CN103495161B - A kind of mixture and preparation method thereof of polynary pneumococcal capsular polysaccharide-protein conjugate - Google Patents
A kind of mixture and preparation method thereof of polynary pneumococcal capsular polysaccharide-protein conjugate Download PDFInfo
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Abstract
The invention discloses a kind of mixtures of polynary pneumococcal capsular polysaccharide-protein conjugate, include the adjuvant that 13 kinds of pneumococcal capsular polysaccharide-protein conjugates and enhancing are immune, each pneumococcal capsular polysaccharide-protein conjugate is passed through the combination of covalent bond and same protein carrier by corresponding Pneumococcal serotype capsular polysaccharide, 13 kinds of pneumococcal capsular polysaccharides are from Pneumococcus serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F bacterium is obtained by purification, the protein carrier is the A chain of the diphtheria toxin muton CRM 197 obtained by gene recombined escherichia coli expression.Also disclose preparation method.The easy purification compared with conventional bacteria ferments full chain diphtheria toxin muton CRM 197, yield is big, at low cost, the experiment proved that mixture of the present invention has immunogenicity, can be used for clinical inoculation.
Description
Technical field:
The present invention relates to a kind of mixtures of 13 with immunogenicity kind pneumococcal capsular polysaccharide-protein conjugate
And preparation method thereof.
Background technique:
The Main victims of pneumococcal disease are children, especially in developing country, there is millions of children every year
Cause suffers from an inflammation of the lungs, meningitis is dead.Currently, vaccine inoculation is one of the effective way for protecting children to invade from pneumococcal bacteria.From
It is seen in chemical structure, pneumococcus possesses a cell surface capsular polysaccharide layer, and function is to aid in pathogenic infection host.Pod
Film polysaccharide can shield bacterial cell surface functional component from being identified by host immune system, prevent complement system by bacterium table
Face protein activation and immunocyte phagocytosis, if bacterium is swallowed, capsular polysaccharide can prevent bacterium to be killed.Different pneumonia balls
With the capsular polysaccharide of different chemical structures in bacteria strain, a variety of different serological type strains are generated.Lung caused by pneumococcus
Scorching or meningitis be by a big chunk strain infection in known 90 kinds of serotype caused by.If with single serotype
Pneumococcal capsular polysaccharide prepares vaccine to prevent pneumococcal infection, and effect is limited；Therefore, to improve preventive effect,
It should be comprising there are many pneumococcal capsular polysaccharides of different serotypes in vaccine.Result in clinical application proves, with a variety of pneumonia
The vaccine prevention significant effect of coccus capsular polysaccharide mixture production.But there is also following defects for such vaccine: first, there is weight
The polysaccharide of multiple chemical structure is the immunogene of 2 class of T cell self, and the not participation of T cell, they are can not to induce immune note
Recall effect, stimulation body generates IgM and IgG2 antibody, and retention time is short in vivo for these antibody, cannot be effectively protected body
From the invasion of bacterium；Second, this kind of vaccine can not induce infant of the age less than 2 years old to generate immune response to prevent disease
Disease, and this crowd is exactly that developing immune system is not perfect, is easy to suffer from the people at highest risk of infectious disease.
