CN1899609A - Pneumococcus polysaccharide protein coupling vaccine and its preparing method - Google Patents

Abstract

The present invention is pneumococus polysaccharide protein coupling vaccine comprising covalently connected pneumococus capsule polysaccharide and recombinant pneumolysin without hemolytic activity modification and its preparation process. The vaccine has pneumolysin without hemolytic activity as protein carrier, no need of eliminating hemolysis toxicity of pneumolysin with formalin and ensured safety, and may be used for infant below 2 yeas old to prevent tympanitis. Owing to the pneumolysin as the self protein, the vaccine has no probable immune interference reaction and strengthened immune protecting effect. The vaccine has cross immunizing protection effect on various kinds of serum type pneumococus and raised immune memory response to pneumococus infection.

Description

Pneumococcus polysaccharide protein coupling vaccine and preparation method thereof

Technical field

The present invention relates to the new generation vaccine category of biomedicine field, be specifically related to adopt technique for gene engineering development Pneumococcus polysaccharide protein coupling vaccine, more specifically relate to modified and Pneumococcus polysaccharide protein coupling vaccine that pneumolysin (albumen) that do not have a hemolytic activity is a carrier, and preparation method thereof and as the application of otitis media vaccine.

Background technology

Otitis media (comprising acute otitis media AOM and secretory otitis media OME) is one of common disease of infant, is the otitis media disease that is caused by antibacterial or virus.Discover that the child in 2-3 year has and suffered from otitis media more than 80% at least 1 time, after birth to 6 months age brackets, about 17% child once had the otitis media history of attack more than 3 times or 3 times, and the birth back has 62% child to suffer from otitis media at least one time to 1 years old age bracket.Acute otitis media will cause about child of about 10% auditory dysesthesia, speech in various degree to form obstacle, and may influence the development of speech and the intelligence development of infant if can not get timely, effectively treatment.Cause the main pathogenic bacterium of otitis media outbreak have streptococcus pneumoniae, can not the typing hemophilus influenza and micrococcus catarrhalis etc.Dowell (Dowell SF, Butler JC, Giebink GS, et al.:Acute otitis media:management and surveillancein an era of pneumococcal resi stance--a report from theDrug-resistant Streptococcus pneumoniae Therapeutic Working Group.Pediatr Infect Dis is J.1999; 18 (1): 1-9) acute otitis media of report 40%-50% is caused by streptococcus pneumoniae.Antibiotic appearance once made complication such as chronic suppurative otitis media, acute mastoiditis obviously reduce, but owing to causing Resistant strain, increases antibiotic abuse in recent years, particularly pneumococcal drug resistance problem is particularly serious, report shows that about 50% streptococcus pneumoniae is insensitive to penicillin or other types antibiotic, and has cross resistance.For reducing the antibiotic use amount and avoiding owing to abuse of antibiotics increases drug-resistance of bacteria, focal issue to otitis media control, transfer to prevention from treatment abroad, adopt the otitis media vaccinoprophylaxis, in the hope of generation (Karma P:Vaccination and otitis media.ORL JOtorhinolaryngol Relat Spec, 2002 of reducing otitis media significantly; 64 (2): 80～85).

Aspect vaccine prevention, succeed in developing multiple vaccine abroad in succession, these vaccines are at pathogen such as streptococcus pneumoniae, hemophilus influenzas and develop, mainly contain following several vaccine: (Giebink GS:Immunoprophylaxis of otitis media.Adv Exp Med Biol, 1991 such as pneumococcal capsular polysaccharide vaccine, hemophilus influenza vaccine and pneumococcal polysaccharide-protein coupling vaccine; 303:149～158).Use 14 valencys, 23 valency pneumococcal capsular polysaccharide vaccines ((the Lee CJ that develop as the U.S., Finland etc. more widely, Banks SD, Li JP.Virulence, Immunityand Vaccine Related to Streptococcus pneumoniae.Critical Reviewsin Microbiology.1991,18 (2): 89～114).Advantage is that valence mumber is many, protect wide, but studies show that, this class vaccine has following problem: (1) capsular polysaccharide right and wrong T cell dependent type antigen, though can induce behind this kind of primary vaccination vaccine to produce protectiveness IgM antibody but tire not highly, and inoculation can not be induced needed T cell memory and IgG antibody efficiently once more; (2) immunne response that is produced is only more effective to adult's infection, and to the coccus of children Streptococcus below 2 years old otitis media weak effect.Because the immune system of child below 2 years old is zoon not as yet, lack the ability that non-T cell dependent form antigen is produced immunne response, seldom maybe can not produce antibody response and cause effective immanoprotection action non-T cell dependent form antigen.But child's otitis media send out well age bracket at 6 months to 1 one full year of life, so such vaccine is to the otitis media weak effect below 1 years old.States such as the nineties America and Europe have begun to develop 7 valency pneumococcal polysaccharide-protein coupling vaccines, polysaccharide and some protein carrier is covalently bound with enhance immunity originality (Overturf GD:Pneumococcal vaccination of children.Semin PediatrInfect Dis, 2002; 13 (3): 155～164).

Polysaccharide vaccine only causes non-T cell dependent form immunne response, can not produce immunological memory, and can not be used for child below 2 years old; But and with the cellular immunization and the immunological memory of the combined vaccine excitating organism behind polysaccharide and the carrier protein couplet; produce T cell dependent type immunne response; make thymus dependent/non-dependent polysaccharide antigen be converted into thymus dependent antigen and the immunological memory effect is arranged; can strengthen the protective immune response of child below 2 years old, and can be used for the infant that the age is less than or equal to 6 months.7 valency pneumoprotein coupling vaccines show in the Preliminary Applications result of Finland, by this vaccine serotype at the infant otitis media that causes of streptococcus pneumoniae reduced by 57% (Snow JB Jr.:Progress in theprevention of otitis media through immunization.Otol Neurotol.2002; 23 (1): 1～2).The child that the otitis media tendency is arranged, its nasopharynx part streptococcus pneumoniae sneak case is serious far beyond normal children, for secretory otitis media (OME), the carrier state that this type of vaccine of prophylactic immunization can be by improving nasopharynx part, reduce the parasitism of pathogen, and help to eliminate middle ear effusion at this place.Kilic etc. are with 23 valency pneumococcal capsular polysaccharide vaccines and Type B hemophilus influenza albumen coupling vaccine therapy secretory otitis media, obtained the effect same (Kilic R with grommet insertion, SafakMA, Ozdek A, et al.:Effect of 23valent pneumococcal polysaccharideand Haemophilus influenza conjugated vaccines on the clinicalcourse of otitis media with effusion.Laryngoscope, 2002; 112 (11): 2042～2045).External pneumococal polysaccharide and phase I clinical trial that can not typing hemophilus influenza albumen coupling vaccine achieve success (Snow JB Jr.:Progress in theprevention of otitis media through immunization Otol Neurotol, 2002; 23 (1): 1～2).

