CN107929728A - A kind of pneumoprotein vaccine and preparation method thereof - Google Patents

Abstract

The invention discloses a kind of pneumoprotein vaccine, is to include one or more pneumoniae capsulars and pneumoniae capsular protein conjugates obtained by carrier protein covalent attachment, carrier protein is agglutinin.Compared with prior art, advantage of the invention is：The protein vaccine administering mode of offer is nose administration, relative to intramuscular injection, nasal-cavity administration mode more convenient and quicker；Protein vaccine provided by the invention can be used as commercially available pneumococcus supplement immune vaccine to use；It when extensive epidemic situation occurs, can temporarily be used as emergency vaccine, immune-base is provided subsequently to carry out commercially available Pnu-Imune 23 inoculation.

Description

A kind of pneumoprotein vaccine and preparation method thereof

Technical field

The present invention relates to a kind of pneumoprotein vaccine, is to be related to a kind of pneumonia available for nasal-cavity administration specifically Pneumococcal proteins vaccine.

Background technology

First, pneumococcal disease epidemiology

High incidence, high case fatality rate, the antibiotic of streptococcus pneumonia property disease (pneumococcal disease, PDs) Drug resistance and serious sequelae, especially disease seriously affect infant and the elderly's generation, make the disease into For one of important public health problem in the whole world, China is also the country occurred frequently of streptococcus pneumonia property disease.Streptococcus pneumonia (Streptococcus pneumoniae, SP, also referred to as Diplococcus pneumopniae, Pneumococcus) is streptococcus, is a kind of mistake The Grain-positive diplococcus of hydrogen oxide enzyme feminine gender, the pod membrane of its mantle is the special construction of bacterium, is present in some bacteriums One layer of mucus material outside cell membrane, can protect bacteria from antibacterial or Fungicidal substance infringement and host phagocytes Phagocytosis, while bacterial adhesion is additionally aided in host cell surface to induce infection, it is the significant surfaces knot of bacterium existence Structure.Streptococcus pneumonia is the principal element for causing the serious diseases such as infantile pneumonia, bacteremia, meningitis, and causes sinusitis With the most common cause of disease of otitis media acuta.According to its capsular polysaccharide antigen sex differernce, 90 various serotypes are had now been found that.Infection Or stimulate the protection antibody of body generation that there is type specificity after inoculation polysaccharide antigen, also some antibody have associated serum type A degree of cross protection.

Streptococcus pneumonia is distributed widely in right boundary, and the mankind are important natural reservoir (of bird flu viruses).Nasopharynx, larynx larynx of the streptococcus pneumonia in people Can normal presence with oral cavity.The pharynx nasalis carrying rate of children is higher than adult.Infant is considered as the major storage of the bacterium Host.Cross-section survey shows, infant's nasopharynx carrying rate developed country can as low as 27%, it is reachable in developing country 85%.General population is compared, streptococcus pneumonia property disease is more easy to hair in less than 5 years old children and over-65s old man crowd.

Streptococcus pneumonia is mainly directly propagated by the respiratory tract spittle or causes autogenous infection by being colonized bacterium.Streptococcus pneumonia Nasal sinus or middle ear, which can locally be sent out, to be caused to infect, and suction lower respiratory tract causes pneumonia.When bacterium invades blood circulation, companion or not Breeding is sent out with other positions, then can cause invasive infection, causes aggressive streptococcus pneumonia property disease (invasive Pneumococcal diseases, IPD).

Streptococcus pneumonia property disease is divided to invasion and two class of Non-Invasive.IPD refers to streptococcus pneumonia intrusion and external environment Without directly communicating, being infected caused by originally sterile position and tissue, mainly including meningitis, Bacteraemic pneumonia, and septicopyemia The rare infection such as disease, pyothorax, osteomyelitis, pericarditis, endocarditis, peritonitis and pyogenic arthritis.80%~90% in IPD The deficiencies of for Bacteraemic pneumonia, 5%~10% being meningitis, pleurisy and arthritis 5%.Non-Invasive streptococcus pneumonia property Disease mainly includes otitis media acuta, nasosinusitis and non-Bacteraemic pneumonia.The streptococcus pneumonia property pneumonia 80% of clinical diagnosis It is Bacteraemic pneumonia for non-Bacteraemic pneumonia, 20%.IPD is common not as Non-Invasive streptococcus pneumonia property disease, still Etiological diagnosis is easier to clearly, therefore IPD incidence is passed through frequently as reflection streptococcus pneumonia property Disease Spectrum and serotype distribution One of important indicator.

