus influenza (Hoemophilusinfluenzatypeb, Hib) infection has become the main cause causing child's morbidity all over the world and death, there are about 30-50 ten thousand child every year and dies from the diseases such as the Hib meningitis caused and pneumonia.
The health of the world mankind in infectious disease still serious threat, especially causes infant morbidity, is in hospital and the major reason of death. Along with the development of science and technology, novel vaccine is constantly studied and exploitation, and it is also increasing that current every country receives the Vaccines classes of people's planned immunization, and China started from 2008, by 5 original Seedlings anti-7 diseases, the Immune Programming was expanded as the anti-l5 of l4 Seedling sick. Vaccines classes increases the childhood vaccination number of times brought and increases and vaccine administrative loads increase etc. so that develop combined vaccine imperative. A lot of vaccine suppliers external at present and some domestic vaccine enterprises are all being devoted to exploitation combined vaccine, and wherein the combined vaccine based on DTaP is again the emphasis of current exploitation.
Using Hib vaccine is the effective measures controlling Hib affecting conditions. Hib vaccine has good immunogenicity, can produce good immunne response after inoculation, can bring out body and produce being effectively protected property bactericidin.
The DTaP vaccine of prevention diphtheria, tetanus and three kinds of diseases of pertussis is widely used in Chinese Infant and Children immunity inoculation, the Hib combined vaccine of the prevention disease such as infant meningitis and pneumonia has been succeeded in developing and has been included in local expansion the Immune Programming, both vaccine immunity programs are identical, at present external existing DTaP-Hib combined vaccine commodity come out, but domestic not yet succeed in developing DTaP-Hib combined vaccine.
The Hib combined vaccine developed at present is safely and effectively, and after inoculation 3 doses, the child of 90%-99% can produce antibody. Therefore, Hib vaccine is included in the planning of infant routine immunization by WHO suggestion. It addition, this vaccine and DTaP vaccine (DTaP) have identical immune programme for children, therefore, develop acellular whooping cough Type B hemophilus influenza combined vaccine (DTaP/Hib) and be significant. The purpose of combined vaccine exploitation is to prevent a greater variety of disease reducing vaccine injection number of times while. Its meaning is possible not only to improve vaccine coverage and rate of vaccination, minimizing multiple injection to the misery of the brought health of baby and father and mother and psychology, the minimizing managerial difficulty of vaccine, reduction inoculation and administration fee use; Also can reduce in production of vaccine must containing preservative and the dosage such as adjuvant, lower the untoward reaction etc. of vaccine.
Summary of the invention
It is an object of the invention to provide the preparation method of a kind of Type B hemophilus influenza capsular polysaccharide and combined vaccine.
For reaching above-mentioned purpose, the preparation method providing a kind of Type B hemophilus influenza capsular polysaccharide in one embodiment of the present of invention, comprise the following steps:
(1) fermentation culture: Type B hemophilus influenza is inoculated into fermentation cylinder for fermentation, and terminates cultivating in the later stage of logarithmic growth or the early stage of resting stage, it is thus achieved that culture fluid;
(2) sterilizing precipitation: by high speed centrifugation after culture fluid sterilizing, collects supernatant, and addition cetyl trimethylammonium bromide fully mixes in supernatant, then stands and is centrifuged, collects precipitate;
(3) Polyose extraction and purification
The precipitate of Type B hemophilus influenza adds in sodium chloride solution and dissociates, centrifugal collection supernatant after dissociating; Supernatant is first concentrated by ultrafiltration, adding ethanol to ethanol mass fraction after concentration is 20%, centrifugal segregation precipitation after stand at low temperature, be then centrifuged in liquid adding dehydrated alcohol to ethanol content is 85%, it is centrifuged after stand at low temperature, collects precipitation and also use dehydrated alcohol and washing with acetone, in precipitation, then add water dissolution, lysate recentrifuge, and centrifugal liquid is carried out dialysis treatment; Concentration obtains rough polysaccharide;
Rough polysaccharide is dissolved in saturated acetic acid sodium solution, adds cold phenol solution extracted several times;Then the centrifugal supernatant of collecting of stirring, and by ultrafiltration or dialysis dephenolize, add ethanol to final concentration of 60%��80%, collected after centrifugation precipitate, precipitate uses dehydrated alcohol and washing with acetone, and then vacuum drying obtains refined polysaccharide;
(4) polysaccharide derives
Refined polysaccharide is dissolved in distilled water and regulates pH value to 10.5��11, activating at temperature 20 DEG C��26 DEG C, being subsequently adding acid solution to pH is 9, is subsequently adding adipic dihydrazide ADH reaction, regulate pH value after completion of the reaction and stir more than 12h to alkalescence, then dialyse, filter, dry; Obtain polysaccharide spin-off;
(5) preparation of GL-PP conjugate
PH value is regulated with carrier protein to 5.5��5.9 after being mixed by polysaccharide spin-off, add 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide EDC in 2 DEG C��8 DEG C reactions, regulate pH value after completion of the reaction to dialyse to 6.7��7.1, dialysis solution uses ultrafilter membrane degerming after column chromatography purification, concentration, obtains Type B hemophilus influenza capsular polysaccharide.