Pneumococcal capsular polysaccharide antigen has determined its serotype specificity, is the basis of distinguishing serotype.According to capsular polysaccharide composition difference, it is 90 kinds of serotypes that streptococcus pneumoniae is divided into, what cause that otitis media takes place only is wherein a part of pod membrane serotype, (Hanage WP such as nearest Hanage, AuranenK, Syrjanen R, et al.Ability of pneumococcal serotypes and clonesto cause acute otitis media:implications for the prevention ofotitis media by conjugate vaccines.Infect Immun.2004Jan; 72 (1): 76-81) studies show that, present several pneumococal polysaccharide serotypes that vaccine comprised cause the odds ratio of acute otitis media obviously to increase unlike other serotype, thereby think that the continuous variation of the streptococcus pneumoniae serotype that causes acute otitis media is the existing unfavorable basic reason of streptococcus pneumoniae coupling vaccine long-term effect.Thereby, if can search out a kind of composition all effective to most of pneumococal polysaccharide serotypes, that can produce the cross immunity effect and make vaccine, be expected to address this problem.

At present, the development of the domestic pneumococcal capsular polysaccharide of carrying out-albumen coupling vaccine is the same with external great majority research, adopt diphtheria or tetanus toxoid as protein carrier, its advantage is that these two kinds of albumen are the vaccine for man composition already, and safety and preparation are out of question.But use these two kinds of carrier proteins also to have following deficiency: 1) the major antigen composition of this type of coupling vaccine is wherein contained serotype capsular polysaccharide, and invalid to other serotype pneumococcal infections that are not included in the vaccine, promptly lack cross immunity protection effect; 2) may with in child's routine immunization widely used diphtheria and tetanus vaccine generation immune interference the reaction.

The method that overcomes above-mentioned weak point is to adopt the albumen of streptococcus pneumoniae self not only to develop pneumolysin proteoglycan coupling vaccine as antigen but also as carrier protein, and alternative this bacterium oneself protein has streptococcus pneumoniae surface protein PspA, pneumolysin (Pneumolysin) etc.

Pneumolysin proteoglycan coupling vaccine is the research of current international forward position, does not domesticly see similar research report as yet.Adopt pneumolysin as the advantage of protein carrier to be: 1) streptococcus pneumoniae of all different serotypes all can produce same pneumolysin albumen, therefore may be all effective with its vaccine to various serotype pneumococcal infections, can induce cross immunity protection effect; 2) but it is as the stronger immanoprotection action of virulence factor induction ratio polysaccharide; 3) use the streptococcus pneumoniae oneself protein to do the vaccination that carrier protein is made, can avoid the immune interference reaction taking place with other vaccines such as diphtheria vaccine, tetanus vaccine inoculation; 4) hemolysin can play immune supplementary function to its polysaccharide antigen as the oneself protein of this bacterium, thereby strengthens the immanoprotection action of polysaccharide; 5) not only can improve the immunological memory of pneumococcal infection had been replied as antigen protein but also as the carrier protein of polysaccharide.

Yet the weak point of pneumolysin proteoglycan combined vaccine is that to have toxicity be the haemolysis activity to pneumolysin.The method of head it off abroad reports and adopt formalin to handle to slough the hemolytic toxicity of pneumolysin, and is covalently bound and prepare GL-PP coupling vaccine with pneumococcal capsular polysaccharide again.The present inventor adopts technique for gene engineering that the aminoacid of pneumolysin c-terminus is excised singly, until its forfeiture hemolytic toxicity, to there be the pneumolysin of hemolytic activity and pneumococcal capsular polysaccharide covalently bound with the amino method of reduction then, prepare Pneumococcus polysaccharide protein coupling vaccine safely and effectively, thereby finished the present invention.

Summary of the invention

The object of the present invention is to provide a kind of reorganization pneumolysin of modifying with no hemolytic activity is the Pneumococcus polysaccharide protein coupling vaccine of carrier protein.

The present invention also aims to provide the preparation method of this Pneumococcus polysaccharide protein coupling vaccine.

Pneumococcus polysaccharide protein coupling vaccine provided by the invention is a pneumococal polysaccharide and modified and do not have the covalently bound Pneumococcus polysaccharide protein coupling vaccine that forms of reorganization pneumolysin of hemolytic activity.

In the Pneumococcus polysaccharide protein coupling vaccine of the present invention, pneumococcal capsular polysaccharide comprises 3,14,6B, 19F and 23F serotype; Modified reorganization pneumolysin has 11 amino acid whose sequences of the pneumolysin carboxy terminal deletion shown in the SEQ ID NO.1.

Pneumococcus polysaccharide protein coupling vaccine of the present invention can be used as the otitis media vaccine.

Pneumococcus polysaccharide protein coupling vaccine provided by the invention is prepared with following method:

(1) obtains gene shown in the SEQ ID NO.2 with primer shown in primer shown in the SEQ ID NO.4 and the SEQ ID NO.7 with PCR amplification;

(2) with restricted enzyme NdeI and XhoI difference digestion step (1) gained gene and prokaryotic expression carrier pET-21a, be converted into after the connection in e. coli bl21 (DE3) host cell, be built into the engineering bacteria of recombinant plasmid transformed; Carry out the cultivation of this project bacterium;

(3) induce step (2) to transform gene shown in the engineering bacterium expression SEQID NO.2 with isopropyl ss-D-thiogalactoside, the expression product that obtains is identified with the SDS-PAGE gel electrophoresis;

(4) with the affine adsorption chromatography purification of Ni-NTA expression product;

(5) with of the expression product mutually coupling of the amino method of reduction with pneumococcal capsular polysaccharide with step (4) gained of purification, and separation and purification, Pneumococcus polysaccharide protein coupling vaccine obtained.

Detailed Description Of The Invention

(streptococcus pneumoniae) hemolysin albumen that streptococcus pneumoniae itself is produced is trace very, the present invention adopts gene engineering method, from streptococcus pneumoniae the clone obtain the pneumolysin gene modified after, carry out purification with escherichia coli expression pneumolysin albumen and to it.

1. the present inventor is according to the pneumolysin gene order (seeing the SEQ ID NO.3 in the sequence table) (GenBank accession No.X52474) of report in 1987 such as Walker, initial design have restricted enzyme NdeI ( CATATG) and XhoI (C TCGAG) primer sequence 4 (SEQ ID NO.4) and the primer sequence 5 (SEQ ID NO.5) of recognition site, the pneumolysin gene that adopts round pcr to obtain having SEQ ID NO.3 sequence from pneumococcal chromosomal DNA amplification, and digest this pneumolysin gene and prokaryotic expression carrier (plasmid) pET-21a respectively with restricted enzyme NdeI and XhoI, construct the gene recombination plasmid that to express pneumolysin after the connection, be converted in e. coli bl21 (DE3) host cell, carry out the cultivation of this host bacterium.