China's epidemiological study shows that drug resistance phenomenon occur more and more in pediatric respiratory tract infection common antibiotics. In one 2 years by a definite date (2005,2006) that 8 cities carry out, the perspective study of less than 5 years old Hospitalized Children is found, aggressive bacterium In strain, there are 55% and 17% pair of penicillin height and intermediate-resistant respectively；451 plants of invasions and Non-Invasive bacterial strain, have respectively 64% and 18% bacterial strain is to penicillin height and intermediate-resistant.Equally, in adult, insensitive penicillin is also one important Problem.2010 Chinese Bacterial resistance surveillance coorporative networks monitoring displays, by U.S. clinical and the Laboratory Standard committee 2008 Amended standard, penicillin-susceptible strain, intermediary's strain and persister are respectively 70.3%, 15.9% and in children's clinical separation strain 13.8%；It is respectively 92.2%, 3.3% and 4.4% in adult.2008 to streptococcus pneumonia penicillin minimum inhibitory concentration After break adjustment, penicillin-susceptible rate greatly improves.In addition, the crossing drug resistant and multidrug resistant of the insensitive strain of China's penicillin show As obvious, cynnematin drug resistance is also being raised, the macrolideses such as non-beta lactam antibiotics especially erythromycin are being resisted The resistant rate of raw element is especially high, considerably beyond western countries.171 be separated in 11 11, city hospitals for 2006~2008 years In IPD plants of strain, the bacterial strain for having 96% and 75% respectively separates erythromycin and sulfamethoxazole drug resistance, 77% meningitis Strain is Penicillin-resistant strain, and 30% meningitis separation strains are to third generation cephalosporin (ceftriaxone) drug resistance.1~60 month, Beijing In age children, there are 87%~94% isolated strains to erythromycin-resistant, 80% pair of tetracycline and sulfamethoxazole drug resistance. Zhou etc. is separated to 140 plants of streptococcus pneumonias from the children with upper respiratory infections of Beijing, to the drug resistance of erythromycin and azithromycin Rate respectively reaches 96.4% and 97.1%, wherein the bacterial strain of 64.3% (90/140) is multidrug resistant strain.In industrialized country, Although possessing suitable antibiosis extract for treating and Intensive Care Therapy, the bacteremic case fatality rate of adult pneumonia coccus reaches 15%~20%, Elderly population is up to 30%~40%.Although tympanitis caused by pneumococcal infection and the nasosinusitis order of severity are relatively light, But more typical hygienic issues are still fallen within the world.To sum up, streptococcus pneumonia is to cause Chinese children and old human hair Disease, dead important pathogen body, therefore be that prevention the important of streptococcus pneumonia property disease is arranged by being inoculated with Streptococcus pneumoniae vaccine Apply, there is Great significance to prevention streptococcus pneumoniae infection.

2nd, the prevention of pneumococcal disease

Vaccine is by pathogenic microorganism (such as bacterium, rickettsia, virus) and its metabolite, by manually subtracting Poison, inactivation or using transgenosis the methods of manufactured active immunity preparation for being used to preventing or treating infectious disease, are biological agents In most commonly seen one kind.S. pneumoniae capsular contains polysaccharide antigen, is its necessary protection sex factor and virulence factor, It is the basis of parting.Capsular polysaccharide (capsular polysaccharide, CP) has good immunogenicity, extensively should Development for polysaccharide vaccine.Johnson etc. shows the system review of 1980~2007 documents global less than 5 years old children are about 70% IPD is caused by 6~11 kinds of serotype.Cause the serotype of Non-Invasive disease more, and less than 5 years old children The most common serotypes of IPD are l, 5,6A, 6B, 14,19F and 23F.IPD caused by the type of wherein 1,5 and 14 accounts for 28% in the whole world~ 43%, account for 30% in 20 poorest nations；IPD caused by 23F and 19F accounts for 9%~18% in the whole world.In Europe, U.S. Continent and Oceania, 18C types are also very common.

