CN103495161A - Mixture of poly-pneumococcal capsular polysaccharide-protein conjugates and preparation method of mixture - Google Patents
Mixture of poly-pneumococcal capsular polysaccharide-protein conjugates and preparation method of mixture Download PDFInfo
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Abstract
The invention discloses a mixture of poly-pneumococcal capsular polysaccharide-protein conjugates. The mixture contains 13 pneumococcal capsular polysaccharide-protein conjugates and an immunity-enhancement adjuvant, wherein each pneumococcal capsular polysaccharide-protein conjugate is formed by combining corresponding serum-type pneumococcal capsular polysaccharide with a same protein carrier through a covalent bond; the 13 pneumococcal capsular polysaccharides are obtained by purifying and extracting 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F bacteria; the protein carrier is A chain of a diphtherin mutant CRM197 obtained from expression of genetic recombinant escherichia coli. Meanwhile, the invention discloses a preparation method of the mixture. The mixture, compared to conventional bacterial fermentation total-chain diphtherin mutant CRM197, is easy to purify, high in yield and low in cost; experiments prove that the mixture disclosed by the invention is immunogenic, and is applicable to clinical inoculation.
Description
Technical field:
The present invention relates to a kind of mixture with immunogenic 13 kinds of pneumococcal capsular polysaccharide-protein conjugates and preparation method thereof.
Background technology:
The main victim of streptococcus pneumoniae property disease is the child, especially in developing country, have every year millions of children because suffering from an inflammation of the lungs, meningitis death.At present, vaccination is one of child protection effective way of avoiding pneumococcal bacteria invasion and attack.From chemical constitution, streptococcus pneumoniae has a cell surface capsular polysaccharide layer, and its function is to help the pathogenic infection host.Capsular polysaccharide can shield bacterial cell function of surface composition and avoid being identified by host immune system, prevents that complement system from being activated by bacterium surface albumen and immunocyte is engulfed, if antibacterial is engulfed, capsular polysaccharide can prevent that antibacterial is killed.The capsular polysaccharide that has different chemical structures in different streptococcus pneumoniae bacterial strains, produce multiple different serological type strain.Pneumonia due to streptococcus pneumoniae or meningitis are that a big chunk strain infection in 90 kinds of known serotypes causes.If prepare vaccine with the prevention pneumococcal infection with single serotype pneumococcal capsular polysaccharide, effect is limited; Therefore, want to improve preventive effect, should include the pneumococcal capsular polysaccharide of multiple different serotypes in vaccine.Result in clinical application proves, remarkable by the vaccine prevention effect of multiple pneumococcal capsular polysaccharide mixture making.But also there is following defect in such vaccine: first, polysaccharide with repetition chemical constitution is the immunogen of T cell self reliance type 2 classes, there is no the participation of T cell, they are can't the induction of immunity memory effect, stimulate body to produce IgM and IgG2 antibody, these antibody retention time in vivo are short, can not effectively protect body to avoid the invasion and attack of antibacterial; The second, this class vaccine can not induce infant that the age is less than 2 years old to produce immunne response to carry out prevent disease, and this crowd just immune system grow imperfection, the high-risk group who easily suffers from infectious disease.
At present, people have started polysaccharide antigen is combined and is prepared new generation vaccine with protein carrier, to improve the immunogenicity of polysaccharide.As John Robbins passes through popular haemophilus b type (Haemophilus influenzae type b, Hib) capsular polysaccharide (Polyribosylribitolphosphate, PRP) covalent bond is connected to protein carrier (tetanus toxoid) and prepares popular haemophilus b type polysaccharide PRP-tetanus toxoid conjugate (PRP-TT).When bacterial polysaccharides is connected to protein carrier with the form of covalent bond; because protein is a kind of T cell dependence antigen; the polysaccharide that covalent bond can be connected is transformed into T cell dependence antigen; thereby stimulate body to produce the specific IgG antibodies that is directed to this polysaccharide, the protection body is not subject to the infection of antibacterial.But because pneumonia or meningitis that streptococcus pneumoniae causes can be by due to multiple different serotypes or strain infection; and between each serotype or bacterial strain due to the difference of the chemical constitution of bacterium surface polysaccharide; its antibody does not have cross-immune reaction; so inoculate single serotype or bacterial strain combined vaccine, can't protect and be vaccinated the infection that human body avoids other serotype or bacterial strain.Therefore, the multivalent pneumococcal polysaccharide-protein combined vaccine is primary study direction from now on, Pfizer Inc.'s develop goes out 13 valency pneumococal polysaccharides-CRM197 combined vaccine, to prevent pneumonia and meningitis for the child, but this vaccine carrier protein CRM197 used is by the wild strain of fermentation culture diphtheria corynebacterium variant, carry out the purification acquisition from its fermentation liquid, it is long that this method prepares the CRM197 production cycle, yield poorly, production cost is high, cause this vaccine product with high costs, can't produce in enormous quantities, general public can't be accepted fancy price, therefore, can't apply widely.
Diphtheria toxin muton CRM 197 and diphtheria toxin, diphtherotoxin structural similarity, contain A chain and B chain.Sudden change because the 52nd aminoacid of the A chain of CRM197 occurs, become glutamic acid by glycine, and become anatoxine.Therefore, diphtheria toxin muton CRM 197 is by Pfizer for developing the protein carrier of polysaccharide-protein combined vaccine, and from the angle of medicine, its advantage is safety, nontoxic; With other protein carrier, as tetanus toxoid, diphtheria toxoid are compared, the surface that CRM197 is this albumen as the advantage of protein carrier has the amino of stabilizing amount, and amino is while being the synthetic conjugate of reduction amine method, the site of protein carrier and polysaccharide combination.And toxoid is processed due to process formaldehyde detoxification, the amino meristic variation of molecular surface is larger.The synthesis condition of setting up stable polysaccharide and protein bound needs carrier protein to have stable amino quantity.
Diphtheria toxin muton CRM 197 is that this variant can be secreted CRM197 to culture supernatant under suitable condition, and be purified into the purity albumen that is appropriate to synthetic combined vaccine protein carrier, is a challenge greatly by fermentation diphtheria toxin, diphtherotoxin variant.In addition, obtain a large amount of CRM197 albumen, need to carry out jumbo bacterial fermentation, its production cost is high.For this reason, obtaining CRM197 albumen by other production ways is the direction that people make great efforts, and by technique for gene engineering, with escherichia expression system, produces the first-selection that CRM197 albumen is existing biotechnology.Yet many results of study show, because the molecular weight of CRM197 albumen reaches 68kDa, with escherichia coli expression, CRM197 molecule great majority out are inclusion bodys of insolubility, the content of solubility is few, and the CRM197 albumen of solubility is the essential condition as the carrier protein of synthetic polysaccharide-protein conjugate.If inclusion body is become to the albumen of solubility, need to adopt the method for degeneration, renaturation to obtain, but the solubility CRM197 protein yield obtained by this technique is still very low, can't reduce the cost of producing vaccine, realizes cheap production in enormous quantities.
Summary of the invention:
Above-mentioned defect in view of prior art exists, the objective of the invention is mixture proposed a kind of polynary pneumococcal capsular polysaccharide-protein conjugate and preparation method thereof.