Currently, people have started polysaccharide antigen preparing new generation vaccine in conjunction with protein carrier, to improve the immune of polysaccharide
Originality.If John Robbins is by by popular influenzae type (Haemophilus influenzae type b, Hib)
Capsular polysaccharide (Polyribosylribitolphosphate, PRP) is covalently bonded to protein carrier (tetanus toxoid)
Prepare popular influenzae type polysaccharide PRP- tetanus toxoid conjugate (PRP-TT).When bacterial polysaccharides are with covalent bond
Form be connected to protein carrier, since protein is a kind of T cell dependence antigen, can by the polysaccharide being covalently keyed turn
Become T cell dependence antigen, so that stimulating body to generate is directed to the specific IgG antibodies of the polysaccharide, protect body not by
The infection of bacterium.But since pneumonia caused by pneumococcus or meningitis can be by a variety of different serotypes or strain infection institutes
It causes, and due to the difference of the chemical structure of bacterial surface polysaccharides between each serotype or bacterial strain, antibody does not have cross immunity anti-
It answers, so serotype or bacterial strain combined vaccine that inoculation is single, can not protect and be vaccinated human body from other serotypes or bacterial strain
Infection.Therefore, multivalent pneumococcal polysaccharide-protein combined vaccine is focus on research direction from now on, and Pfizer Inc. is
13 valence pneumococal polysaccharide-CRM197 combined vaccines are successfully developed, to prevent pneumonia and meningitis for children, but it is this
Carrier protein CRM197 used in vaccine is purified from its fermentation liquid by fermented and cultured corynebacterium diphtheriae variant open country strain
It obtaining, this method causes the vaccine product with high costs come long, the low output for preparing the CRM197 production cycle, high production cost,
It can not produce in enormous quantities, general public can not receive fancy price；Therefore, it is impossible to widely promote and apply.
Diphtheria toxin muton CRM 197 is similar with diphtheria toxin structure, contains A chain and B chain.Due to the A chain of CRM197
The mutation that 52nd amino acid occurs, becomes glutamic acid by glycine, and becomes anatoxine.Therefore, diphtheria toxin is mutated
Body CRM197 is used to develop the protein carrier of polysaccharide-protein combined vaccine, from the perspective of drug, advantage by Pfizer
It is safety, it is nontoxic；It compares with other protein carriers, such as tetanus toxoid, diphtheria toxoid, CRM197 is as protein carrier
The advantages of be the albumen surface have stabilizing amount amino, and amino be reduction amine method synthesis conjugate when, protein carrier
The site combined with polysaccharide.And toxoid is since by the processing of formaldehyde detoxification, the amino amount variation of molecular surface is larger.It establishes
The synthesis condition that stable polysaccharide and protein combine needs carrier protein to have stable amino amount.
Diphtheria toxin muton CRM 197 is by the diphtheria toxin variant that ferments, which under suitable conditions can
Enough secretion CRM197 are purified into that be appropriate to the purity albumen of synthesis combined vaccine protein carrier be a pole to culture supernatant
Big challenge.In addition, to obtain a large amount of CRM197 albumen, need to carry out the bacterial fermentation of large capacity, high production cost.For
This, it is the direction that people make great efforts that CRM197 albumen is obtained by other production ways, passes through technique for gene engineering large intestine bar
Bacterium expression system produces the first choice that CRM197 albumen is existing biotechnology.However many results of study show due to
The molecular weight of CRM197 albumen reaches 68kDa, is insolubility with most of the CRM197 molecule that Bacillus coli expression comes out
Inclusion body, soluble content is few, and soluble CRM197 albumen is used as the carrier protein of synthesis polysaccharide-protein conjugate
Necessary condition.If inclusion body is become soluble albumen, need to obtain using the method for denaturation, renaturation, but logical
The soluble CRM197 protein yield for crossing technique acquisition is still very low, can not reduce the cost of production vaccine, realize cheap
Produce in enormous quantities.
Summary of the invention:
In view of drawbacks described above of the existing technology, the purpose of the present invention is to propose to a kind of polynary pneumococcal capsule is more
Sugar-protein conjugate mixture and preparation method thereof.
The purpose of the invention will be achieved through the following technical solutions:
A kind of mixture of polynary pneumococcal capsular polysaccharide-protein conjugate is more comprising 13 kinds of pneumococcal capsules
The adjuvant that sugar-protein conjugate and enhancing are immunized, each pneumococcal capsular polysaccharide-protein conjugate is by corresponding to
Pneumococcal serotype capsular polysaccharide pass through the combination of covalent bond and same protein carrier, 13 kinds of pneumococcal capsules
Polysaccharide is to pass through purification from Pneumococcus serotypes 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F bacterium
It obtains, the protein carrier is the A chain of the diphtheria toxin muton CRM 197 obtained by gene recombined escherichia coli expression.