Induce conversion host bacterium to express the pneumolysin gene with isopropyl ss-D-thiogalactoside (IPTG), expression product is identified with sodium lauryl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE), comprise what of expressing quantity, and the form of expressing, i.e. solubility or insolubility.

Adopt the broken bacterium of ultrasound wave behind a large amount of host of cultivation bacterium,, obtain having 6 group amino acid whose gene recombinaton pneumolysins (Pn) that come from prokaryotic expression carrier pET-21a coding at its c-terminus through the affine adsorption chromatography purification of Ni-NTA.

2. the present inventor is after obtaining the gene DNA of pneumolysin, designed have restricted enzyme NdeI ( CATATG) and XhoI ( CTCGAG) primer sequence 4 (SEQID NO.4) and the primer sequence 6 (SEQ ID NO.6) of recognition site, adopt round pcr to obtain the gene of 5 amino acid whose pneumolysins of coding carboxy terminal deletion from the gene DNA amplification of pneumolysin, and digest this PCR product and prokaryotic expression carrier pET-21a respectively with restricted enzyme NdeI and XhoI, be converted into then in e. coli bl21 (DE3) host cell, construct the gene recombination plasmid that can be expressed in 5 amino acid whose pneumolysins of carboxy terminal deletion.Inducing conversion host bacterium to express this gene expression product with isopropyl ss-D-thiogalactoside (IPTG) identifies with sodium lauryl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE), comprise what of expressing quantity, and the form of expressing, be solubility or insolubility, with and hemolytic activity.

Adopt the broken bacterium of ultrasound wave behind a large amount of host of cultivation bacterium, obtain at 5 aminoacid of carboxy terminal deletion through the affine adsorption chromatography purification of Ni-NTA but have 6 amino acid whose gene recombinaton pneumolysins of group that come from prokaryotic expression carrier pET-21a coding.This gene recombinaton pneumolysin still has hemolytic activity.

3. the present inventor is after obtaining the gene DNA of pneumolysin, designed have restricted enzyme NdeI ( CATATG) and XhoI ( CTCGAG) primer sequence 4 (SEQID NO.4) and the primer sequence 7 (SEQ ID NO.7) of recognition site, adopt round pcr to obtain having the gene of 11 amino acid whose pneumolysins of coding carboxy terminal deletion (protein shown in the SEQ ID NO.1 sequence) of SEQ ID NO.2 sequence the sequence table from pneumococcal chromosomal DNA amplification, and digest this PCR product and prokaryotic expression carrier pET-21a respectively with restricted enzyme NdeI and XhoI, be converted into then in e. coli bl21 (DE3) host cell, construct the gene recombination plasmid that to express 11 amino acid whose pneumolysins of carboxy terminal deletion.Induce conversion host bacterium to express this gene with isopropyl ss-D-thiogalactoside (IPTG), expression product is identified with sodium lauryl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE), comprise what of expressing quantity, and the form of expressing, be solubility or insolubility, with and hemolytic activity.

Adopt the broken bacterium of ultrasound wave behind a large amount of host of cultivation bacterium, obtain at 11 aminoacid of carboxy terminal deletion through the affine adsorption chromatography purification of Ni-NTA but have 6 amino acid whose gene recombinaton pneumolysins of group that come from prokaryotic expression carrier pET-21a coding.This gene recombinaton pneumolysin does not have hemolytic activity, but keeps immunogenicity.

Then, the amino method of employing reduction will not have hemolytic activity, 11 amino acid whose pneumolysins of carboxy terminal deletion and pneumococcal capsular polysaccharide are covalently bound, after soon pneumococcal capsular polysaccharide will produce active reactive group with the sodium periodate oxidation, 11 the amino acid whose pneumolysin albumen of carboxy terminal deletion and the cross-linking agent Cyanborohydride that add gene recombinaton, the two is crosslinked for stirring reaction 5 angels, reuse Sepharose 4B post carries out chromatography to this conjugate, obtain the higher conjugate of purity and also concentrated, prepare Pneumococcus polysaccharide protein coupling vaccine.

The present invention adopts technique for gene engineering that the c-terminus aminoacid of pneumolysin is excised singly, until its forfeiture hemolytic toxicity, it is covalently bound to adopt the amino method of reduction will lose the pneumolysin and the pneumococcal capsular polysaccharide of hemolytic activity then, makes GL-PP coupling vaccine.This vaccine is a protein carrier with the pneumolysin of no hemolytic activity, has avoided removing with formalin the step of pneumolysin hemolytic toxicity, has guaranteed the safety of vaccine, can be used for the prevention of otitis media of infant below 2 years old; Because hemolysin is pneumococcal oneself protein, thereby can avoid and other vaccines such as diphtheria vaccine, the contingent immune interference reaction in tetanus vaccine inoculation back; Hemolysin is that pneumococcal oneself protein can play supplementary function to its polysaccharide antigen in immunity, thereby strengthens the immanoprotection action of polysaccharide antigen; Pneumococcal infection has cross immunity protection effect to this vaccine to various serotypes, and can improve the immunological memory of pneumococcal infection is from now on replied.

Description of drawings

Fig. 1 is the amplification of pneumolysin encoding gene (Pn gene).

Fig. 2 shows the double digestion qualification result of Pn gene and expression vector pET-21a recombiant plasmid.

Fig. 3 shows the amplification of 5 amino acid whose Pn genes of carboxy terminal deletion.

Fig. 4 shows the amplification of 11 amino acid whose Pn genes of carboxy terminal deletion.

Fig. 5 shows that c-terminus lacks the double digestion qualification result of 5 or 11 amino acid whose Pn genes and expression vector pET-21a recombiant plasmid respectively.

Fig. 6 shows the result of pneumolysin (Pn) protein expression.

Fig. 7 shows the result of Pn protein purification.

Fig. 8 shows the result of 11 amino acid whose Pn protein purifications of carboxy terminal deletion.

Fig. 9 shows the immunoreactive Western-blot figure of 5 and 11 amino acid whose Pn protein-specifics of carboxy terminal deletion.

Figure 10 is the Sepharose 4B sieve chromatography collection of illustrative plates of gene recombinaton pneumolysin albumen (rPn).

Figure 11 is the Sepharose4B sieve chromatography collection of illustrative plates of pneumococcal capsular polysaccharide 19F serotype (PS19F).