The commercially available Streptococcus pneumoniae vaccine in China has two kinds at present, i.e. PCV (pneumococcal conjugate ) and PPV (pneumococcal polysaccharide vaccine) vaccine.PPV is by streptococcus pneumoniae capsular polysaccharide As antigen, blood serum induced type specificity IgG (based on IgG2 hypotypes), IgM and IgA antibody, strengthen opsonophagocytosis, promote white Cell and phagocyte kill streptococcus pneumonia, have specific protective effect to streptococcus pneumoniae infection.PCV is by pneumonia chain Streptococcus polysaccharides are incorporated into the protein carrier with immunogenicity by chemical method, can preferably strengthen antibody response and induce immune Memory.

Since polysaccharide vaccine is containing only polysaccharide antigen (a kind of T-independent antigen), the B lymphs of maturation can be stimulated thin Born of the same parents, but not stimulate T lymphocytes, it is impossible to produce immunological memory.Infants Below immune function development in 2 years old is not perfect, right The immune response of polysaccharide is very poor, so polysaccharide vaccine cannot effectively induce the infant of less than 2 years old to produce protective immune response. Combined vaccine is adsorbed in adjuvant, polysaccharide antigen and carrier egg after being combined by the polysaccharide antigen of common causative serotype with carrier protein White Binding change antigenic property, from T-independent antigen (polysaccharide) is converted to T cell dependence antigen (polysaccharide egg White conjugate), so as to stimulate t helper cell, induction produces immunological memory, when the polysaccharide antigen identical with vaccine component pierces again The immune response of enhancing can be produced when sharp.Shortly effective be immunized can be produced to T cell dependence antigen after baby due should Answer, combined vaccine energy effective stimulus infant immunisation system, produces enough protection antibodies, and inducing immunological memory.WHO is pushed away Recommend and PCV is included into the Immune Programming, especially country of those less than 5 years old child mortalities more than 50/1000 life birth baby.

The PCV listed in the world has PCV7, PCV10 and PCV13, PCV7 containing 7 kinds (4,6B, 9V, 14,18C, 19F and 23F), PCV13 containing 13 kinds (1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F), PCV10 containing 10 kinds (1,4,5, 6B, 7F, 9V, 14,18C, 19F and 23F) streptococcus pneumoniae polysaccharides antigen.

What China listed at present is that (common name of State Food and Drug Administration's approval is " 7 valency pneumococcus to PCV7 Combined vaccine ") and PPV23 (general entitled " the 23 valency pneumococal polysaccharide epidemic diseases of State Food and Drug Administration's approval Seedling ").PCV7 is per 0.5mL agent containing 2 μ g serotypes 4,9V, 14, the monose of 19F and 23F, the oligosaccharides of 2 μ g serotypes 18C, 4 μ g blood The polysaccharide of clear type 6B；CRM197 carrier proteins 20 μ g, Aluminium phosphate adjuvant 0.125mg, without preservative.But in vaccine inoculation crowd Have and PCV7 is disabled to diphtheria toxoid allergy sufferers, this undoubtedly had limitation to less than 2 years old people at highest risk diphtheria toxoid autopath Effect.Due to the limitation of polysaccharide vaccine, PPV23 is mainly used for the streptococcus pneumonia property disease of adult and more than 2 years old people at highest risk Disease.PPV23 include 23 kinds purifying streptococcus pneumoniae polysaccharides antigens, i.e. serotype l, 2,3,4,5,6B, 7F, 8,9N, 9V, l0A, 11A, 12F, 14,15B, l7F, 18C, 19A, 19F, 20,22F, 23F and 33F.Every dose (0.5mL) contains above-mentioned various 25 μ of capsular saccharides G, no adjuvant.For capsular polysaccharide vaccine since its component is single, there is no the material for easily causing immune side reaction, makes such vaccine more Safely, effectively, it has also become one of most vaccine of application.

WHO suggests, when baby is inoculated with pneumococcal conjugated vaccine, using 3 doses of fundamental immunity schemes (i.e. 3p+0 schemes)； Alternatively, 1 dose of booster immunization scheme (2p+1 schemes) also can be added using 2 doses of alternative fundamental immunities.Selection 3p+0 schemes and During 2p+1, epidemiology, the arrangement of time of each dose inoculation of possible rate of vaccination and vaccine of pneumoccoccosis are referred to.