Preferably, using fermentor cultivation bacterium solution in step (1), inoculate initial concentration A600=0.18, cultivating pH is 7.5, and cultivation temperature is 36 DEG C, and incubation time is 10h��15h; Results bacterial concentration A600 >=4.0.
Preferably, in step (2), the final concentration volume fraction of cetyl trimethylammonium bromide is 0.1%.
Preferably, in step (3), sodium chloride adds the final concentration of 10mg/ml of postprecipitation thing polysaccharide.
Preferably, adding ethanol to ethanol mass fraction in step (3) is 20%, and after standing more than 12h at 4 DEG C, centrifugal segregation precipitates.
Preferably, in step (4) addition is refined polysaccharide 3 times��4 times of adipic dihydrazide ADH.
Preferably, the content of step (4) refined polysaccharide is 10mg/10ml.
Preferably, in step (5), carrier protein is tetanus toxoid protein.
Another object of the present invention is open a kind of combined vaccine containing Type B hemophilus influenza capsular polysaccharide, and combined vaccine includes Type B hemophilus influenza capsular polysaccharide, acellular pertussis vaccine, diphtheria toxoid vaccine and tetanus toxoid vaccine.
The preparation method that it is a further object to provide this vaccine, Type B hemophilus influenza capsular polysaccharide stock solution is added after adding aluminium hydroxide absorption including by acellular pertussis stock solution, Toxoidum Diphthericum Purificatum, tetanus toxoid purified, then normal saline dilution is used, add thimerosal and regulate pH value to 5.8, add 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide EDC and react 1 hour; Regulate pH to 7.0 again, be then cooled to 4 DEG C, degerming after finally dialysis, ultrafiltration concentration, in 4 DEG C of preservations.
In sum, the invention have the advantages that
Preparation method disclosed by the invention, prior art is optimized, the Type B hemophilus influenza capsular polysaccharide of high-purity, low impurity can be obtained, the present invention is by forming polyvalent vaccine with multicomponent vaccine combination simultaneously, multiple disease can be carried out immunity simultaneously, reduce inoculation times, reduce medical-risk, it is possible to once inoculation obtains better immune effect.
Detailed description of the invention
Embodiment 1
The preparation method of a kind of Type B hemophilus influenza capsular polysaccharide, comprises the following steps:
(1) Type B hemophilus influenza seed lot strain is set up
Adopt Type B hemophilus influenza CMCC58547 or CMCC58534 bacterial strain, derive from Nat'l Pharmaceutical & Biological Products Control Institute. After main seed lot breakdown, passage number is less than 5 generations, must not exceed for 5 generations to inoculation fermentation tank subculture number of times after working seed lots breakdown.
(2) production seed is prepared
Breakdown working seed lots strain, is seeded on liquid or solid Hib synthetic medium, the production seed that preparation quantity is suitable.
(3) production culture medium is prepared
Adopt Hib synthetic medium, harmful or other allergenic agent must not be contained.
(3) fermentation culture: by 300 liters of fermentor cultivation 100L bacterium solution, inoculates initial concentration A600=0.18, cultivate pH be 7.5, cultivation temperature be 36 DEG C, cultivate 10��15 hours, results bacterial concentration A600 >=4.0. In incubation, sampling carries out pure bacterium test, gram stain smear microscopy, as found pollution microbes, should discard.
(4) sterilizing precipitation: when the early stage in later stage or resting stage that antibacterial is in logarithmic growth terminates cultivating, sampling carries out bacterial concentration mensuration and pure bacterium checks, then in culture, formalin or the sodium deoxycholate solution sterilization of suitable concentration are added, to guarantee that sterilization damages Hib capsular polysaccharide completely and not and is advisable. High speed centrifugation after culture fluid sterilizing, collects supernatant, and to add cetyl trimethylammonium bromide CTAB in supernatant to whole content be 0.1% fully mix, and then stands and is centrifuged, collects precipitate.