Purpose of the present invention will be achieved by the following technical programs:
A kind of mixture of polynary pneumococcal capsular polysaccharide-protein conjugate, the conjugate that comprises 13 kinds of pneumococcal capsular polysaccharide-protein and the immune adjuvant of enhancing, the conjugate of each pneumococcal capsular polysaccharide-protein is the combination by covalent bond and same protein carrier by corresponding serotype pneumococcal capsular polysaccharide, described 13 kinds of pneumococcal capsular polysaccharides are from streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F antibacterial obtain by purification, described protein carrier is to be expressed the A chain of the diphtheria toxin muton CRM 197 obtained by gene recombined escherichia coli.
Further, the nucleotides sequence of the A chain of described gene recombinaton diphtheria toxin muton CRM 197 is classified as:
1 GGCGCTGACG ATGTTGTTGA CTCCTCGAAA TCCTTTGTTA TGGAAAACTT CTCCTCCTAC
61 CACGGCACGA AACCGGGCTA TGTTGACTCT ATTCAGAAAG GCATCCAAAA ACCGAAGAGT
121 GGCACCCAGG GTAACTACGA TGACGATTGG AAGGAATTCT ACTCCACGGA CAATAAGTAT
181 GATGCGGCCG GCTACTCGGT CGACAACGAA AATCCGCTGA GCGGTAAAGC AGGCGGTGTG
241 GTTAAGGTTA CCTATCCGGG TCTGACGAAA GTTCTGGCGC TGAAGGTCGA TAACGCCGAA
301 ACCATTAAAA AGGAACTGGG CCTGTCACTG ACCGAACCGC TGATGGAACA AGTGGGTACG
361 GAAGAATTTA TCAAACGTTT CGGCGATGGT GCATCCCGCG TCGTGCTGTC ACTGCCGTTT
421 GCTGAAGGCA GCTCTAGTGT GGAATACATT AACAATTGGG AACAAGCAAA AGCTCTGAGC
481 GTTGAACTGG AAATCAATTT CGAAACGCGT GGCAAACGCG GTCAAGATGC TATGTATGAA
541 TATATGGCTC AGGCGTGTGC GGGCAATCGT GTGCGTCGCT AA。
Further, described adjuvant is aluminum phosphate or aluminium hydroxide;
The preparation method of the mixture of described polynary pneumococcal capsular polysaccharide-protein conjugate is as follows:
The first, the preparation of the A catenin of diphtheria toxin muton CRM 197
A chain nucleotide sequence with the synthetic CRM197 of PCR method, include the restriction endonuclease recognition sequence of NcoI enzyme action recognition site CCATGG and the restriction endonuclease recognition sequence that contains BamHI enzyme action recognition site GGATCC, the recognition sequence of above-mentioned two restricted enzyme is held and is connected with the 5 ' end and 3 ' of the A chain gene sequence of diphtheria toxin muton CRM 197 respectively, obtains the recombination fragment of the A chain of diphtheria toxin muton CRM 197;
By the A chain nucleotide gene fragment of the synthetic CRM197 of Nco I/Bam H digestion with restriction enzyme PCR method, after purification, in gene fragment clone to an expression vector that digestion is obtained, obtain the plasmid of the A catenin of expressing diphtheria toxin muton CRM 197;
The plasmid of the A catenin of the expression diphtheria toxin muton CRM 197 that step 2 is obtained proceeds in escherichia coli expression host BL21, obtain the escherichia coli expression bacterium of the A catenin of expressing diphtheria toxin muton CRM 197, the albumen after being cultivated, induce by the escherichia coli of the A catenin to diphtheria toxin muton CRM 197, separate from thalline, purification obtained the A chain of diphtheria toxin muton CRM 197.
The preparation of the second, 13 kind of serotype pneumococcal capsular polysaccharide
By 13 kinds of serotype streptococcus pneumoniae are fermented respectively, bacterium liquid is got centrifuged supernatant after separating, and carries out the purification of capsular polysaccharide, obtains corresponding serotype pneumococcal capsular polysaccharide;
The 3rd, 13 serotype pneumococcal capsular polysaccharide-protein conjugates synthetic
The A catenin covalent bond of the diphtheria toxin muton CRM 197 that the pneumococcal capsular polysaccharide of 13 kinds of purification of the second acquisition is obtained with first step respectively is connected, and obtains 13 kinds of pneumococcal capsular polysaccharide-protein conjugates;
The 4th, the preparation of the mixture of 13 kinds of pneumococcal capsular polysaccharide-protein conjugates
The 3rd 13 kinds of pneumococcal capsular polysaccharide-protein conjugates that obtain are carried out to even mixing with the adjuvant that strengthens the conjugate immunity and obtain object.
Outstanding effect of the present invention is:
(1) in mixture of the present invention, 13 kinds of different serotypes pneumococcal capsular polysaccharides have been contained the streptococcus pneumoniae that global major part causes human diseases, and immunogenicity is good;
(2) experiment showed, that usining the mixture of polynary pneumococcal capsular polysaccharide-protein conjugate prepared as protein carrier by the A chain of diphtheria toxin muton CRM 197 has immunogenicity, can be used for clinical inoculation;
(3) by the A catenin of the diphtheria toxin muton CRM 197 of expressing in gene recombined escherichia coli, with the corresponding CRM197 albumen that the wild strain fermentation of traditional antibacterial obtains, compare, not only output is high, easy purification, and also preparation cost is cheap.
In order to further illustrate the specific embodiment of the present invention, below illustrate embodiments of the present invention.
The accompanying drawing explanation:
Fig. 1 is the A catenin plasmid electrophoretogram of the expression diphtheria toxin muton CRM 197 with after Nco I and Bam HI digestion with restriction enzyme;
In figure, 1 swimming lane is plasmid, and 2 swimming lanes are the A catenin plasmid electrophoretogram of the expression diphtheria toxin muton CRM 197 with after Nco I and Bam HI digestion with restriction enzyme, 3 swimming lane molecular weight Marker, and 4 marker locations are 5000bp, 5 marker locations are 500bp;
The A catenin that Fig. 2 is diphtheria toxin muton CRM 197 is expressed bacterium cultured products and the SDS-PAGE glue collection of illustrative plates that contrasts the escherichia coli cultured products, and in figure, 6 swimming lanes are not contain the escherichia coli culture fluid of the A chain plasmid of diphtheria toxin muton CRM 197; The inoculum that 7 swimming lanes are the A chain plasmid escherichia coli expression bacterium that contains diphtheria toxin muton CRM 197; The supernatant of the broken centrifugal rear acquisition of bacterium of the inoculum that 8 swimming lanes are the A chain plasmid escherichia coli expression bacterium that contains diphtheria toxin muton CRM 197; The broken bacterium centrifugal sediment of the inoculum that 9 swimming lanes are the A chain plasmid escherichia coli expression bacterium that contains diphtheria toxin muton CRM 197; 10 refer to the A chain of diphtheria toxin, diphtherotoxin variant CRM197;
Fig. 3 is the Western blot calibrating electrophoretogram corresponding with Fig. 2, and in figure, 15 refer to the hybridization signal of the A chain warp Western blot of corresponding diphtheria toxin, diphtherotoxin variant CRM197;
After the A catenin renaturing inclusion bodies of Fig. 4 for the diphtheria toxin muton CRM 197 of expression acquisition, the SDS-PAGE electrophoretogram carried out; In figure, the inclusion body of the broken centrifugal rear acquisition of bacterium of the inoculum that 16 swimming lanes in the SDS-PAGE part are the A chain plasmid escherichia coli expression bacterium that contains diphtheria toxin muton CRM 197, the supernatant of centrifugal acquisition after renaturation; The inclusion body of the broken centrifugal rear acquisition of bacterium of the inoculum that 17 swimming lanes in the SDS-PAGE part are the A chain plasmid escherichia coli expression bacterium that contains diphtheria toxin muton CRM 197, the precipitate of centrifugal acquisition after renaturation; 18 swimming lanes in the PVDF part are that the corresponding Western blot of SDS-PAGE part analyzes collection of illustrative plates with 19 swimming lanes; 20 refer to the A chain of diphtheria toxin, diphtherotoxin variant CRM197, the 21st, and the hybridization signal of A chain on celluloid of diphtheria toxin, diphtherotoxin variant CRM197;
Fig. 5 is for expressing the A catenin inclusion body of the diphtheria toxin muton CRM 197 obtained, the centrifuged supernatant obtained after renaturation, the two-way immunodiffusion figure carried out; Wherein, 22 holes are anti-CRM197 albumen serum, and 23 holes are for expressing the A catenin of the diphtheria toxin muton CRM 197 obtained.