Further, the nucleotide sequence of the A chain of the genetic recombination diphtheria toxin muton CRM 197 are 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。
Further, the adjuvant is aluminum phosphate or aluminium hydroxide；
The mixture of the polynary pneumococcal capsular polysaccharide-protein conjugate the preparation method is as follows:
First, the preparation of the A catenin of diphtheria toxin muton CRM 197
Step 1, the building of the A catenin genetic fragment of diphtheria toxin muton CRM 197
It include the limitation of NcoI digestion recognition site CCATGG with the A chain nucleotide sequence of PCR method synthesis CRM197
Property endonuclease recognition sequence and the restriction endonuclease recognition sequence containing BamHI digestion recognition site GGATCC, it is above-mentioned two
The identification sequence of restriction enzyme connects with 5 ' ends of the A chain gene sequence of diphtheria toxin muton CRM 197 and 3 ' ends respectively
It connects, obtains the recombination segment of the A chain of diphtheria toxin muton CRM 197；
Step 2, the plasmid construction of the A catenin of diphtheria toxin muton CRM 197 is expressed
It is pure with the A chain nucleotide gene segment of Nco I/Bam H digestion with restriction enzyme PCR method synthesis CRM197
After change, the gene fragment clone that digestion is obtained obtains the A of expression diphtheria toxin muton CRM 197 into an expression vector
The plasmid of catenin；
Step 3, the preparation of the A catenin of diphtheria toxin muton CRM 197
The plasmid of the A catenin for the expression diphtheria toxin muton CRM 197 that step 2 obtains is transferred to Bacillus coli expression
In host BL21, the Bacillus coli expression bacterium of the A catenin of expression diphtheria toxin muton CRM 197 is obtained, by diphtheria poison
It after the Escherichia coli of the A catenin of plain mutant CRM 197 are cultivated, induced, is separated from thallus, purifying obtains diphtheria poison
The albumen of the A chain of plain mutant CRM 197.
Second, the preparation of 13 kinds of Pneumococcal serotype capsular polysaccharides
By fermenting respectively to 13 kinds of Pneumococcal serotypes, after bacterium solution separation, centrifuged supernatant is taken, carries out pod membrane
The purifying of polysaccharide obtains corresponding Pneumococcal serotype capsular polysaccharide；
Third, 13 Pneumococcal serotype capsular polysaccharide-protein conjugate synthesis
The diphtheria toxin that the pneumococcal capsular polysaccharide for 13 kinds of purifying that second obtains is obtained with first step respectively is dashed forward
The A catenin of variant CRM197 is covalently keyed, and obtains 13 kinds of pneumococcal capsular polysaccharide-protein conjugates；
4th, the preparation of 13 kinds of pneumococcal capsular polysaccharide-protein conjugate mixtures
The adjuvant of 13 kinds of pneumococcal capsular polysaccharide-protein conjugates and enhancing conjugate immunity that third is obtained
Uniformly mix up to object.
Protrusion effect of the invention are as follows:
(1) in inventive mixture 13 kinds of different serotypes pneumococcal capsular polysaccharides cover the whole world largely lead to people
The pneumococcus of class disease, immunogenicity are good；
(2) it is demonstrated experimentally that the polynary pneumococcus prepared using the A chain of diphtheria toxin muton CRM 197 as protein carrier
Capsular polysaccharide-protein conjugate mixture has immunogenicity, can be used for clinical inoculation；
(3) by the A catenin for the diphtheria toxin muton CRM 197 expressed in gene recombined escherichia coli, with tradition
The corresponding CRM197 albumen that strain fermentation in bacterium open country obtains is compared, and not only yield is high, is easy purifying, and preparation cost is cheap.