(5) Polyose extraction and purification
Adding in the precipitate of Type B hemophilus influenza in the sodium chloride solution of 0.6mol/L makes sludge concentration dissociate after reaching 10mg/ml, and after dissociating, under rotating speed 6500r/min, centrifugal 30min collects supernatant; Supernatant is first concentrated by ultrafiltration, adding ethanol to ethanol mass fraction after concentration is 20% precipitation, after standing 14h at 4 DEG C, centrifugal 15min removes precipitation, be then centrifuged in liquid adding dehydrated alcohol to ethanol content is 85%, it is centrifuged after 4 DEG C of standing 3h, collects precipitation and also use dehydrated alcohol and washing with acetone, in precipitation, then add water dissolution, lysate recentrifuge, and centrifugal liquid is carried out dialysis treatment; Concentration obtains rough polysaccharide.
Rough polysaccharide is dissolved in saturated acetic acid sodium solution, adopts phenol lifting manipulation, add cold phenol solution extracted several times. Then the centrifugal supernatant of collecting of stirring, and by ultrafiltration or dialysis dephenolize, add ethanol to final concentration of 80%, collected after centrifugation precipitate, precipitate uses dehydrated alcohol and washing with acetone, and then vacuum drying obtains refined polysaccharide;
(6) polysaccharide derives
Refined polysaccharide is dissolved in distilled water to content be 10mg/ml, regulate pH value to 10.8, priming reaction 30min at temperature 23 DEG C, being subsequently adding acid solution to pH is 9, being subsequently adding adipic dihydrazide ADH and react 6min, the addition of adipic dihydrazide ADH is 3.4 times of refined polysaccharide. Adjustment pH value is to 7.5 after completion of the reaction, and stirs more than 12h after reaction 15min at 23 DEG C, then dialyses, filters, dries; Obtain polysaccharide spin-off.
(7) preparation of GL-PP conjugate
PH value will be regulated to 5.9 after polysaccharide spin-off and carrier protein tetanus toxoid protein mixed in equal amounts, add equivalent 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide EDC and react 1h in 2 DEG C��8 DEG C, regulate pH value after completion of the reaction to 7, and at 4 DEG C, dialysis more than 12h in 0.2mol/LNaCl solution, dialysis solution uses ultrafilter membrane degerming after column chromatography purification, concentration, obtains Type B hemophilus influenza capsular polysaccharide.
Embodiment 2
The preparation method of a kind of Type B hemophilus influenza capsular polysaccharide, comprises the following steps:
(1) fermentation culture: by 300 liters of fermentor cultivation 100L bacterium solution, inoculates initial concentration A600=0.18, cultivate pH be 7.5, cultivation temperature be 36 DEG C, cultivate 10��15 hours, results bacterial concentration A600 >=4.0.In incubation, sampling carries out pure bacterium test, gram stain smear microscopy, as found pollution microbes, should discard.
(2) sterilizing precipitation: when the early stage in later stage or resting stage that antibacterial is in logarithmic growth terminates cultivating, sampling carries out bacterial concentration mensuration and pure bacterium checks, then in culture, formalin or the sodium deoxycholate solution sterilization of suitable concentration are added, to guarantee that sterilization damages Hib capsular polysaccharide completely and not and is advisable. High speed centrifugation after culture fluid sterilizing, collects supernatant, and to add cetyl trimethylammonium bromide CTAB in supernatant to whole content be 0.1% fully mix, and then stands and is centrifuged, collects precipitate. Cetyl trimethylammonium bromide can precipitate accounting and acidic polysaccharide, the i.e. capsular polysaccharide of the present invention.