Figure 12 is the sieve chromatography collection of illustrative plates of pneumococcal capsular polysaccharide 19F serotype and gene recombinaton pneumolysin protein conjugates (rPn-19F).

Figure 13 shows inductive serological specificity antibody mediated immunity responsing reaction (ELISA method mensuration) result behind the rPn-19F inoculation mice.

The specific embodiment

Below the present invention is further elaborated with embodiment.These embodiment only are used to illustrate the present invention, and scope of the present invention are not constituted any restriction.The main genetic engineering molecular biology method that adopts routine among the embodiment, these methods are well known to those of ordinary skill in the art, J. Sa nurse Brooker for example, D.W. the Russell is outstanding, Huang Peitang etc. translate: " molecular cloning experiment guide " (third edition, in August, 2002, Science Press publishes, Beijing) in relevant chapters and sections.Those of ordinary skills are according to embodiment once, the slightly modified and conversion be not difficult as the case may be and successfully implement the present invention, and these are revised and conversion all belongs to the scope of the application's claim.

Among the embodiment primer of PCR by Shanghai give birth to that worker's biotechnology company limited is synthetic, purification and mass spectrography identify correct; Reagent such as various restricted enzyme, other modification enzymes and correlated response buffer are available from Shanghai TaKaRa company; Chemical reagent is all available from Shanghai chemical reagents corporation.Streptococcus pneumoniae serotype 19F capsular polysaccharide is provided by Chengdu Inst. of Biological Products.

The clone and the amplification of embodiment 1 pneumolysin encoding gene (Pn gene)

The following primer a of pneumolysin sequence (GenBank accession no.X52474) design (SEQ ID NO. according to bibliographical information 4) and primer b (SEQ ID NO. 5):

Primer a:5 ' CG C ATA TGG CAA ATA AAG CAG TAA A 3 '

Primer b:5 ' GC C TCG AGC TAG TCA TTT TCT ACC TT 3 '

Primer a 5 ' end contain a NdeI ( CATATG) restriction endonuclease recognition sequence; Primer b 5 ' end contain an XhoI ( CTCGAG) restriction endonuclease recognition sequence.

Adopt primer a and primer b, the utilization PCR method increases from streptococcus pneumoniae DNA genome and obtains the Pn gene, the PCR condition enactment is: 94 ℃ * 5 minutes → (94 ℃ * 1 minute, 60 ℃ * 1 minute, 72 ℃ * 1 minute 30 seconds) * 35 take turns circulation → last and extended 8 minutes for 72 ℃, obtain the pneumolysin gene DNA sequence.After amplified reaction finishes, identify the PCR product,, then it is reclaimed and the row agarose gel electrophoresis inspection if above-mentioned pcr amplified fragment size is consistent with expection with 1% agarose gel electrophoresis and ethidium bromide staining method.

Fig. 1 shows the amplification of Pn gene.Wherein, swimming lane M is the DL2000 labelling; Swimming lane 1 is made up of 1,416 base pair (bp) for the Pn gene of amplification.

The structure of embodiment 2 recombinants

With behind the NdeI/XhoI double digestion, adopt the method for low-melting agarose gel electrophoresis to reclaim the pneumolysin gene dna fragmentation of amplification among the embodiment 1, connect same then through double digestion and in the pET-21a plasmid after reclaiming.With this recombinant plasmid transformed BL21 (DH3) competence Bacillus coli cells, transform back culture of bacteria extracting recombinant plasmid dna and do the double digestion evaluation; And measure its DNA sequence (going up the biological company limited of sea base health measures), identical with the pneumolysin sequence of bibliographical information.

Fig. 2 shows the double digestion qualification result of recombiant plasmid.Wherein, swimming lane M is molecular weight marker DL-15000; Swimming lane 1 is the NdeI/XhoI double digestion result of recombiant plasmid; Swimming lane 2 is the recombiant plasmid of enzyme action not; Swimming lane 3 is the NdeI/XhoI double digestion result of empty plasmid pET-21a.The result is presented at the Pn gene that one 1,416 base pair (bp) is arranged in the swimming lane 1.

Embodiment 3 induces the purification of expression of pneumolysin encoding gene and expression product:

In the host bacterium, induce the pneumolysin expression of gene with isopropyl ss-D-thiogalactoside (IPTG), expression product is identified with sodium lauryl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE), comprise what of expressing quantity, and the form of expressing, i.e. solubility or insolubility.Contain in the LB culture medium of kanamycin at 200mL,, cultivate on 37 ℃ of shaking tables, treat bacterial growth to exponential phase, i.e. bacterium liquid OD with the host bacterium of 1: 100 ratio inoculation expression Pn _600nmIn the time of in the scope of 0.6-0.8, IPTG with 1: 1000 ratio adding 1M induces (result that Fig. 6 shows the Pn protein expression), on shaking table, cultivated 4 hours again, getting 2mL bacterium liquid does and induces evaluation, centrifugal 15 minutes of residue bacterium liquid 10000rpm, collecting bacterial sediment, is that the binding buffer liquid of 20mL, pH8.0 is resuspended with cumulative volume.Through freeze thawing, promptly broken antibacterial---ultrasonic power is 375W in Ultrasonic Cell Disruptor after-20 ℃ melt in 37 ℃ of constant temperature water baths after freezing again, each 4 seconds of time, interval 3 seconds, totally 10 times.Freeze thawing and ultrasonication step repeat 5 times.13000rpm is centrifugal 30 minutes subsequently, and get the supernatant suspension and make affinitive layer purification with immobilization Ni resin, because the c-terminus of gene recombinaton Pn has 6 histidine, fixed nickel ion sequestration in their energy and the Ni resin.5mL supernatant suspension behind the broken bacterium is slowly added in the resin, and the rotation mixing is adorned post and is collected effluent after 90 minutes.For identifying that whether pneumolysin albumen is by the Ni resin absorption.With the resuspended washing resin of binding buffer liquid 4mL of pH8.0, mixing left standstill after 2 minutes, inhaled and removed supernatant.Repeated washing 3 times.The eluent that contains 20mM, 50mM, 100mM, 150mM, 200mM, 250mM, 300mM imidazoles that adds each 8mL successively carries out eluting, collects effluent respectively.Can be observed by SDS-PAGE, contain in the eluent of 100mM (the 5th swimming lane) and 150mM (the 6th swimming lane) imidazoles pneumolysin albumen can purification more than 90%.