Specific embodiment in order to further illustrate the present invention, is exemplified below embodiments of the present invention.
Detailed description of the invention:
Fig. 1 is the A chain with the expression diphtheria toxin muton CRM 197 after Nco I and Bam HI digestion with restriction enzyme
Albumen plasmid electrophoretogram；
In figure, 1 swimming lane is plasmid, and 2 swimming lanes are with the expression diphtheria poison after Nco I and Bam HI digestion with restriction enzyme
The A catenin plasmid electrophoretogram of plain mutant CRM 197,3 swimming lane molecular weight Marker, 4 marker locations are 5000bp, 5 mark positions
It is set to 500bp；
Fig. 2 is that the A catenin expression bacterium cultured products of diphtheria toxin muton CRM 197 are produced with Escherichia coli culture is compareed
The SDS-PAGE glue map of object, in figure, 6 swimming lanes are that the Escherichia coli of the A chain plasmid without diphtheria toxin muton CRM 197 are trained
Nutrient solution；7 swimming lanes are the inoculum that the A chain plasmids E. coli containing diphtheria toxin muton CRM 197 expresses bacterium；8 swimming lanes
It is obtained after the broken bacterium centrifugation of inoculum for expressing bacterium for the A chain plasmids E. coli containing diphtheria toxin muton CRM 197
Supernatant；9 swimming lanes are the broken bacterium of inoculum that the A chain plasmids E. coli containing diphtheria toxin muton CRM 197 expresses bacterium
Centrifugal sediment；10 refer to the A chain of diphtheria toxin variant CRM197；
Fig. 3 is that Western blot corresponding with Fig. 2 examines and determine electrophoretogram, and in figure, 15 refer to corresponding diphtheria toxin variation
The hybridization signal of the A chain warp Western blot of body CRM197；
Fig. 4 is the SDS- of progress after the A catenin renaturing inclusion bodies for the diphtheria toxin muton CRM 197 that expression obtains
PAGE electrophoretogram；In figure, 16 swimming lanes in the part SDS-PAGE are the A chain plasmid large intestine containing diphtheria toxin muton CRM 197
The inoculum of bacillus expression bacterium breaks the inclusion body obtained after bacterium centrifugation, and the supernatant of acquisition is centrifuged after renaturation；SDS-
17 swimming lanes in the part PAGE are the Bacteria Culture that the A chain plasmids E. coli containing diphtheria toxin muton CRM 197 expresses bacterium
Liquid breaks the inclusion body obtained after bacterium centrifugation, and the sediment of acquisition is centrifuged after renaturation；18 swimming lanes and 19 swimming lanes in the part PVDF
It is the corresponding Western blot analysis map in the part SDS-PAGE；20 refer to the A chain of diphtheria toxin variant CRM197,21
It is hybridization signal of the A chain of diphtheria toxin variant CRM197 on nitrocellulose；
Fig. 5 is the A catenin inclusion body for the diphtheria toxin muton CRM 197 that expression obtains, the centrifugation obtained after renaturation
Supernatant, the two-way Immune proliferation figure of progress；Wherein, 22 holes are anti-CRM197 albumen serum, and 23 holes are the diphtheria that expression obtains
The A catenin of Toxin mutants CRM197.
Specific embodiment:
Be exemplified below to a specific embodiment of the invention, for embodiment be only to product of the present invention and its side
Method makees generality illustration, helps to more fully understand the present invention, but be not limiting upon protection scope of the present invention.In embodiment
The experimental method is unless otherwise specified conventional method；The reagent and material unless otherwise specified can be from business
Approach obtains.