The specific embodiment:
Below illustrate the specific embodiment of the present invention, for embodiment product of the present invention and method thereof are made to the generality illustration, contribute to understand better the present invention, but can't limit the scope of the invention.Described experimental technique, if no special instructions, be conventional method in an embodiment; Described reagent and material, if no special instructions, all can obtain from commercial channels.
Embodiment 1: a kind of mixture of polynary pneumococcal capsular polysaccharide-protein conjugate, the conjugate that comprises 13 kinds of pneumococcal capsular polysaccharide-protein and aluminum phosphate adjuvant, the conjugate of each pneumococcal capsular polysaccharide-protein is the combination by covalent bond and same protein carrier by corresponding serotype pneumococcal capsular polysaccharide, described 13 kinds of pneumococcal capsular polysaccharides are from streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, in 19F and 23F inoculum, by purification, obtain, described protein carrier is to be expressed the A catenin of the diphtheria toxin muton CRM 197 obtained by gene recombined escherichia coli, the nucleotides sequence of the A catenin of described gene recombinaton diphtheria toxin muton CRM 197 is classified as:
1 GGCGCTGACG ATGTTGTTGA CTCCTCGAAA TCCTTTGTTA TGGAAAACTT CTCCTCCTAC
61 CACGGCACGA AACCGGGCTA TGTTGACTCT ATTCAGAAAG GCATCCAAAA ACCGAAGAGT
121 GGCACCCAGG GTAACTACGA TGACGATTGG AAGGAATTCT ACTCCACGGA CAATAAGTAT
181 GATGCGGCCG GCTACTCGGT CGACAACGAA AATCCGCTGA GCGGTAAAGC AGGCGGTGTG
241 GTTAAGGTTA CCTATCCGGG TCTGACGAAA GTTCTGGCGC TGAAGGTCGA TAACGCCGAA
301 ACCATTAAAA AGGAACTGGG CCTGTCACTG ACCGAACCGC TGATGGAACA AGTGGGTACG
361 GAAGAATTTA TCAAACGTTT CGGCGATGGT GCATCCCGCG TCGTGCTGTC ACTGCCGTTT
421 GCTGAAGGCA GCTCTAGTGT GGAATACATT AACAATTGGG AACAAGCAAA AGCTCTGAGC
481 GTTGAACTGG AAATCAATTT CGAAACGCGT GGCAAACGCG GTCAAGATGC TATGTATGAA
541 TATATGGCTC AGGCGTGTGC GGGCAATCGT GTGCGTCGCT AA。
Embodiment 2: change the adjuvant in embodiment 1 into aluminium hydroxide.
Described in embodiment 1, the preparation method of the mixture of polynary pneumococcal capsular polysaccharide-protein conjugate is as follows:
The first, the preparation of the A catenin of diphtheria toxin muton CRM 197
1, diphtheria toxin muton CRM 197 protein A chain amino acid sequence is confirmed
The diphtheria toxin muton CRM 197 protein A chain of DNA recombinant expression is that the amino acid polypeptide of 1 to 193 part in the diphtheria toxin, diphtherotoxin aminoacid sequence forms, and from GenBank, obtains the gene order that CRM197 is complete (HW71379), and its aminoacid sequence is as follows:
1 GADDVVDSSK SFVMENFSSY HGTKPGYVDS IQKGIQKPKS GTQGNYDDDW KEFYSTDNKY
61 DAAGYSVDNE NPLSGKAGGV VKVTYPGLTK VLALKVDNAE TIKKELGLSL TEPLMEQVGT
121 EEFIKRFGDG ASRVVLSLPF AEGSSSVEYI NNWEQAKALS VELEINFETR GKRGQDAMYE
181 YMAQACAGNR VRR
The original glycine (G) of encoding of the 52nd amino acids in this protein nucleic acid sequence, become glutamic acid (E);
2, gene optimization design
In order to improve the expression of escherichia expression system to these albumen, need to be optimized its nucleotide sequence, after the optimization of diphtheria toxin, diphtherotoxin G52E fragment, nucleotide sequence is as follows:
1 GGCGCTGACG ATGTTGTTGA CTCCTCGAAA TCCTTTGTTA TGGAAAACTT CTCCTCCTAC
61 CACGGCACGA AACCGGGCTA TGTTGACTCT ATTCAGAAAG GCATCCAAAA ACCGAAGAGT
121 GGCACCCAGG GTAACTACGA TGACGATTGG AAGGAATTCT ACTCCACGGA CAATAAGTAT
181 GATGCGGCCG GCTACTCGGT CGACAACGAA AATCCGCTGA GCGGTAAAGC AGGCGGTGTG
241 GTTAAGGTTA CCTATCCGGG TCTGACGAAA GTTCTGGCGC TGAAGGTCGA TAACGCCGAA
301 ACCATTAAAA AGGAACTGGG CCTGTCACTG ACCGAACCGC TGATGGAACA AGTGGGTACG
361 GAAGAATTTA TCAAACGTTT CGGCGATGGT GCATCCCGCG TCGTGCTGTC ACTGCCGTTT
421 GCTGAAGGCA GCTCTAGTGT GGAATACATT AACAATTGGG AACAAGCAAA AGCTCTGAGC
481 GTTGAACTGG AAATCAATTT CGAAACGCGT GGCAAACGCG GTCAAGATGC TATGTATGAA
541 TATATGGCTC AGGCGTGTGC GGGCAATCGT GTGCGTCGCT AA
Restriction endonuclease recognition sequence is connected confirmation with gene: Nco I enzyme action recognition site CCATGG, BamHI enzyme action recognition site GGATCC, (through the G52E gene order is analyzed, in sequence, without Nco I and BamHI restriction enzyme site), the synthetic complete sequence of G52E gene is as follows:
1 GGCGCTGACG ATGTTGTTGA CTCCTCGAAA TCCTTTGTTA TGGAAAACTT CTCCTCCTAC
61 CACGGCACGA AACCGGGCTA TGTTGACTCT ATTCAGAAAG GCATCCAAAA ACCGAAGAGT
121 GGCACCCAGG GTAACTACGA TGACGATTGG AAGGAATTCT ACTCCACGGA CAATAAGTAT
181 GATGCGGCCG GCTACTCGGT CGACAACGAA AATCCGCTGA GCGGTAAAGC AGGCGGTGTG
241 GTTAAGGTTA CCTATCCGGG TCTGACGAAA GTTCTGGCGC TGAAGGTCGA TAACGCCGAA
301 ACCATTAAAA AGGAACTGGG CCTGTCACTG ACCGAACCGC TGATGGAACA AGTGGGTACG
361 GAAGAATTTA TCAAACGTTT CGGCGATGGT GCATCCCGCG TCGTGCTGTC ACTGCCGTTT
421 GCTGAAGGCA GCTCTAGTGT GGAATACATT AACAATTGGG AACAAGCAAA AGCTCTGAGC
481 GTTGAACTGG AAATCAATTT CGAAACGCGT GGCAAACGCG GTCAAGATGC TATGTATGAA
541 TATATGGCTC AGGCGTGTGC GGGCAATCGT GTGCGTCGCT AA
3, host and carrier are selected
The carrier of e. coli bl21 expression system is to obtain by transformation, e. coli bl21 is current general escherichia coli recipient bacterium, be the lysogenic bacterium of the high efficient expression of a kind of energy, contain the T7 pol gene, the Lac UV5 promoter that t7 rna polymerase is induced by IPTG is controlled; Therefore, restructuring G52E albumen need be take IPTG as derivant.Multiple clone site in carrier contains Nco I, BamHI, and do not contain the point of contact of these two restricted enzyme in genes of interest, so, at this target gene 5 ' hold 3 ' add corresponding restriction endonuclease recognition sequence, thereby be cloned in carrier, form recombiant plasmid;
4, gene is synthetic
The gene of diphtheria toxin, diphtherotoxin variant CRM197 protein A chain is synthetic by DNA synthesizer, and carry out the gene confirmation by following detection method, enzyme action is identified: the diphtheria toxin, diphtherotoxin variant CRM197 protein A chain of getting recovery is expressed strain, be seeded on the LB minimal medium plate that contains the 50mg/ml kanamycin, picking list bacterium colony after 37 ℃ of overnight incubation, alkaline lysis method of extracting plasmid DNA, by Nco I and Bam HI double digestion recombiant plasmid, as shown in Figure 1, electrophoresis showed obtains about 5396bp and 593bp two bands, wherein, Lane1 is expression plasmid, Lane2 is for using Nco I and the postdigestive expression plasmid of Bam HI, Lane M is ladder, with expection target DNA fragment size, be consistent,