The clone and the amplification of 5 amino acid whose pneumolysin encoding genes of embodiment 4 carboxy terminal deletions

Pneumolysin sequence (GenBank accession no.X52474) according to bibliographical information designs following primer C (SEQ ID NO.4) and primer sequence d (SEQ ID NO.6):

Primer c:5 ' CAT ATGGCA AAT AAA GCA GTA AAT GA 3 '

Primer d:5 ' CTC GAGATC CTC TAC CTG AGG ATA GA 3 '

Primer c 5 ' end contain a NdeI ( CAT ATG) restriction endonuclease recognition sequence; 5 ' the end of primer d contains the restriction endonuclease recognition sequence of an XhoI (CTC GAG).

Adopt primer c and primer d, the utilization PCR method amplifies 5 amino acid whose Pn encoding genes of carboxy terminal deletion from streptococcus pneumoniae DNA genome, the PCR condition enactment is: 94 ℃ * 5 minutes → (94 ℃ * 1 minute, 60 ℃ * 1 minute, 72 ℃ * 1 minute 30 seconds) * 35 take turns circulation → last and extended 8 minutes for 72 ℃, obtain the pneumolysin gene DNA sequence.After amplified reaction finishes, identify the PCR product,, then it is reclaimed and the row agarose gel electrophoresis inspection if above-mentioned pcr amplified fragment size is consistent with expection with 1% agarose gel electrophoresis and ethidium bromide staining method.

Fig. 3 shows the amplification of 5 amino acid whose Pn encoding genes of carboxy terminal deletion.Wherein, swimming lane M is the DL2000 labelling; Swimming lane 1 is made up of 1,398 base pair (bp) for 5 amino acid whose Pn encoding genes of c-terminus forfeiture of amplification.

5 amino acid whose pneumolysin encoding gene construction of recombinant plasmid of embodiment 5 carboxy terminal deletions:

With behind the NdeI/XhoI double digestion, adopt the low melting-point agarose gel electrophoresis to reclaim the pneumolysin gene dna fragmentation of amplification among the embodiment 4, connect same then through double digestion and in the pET-21a plasmid after reclaiming.With this recombinant plasmid transformed BL21 (DH3) competence Bacillus coli cells, cultivate transformed bacteria extracting recombinant plasmid dna and do the double digestion evaluation; And measure its DNA sequence (go up sea base health biological company limited measure), and compare with the pneumolysin encoding gene of bibliographical information, in 15 bases of carboxy terminal deletion, remainder is identical.

The clone and the amplification of 11 amino acid whose pneumolysin encoding genes of embodiment 6 carboxy terminal deletions

Pneumolysin sequence (GenBank accession no.X52474) design following primer e (SEQ ID NO.4) and primer f (SEQ ID NO.7) according to bibliographical information:

Primer e:5 ' CAT ATGGCA AAT AAA GCA GTA AAT GA 3 '

Primer f:5 ' CTC GAGGAG AGT TGT TCC CCA AAT AG 3 '

Primer e 5 ' end contain a NdeI ( CAT ATG) restriction endonuclease recognition sequence; The restriction endonuclease recognition sequence that contains an XhoI (CTC GAG) at the 5 ' end of primer f.

Adopt primer e and primer f, the utilization PCR method amplifies 11 amino acid whose Pn encoding genes of carboxy terminal deletion from streptococcus pneumoniae DNA genome, the PCR condition enactment is: 94 ℃ * 5 minutes → (94 ℃ * 1 minute, 60 ℃ * 1 minute, 72 ℃ * 1 minute 30 seconds) * 35 take turns circulation → last and extended 8 minutes for 72 ℃, obtain the pneumolysin gene DNA sequence.After amplified reaction finishes, identify the PCR product,, then reclaim and do the agarose gel electrophoresis inspection if above-mentioned pcr amplified fragment size is consistent with expection with 1% agarose gel electrophoresis and ethidium bromide staining method.

Fig. 4 is the amplification of 11 amino acid whose Pn encoding genes of carboxy terminal deletion.Wherein, swimming lane M is molecular weight marker DL2000; Swimming lane 1 is made up of 1,380 base pair (bp) for 11 amino acid whose Pn encoding genes of carboxy terminal deletion of amplification.

11 amino acid whose pneumolysin encoding gene construction of recombinant plasmid of embodiment 7 carboxy terminal deletions:

With behind the NdeI/XhoI double digestion, adopt the method for low-melting agarose gel electrophoresis to reclaim the pneumolysin gene dna fragmentation of amplification among the embodiment 4, connect same then through double digestion and in the pET-21a plasmid after reclaiming.With this recombinant plasmid transformed BL21 (DH3) competence Bacillus coli cells, cultivate transformed bacteria extracting recombinant plasmid dna and do the double digestion evaluation; And measure its DNA sequence (go up sea base health biological company limited measure) and be sequence shown in the SEQ ID NO.2, compare with the pneumolysin encoding gene of bibliographical information, in 33 bases of carboxy terminal deletion, remainder is identical.

Fig. 5 is the double digestion qualification result of 5 of carboxy terminal deletions or 11 amino acid whose pneumolysin encoding gene recombiant plasmid.Wherein, swimming lane M is molecular weight marker DL-15000; Swimming lane 1 is the NdeI/XhoI double digestion result of 11 amino acid whose recombiant plasmid of carboxy terminal deletion, and the dna fragmentation of a 1380bp is arranged; Swimming lane 2 is the NdeI/XhoI double digestion result of 5 amino acid whose recombiant plasmid of carboxy terminal deletion, and the dna fragmentation of a 1398bp is arranged.

The abduction delivering and the purification of 5 or 11 amino acid whose pneumolysins of embodiment 8 carboxy terminal deletions

Press the same method of embodiment 3, the coding recombination of embodiment 5 and 5 or 11 amino acid whose pneumolysins of 7 resulting carboxy terminal deletions is carried out abduction delivering and purification.

Fig. 8 A is the abduction delivering and the purification result of 11 amino acid whose pneumolysins of disappearance.Wherein, chant 1 and be whole cell lysate before inducing; Chant 2 for inducing back whole cell lysate; Chant 3 and be cellular lysate supernatant to be purified; Chant 4 and be the precipitation of the cellular lysate after the fragmentation; Chant 5 and wear liquid for the stream behind the application of sample; Chant 6 for the imidazoles eluent of 20mM; Chant 7 for the imidazoles eluent of 50mM; Chant 8 for the imidazoles eluent of 100mM; Chant 9 for the imidazoles eluent of 150mM;

Fig. 8 B is the abduction delivering and the purification result of 5 amino acid whose pneumolysins of disappearance.Wherein, chant 1 and be whole cell lysate before inducing; Chant 2 for inducing back whole cell lysate; Chant 3 for the imidazoles eluent of 150mM.