In an extensive work place study in the U.S., by vaccine serotype induction for aggressive pneumococcal disease Protective immunity in the children for being vaccinated with least 3 pins time up to 97.4% (95% credibility interval：82.7%~ 99.9%) [use " meeting program analysis " (effective analysis of cases, per-protocol analysis)], be at least inoculated with 1 pin Up to 93.9% (95% credibility interval in secondary children：79.6%~98.5%) [use " Intentionality Treatment analysis " (intention-to-treat analysis)].In addition, after the secondary vaccine of inoculation at least 1 pin, the aggressive pneumonia of report Coccus disease totally reduces 89.1% (95% credibility interval：73.7%~95.8%).Connect by PCV7 introduction National Immunizations After kind of planning 1 year, aggressive pneumococcal disease caused by the Pneumococcus serotypes contained in the vaccine all 1 years old with Incidence in lower inoculator reduces 100% (95% credibility interval：87.3%~100%).After the vaccine is introduced 3 years, 1 The incidence of all invasion pneumococcal diseases reduces 84.1% in year following children；Reduced at 20~39 years old in adult 52%, 27% was reduced in individual more than 60 years old.Follow-up monitoring shows, after the vaccine is introduced 2 years, aggressive pneumococcus Incidence of the disease below 5 years old in children reduces 75%.More than 5 years old, aggressive pneumonia in the crowd of non-vaccine inoculation The decline of coccus disease incidence is probably to reduce because of the spread of germs from less than 5 years old, the infant of vaccine inoculation, I.e. so-called " indirectly immune " or " herd immunity " phenomenon, there are about 68% in all aggressive pneumococcal diseases being prevented from can Be attributed to the fact that this indirect effect^[1,2,3].And China is relatively fewer on the information that PCV7 immunity inoculations lapse to.

The routine immunization that the abundant youngster TM (septivalency pneumococcal conjugated vaccine) that the Wyeth of China's listing develops recommends connects Planting program is：3rd, 4,5 monthly ages carried out fundamental immunity, 12~15 monthly age booster immunizations, were inoculated with the monthly age first with specific reference to children and connect Kind, such as 3~6 month infants：3 doses, every dose of 0.5mL of basic immunity, is seeded in 3 monthly ages, immune programme 3,4, May first Age is each one, and inoculation every time is at least spaced 1 month, it is proposed that the 4th dose is inoculated with 12~15 monthly ages；7~11 month infants：Exempt from basis Epidemic disease is inoculated with 2 doses, every dose of 0.5mL, and inoculation every time is at least spaced 1 month, it is proposed that the 3rd dose is inoculated with after 12 monthly ages, is connect with the 2nd time Kind is at least spaced 2 months；12~23 monthly age childs：2 doses, every dose of 0.5mL are inoculated with, inoculation every time is at least spaced 2 months；24 monthly ages ~5 years old children：1 dose of inoculation.But since intramuscular injection that may be present is reacted, inoculation crowd Willingness is not high, frequently results in immune Scheme can not perform according to plan, cause vaccine protection to weaken.

3rd, vaccine nasal drug delivery system progress

The common dosage forms of vaccine predominantly take orally and injection, due to the molecular activity requirement of vaccine, current vaccine dose Type research is concentrated mainly in injection.Vaccine is inoculated with by the way of subcutaneously or intramuscularly injecting mostly at present, and injection inoculation leads to It often inducible system can effectively be immunized, but mucosa-immune can not be induced, cannot produce mucosal secretory in vaccinee's body is immunized Globulin (slgA) antibody.Meanwhile injection inoculation mode there is transfer period it is long, often, kind of the rate that stops is high and easily occurs The compliance difference greatly limitation vaccine inoculation of the problems such as cross-infection, especially patient child.In recent years, nasal-cavity administration is as a kind of Noninvasive vaccination approach is increasingly becoming the hot spot of research, and nasal drug delivery system refers to after nasal-cavity administration, through schneiderian membrance Absorb so as to play a kind of preparation of locally or systemically treatment or prevention effect.As new auxiliary material and the gradual of new technology are applied, Nasal cavity can avoid destruction and the first pass effect of liver of gastrointestinal enzyme with it, and drug absorption is rapid, rapid-action, be easy to be suffered from The features such as person receives and compliance is good, has been counted as the optimal method of administration of many medicines such as vaccine, hormone, polypeptide.At present, There is the nasal-cavity administration launch of some protein, polypeptide drug, such as the calcitonin of Novartis, Switzerland (Calcitonin), the minirin (Desmopressin) of Ferring companies of Germany and the cloth of Aventis companies of France Auspicious (Buserelin) etc. is given up, the bacterin preparation listing of existing minority nasal-cavity administration, the Medlm- being had been approved by such as U.S. FDA Nasal spray administration live influenza virus vaccine (influenza virus vaccine live, the trade name of mune companies： FluMist) etc..But there is vaccine nasal-cavity administration absorption to be easily obstructed, irritation on mucous membrane is strong, is easily degraded by enzyme, can not keep slow The problems such as releasing long-acting^[4,5]。