5, the conversion of recombiant plasmid and expression calibrating
Get the instantaneous 40 μ L distilled waters that add after centrifugal of plasmid dry powder and dissolve, after dissolving plasmid concentration be 100ng/ μ L.Getting a pipe competent cell is placed on ice, add 5 μ L to connect product, mix, place 20min on ice, after 42 ℃ of thermal shock 90s, be placed in rapidly 3min on ice, competent cell after previous step is processed joins rapidly in the 1mL LB fluid medium of 37 ℃ of preheatings, and shaken cultivation 1h in 37 ℃ of shaking tables, draw 100～300 μ L bacterium liquid, with spreading rod, bacterium liquid is evenly coated on the LB culture medium flat plate that contains kanamycin, be inverted overnight incubation for 37 ℃.Experimental result: after 37 ℃ of inversion overnight incubation, occur smooth surface on culture dish, neat in edge, medium sized faint yellow bacterium colony;
6, the screening of positive colony calibrating
Clear bright mellow and full single bacterium colony on picking LB flat board, be seeded in the aseptic and LB culture medium that contains kanamycin of 5ml, and under 37 ℃, 180rpm shakes speed and cultivates.Switching 5ml bacterium liquid contains in kanamycin LB culture medium to 500ml, and under 37 ℃, 180rpm shakes speed and cultivates, bacterium liquid OD
600during to 1.0 left and right (about 4h), the IPTG that adds 1.0M is 1.0mmol/L to final concentration, and under 20 ℃, 180rpm shakes speed and cultivates 6h.4 ℃, 4000rpm, centrifugal 30min, collect thalline.The thalline quality of weighing, as a volume, add the PBS of 5 times of thalline volumes resuspended.In bacterium liquid, add 5 X SDS-PAGE loading buffer to working concentration, boil 5-8min, 1000rpm, centrifugal 2min, get the supernatant loading, and SDS-PAGE detects expression, is accredited as the bacterial strain amplification culture of positive colony, saves as primordial seed;
7, protein expression analysis
Get primordial seed and be seeded in the LB culture medium that contains the 50mg/ml kanamycin, 37 ℃ of concussions are cultured to OD
600approximately 0.6 o'clock, by final concentration, 1mmol/L added IPTG, and after continuing to cultivate 2h, centrifugal collection thalline, add appropriate PBS, ultrasonication, and the centrifugal supernatant of collecting respectively, precipitation is carried out SDS-PAGE.And carry out Western blot analysis, the electrophoretogram result as shown in Figure 2, wherein, contrast applied sample amount 10 μ L, full bacterium applied sample amount 10 μ L, supernatant applied sample amount 20 μ L, precipitation applied sample amount 10 μ L, Western blot verification result as shown in Figure 3, wherein, contrast applied sample amount 5 μ L, full bacterium applied sample amount 5 μ L, supernatant applied sample amount 10 μ L, precipitation applied sample amount 5 μ L;
The above results is analyzed, through shake-flask culture, ultrasonication after the collection thalline, difference collecting precipitation and supernatant, electrophoresis result shows that the destination protein major part exists with the inclusion body form, with the soluble protein form, exist on a small quantity, Western blot shows that soluble protein and inclusion body are by hybridization signal;
8, renaturing inclusion bodies
8.1 thalline washing
Weigh the 40g wet thallus in the 500ml centrifuge tube, add the resuspended thalline of 400ml1xPBS, stirring suspension on magnetic stirring apparatus (about 10min), under 4 ℃, 9000rpm, centrifugal 15min, abandon supernatant, collects thalline, repeats above step twice;
8.2 bacterial cell disruption
In the thalline of small lot, add 400ml1xPBS to the thalline centrifuge tube, thalline/buffer (w/v)=1:10 carries out fragmentation, ultrasound condition: ice-water bath, ultrasonic 5 seconds, 5 seconds, gap, broken total time 30-40min on Ultrasound Instrument; Under 4 ℃, 9000rpm, centrifugal 30min; Abandon supernatant, collecting precipitation, add 400ml lavation buffer solution A, under room temperature on magnetic stirring apparatus stirring suspension (about 30min), under 4 ℃, 9000rpm, centrifugal 30min, abandon supernatant, collecting precipitation (being inclusion body), repeat above step, change successively lavation buffer solution B, C, D, deposit buffer;
8.3 renaturing inclusion bodies
Add the inclusion body (according to inclusion body weight/degeneration buffer volume (w/v)=1:30) after 240ml degeneration buffer extremely washs, under room temperature on magnetic stirring apparatus stirring and dissolving fully (about 30min), under 25 ℃, the centrifugal 30min of 12000rpm, collect supernatant, abandon precipitation, centrifuged supernatant is transferred in the 6-8KD bag filter, the sealing bag filter, put bag filter in 2L renaturation buffer 1, under room temperature, stir dialysed overnight on magnetic stirring apparatus, proceed to bag filter in 2L renaturation buffer 2 next day, stirring at room dialysis 8-10h, bag filter is proceeded in 2L renaturation buffer 3, the stirring at room dialysed overnight, proceed to bag filter in 2L renaturation buffer 4 next day, stirring at room dialysis 8-10h, bag filter is proceeded in 2L renaturation buffer 5, the stirring at room dialysed overnight.Proceed to bag filter in 2L deposit buffer next day, and stirring at room dialysis 8-10h changes deposit buffer secondary, the room temperature dialysed overnight, get the 1ml dialysis solution, the centrifugal 10min of room temperature 12000rpm, collect supernatant, survey protein concentration, experimental result, as shown in the SDS-PAGE electrophoresis detection in Fig. 4 left side, is got appropriate dialysis solution, the centrifugal 10min of room temperature 12000rpm, collect supernatant, sample detection.Carry out Western blot hybrid experiment, result is as shown in Fig. 4 right side;
Carry out two-way immunodiffusion test: get the step centrifuged supernatant, send test set to carry out two-way immunodiffusion test, as shown in Figure 5, testing result shows obvious immuning lines, wherein, No. 1 hole is the diphtheria toxin, diphtherotoxin variant CRM197 protein A chain after renaturation, and No. 2 holes are the diphtheria toxoid antiserum.