Embodiment 1: a kind of mixture of polynary pneumococcal capsular polysaccharide-protein conjugate includes 13 kinds of pneumonia balls
Bacterium capsular polysaccharide-protein conjugate and Aluminium phosphate adjuvant, each pneumococcal capsular polysaccharide-protein conjugate are
Pass through the combination of covalent bond and same protein carrier, 13 kinds of pneumonia balls by corresponding Pneumococcal serotype capsular polysaccharide
Bacterium capsular polysaccharide is from Pneumococcus serotypes 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F inoculum
In by purification obtain, the protein carrier be by gene recombined escherichia coli expression obtain diphtheria toxin mutation
The A catenin of CRM197, the nucleotide sequence of the A catenin of the genetic recombination diphtheria toxin muton CRM 197 are 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。
Embodiment 2: the adjuvant in embodiment 1 is changed to aluminium hydroxide.
The mixture of polynary pneumococcal capsular polysaccharide-protein conjugate described in embodiment 1 the preparation method is as follows:
First, the preparation of the A catenin of diphtheria toxin muton CRM 197
1, diphtheria toxin muton CRM 197 albumin A chain amino acid sequence confirms
The diphtheria toxin muton CRM 197 albumin A chain of DNA recombinant expression is by 1 in diphtheria toxin amino acid sequence
Amino acid polypeptide to 193 parts forms, and obtains the complete gene order of CRM197 (HW71379) from GenBank, amino acid
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 52nd amino acids in the protein nucleic acid sequence encode original glycine (G), become glutamic acid (E)；
2, gene optimization designs
In order to improve expression of the escherichia expression system to these albumen, need to optimize its nucleic acid sequence, it is white
Nucleic acid sequence is as follows after the optimization of larynx toxin G52E segment:
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 connect confirmation: Nco I digestion recognition site CCATGG, BamHI digestion with gene
Recognition site GGATCC, (by analyzing G52E gene order, in sequence, no Nco I and BamHI restriction enzyme site),
G52E gene chemical synthesis complete 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
3, host and carrier selection
The carrier of e. coli bl21 expression system is obtained by transformation, and e. coli bl21 is large intestine general at present
Bacillus recipient bacterium is a kind of lysogenic bacterium of energy high efficient expression, and containing T7 pol gene, t7 rna polymerase is lured by IPTG
The Lac UV5 promoter control led；Therefore, recombination G52E albumen need to be using IPTG as inducer.Multiple cloning sites in carrier contain
There are Nco I, BamHI, and do not contain the point of contact of the two restriction enzymes in target gene, so at the target gene 5 ' end
3 ' are added corresponding restriction endonuclease recognition sequence, form recombinant plasmid to be cloned into carrier；
4, gene chemical synthesis
The gene of diphtheria toxin variant CRM197 albumin A chain be synthesized by DNA synthesizer, and with following detection method into
Row gene confirmation, digestion identification: take recovery diphtheria toxin variant CRM197 albumin A chain express strain, be seeded to containing
On the LB minimal medium plate of 50mg/ml kanamycins, picking single colonie after 37 DEG C of overnight incubations, alkaline lysis method of extracting plasmid
DNA, by Nco I and Bam HI double digestion recombinant plasmid, as shown in Figure 1, electrophoresis showed obtains about 5396bp and 593bp two
Band, wherein Lane1 is expression plasmid, and Lane2 is with the postdigestive expression plasmid of Nco I and Bam HI, and Lane M is
Ladder is consistent with expected purpose DNA fragmentation size；
5, the conversion and expression calibrating of recombinant plasmid
The dissolution of 40 μ L distilled waters is added after taking plasmid dry powder brief centrifugation, dissolved plasmid concentration is 100ng/ μ L.