(3) Polyose extraction and purification
Adding in the precipitate of Type B hemophilus influenza in the mixed solution of the sodium chloride of 0.6mol/L and the manganese chloride solution of equivalent makes sludge concentration dissociate after reaching 10mg/ml, and after dissociating, under rotating speed 6500r/min, centrifugal 30min collects supernatant; Supernatant is first concentrated by ultrafiltration, adding ethanol to ethanol mass fraction after concentration is 20% precipitation, after standing 14h at 4 DEG C, centrifugal 15min removes precipitation, be then centrifuged in liquid adding dehydrated alcohol to ethanol content is 85%, it is centrifuged after 4 DEG C of standing 3h, collects precipitation and also use dehydrated alcohol and washing with acetone, in precipitation, then add water dissolution, lysate recentrifuge, and centrifugal liquid is carried out dialysis treatment; Concentration obtains rough polysaccharide. By using calcium chloride and manganese chloride that precipitate is dissociated in the present embodiment, dissociation solution Nucleic Acid after detection embodiment 1 under the same terms and technique is dissociated is apparently higher than the present embodiment, compare embodiment 1 reduce by 12%��14% adding dissociation solution Nucleic Acid after manganese chloride, the accounting do not dissociated is retained in precipitation, the purity making follow-up enucleation acid technique is higher, lower containing nucleic acid impurities.
Rough polysaccharide is dissolved in saturated acetic acid sodium solution, adopts phenol lifting manipulation, add cold phenol solution extracted several times. Then the centrifugal supernatant of collecting of stirring, and by ultrafiltration or dialysis dephenolize, add ethanol to final concentration of 80%, collected after centrifugation precipitate, precipitate uses dehydrated alcohol and washing with acetone, and then vacuum drying obtains refined polysaccharide;
(4) polysaccharide derives
Refined polysaccharide is dissolved in distilled water to content be 10mg/ml, regulate pH value to 10.8, priming reaction 30min at temperature 23 DEG C, being subsequently adding acid solution to pH is 9, being subsequently adding adipic dihydrazide ADH and react 6min, the addition of adipic dihydrazide ADH is 3.4 times of refined polysaccharide. Adjustment pH value is to 7.5 after completion of the reaction, and stirs more than 12h after reaction 15min at 23 DEG C, then dialyses, filters, dries; Obtain polysaccharide spin-off.
(5) preparation of GL-PP conjugate
PH value will be regulated to 5.9 after polysaccharide spin-off and carrier protein tetanus toxoid protein mixed in equal amounts, add equivalent 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide EDC and react 1h in 2 DEG C��8 DEG C, regulate pH value after completion of the reaction to 7, and at 4 DEG C, dialysis more than 12h in 0.2mol/LNaCl solution, dialysis solution uses ultrafilter membrane degerming after column chromatography purification, concentration, obtains Type B hemophilus influenza capsular polysaccharide.
Embodiment 3
A kind of combined vaccine containing Type B hemophilus influenza capsular polysaccharide includes Type B hemophilus influenza capsular polysaccharide, acellular pertussis vaccine, diphtheria toxoid vaccine and tetanus toxoid vaccine; Its preparation method is:
Type B hemophilus influenza capsular polysaccharide stock solution is added after adding aluminium hydroxide absorption including by acellular pertussis stock solution, Toxoidum Diphthericum Purificatum, tetanus toxoid purified, then normal saline dilution is used, add thimerosal to every milliliter containing thimerosal ten thousand/, regulate pH value to 5.8, add 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide EDC and react 1 hour;Regulate pH to 7.0 again, be then cooled to 4 DEG C, degerming after finally dialysis in 0.2mol/LNaCl solution, ultrafiltration concentration, in 4 DEG C of preservations.
Embodiment 4: potency test
Choosing body weight is 12��14 grams of NIH (or BALb/c) mices 10, separately takes and is only used as comparison with batch mice 10. One group carries out subcutaneous injection with the vaccine on mouse of embodiment 3 preparation, and matched group injects 0.85% sodium chloride solution; And in subcutaneous injection on the 1st, 14th twice, per injection dosage is the Hib combined vaccine containing 2.5 �� g polysaccharide, take a blood sample in the 21st��28 Nikkei retroorbital venous, measure anti-HibIgG antibody with ELISA method, obtain Cutoff value with the A value of 0.85% sodium chloride solution control group mice serum. Detection learns that vaccine group has the anti-HibIgG antibody horizontal of serum of more than 80% mice higher than Cutoff value, illustrates that the vaccine of the present invention is that tool is virtuous.