9, CRM197 protein A chain is expressed Escherichia coli fermentation
From colibacillus engineering work seed bank cryogenic refrigerator, take out a work seed pipe, at room temperature thaw; Be transferred in the culture medium of 50 milliliters the bacterium liquid in the seed pipe is aseptic, at 37 ℃, in the shaking table that to shake speed be 180rpm, be cultured to OD
600=1.0 left and right; By in the culture medium of bacterium liquid aseptic inoculation to 1 liter, at 37 ℃, in the shaking table that to shake speed be 180rpm, be cultured to OD
600=1.0 left and right; The inoculation seed liquor rises in 20 liters of culture medium in fermentation tank to 50-, under 37 ℃, under the 180rpm condition, is fermented, and works as OD
600during to 7-8, add IPTG to induce recombiant protein synthetic in antibacterial; After fermentation to 14 hour, stop fermentation, centrifugal, collect thalline stand-by;
10, CRM197 protein A chain purification
Weigh the 3g wet thallus in the 50ml centrifuge tube, add the resuspended thalline of 30ml1xPBS, stir 3min on magnetic stirring apparatus; Under 4 ℃, 4000rpm, centrifugal 10min, abandon supernatant, collects thalline; Repeat this step twice; Add 30ml1xPBS to the thalline centrifuge tube, carry out fragmentation on Ultrasound Instrument; Under 4 ℃, 10000rpm, centrifugal 10min; Collecting precipitation, abandon supernatant; Add the 30ml1xPBS buffer, stir 3min on magnetic stirring apparatus; Under 4 ℃, 4000rpm, centrifugal 10min; Abandon supernatant, collect inclusion body, add the inclusion body after 90ml degeneration buffer extremely washs, under 25 ℃, the centrifugal 30min of 10000rpm, collect supernatant, abandons precipitation; Centrifuged supernatant is transferred in the 6-8KD bag filter to the sealing bag filter; Put bag filter in 2L renaturation buffer 1, under room temperature, stir dialysed overnight on magnetic stirring apparatus; Proceed to bag filter in 2L renaturation buffer 2 next day, stirring at room dialysis 8-10h; Bag filter is proceeded in 2L renaturation buffer 3 to the stirring at room dialysed overnight; Proceed to bag filter in 2L renaturation buffer 4 next day, stirring at room dialysis 8-10h; Bag filter is proceeded in 2L renaturation buffer 5 to the stirring at room dialysed overnight; Proceed to bag filter in 2L deposit buffer next day, stirring at room dialysis 8-10h; Change deposit buffer secondary, the room temperature dialysed overnight; Get the 1ml dialysis solution, the centrifugal 10min of room temperature 12000rpm, collect supernatant, surveys protein concentration; DEAE glue post by the protein solution loading to pre-balance, use Gradient elution, and collect the destination protein peak; Then loading to Phenyl drainage column is further purified, and collects eluting peak; Last loading SP agarose gel post, collect eluting peak; The purification destination protein collect obtained is gone in bag filter, dialyses in the sodium chloride of 0.15M, after completing, be transferred under 4 ℃, store stand-by;
The preparation of the second, 13 kind of serotype pneumococcal capsular polysaccharide
By 13 kinds of serotype streptococcus pneumoniae are fermented respectively, bacterium liquid is got centrifuged supernatant after separating, and carries out the purification of capsular polysaccharide, obtains corresponding serotype pneumococcal capsular polysaccharide;
The 3rd, 13 kinds of serotype pneumococcal capsular polysaccharide-protein conjugates synthetic
The A catenin covalent bond of the diphtheria toxin muton CRM 197 that the pneumococcal capsular polysaccharide of 13 kinds of purification of the second acquisition is obtained with first step respectively is connected, and obtains 13 kinds of pneumococcal capsular polysaccharide-protein conjugates;
1) streptococcus pneumoniae serotype 1 capsular polysaccharide-CRM197 protein A catenin conjugate is synthetic
Take the Pn1 degradation of polysaccharide of 5mg in reaction bulb, the 1mol/L NaCl that measures 0.5ml adds in reaction bulb; Magnetic agitation is dissolved polysaccharide fully.Record the initial pH of polysaccharide solution, measure respectively appropriate CDAP solution, add in reaction bulb, stirring reaction 1.5min under room temperature, the pH of mensuration solution during 30s.1.5min after, pH to 9.5 with 0.2mol/L NaOH regulator solution, stirring reaction 3min under room temperature, maintain pH 9.5 with 0.2mol/L NaOH, cross after 3min immediately to the CRM197 protein A chain that adds 5mg in reaction bulb, at room temperature (25 ℃) stirring reaction 1h, measure 37.5 μ l2mol/L lysines and add in reaction bulb, with 0.1N HCl regulator solution pH to 9.0.Stirring reaction 30min under room temperature, reaction bulb is transferred to reaction under 4 ℃ to spend the night, reactant mixture is transferred in bag filter (MWCO6-8000), under 4 ℃, to 0.85%NaCl solution dialysis 3 times, 6L/ time, by reaction mixture 10000rpm, after centrifugal 10min, get supernatant after dialysis finishes, adopt the polysaccharide conjugate after Sepharose CL-4B gel column purification is dialysed, and collect the conjugate peak, sample censorship;
2) streptococcus pneumoniae serotype 3 capsular polysaccharides-CRM197 protein A link compound is synthetic
Take the Pn3 degradation of polysaccharide of 5mg in reaction bulb, measure 0.68ml1mol/L NaCl and add in reaction bulb; Magnetic agitation is dissolved polysaccharide fully, records the initial pH of polysaccharide solution, measures respectively appropriate CDAP solution, adds in reaction bulb stirring reaction 1.5min under room temperature, the pH of mensuration solution during 30s.1.5min after, pH to 9.5 with 0.2mol/L NaOH regulator solution, stirring reaction 3min under room temperature, maintain pH 9.5 with 0.2mol/L NaOH, cross after 3min immediately to adding 3mg CRM197 protein A chain, (25 ℃) stirring reaction 1h at room temperature, measuring 37.5 μ l2mol/L lysines adds in reaction bulb, with 0.1mol/L HCl regulator solution pH to 9.0, stirring reaction 30min under room temperature, reaction bulb is transferred to reaction under 4 ℃ to spend the night, reactant mixture is transferred in bag filter (MWCO6-8000), under 4 ℃, to 0.85%NaCl solution dialysis 3 times, 6L/ time, after dialysis finishes by reaction mixture 10000rpm, get supernatant after centrifugal 10min, adopt the polysaccharide conjugate after Sepharose CL-4B gel column purification is dialysed, and collection conjugate peak, the sampling censorship,