The mensuration of embodiment 9 pneumolysins and 5 or 11 amino acid whose pneumolysin hemolytic activities of carboxy terminal deletion

Will be O.8ml Freshman or rabbit erythrocyte add among the 39.2mlPBS (pH7.2), centrifugal 2 minutes of 3000r.p.m. abandons behind the supernatant resuspendedly with 39.2mlPBS, repeats 3 times the erythrocyte with the flush away fragmentation, makes 2% people or rabbit erythrocyte suspension.Respectively 160ngPn, 5 amino acid whose Pn of 160ng carboxy terminal deletion and 11 amino acid whose Pn of 40ng carboxy terminal deletion are added among the 1.8ml PBS, 10 times of doubling dilution to the 10 pipes, mix with 1ml 2% red blood cell suspension respectively, 37 ℃ of water-baths are hatched after 20 minutes with 10, centrifugal 2 minutes of 000r.p.m..Get and respectively manage supernatant and survey its OD at 541nm.Negative control is the mixed liquor of 1ml PBS and 1ml 2% red blood cell suspension, and PBS can not make erythroclasis; Positive control is the mixed liquor of 1ml deionized water and 1ml 2% red blood cell suspension, because the osmotic pressure principle, deionized water can make erythroclasis.The results are shown in Table 1.

5,11 aminoacid of table 1 pneumolysin and carboxy terminal deletion

The mensuration of pneumolysin hemolytic activity

11 amino acid whose Pn albumen of carboxy terminal deletion do not have hemolytic activity as can be seen from the above table.

Annotate: PN, pneumolysin; Aa, aminoacid.

5 or 11 amino acid whose streptococcus pneumoniae of embodiment 10 pneumolysins and carboxy terminal deletion

The immunity printing and dyeing of hemolysin are measured

After antigen made polyacrylamide gel electrophoresis (SDS-PAGE) and lose shape, transfer on the nitrocellulose filter, and dye with Ponceaux; After treating that the antigen band manifests, water stops dyeing, downcuts protein standard molecular weight band (Mark) and preserves; Remainder places on the shaking table with 5% defatted milk powder and sealed 1 hour.Then with 3%PBS-Tween20 washing 3 times, 5 minutes/time; The mice serum that adds dilution in 1: 1000 shakes reaction 1.5 hours; After washing 3 times, add alkali phosphatase (AP) the labelling goat-anti mice total IgG antibody (SigmanA3688) of dilution in 1: 8000, shake reaction 1.5 hours; Adding AP substrate after washing 3 times (BCIP/NBT: AP Buffer=4: 1) the liquid colour developing; At last with the PBS color development stopping that contains 1mM EDTA.

Fig. 9 is the specific immune response Western-blot of 11 amino acid whose pneumolysins of disappearance.Wherein, chant 1 low-molecular-weight standard protein; Chant 2 and be Pn albumen; Chant 3 and be 5 amino acid whose Pn albumen of disappearance; Chant 4 and be 11 amino acid whose Pn albumen of disappearance.

Crosslinked (is example with serotype 19F) of the gene recombinaton pneumolysin (rPN) of embodiment 11 pneumococcal capsular polysaccharides and no hemolytic toxicity

Pneumococcal capsular polysaccharide 19F solution and the 0.2ml concentration that with 4ml concentration is 5mg/ml is that the sodium periodate solution of 40mmol/L mixed back lucifuge oxidation after 10 minutes, gets 6.2 μ lEthyeuglycol and adds in the above-mentioned mixed solution and stop oxidation.PBS solution with 1000ml 0.15M was dialysed 1 hour, changed liquid altogether three times.The pneumolysin albumen rPN that in above-mentioned mixed solution, adds the 10mg gene recombinaton after dialysis finishes, be settled to 20ml, stir and add the cross-linking agent Cyanborohydride that 5ml concentration is 100mmol/l (being that final concentration is 20mmol/l) after 30 minutes again, stirred 5 days.Adopt Sepharose 4B post that conjugate is carried out sieve chromatography, to obtain the higher conjugate of purity and to be concentrated, the results are shown in Figure 10-12, the peak of simple pneumolysin albumen in conjugate peak or polysaccharide 19F obviously moves forward among the figure.

Experimental example 12 pneumococcal capsular polysaccharides-gene recombinaton pneumolysin protein conjugate inoculation mice inductive serological specificity antibody mediated immunity responsing reaction (ELISA method mensuration)

Material and method:

1. laboratory animal

After 8 week ages female BALB/C mice random packet, raise at SPF (Specific Pathogen Free) the level Animal House that The 2nd Army Medical College animal center in Shanghai provides.

2. the immunity inoculation of laboratory animal

With 20 of female BALB/C mice, be divided into 4 groups at random, every group 5: first group of matched group (not immune), second group of (usefulness 19F-rPN conjugate immunity, wherein 19F dosage is 1ug/ time), the 3rd group (with the immunity of 19F-rPN conjugate, 19F dosage is 3ug/ time), the 4th group (with the immunity of 19F-rPN conjugate, the 19F amount is 10ug/ time).Three groups of the mixed solutions in back respectively at lumbar injection 0.5ml conjugate and aluminum hydroxide adjuvant, every two all immunity once, totally three times, after the last immunity the 7th day, to put to death after the Animal Anesthesia and gather in the crops whole blood, the serum after centrifugal is in-20 ℃ of preservations.

3. adopt conventional ELISA indirect method to carry out anti-polysaccharide 19F and anti-pneumolysin detection of antibodies

Pneumolysin albumen (5 μ g/ml) with pure protein conjugate of pneumococcal capsular polysaccharide 19F-ovum gallinaceum (5 μ g/ml) or purification wraps by 96 hole ELISA Plate (50 μ l/ hole), and 4 ℃ of placements are spent the night, 0.1%PBS-polysorbas20 washing 3 times; Every hole was sealed 1 hour for 37 ℃ with 0.5% bovine serum albumin of 100 μ l, PBS-polysorbas20 washing 3 times; Mice serum was since 1: 50 doubling dilution to 1: 3200, add respectively in (50 μ l/ hole) in each hole, hatched 2 hours for 37 ℃, add after washing 3 times by PBS-polysorbas20 buffer 1: 10, and the alkaline phosphatase labelling goat-anti mice total IgG antibody of 000 dilution (Sigma, Cat#A3688), hatched 1.5 hours for 37 ℃, add the substrate colour developing after washing 3 times, use the NaOH cessation reaction of 3M after 30 minutes, and make OD with microplate reader _405nmDetect.The results are shown in Figure 13 and Figure 14.

The result of this experimental example shows: 19F-rPN GL-PP combined vaccine can be induced the specific antibody of anti-polysaccharide 19F and pneumolysin simultaneously in the mice body.

Sequence table

SEQUENCE?LISTING

＜110〉Subsidiary Hospital of Eye-Ear-Throat Department, Fudan Univ.