Claims (10)
1. a preparation method for Type B hemophilus influenza capsular polysaccharide, comprises the following steps:
(1) fermentation culture: Type B hemophilus influenza is inoculated into fermentation cylinder for fermentation, and terminates cultivating in the later stage of logarithmic growth or the early stage of resting stage, it is thus achieved that culture fluid;
(2) sterilizing precipitation: by high speed centrifugation after culture fluid sterilizing, collects supernatant, and addition cetyl trimethylammonium bromide fully mixes in supernatant, then stands and is centrifuged, collects precipitate;
(3) Polyose extraction and purification
The precipitate of Type B hemophilus influenza adds in sodium chloride solution and dissociates, centrifugal collection supernatant after dissociating; Supernatant is first concentrated by ultrafiltration, adding ethanol to ethanol mass fraction after concentration is 20%, centrifugal segregation precipitation after stand at low temperature, be then centrifuged in liquid adding dehydrated alcohol to ethanol content is 85%, it is centrifuged after stand at low temperature, collects precipitation and also use dehydrated alcohol and washing with acetone, in precipitation, then add water dissolution, lysate recentrifuge, and centrifugal liquid is carried out dialysis treatment; Concentration obtains rough polysaccharide;
Rough polysaccharide is dissolved in saturated acetic acid sodium solution, adds cold phenol solution extracted several times; Then the centrifugal supernatant of collecting of stirring, and by ultrafiltration or dialysis dephenolize, add ethanol to final concentration of 60%��80%, collected after centrifugation precipitate, precipitate uses dehydrated alcohol and washing with acetone, and then vacuum drying obtains refined polysaccharide;
(4) polysaccharide derives
Refined polysaccharide is dissolved in distilled water and regulates pH value to 10.5��11, activating at temperature 20 DEG C��26 DEG C, being subsequently adding acid solution to pH is 9, is subsequently adding adipic dihydrazide ADH reaction, regulate pH value after completion of the reaction and stir more than 12h to alkalescence, then dialyse, filter, dry; Obtain polysaccharide spin-off;
(5) preparation of GL-PP conjugate
PH value is regulated with carrier protein to 5.5��5.9 after being mixed by polysaccharide spin-off, add 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide EDC in 2 DEG C��8 DEG C reactions, regulate pH value after completion of the reaction to dialyse to 6.7��7.1, dialysis solution uses ultrafilter membrane degerming after column chromatography purification, concentration, obtains Type B hemophilus influenza capsular polysaccharide.
2. the method for claim 1, it is characterised in that: using fermentor cultivation bacterium solution in described step (1), inoculate initial concentration A600=0.18, cultivating pH is 7.5, and cultivation temperature is 36 DEG C, and incubation time is 10h��15h; Results bacterial concentration A600 >=4.0.
3. method as claimed in claim 1, it is characterised in that: in described step (2), the final concentration volume fraction of cetyl trimethylammonium bromide is 0.1%.
4. method as claimed in claim 1, it is characterised in that: in described step (3), sodium chloride adds the final concentration of 10mg/ml of postprecipitation thing polysaccharide.
5. method as claimed in claim 1, it is characterised in that: adding ethanol to ethanol mass fraction in described step (3) is 20%, stands the rear centrifugal segregation precipitation of more than 12h at 4 DEG C.
6. method as claimed in claim 1, it is characterised in that: in described step (4) addition is refined polysaccharide 3 times��4 times of adipic dihydrazide ADH.
7. method as claimed in claim 1, it is characterised in that: the content of described step (4) refined polysaccharide is 10mg/10ml.
8. method as claimed in claim 1, it is characterised in that: in described step (5), carrier protein is tetanus toxoid protein.
9. the combined vaccine of the Type B hemophilus influenza capsular polysaccharide prepared containing arbitrary described method in claim 1��8, it is characterised in that: described combined vaccine includes Type B hemophilus influenza capsular polysaccharide, acellular pertussis vaccine, diphtheria toxoid vaccine and tetanus toxoid vaccine.
10. the method for vaccine described in preparation claim 9, it is characterized in that: include after adding aluminium hydroxide absorption, acellular pertussis stock solution, Toxoidum Diphthericum Purificatum, tetanus toxoid purified are added Type B hemophilus influenza capsular polysaccharide stock solution, then normal saline dilution is used, add thimerosal and regulate pH value to 5.8, add 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide EDC and react 1 hour; Regulate pH to 7.0 again, be then cooled to 4 DEG C, degerming after finally dialysis, ultrafiltration concentration, in 4 DEG C of preservations.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109602902A (en) * | 2018-11-27 | 2019-04-12 | 广东渔跃生物技术有限公司 | One boar Pasteurella polysaccharide protein Conjugate vaccines and preparation method thereof |
| CN116920083A (en) * | 2023-07-27 | 2023-10-24 | 北京百晖生物科技有限公司 | Hib conjugate vaccine |
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