3) streptococcus pneumoniae serotype 4 capsular polysaccharides-CRM197 protein A link compound is synthetic
Take the 5mg activated polysaccharide to reaction bulb, measure the 0.5M sodium phosphate buffer of 100 μ l, add in reaction bulb, measure the CRM197 protein A chain of 5mg to reaction bulb, magnetic agitation is dissolved polysaccharide fully; Measure the 0.5ml pure water and add in reaction bulb, magnetic agitation mixes; Take the NaBH of 5.0mg
3(CN), add in reaction bulb, the dry bath that reaction system is placed in to 30 ℃ reacts 12h, after reaction finishes, the 0.15MNaCl solution that measures 1.5ml adds in reaction bulb, takes the sodium borohydride of 2.5mg, add in reaction bulb, reaction system is placed under 22 ℃ reacts 5h; Reactant mixture is transferred to bag filter (MWCO12-14Kd), under 4 ℃, to 0.15M sodium chloride solution dialysis 3 times, the liquid measure of dialysing 6L at every turn; By reaction mixture 10000rpm, after centrifugal 10min, get supernatant after dialysis finishes, adopt the polysaccharide conjugate after Sepharose CL-4B gel column purification is dialysed, and collect the conjugate peak, the sampling censorship;
4) streptococcus pneumoniae serotype 5 capsular polysaccharides-CRM197 protein A link compound is synthetic
Take the 5.0mg activated polysaccharide, be added in reaction bulb, to the 0.5M sodium phosphate buffer that adds 100 μ l in reaction bulb; Take the CRM197 protein A chain of 4.0mg, add in reaction bulb, measure the 0.5ml pure water and add in reaction bulb, magnetic agitation makes reactants dissolved, the pH of assaying reaction system; Take 5.0mgNaBH3(CN), add in reaction bulb; Reaction system is placed under room temperature and reacts 48 hours; Take the NaBH of 2.5mg, be dissolved in 10 μ l pure water, after mixing dissolve complete with liquid-transfering gun, add in reaction bulb; Reaction system is placed in to 23 ℃ of lower stirring reactions 5 hours; Reactant mixture is transferred in bag filter (MWCO6-8Kd), under 4 ℃, to 0.15M sodium chloride solution dialysis 3 times, within every 5 hours, changes liquid once; By reaction mixture 10000rpm, after centrifugal 10min, get supernatant after dialysis finishes, adopt the polysaccharide conjugate after Sepharose CL-4B gel column purification is dialysed, and collect the conjugate peak, the sampling censorship;
5) streptococcus pneumoniae serotype 6A capsular polysaccharide-CRM197 protein A link compound is synthetic
Take 6.0mg activation Pn6A polysaccharide, be dissolved in the 1mL purified water, be stirred to and dissolve the rear original ph of surveying fully; With 0.1M NaOH conditioned reaction liquid pH value to 7.0; To the CRM197 protein A chain that adds 4mg in reaction system, stirring and evenly mixing; Take the NaBH of 5.0mg
3(CN), add in above-mentioned reaction bulb, room temperature reaction 18 hours; Sampling censorship after reaction finishes; Take the NaBH of 2.7mg, add in above-mentioned reaction bulb, room temperature reaction 5 hours; Sampling censorship after reaction finishes; Reactant mixture is transferred to bag filter, under 4 ℃, to 0.15M sodium chloride solution dialysis 5 times, 6L/ time, by reaction mixture 10000rpm, after centrifugal 10min, get supernatant after dialysis finishes, adopt the polysaccharide conjugate after Sepharose CL-4B gel column purification is dialysed, and collect the conjugate peak, sample censorship;
6) streptococcus pneumoniae serotype 6B capsular polysaccharide-CRM197 protein A link compound is synthetic
The Pn6B that takes 5.0mg is dissolved in the 1mL purified water, is stirred to and dissolves the rear original ph of surveying fully; With 0.1M NaOH conditioned reaction liquid pH value to 7.0; To the CRM197 protein A chain that adds 2.5mg in reaction system, stirring and evenly mixing; Take the NaBH of 5.0mg
3(CN), add in above-mentioned reaction bulb, room temperature reaction 20 hours; Take the NaBH of 2.5mg, add in above-mentioned reaction bulb, room temperature reaction 6 hours; Reactant mixture is transferred to bag filter, under 4 ℃, to 0.15M NaCl solution dialysis 5 times, 6L/ time; By reaction mixture 10000rpm, after centrifugal 10min, get supernatant after dialysis finishes, adopt the polysaccharide conjugate after Sepharose CL-4B gel column purification is dialysed, and collect the conjugate peak, the sampling censorship;
7) streptococcus pneumoniae serotype 7F capsular polysaccharide-CRM197 protein A link compound is synthetic
Take the Pn7F polysaccharide of 10.0mg, be dissolved in the 1mL purified water, be stirred to fully and dissolve; Drip respectively 0.1M NaOH solution to polysaccharide solution, regulate pH to 7.0; To the CRM197 protein A chain that adds 3.5mg in reaction system, stirring and evenly mixing; Take the NaBH of 5.0mg
3(CN), add in above-mentioned reaction bulb, room temperature reaction 20 hours; Add 990 μ l pure water, stirring and evenly mixing in reaction bulb; Take the NaBH of 2.5mg, add in above-mentioned reaction bulb, room temperature reaction 6 hours; Reactant mixture is transferred to bag filter (MWCO6000-8000), under 4 ℃, to 5mM succinate/0.9% sodium chloride buffer dialysis 5 times, 6L/ time; By reaction mixture 10000rpm, after centrifugal 10min, get supernatant after dialysis finishes, adopt the polysaccharide conjugate after Sepharose CL-4B gel column purification is dialysed, and collect the conjugate peak, the sampling censorship.