The content of the invention

In view of the above-mentioned problems existing in the prior art, the object of the present invention is to provide a kind of pneumoprotein vaccine, with The form of nasal-cavity administration is inoculated with, which does not contain the high molecular component such as animal derived, food-borne, and is easy to absorb, from And possess splendid economic benefit and environmental benefit.

For achieving the above object, the technical solution adopted by the present invention is as follows：

A kind of pneumoprotein vaccine, is to include one or more pneumoniae capsulars with carrier protein covalently to connect Pneumoniae capsular-protein conjugates obtained by connecing, carrier protein are agglutinin.

Agglutinin (lectin) is the glycoprotein or the special body (sugar-specifity) of sugar of non-immunogenic, 19th century Found by Stillmark, alternative identification cell membrane surface glycan molecule or acceptor spline structure, so that with glycosylating biomembrane knot Close (internalization process of lysosome transfer mode), serve as lysosome sample " adjuvant " (drug delivery of delivery system adjuvant).Modification of the agglutinin to particulate delivery system is except the suction-operated (adsorption through gap on particulate Process), can also be by loading the covalent bonding (covalent linkage) of amino, carboxyl or carboxyl on carrier material.

Preferably, pneumoniae capsular-protein conjugates molecular weight is 30~1000kDa.

Preferably, agglutinin is selected from phytolectin, as pinellia agglutinin, bupleurum lectin, wheat germ agglutinin, castor-oil plant coagulate Collection element or horse crab agglutinin.

Preferably, pneumoniae capsular, preferably separates the capsular polysaccharide on purification Pneumococcal serotype pod membrane, The serotype of the pneumococcus include 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and/or 23F.

Preferably, the mass ratio of S. pneumoniae capsular saccharide and carrier protein is (0.5~2)：1, be preferably (0.5~1.5)： 1。

Preferably, the preparation method of above-mentioned pneumoprotein vaccine includes the following steps：

Step a) is prepared and separating-purifying carrier protein；

Capsular saccharides on step b) difference separating-purifying various serotype pneumococcal capsules；

Capsular saccharides obtained by step c) activation steps b, with step a gained agglutinin covalent couplings；

Coupled body, obtains protein vaccine stoste obtained by step d) separating-purifying steps c.

It is highly preferred that the preparation method of above-mentioned pneumoprotein vaccine includes the following steps：

Step a) is prepared and separating-purifying agglutinin；

Capsular saccharides on step b) difference separating-purifying various serotype pneumococcal capsules；

Capsular saccharides obtained by step c) activation steps b, under ultrasound condition with step a obtained by agglutinin covalent coupling；

Coupled body, obtains protein vaccine stoste obtained by step d) separating-purifying steps c.

It is highly preferred that the method that above-mentioned carrier protein preparation method can refer to phytolectin separating-purifying in the prior art Prepare, for details, reference can be made to following embodiments, be not intended as the present invention whether achievable key factor.

It is highly preferred that the separation purifying technique of capsular saccharides can be operated according to the prior art in step b.

It is highly preferred that the concrete operations that step c prepares the S. pneumoniae capsular saccharide of activation are：In capsular saccharides obtained by step b Physiological saline is added, CDAP activation 3min is added, adds isometric ADH solution reactions 30min, resulting solution is through being concentrated by ultrafiltration It is spare afterwards；It is preferred that pH is 7.5~9.5 in this operation.