It takes a pipe competent cell to be placed on ice, 5 μ L connection products is added, mix, place 20min on ice, it is fast after 42 DEG C of thermal shock 90s
Speed is placed in 3min on ice, by the 1mL LB liquid mediums of previous step treated competent cell is quickly adding into 37 DEG C of preheatings
In, the shaken cultivation 1h in 37 DEG C of shaking tables, draw 100~300 μ L bacterium solutions, with spreading rod by bacterium solution be spread evenly across containing card that
On the LB culture medium flat plate of mycin, 37 DEG C of inversion overnight incubations.Experimental result: after 37 DEG C of inversion overnight incubations, go out on culture dish
Existing surface is smooth, neat in edge, medium sized faint yellow bacterium colony；
6, the screening calibrating of positive colony
Clear bright mellow and full single colonie on picking LB plate, is seeded in the sterile and containing kanamycin LB culture medium of 5ml,
At 37 DEG C, 180rpm shakes fast culture.5ml bacterium solution of transferring is into 500ml LB culture medium containing kanamycin, at 37 DEG C,
180rpm shakes fast culture, bacterium solution OD600When to 1.0 or so (about 4h), it is added the IPTG to final concentration of 1.0mmol/L of 1.0M, 20
At DEG C, 180rpm shakes speed culture 6h.4 DEG C, 4000rpm, it is centrifuged 30min, collects thallus.Weighing thallus quality, as an individual
Product, the PBS that 5 times of thallus volumes are added are resuspended.5 X SDS-PAGE loading buffer are added into bacterium solution to working concentration,
5-8min, 1000rpm are boiled, 2min is centrifuged, takes supernatant loading, SDS-PAGE detects expression, is accredited as positive colony
Bacterial strain expands culture, saves as primordial seed；
7, protein expression analysis
Primordial seed is taken to be seeded in the LB culture medium containing 50mg/ml kanamycins, 37 DEG C of shake cultures to OD600About
When 0.6, IPTG is added by final concentration 1mmol/L, continues after cultivating 2h, thalline were collected by centrifugation, it is added appropriate PBS, ultrasonication,
Supernatant is collected in centrifugation respectively, and precipitating carries out SDS-PAGE.And carry out Western blot analysis, electrophoretogram result as shown in Fig. 2,
Wherein, applied sample amount 10 μ L, complete 10 μ L of bacterium applied sample amount, 20 μ L of supernatant applied sample amount are compareed, 10 μ L, Western blot of applied sample amount is precipitated
Verification result is as shown in Figure 3, wherein control applied sample amount 5 μ L, complete 5 μ L of bacterium applied sample amount, 10 μ L of supernatant applied sample amount precipitate applied sample amount 5
μL；
The above results are analyzed, through shaking flask culture, ultrasonication after thallus is collected, collects precipitating and supernatant respectively,
Electrophoresis result shows that destination protein largely exists with inclusion bodies, exists in the form of soluble protein on a small quantity, Western
Blot shows soluble protein and inclusion body by hybridization signal；
8, renaturing inclusion bodies
8.1 cell washing
40g wet thallus weigh in 500ml centrifuge tube, 400ml1xPBS is added, thallus is resuspended, stirred on magnetic stirring apparatus
Suspension (about 10min) is mixed, at 4 DEG C, 9000rpm is centrifuged 15min, abandons supernatant, collects thallus, repeats above step twice；
8.2 bacterial cell disruption
400ml1xPBS is added in the thallus of small lot to thallus centrifuge tube, thallus/buffer (w/v)=1:10, in ultrasound
Be crushed on instrument, ultrasound condition: ice-water bath, ultrasound 5 seconds, gap 5 seconds are crushed total time 30-40min；At 4 DEG C,
9000rpm is centrifuged 30min；Supernatant is abandoned, precipitating is collected, 400ml washing buffer A is added, at room temperature on magnetic stirring apparatus
Stirring suspension (about 30min), at 4 DEG C, 9000rpm is centrifuged 30min, abandons supernatant, collects precipitating (i.e. inclusion body), repeats
Above step successively replaces washing buffer B, C, D, stock buffer；