8) streptococcus pneumoniae serotype 9V capsular polysaccharide-CRM197 protein A link compound is synthetic
Take the Pn9V activated polysaccharide of 10mg, measure the sodium phosphate buffer of 125 μ l, measure 125 μ l pure water and add in reaction bulb, magnetic agitation is dissolved polysaccharide fully; Measure the CRM197 protein A chain of 15mg to adding in reaction bulb, stirring and dissolving is complete; Take the NaBH of 10mg
3(CN), add in reaction bulb; Reaction system is placed under 22 ℃ and reacts 48 hours; Take the NaBH of 2.5mg
4, adding in reaction bulb, reaction is 5 hours under 22 ℃; By reaction mixture 10000rpm, get supernatant after centrifugal 10min, adopt the AKTA system, the polysaccharide conjugate after the dialysis of Sepharose CL-4B gel column purification, and collect in conjunction with peak;
9) streptococcus pneumoniae serotype 14 capsular polysaccharides-CRM197 protein A link compound is synthetic
Take the Pn14 activated polysaccharide of 5mg, measure the CRM197 protein A chain of 1ml3.9mg, add in reaction bulb, magnetic agitation is dissolved polysaccharide fully; Add weak reductant NaBH
3(CN), after 5mg, under 22 ℃, reaction is 48 hours; Add strong reductant NaBH
42.5mg under room temperature, reaction is 4 hours; Reaction mixture is transferred in bag filter (MWCO12-14Kd), with the dialysis solution rinse reaction bulb of 2ml; Under 4 ℃, to 0.15M sodium chloride solution dialysis 3 times, 6L/ time, within every 5 hours, change liquid once.Collect dialysis solution after dialysis finishes, 10000rpm, get supernatant after centrifugal 10min, the polysaccharide conjugate after the dialysis of employing Sepharose CL-4B gel column purification, and collect the conjugate peak;
10) streptococcus pneumoniae serotype 18C capsular polysaccharide-CRM197 protein A link compound is synthetic
Take the Pn18C degradation of polysaccharide of 5mg, dissolve with the 1mL1M sodium chloride solution; Survey its original ph after dissolve complete; Add appropriate CDAP solution, under room temperature, stir 1.5min, add the pH to 9.0 of 0.2M NaOH solution regulator solution, afterwards in room temperature reaction 3min; Add the CRM197 protein A chain of 10mg, under 25 ℃, react 45min; Reaction adds 37.5 μ l2M lysine solutions after finishing; Under 25 ℃, reaction 30min is placed on 4 ℃ of reactions and spends the night; Reaction mixture is gone in bag filter (MWCO6000-8000), under 4 ℃, to 0.85% sodium chloride solution dialysis, change liquid 3 times, 6L/ time, within every 5 hours, change liquid once, collect dialysis solution after dialysis finishes, the centrifugal 10min of 10000rpm, get supernatant, adopt the CL-4B gel-purified this in conjunction with polysaccharide, and collect the conjugate peak; Detect albumen and polyoses content in conjugate solution;
11) streptococcus pneumoniae serotype 19A capsular polysaccharide-CRM197 protein A link compound is synthetic
Take 10.0mg oxidation Pn19A polysaccharide, be dissolved in the buffer of 0.5mL, put bar magnet in reaction bulb, be stirred to polysaccharide and dissolve fully; The CRM197 protein A chain that adds 10mg; Stirring and evenly mixing, take the NaBH of 5.0mg
3(CN), be added in reaction bulb, at room temperature react 20 hours; Take the NaBH of 2.5mg
4, add in reaction bulb, at room temperature react 5 hours; Reaction mixture is gone in bag filter (MWCO6000-8000), under 4 ℃, to 0.85% sodium chloride solution dialysis, change liquid 3 times, 6L/ time, within every 5 hours, change liquid once; Collect dialysis solution after dialysis finishes, the centrifugal 10min of 10000rpm, get supernatant, adopt the CL-4B gel-purified this in conjunction with polysaccharide, and collect the conjugate peak; Detect albumen and polyoses content in conjugate solution;
12) streptococcus pneumoniae serotype 19F capsular polysaccharide-CRM197 protein A link compound is synthetic
Take 5.2mg oxidation Pn19F polysaccharide, be dissolved in the 1ml pure water, put bar magnet in reaction bulb, be stirred to polysaccharide under room temperature and dissolve fully on magnetic stirring apparatus; The CRM197 protein A chain that adds 3.0mg; After stirring and evenly mixing, take the NaBH of 4.9mg
3(CN), add and put in reaction bulb, keep stirring on magnetic stirring apparatus always; Under 18 ℃ of room temperatures, reaction is 24 hours; Take the NaBH of 2.5mg
4, be added to reaction bulb; Under 18 ℃ of calorstats, reaction is 5 hours; Reactant mixture is transferred to bag filter (MWCO12-14000), and under 4 ℃, to buffer dialysis 5 times, the liquid measure of at every turn dialysing 6L, change liquid once in every 5 hours; Collect dialysis solution after dialysis finishes, the centrifugal 10min of 10000rpm, get supernatant, adopt the CL-4B gel-purified this in conjunction with polysaccharide, and collect the conjugate peak; Detect albumen and polyoses content in conjugate solution;
13) streptococcus pneumoniae serotype 23F capsular polysaccharide-CRM197 protein A link compound is synthetic
Take 4.9mg oxidation Pn23F polysaccharide, be dissolved in the 1ml pure water, put bar magnet in reaction bulb, be stirred to polysaccharide under room temperature and dissolve fully on magnetic stirring apparatus; The CRM197 protein A chain that adds 5.0mg; Take the NaBH of 5.1mg
3(CN), add and put in reaction bulb, keep stirring on magnetic stirring apparatus always; Under 18 ℃ of calorstats, reaction is 17 hours; Take the NaBH of 2.5mg
4, be added to reaction bulb; Under 18 ℃ of calorstats, reaction is 5 hours; Reactant mixture is transferred to bag filter (MWCO12-14000), and under 4 ℃, to the dialysis of 0.15M sodium chloride solution, the liquid measure of at every turn dialysing 6L, change liquid once in every 5 hours, totally 5 times; Collect dialysis solution after dialysis finishes, the centrifugal 10min of 10000rpm, get supernatant, adopt the CL-4B gel-purified this in conjunction with polysaccharide, and collect the conjugate peak; Detect albumen and polyoses content in conjugate solution;
The 4th, the preparation of the mixture of polynary pneumococcal capsular polysaccharide-protein conjugate
With the Labscale ultrafiltration system of Millipore by 1,3,4,5,6A, 7F, 9V, 14,18C, 19A, 19F and 23F serotype conjugate solution are concentrated into polysaccharide concentration and are approximately 40mg/ml; 6B serotype conjugate solution concentration is concentrated into polysaccharide concentration and is approximately 80mg/ml; According to following form calculate add respective volume monovalent serum type conjugate solution to office preparation bottle:
In connection with 0.22 μ m film aseptic filtration for the thing mixed liquor; Add aluminum phosphate colloid, ultimate density is 125mg/ml; Be settled to final volume with buffer; Fill, the 0.5ml/ bottle;
The 5th, the mixture immunogenicity experiments of polynary pneumococcal capsular polysaccharide-protein conjugate:
(1) the injection mice detects the A catenin conjugate mixture preparation of 13 valency pneumococal polysaccharide-CRM197:
The CM57 that gets 5-6 week is 70 of mices, polynary pneumococal polysaccharide prepared by every injected in mice-CRM197 protein A chain combination vaccine, injection capacity be 0.1ml/ mice/time, the injection mice is divided into three groups, the polynary pneumococal polysaccharide of one group of injection the present invention preparation-CRM197 protein A chain vaccine, the PVC13(Pfizer of clinical use on another group injection market) in contrast, the 3rd group is blank to vaccine, and concrete vaccinate and collection immune serum flow sheet are as follows:
Gather mouse blood to centrifuge tube, at room temperature static 2 hours, under the 10000rpm condition centrifugal 10 minutes, draw centrifugal supernatant serum with liquid-transfering gun, be stored in 4 ℃ of refrigerators, to be checked;
(2) the ELISA method detects polysaccharide antibody titre in mice serum
Prepare respectively in 13 kinds of different serotypes pneumococal polysaccharide storing solution 1mg/ml(1 * PBS solution), be stored in refrigerator, dilute serotype pneumococal polysaccharide storage liquid to be checked to coated buffer 2-4 μ g/ml, add the coated solution of 100 μ l and be coated with elisa plate in each hole, at room temperature overnight incubation, wash 4 times with washing the plate buffer, add 100 μ l sealing buffer, at room temperature hatch 2 hours, wash 4 times with washing the plate buffer, can preserve one week under 4 ℃; The corresponding test serum 1:10 of injected in mice combined vaccine and control sample acquisition is diluted to working prototype serum, the dilution suitable multiple, join in elisa plate the first round, cumulative volume 200 μ l, start to carry out two times of serial dilutions downwards from first row, at room temperature hatch 2 hours, with washing the plate buffer, wash 4 times, add 100 μ l alkali phosphatase enzyme mark sheep anti-mouse antibodies (1:2000 dilution), at room temperature hatch 4 hours, wash 4 times with washing the plate buffer, add 100 μ l phosphoric acid-4-nitro phenyl ester disodium salt substrate solution, at 405nm, read dish;
Result shows, the immunogenicity of combined vaccine of the present invention is good, and the IgG antibody titer of injecting anti-specificity pneumococal polysaccharide in the mice serum after three pins is showing higher than IgG antibody titer in the serum of injection one pin and two pins; Inject each serotype antibody titer after three pins higher than more than 4 times of one pin of injection, meet the standard that WHO improves for the combined vaccine titre; With clinical use PVC13 vaccine on market, compare, the titre of each serotype antibody, after injection three pins, the mouse resisting anteserum titre approaches even better.