It is highly preferred that the concrete operations for activating S. pneumoniae capsular saccharide coupling agglutinin are by step c：Under ultrasound condition, ADH- capsular saccharide derivatives are added into physiological saline, add EDAC to final concentration of 0.1mol/L, when reaction 3 is small at room temperature, instead Thing is answered to be dialysed with 4 DEG C of normal saline dialysis films, adding agglutinin, (agglutinin is 1.5 with capsular saccharides mass ratio：1)；It is preferred that this PH is 4.5~6.5 in operation.

Further, ultrasonic frequency range is 15~30kHz.

It is highly preferred that step d concrete operations are：Step c gained reactants centrifuge 2min through 5000rpm/min, take supernatant Liquid layer is analysed, and aseptic filtration, obtains protein vaccine solution；It is preferred that supernatant layer is through mannose-Sepharose gel chromatographies.

Compared with prior art, the present invention has the advantage that：

1st, agglutinin is the common adjuvant of delivery system, but protein vaccine provided by the invention is proposed with plant lectin first Element prepares Pnu-Imune 23 for protein carrier, lays the first stone for the feasible direction of pneumovax；

2nd, protein vaccine administering mode provided by the invention is nose administration, relative to intramuscular injection, nasal-cavity administration mode More convenient and quicker；

3rd, protein vaccine provided by the invention can be used as commercially available pneumococcus supplement immune vaccine to use；

4th, protein vaccine provided by the invention can improve commercially available Pnu-Imune 23 antibody concentration and Conversion rate, reduce vaccine The probability of inoculation failure；

5th, when extensive epidemic situation occurs, can temporarily be used as emergency vaccine, subsequently to carry out commercially available pneumococcus Vaccine inoculation provides immune-base.

Embodiment

The present invention is made further in detail, intactly to illustrate with reference to embodiment and comparative example.Occur in embodiment Reagent or instrument are operated in a specific embodiment but specified otherwise, is commercially available according to its specification, herein not Repeat.

The percentage sign " % " arrived involved in the present invention, if not specified, refers to mass percent；But the percentage of solution Than unless otherwise specified, referring to the grams containing solute in 100mL solution.

Embodiment 1

First, prepared by agglutinin

Take 500 grams that come from Shanghai Univ. of Traditional Chinese Medicine fresh tuber of pinellia stem tubers to clean and grind rear 6000rpm/min centrifugations 10min, supernatant after purification, collect 60%~95% saturated ammonium sulphate through 20%~95% ammonium sulfate fractionation of saturation degree PTA, active component through mannose-Sepharose gel chromatographies, obtains pinellia agglutinin protein liquid by several times, spare.Also or by aggegation Element is cloned into Escherichia coli and is expressed and isolated and purified, spare.

2nd, S. pneumoniae capsular saccharide is prepared

1. choose 7 kinds of serotypes (4,6B, 9V, 14,18C, 19F and 23F) pneumococcus culture；

2. the capsular saccharides that antigenicity is strong in any of the above Pneumococcal serotype are purified respectively：Centrifuged after pneumococcus inactivation Collect supernatant, through being concentrated by ultrafiltration, the precooling second that volume fraction is 70% is separately added into according to each Pneumococcus serotypes characteristic Alcohol, is collected by centrifugation, and obtains rough polysaccharide；Rough polysaccharide is dissolved in sodium acetate solution, then by 1：2 ratios are mixed with cold phenol, from The heart removes removing protein, and phenol carries 5-6 times repeatedly, collects supernatant, is dialysed with distilled water, and liquid adds 2mol/L calcium chloride solutions after dialysis, Ethanol stirring is added, centrifugation removes nucleic acid, collects supernatant, adds ethanol (final concentration 80% stirs), precipitation is collected by centrifugation, uses second Alcohol, acetone washing precipitate, and are refined capsular saccharides after dehydration and drying, put -20 DEG C and save backup.