＜120〉Pneumococcus polysaccharide protein coupling vaccine and preparation method thereof

<130>051001

<160>7

<170>PatentIn?version?3.3

<210>1

<211>460

<212>PRT

＜213〉synthetic

<400>1

Met?Ala?Asn?Lys?Ala?Val?Asn?Asp?Phe?Ile?Leu?Ala?Met?Asn?Tyr?Asp

1 5 10 15

Lys?Lys?Lys?Leu?Leu?Thr?His?Gln?Gly?Glu?Ser?Ile?Glu?Asn?Arg?Phe

20 25 30

Ile?Lys?Glu?Gly?Asn?Gln?Leu?Pro?Asp?Glu?Phe?Val?Val?Ile?Glu?Arg

35 40 45

Lys?Lys?Arg?Ser?Leu?Ser?Thr?Asn?Thr?Ser?Asp?Ile?Ser?Val?Thr?Ala

50 55 60

Thr?Asn?Asp?Ser?Arg?Leu?Tyr?Pro?Gly?Ala?Leu?Leu?Val?Val?Asp?Glu

65 70 75 80

Thr?Leu?Leu?Glu?Asn?Asn?Pro?Thr?Leu?Leu?Ala?Val?Asp?Arg?Ala?Pro

85 90 95

Met?Thr?Tyr?Ser?Ile?Asp?Leu?Pro?Gly?Leu?Ala?Ser?Ser?Asp?Ser?Phe

100 105 110

Leu?Gln?Val?Glu?Asp?Pro?Ser?Asn?Ser?Ser?Val?Arg?Gly?Ala?Val?Asn

115 120 125

Asp?Leu?Leu?Ala?Lys?Trp?His?Gln?Asp?Tyr?Gly?Gln?Val?Asn?Asn?Val

130 135 140

Pro?Ala?Arg?Met?Gln?Tyr?Glu?Lys?Ile?Thr?Ala?His?Ser?Met?Glu?Gln

145 150 155 160

Leu?Lys?Val?Lys?Phe?Gly?Ser?Asp?Phe?Glu?Lys?Thr?Gly?Asn?Ser?Leu

165 170 175

Asp?Ile?Asp?Phe?Asn?Ser?Val?His?Ser?Gly?Glu?Lys?Gln?Ile?Gln?Ile

180 185 190

Val?Asn?Phe?Lys?Gln?Ile?Tyr?Tyr?Thr?Val?Ser?Val?Asp?Ala?Val?Lys

195 200 205

Asn?Pro?Gly?Asp?Val?Phe?Gln?Asp?Thr?Val?Thr?Val?Glu?Asp?Leu?Lys

210 215 220

Gln?Arg?Gly?Ile?Ser?Ala?Glu?Arg?Pro?Leu?Val?Tyr?Ile?Ser?Ser?Val

225 230 235 240

Ala?Tyr?Gly?Arg?Gln?Val?Tyr?Leu?Lys?Leu?Glu?Thr?Thr?Ser?Lys?Ser

245 250 255

Asp?Glu?Val?Glu?Ala?Ala?Phe?Glu?Ala?Leu?Ile?Lys?Gly?Val?Lys?Val

260 265 270

Ala?Pro?Gln?Thr?Glu?Trp?Lys?Gln?Ile?Leu?Asp?Asn?Thr?Glu?Val?Lys

275 280 285

Ala?Val?Ile?Leu?Gly?Gly?Asp?Pro?Ser?Ser?Gly?Ala?Arg?Val?Val?Thr

290 295 300

Gly?Lys?Val?Asp?Met?Val?Glu?Asp?Leu?Ile?Gln?Glu?Gly?Ser?Arg?Phe

305 310 315 320

Thr?Ala?Asp?His?Pro?Gly?Leu?Pro?Ile?Ser?Tyr?Thr?Thr?Ser?Phe?Leu

325 330 335

Arg?Asp?Asn?Val?Val?Ala?Thr?Phe?Gln?Asn?Ser?Thr?Asp?Tyr?Val?Glu

340 345 350

Thr?Lys?Val?Thr?Ala?Tyr?Arg?Asn?Gly?Asp?Leu?Leu?Leu?Asp?His?Ser

355 360 365

Gly?Ala?Tyr?Val?Ala?Gln?Tyr?Tyr?Ile?Thr?Trp?Asp?Glu?Leu?Ser?Tyr

370 375 380

Asp?His?Gln?Gly?Lys?Glu?Val?Leu?Thr?Pro?Lys?Ala?Trp?Asp?Arg?Asn

385 390 395 400

Gly?Gln?Asp?Leu?Thr?Ala?His?Phe?Thr?Thr?Ser?Ile?Pro?Leu?Lys?Gly

405 410 415

Asn?Val?Arg?Asn?Leu?Ser?Val?Lys?Ile?Arg?Glu?Cys?Thr?Gly?Leu?Ala

420 425 430

Trp?Glu?Trp?Trp?Arg?Thr?Val?Tyr?Glu?Lys?Thr?Asp?Leu?Pro?Leu?Val

435 440 445

Arg?Lys?Arg?Thr?Ile?Ser?Ile?Trp?Gly?Thr?Thr?Leu

450 455 460

<210>2

<211>1380

<212>DNA

＜213〉synthetic

<400>2

atggcaaata?aagcagtaaa?tgactttata?ctagctatga?attacgataa?aaagaaactc 60

ttgacccatc?agggagaaag?tattgaaaat?cgtttcatca?aagagggtaa?tcagctaccc 120

gatgagtttg?ttgttatcga?aagaaagaag?cggagcttgt?cgacaaatac?aagtgatatt 180

tctgtaacag?ctaccaacga?cagtcgcctc?tatcctggag?cacttctcgt?agtggatgag 240

accttgttag?agaataatcc?cactcttctt?gcggttgatc?gtgctccgat?gacttatagt 300

attgatttgc?ctggtttggc?aagtagcgat?agctttctcc?aagtggaaga?ccccagcaat 360

tcaagtgttc?gcggagcggt?aaacgatttg?ttggctaagt?ggcatcaaga?ttatggtcag 420

gtcaataatg?tcccagctag?aatgcagtat?gaaaaaataa?cggctcacag?catggaacaa 480

ctcaaggtca?agtttggttc?tgactttgaa?aagacaggga?attctcttga?tattgatttt 540

aactctgtcc?attcaggtga?aaagcagatt?cagattgtta?attttaagca?gatttattat 600

acagtcagcg?tagacgctgt?taaaaatcca?ggagatgtgt?ttcaagatac?tgtaacggta 660

gaggatttaa?aacagagagg?aatttctgca?gagcgtcctt?tggtctatat?ttcgagtgtt 720

gcttatgggc?gccaagtcta?tctcaagttg?gaaaccacga?gtaagagtga?tgaagtagag 780

gctgcttttg?aagctttgat?aaaaggagtc?aaggtagctc?ctcagacaga?gtggaagcag 840

attttggaca?atacagaagt?gaaggcggtt?attttagggg?gcgacccaag?ttcgggtgcc 900

cgagttgtaa?caggcaaggt?ggatatggta?gaggacttga?ttcaagaagg?cagtcgcttt 960

acagcagatc?atccaggctt?gccgatttcc?tatacaactt?cttttttacg?tgacaatgta 1020

gttgcgacct?ttcaaaacag?tacagactat?gttgagacta?aggttacagc?ttacagaaac 1080

ggagatttac?tgctggatca?tagtggtgcc?tatgttgccc?aatattatat?tacttgggat 1140

gaattatcct?atgatcatca?aggtaaggaa?gtcttgactc?ctaaggcttg?ggacagaaat 1200

gggcaggatt?tgacggctca?ctttaccact?agtattcctt?taaaagggaa?tgttcgtaat 1260

ctctctgtca?aaattagaga?gtgtaccggg?cttgcctggg?aatggtggcg?tacggtttat 1320

gaaaaaaccg?atttgccact?agtgcgtaag?cggacgattt?ctatttgggg?aacaactctc 1380