(3) opsono-cytophagic test (Opsonophagocytic Assay, OPA)
Opsono-cytophagic test is to estimate pneumococcal Polysaccharide Conjugate Vaccine fungicidal effectiveness method, according to UAB-MOPA " the many types of opsonophagocytosis bactericidal assay of streptococcus pneumoniae capsular polysaccharide specific antibody method ", the mouse immune serum obtained is tested;
OPA titre result is as following table:
| Serotype | Matched group | Test group (inject three pins after serum) |
| 1 | 16 | 2048 |
| 3 | 16 | 1024 |
| 4 | 32 | 2048 |
| 5 | 16 | 2048 |
| |
16 | 1024 |
| |
8 | 1024 |
| 7F | 32 | 2048 |
| |
16 | 1024 |
| 14 | 16 | 4096 |
| |
8 | 2048 |
| |
8 | 1024 |
| |
16 | 2048 |
| |
16 | 2048 |
Result of the test is visible, injects the mice serum after three pins and does not inject polynary polysaccharide conjugate vaccine group mice serum relatively, and the OPA titre of its antibody is significantly improved.
The present invention still has numerous embodiments, and all employing equivalents or equivalent transformation and all technical schemes of forming, within all dropping on protection scope of the present invention.
Claims (4)
1. the mixture of a polynary pneumococcal capsular polysaccharide-protein conjugate, it is characterized in that, the conjugate that comprises 13 kinds of pneumococcal capsular polysaccharide-protein and the immune adjuvant of enhancing, the conjugate of each pneumococcal capsular polysaccharide-protein is the combination by covalent bond and same protein carrier by corresponding serotype pneumococcal capsular polysaccharide, described 13 kinds of pneumococcal capsular polysaccharides are from streptococcus pneumoniae serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F antibacterial obtain by purification, described protein carrier is to be expressed the A chain of the diphtheria toxin muton CRM 197 obtained by gene recombined escherichia coli.
2. the mixture of polynary pneumococcal capsular polysaccharide-protein conjugate according to claim 1, is characterized in that, the nucleotides sequence of the A chain gene of described gene recombinaton diphtheria toxin muton CRM 197 is classified as:
1 GGCGCTGACG ATGTTGTTGA CTCCTCGAAA TCCTTTGTTA TGGAAAACTT CTCCTCCTAC
61 CACGGCACGA AACCGGGCTA TGTTGACTCT ATTCAGAAAG GCATCCAAAA ACCGAAGAGT
121 GGCACCCAGG GTAACTACGA TGACGATTGG AAGGAATTCT ACTCCACGGA CAATAAGTAT
181 GATGCGGCCG GCTACTCGGT CGACAACGAA AATCCGCTGA GCGGTAAAGC AGGCGGTGTG
241 GTTAAGGTTA CCTATCCGGG TCTGACGAAA GTTCTGGCGC TGAAGGTCGA TAACGCCGAA
301 ACCATTAAAA AGGAACTGGG CCTGTCACTG ACCGAACCGC TGATGGAACA AGTGGGTACG
361 GAAGAATTTA TCAAACGTTT CGGCGATGGT GCATCCCGCG TCGTGCTGTC ACTGCCGTTT
421 GCTGAAGGCA GCTCTAGTGT GGAATACATT AACAATTGGG AACAAGCAAA AGCTCTGAGC
481 GTTGAACTGG AAATCAATTT CGAAACGCGT GGCAAACGCG GTCAAGATGC TATGTATGAA
541 TATATGGCTC AGGCGTGTGC GGGCAATCGT GTGCGTCGCT AA。
3. the mixture of polynary pneumococcal capsular polysaccharide-protein conjugate according to claim 1, is characterized in that, described adjuvant is aluminum phosphate or aluminium hydroxide.
4. the preparation method of the mixture of described polynary pneumococcal capsular polysaccharide-protein conjugate is as follows:
The first, the preparation of the A catenin of diphtheria toxin muton CRM 197
Step 1, the structure of the A catenin genetic fragment of diphtheria toxin muton CRM 197
A chain nucleotide sequence with the synthetic CRM197 of PCR method, include the restriction endonuclease recognition sequence of Nco I enzyme action recognition site CCATGG and the restriction endonuclease recognition sequence that contains Bam H enzyme action recognition site GGATCC, the recognition sequence of above-mentioned two restricted enzyme is held and is connected with the 5 ' end and 3 ' of the A chain gene sequence of diphtheria toxin muton CRM 197 respectively, obtains the recombination fragment of the A chain of diphtheria toxin muton CRM 197;
Step 2, the plasmid construction of the A catenin of expression diphtheria toxin muton CRM 197
By the A chain nucleotide gene fragment of the synthetic CRM197 of Nco I/Bam H digestion with restriction enzyme PCR method, after purification, in gene fragment clone to an expression vector that digestion is obtained, obtain the plasmid of the A catenin of expressing diphtheria toxin muton CRM 197;
Step 3, the preparation of the A catenin of diphtheria toxin muton CRM 197
The plasmid of the A catenin of the expression diphtheria toxin muton CRM 197 that step 2 is obtained proceeds in escherichia coli expression host BL21, obtain the escherichia coli expression bacterium of the A catenin of expressing diphtheria toxin muton CRM 197, the A catenin after being cultivated, induce by the escherichia coli of the A catenin to diphtheria toxin muton CRM 197, separate from thalline, purification obtained diphtheria toxin muton CRM 197.
The preparation of the second, 13 kind of serotype pneumococcal capsular polysaccharide
By 13 kinds of serotype streptococcus pneumoniae are fermented respectively, bacterium liquid is got centrifuged supernatant after separating, and carries out the purification of capsular polysaccharide, obtains corresponding serotype pneumococcal capsular polysaccharide;
The 3rd, 13 serotype pneumococcal capsular polysaccharide-protein conjugates synthetic
The A catenin covalent bond of the diphtheria toxin muton CRM 197 that the pneumococcal capsular polysaccharide of 13 kinds of purification of the second acquisition is obtained with first step respectively is connected, and obtains 13 kinds of pneumococcal capsular polysaccharide-protein conjugates;
The 4th, the preparation of the mixture of 13 valency pneumococcal capsular polysaccharide-protein conjugates
The 3rd 13 kinds of pneumococcal capsular polysaccharide-protein conjugates that obtain are carried out to even mixing with the adjuvant that strengthens the conjugate immunity and obtain object.
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