3rd, the preparation of protein vaccine

1. S. pneumoniae capsular saccharide activates：S. pneumoniae capsular saccharide (serotype 4,9V, 14,19F prepared by above-mentioned steps With the monose 4g of 23F, the oligosaccharides 4g of serotype 18C, the polysaccharide 8g of serotype 6B) add 5mg/mL (physiological saline) solution In 500mL, CDAP activation is added, controls pH to 8.5 ± 0.5, reacts 3min, adds isometric ADH solution, controls pH to 8.5 Through ADH- capsular saccharide derivatives are concentrated by ultrafiltration to obtain after ± 0.5, reaction 30min；

2. activate S. pneumoniae capsular saccharide coupling agglutinin：Under ultrasound condition, ADH- capsular saccharide derivatives are made into 5mg/ (physiological saline) solution 500m L of mL, add EDAC to final concentration of 0.1mol/L, mix control pH to 5.5 ± 0.5, room temperature It is lower reaction 3 it is small when, reactant is dialysed (4 DEG C of normal saline dialysis) with dialysis membrane, add agglutinin (agglutinin and capsular saccharides Mass ratio is 1.5：1)；

3. protein vaccine isolates and purifies：Reactant through 5000rpm/min centrifuge 2min, take supernatant through mannose- Sepharose gel chromatographies, aseptic filtration obtain protein vaccine solution, take 0.5mL spare, and numbering is sample 1；

It is 4. lyophilized：Specific steps refer to Chinese invention patent application CN 106063933A, and this will not be repeated here.

Above-mentioned protein vaccine solutions for administration mode is nasal mucosa medicine administration, specifically refers to prior art preparation, such as freeze-dried powder Isolate with physiological saline through film and load spray bottle, rupture of membranes mixes before use, disposably sprays into nasal cavity.

Embodiment 2

The difference of the present embodiment and embodiment 1 is only that agglutinin is by the bupleurum lectin after sepharose 4B detoxification, institute It is sample 2 to obtain protein vaccine solution numbers.

Embodiment 3

The difference of the present embodiment and embodiment 1 be only that vaccine capsular saccharide serotypes and dosage for 1,4,5,7F, 9V, 14, 18C, 19F and 23F each 4g, 6B 8g.

Embodiment 4

The difference of the present embodiment and embodiment 1 be only that vaccine capsular saccharide serotypes and dosage for 1,3,4,5,6A, 7F, 9V, 14,18C, 19A, 19F and 23F each 4g, 6B 8g.

Vaccine evaluation

Booster immunization recruitment evaluation experiment is carried out to the protein vaccine that embodiment provides below：

8 week old, 15~20g of weight female mices 100 are chosen, is randomly divided into 5 groups, each identical immune group uses 20 Mouse, sprays nose, negative control group is with reference to WHO Immunization programmes and dosage difference mouse peritoneal injection Prevnar7 or sample 1,2 Physiological saline sprays nose, is specifically shown in Table 1.

Table 1

Take a blood sample respectively at 11 monthly age of mouse and 13 monthly ages, using ELISA method detection antibody average titer and calculate Conversion rate, It the results are shown in Table 2~table 5.

2 11 monthly age of table mouse antibodies average titer

3 11 monthly age of table Positive seroconversion rate (%)

4 13 monthly age of table mouse antibodies average titer

5 13 monthly age of table Positive seroconversion rate (%)

Respectively with 4,6B, 9V, 14,18C, 19F and 23F type 14 monthly age of Pneumococcal challenge mouse, persistently attack 7 days, adopt Microflora in blood is surveyed in blood examination, and testing result is shown in Table 6.

Table 6

Free saccharide content detection is carried out to the protein vaccine that embodiment provides below, the results are shown in Table 7：

Free capsular saccharides assay result (mg/mL) in 7 solution of table

From 1~table of table 5, B groups mouse easily occurs not due to adding a pin vaccine, mouse on the basis of Immunization programme Good reaction, sporadic deaths, therefore excess injection should not be carried out；C group mouse internal antibodies concentration is less than B groups, but is higher than other groups, Mouse antibodies Conversion rate is high, and mouse has no dead；D group mouse internal antibody concentration is most weak, and Conversion rate is up to more than 70%, mouse Have no dead；E groups mouse antibody concentration is less than B groups higher than D groups, and Conversion rate is high, and mouse has no dead.In addition, can by table 6~7 Know, protein vaccine provided by the invention, which is used alone, is relatively combined bacterial immune effect the time difference, and sugar-albumen composition is unstable It is fixed, this is because single structure of the compound of agglutinin-sugar without fixation, once compound is formed, the molecule position participated in Put and conformation be still change (sugar and the combination of albumen are reversible), therefore needs face and matched somebody with somebody with facing, in 3 days must use finish, swum after 7 days From sugared content close to the half of capsular saccharides total content, it is more difficult to evoke recipient immune reaction.From above-mentioned data, this hair The protein vaccine of bright offer can be used as additional project, can pole as Immunization programme but the not up to booster injection of immune effect is completed It is big to improve immune effect.In addition, when there is extensive epidemic situation, protein vaccine provided by the invention can be used as emergency plan to carry out Fundamental immunity.