<210>3

<211>1416

<212>DNA

＜213〉streptococcus pneumoniae

<400>3

atggcaaata?aagcagtaaa?tgactttata?ctagctatga?attacgataa?aaagaaactc 60

ttgacccatc?agggagaaag?tattgaaaat?cgtttcatca?aagagggtaa?tcagctaccc 120

gatgagtttg?ttgttatcga?aagaaagaag?cggagcttgt?cgacaaatac?aagtgatatt 180

tctgtaacag?ctaccaacga?cagtcgcctc?tatcctggag?cacttctcgt?agtggatgag 240

accttgttag?agaataatcc?cactcttctt?gcggttgatc?gtgctccgat?gacttatagt 300

attgatttgc?ctggtttggc?aagtagcgat?agctttctcc?aagtggaaga?ccccagcaat 360

tcaagtgttc?gcggagcggt?aaacgatttg?ttggctaagt?ggcatcaaga?ttatggtcag 420

gtcaataatg?tcccagctag?aatgcagtat?gaaaaaataa?cggctcacag?catggaacaa 480

ctcaaggtca?agtttggttc?tgactttgaa?aagacaggga?attctcttga?tattgatttt 540

aactctgtcc?attcaggtga?aaagcagatt?cagattgtta?attttaagca?gatttattat 600

acagtcagcg?tagacgctgt?taaaaatcca?ggagatgtgt?ttcaagatac?tgtaacggta 660

gaggatttaa?aacagagagg?aatttctgca?gagcgtcctt?tggtctatat?ttcgagtgtt 720

gcttatgggc?gccaagtcta?tctcaagttg?gaaaccacga?gtaagagtga?tgaagtagag 780

gctgcttttg?aagctttgat?aaaaggagtc?aaggtagctc?ctcagacaga?gtggaagcag 840

attttggaca?atacagaagt?gaaggcggtt?attttagggg?gcgacccaag?ttcgggtgcc 900

cgagttgtaa?caggcaaggt?ggatatggta?gaggacttga?ttcaagaagg?cagtcgcttt 960

acagcagatc?atccaggctt?gccgatttcc?tatacaactt?cttttttacg?tgacaatgta 1020

gttgcgacct?ttcaaaacag?tacagactat?gttgagacta?aggttacagc?ttacagaaac 1080

ggagatttac?tgctggatca?tagtggtgcc?tatgttgccc?aatattatat?tacttgggat 1140

gaattatcct?atgatcatca?aggtaaggaa?gtcttgactc?ctaaggcttg?ggacagaaat 1200

gggcaggatt?tgacggctca?ctttaccact?agtattcctt?taaaagggaa?tgttcgtaat 1260

ctctctgtca?aaattagaga?gtgtaccggg?cttgcctggg?aatggtggcg?tacggtttat 1320

gaaaaaaccg?atttgccact?agtgcgtaag?cggacgattt?ctatttgggg?aacaactctc 1380

tatcctcagg?tagaggataa?ggtagaaaat?gactag 1416

<210>4

<211>25

<212>DNA

＜213〉synthetic

<400>4

cgcatatggc?aaataaagca?gtaaa 25

<210>5

<211>26

<212>DNA

＜213〉synthetic

<400>5

gcgagctcct?agtcattttc?tacctt 26

<210>6

<211>26

<212>DNA

＜213〉synthetic

<400>6

ctcgagatcc?tctacctgag?gataga 26

<210>7

<211>26

<212>DNA

＜213〉synthetic

<400>7

ctcgaggaga?gttgttcccc?aaatag 26

Claims (5)

1. Pneumococcus polysaccharide protein coupling vaccine, described vaccine is by reorganization pneumolysin covalently bound form of pneumococcal capsular polysaccharide with no hemolytic activity modification.

2. Pneumococcus polysaccharide protein coupling vaccine as claimed in claim 1, the reorganization pneumolysin of wherein said modification has sequence shown in the SEQ ID NO.1.

3,14,6B, 19F and 23F 3. Pneumococcus polysaccharide protein coupling vaccine as claimed in claim 1, wherein said pneumococcal capsular polysaccharide antigen comprises its following serotype:.

4. Pneumococcus polysaccharide protein coupling vaccine as claimed in claim 1, described vaccine are the otitis media vaccines.

5. the preparation method of Pneumococcus polysaccharide protein coupling vaccine according to claim 1 is characterized in that this method may further comprise the steps:

(1) obtains gene shown in the SEQ ID NO.2 with primer shown in primer shown in the SEQ ID NO.4 and the SEQ ID NO.7 with PCR amplification;

(2) with restricted enzyme Nde I and Xho I difference digestion step (1) gained gene and prokaryotic expression carrier pET-21a, be converted into after the connection in e. coli bl21 (DE3) host cell, be built into the engineering bacteria of recombinant plasmid transformed; Carry out the cultivation of this project bacterium;

(3) induce step (2) to transform gene shown in the engineering bacterium expression SEQIDNO.2 with isopropyl ss-D-thiogalactoside, the expression product that obtains is identified with the SDS-PAGE gel electrophoresis;

(4) with the affine adsorption chromatography purification of Ni-NTA expression product;

(5) with of the expression product mutually coupling of the amino method of reduction with pneumococcal capsular polysaccharide with step (4) gained of purification, and separation and purification, Pneumococcus polysaccharide protein coupling vaccine obtained.

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