Bibliography

[1]QIN Ying,et al.,Technical guideline on application of pneumococcal Vaccine in China (2012), Chin J Epidemiol, November 2012, Vo1.33, No.11：1101-1110.

Qin Ying etc., streptococcus pneumonia property disease related vaccines application technology guide (2012 editions), Chinese epidemiology magazine, In November, 2012 o. 11th of volume 33：1101-1110.

[2] wise man etc., capsular polysaccharide and its Advances on Vaccine [J] animal medicines progress, 2015,36 (11) are opened：83-87.

[3] position paper (2012 year) of the World Health Organization on Pnu-Imune 23, Weekly Epidemiological Record.No.12,2007,pp.93-104.

[4]CAI Xin-jun,et al.,Development of Research on Vaccine Microparticle Nasal Drug Delivery System, Progress in Pharmaceutical Sciences, 2007, Vo1.31, No.9：391-397.

Cai Xinjun etc., the progress of Vaccine Microparticle Nasal Drug Delivery System, pharmacy progress, the 9th phase of volume 31 in 2007： 391-397.

[5] Hu Yin etc., progress [J] Chinese Medicine biotechnologys of nasal drug delivery system, 2008.10,3 (5)： 381-384.

Claims (10)

1. a kind of pneumoprotein vaccine, it is characterized in that：Including one or more pneumoniae capsulars and carrier protein Pneumoniae capsular-protein conjugates obtained by covalent attachment, carrier protein are agglutinin.

2. the protein vaccine described in claim 1, it is characterized in that：Agglutinin is selected from phytolectin.

3. the protein vaccine described in claim 1, it is characterized in that：The serotype of pneumococcus include 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and/or 23F.

4. the protein vaccine described in claim 1, it is characterized in that：The mass ratio of S. pneumoniae capsular saccharide and carrier protein is (0.5 ~2)：1.

5. a kind of preparation method of pneumoprotein vaccine, it is characterized in that, include the following steps：

Step a) is prepared and separating-purifying carrier protein；

Capsular saccharides on step b) difference separating-purifying various serotype pneumococcal capsules；

Capsular saccharides obtained by step c) activation steps b, with step a gained agglutinin covalent couplings；

Coupled body, obtains protein vaccine stoste obtained by step d) separating-purifying steps c.

6. the preparation method of the protein vaccine described in claim 5, it is characterized in that, include the following steps：

Step a) is prepared and separating-purifying agglutinin；

Capsular saccharides on step b) difference separating-purifying various serotype pneumococcal capsules；

Capsular saccharides obtained by step c) activation steps b, under ultrasound condition with step a obtained by agglutinin covalent coupling；

Coupled body, obtains protein vaccine stoste obtained by step d) separating-purifying steps c.

7. the preparation method of the protein vaccine described in claim 6, it is characterized in that, step c prepares the pneumococcal capsule of activation Sugar concrete operations be：Physiological saline is added in capsular saccharides obtained by step b, CDAP activation 3min is added, adds isometric ADH Solution reaction 30min, resulting solution are spare after ultrafiltration concentration.

8. the preparation method of the protein vaccine described in claim 7, it is characterized in that, step c will activate S. pneumoniae capsular saccharide idol Connection agglutinin concrete operations be：Under ultrasound condition, ADH- capsular saccharide derivatives are added into physiological saline, it is dense to end to add EDAC Spend for 0.1mol/L, react at room temperature 3 it is small when, reactant is dialysed with 4 DEG C of normal saline dialysis films, addition agglutinin.

9. the preparation method of the protein vaccine described in claim 6, it is characterized in that：Ultrasonic frequency range is 15~30kHz.

10. the preparation method of the protein vaccine described in claim 6, step d concrete operations are：Step c gained reactants pass through 5000rpm/min centrifuges 2min, takes supernatant to chromatograph, aseptic filtration, obtains protein vaccine solution.