8.3 renaturing inclusion bodies
Inclusion body of the 240ml denaturation buffer to after washing is added (according to forgiving body weight/denaturation buffer volume (w/
V)=1:30), completely (about 30min), at 25 DEG C, 12000rpm is centrifuged stirring and dissolving on magnetic stirring apparatus at room temperature
30min, collect supernatant, abandon precipitating, centrifuged supernatant is transferred in 6-8KD bag filter, close bag filter, set bag filter in
In 2L renaturation buffer 1, at room temperature, dialysed overnight is stirred on magnetic stirring apparatus, bag filter is transferred to 2L renaturation and buffered by next day
In liquid 2, dialysis 8-10h is stirred at room temperature, bag filter is transferred in 2L renaturation buffer 3, dialysed overnight is stirred at room temperature, next day will be saturating
Analysis bag is transferred in 2L renaturation buffer 4, and dialysis 8-10h is stirred at room temperature, bag filter is transferred in 2L renaturation buffer 5, room temperature is stirred
Mix dialysed overnight.Bag filter is transferred in 2L stock buffer by next day, and dialysis 8-10h is stirred at room temperature, replaces stock buffer two
Secondary, room temperature dialysed overnight takes 1ml dialyzate, and room temperature 12000rpm is centrifuged 10min, collects supernatant, surveys protein concentration, experiment
As a result shown in the SDS-PAGE electrophoresis detection on the left of Fig. 4, appropriate dialyzate is taken, room temperature 12000rpm is centrifuged 10min, in collection
Clearly, sample detection.Western blot hybrid experiment is carried out, as a result as shown in the right side Fig. 4；
It carrying out two-way immunodiffusion test: taking step centrifuged supernatant, inspection survey group carries out two-way immunodiffusion test,
As shown in figure 5, there are apparent immuning lines in testing result display, wherein No. 1 hole is the diphtheria toxin variant after renaturation
CRM197 albumin A chain, No. 2 holes are diphtheria toxoid antiserum.
9, CRM197 albumin A chain expresses Escherichia coli fermentation
From colibacillus engineering work seed bank low temperature refrigerator, a work seed pipe is taken out, is thawed at room temperature；It will
In the sterile culture medium for being transferred to 50 milliliters of bacterium solution in seed pipe, at 37 DEG C, shake speed be 180rpm shaking table in cultivate to
OD600=1.0 or so；By in the culture medium of bacterium solution aseptic inoculation to 1 liter, at 37 DEG C, shake cultivated in the shaking table that speed is 180rpm to
OD600=1.0 or so；Be inoculated with seed liquor to 50- rise fermentor in 20 liters of culture mediums in, at 37 DEG C, under the conditions of 180rpm into
Row fermentation, works as OD600When to 7-8, IPTG induction recombinant protein is added and is synthesized in bacterium；It ferments to after 14 hours, stops hair
It is stand-by to collect thallus for ferment, centrifugation；
10, CRM197 albumin A chain purifies
3g wet thallus weigh in 50ml centrifuge tube, 30ml1xPBS is added, thallus is resuspended, stirred on magnetic stirring apparatus
3min；At 4 DEG C, 4000rpm is centrifuged 10min, abandons supernatant, collects thallus；Repeat the step twice；30ml1xPBS is added extremely
Thallus centrifuge tube, is crushed in Ultrasound Instrument；At 4 DEG C, 10000rpm is centrifuged 10min；Precipitating is collected, supernatant is abandoned；Add
Enter 30ml1xPBS buffer, 3min is stirred on magnetic stirring apparatus；At 4 DEG C, 4000rpm is centrifuged 10min；Supernatant is abandoned,
Inclusion body is collected, inclusion body of the 90ml denaturation buffer to after washing is added, at 25 DEG C, 10000rpm is centrifuged 30min, collects
Supernatant abandons precipitating；Centrifuged supernatant is transferred in 6-8KD bag filter, bag filter is closed；Bag filter is set to buffer in 2L renaturation
In liquid 1, at room temperature, dialysed overnight is stirred on magnetic stirring apparatus；Bag filter is transferred in 2L renaturation buffer 2 by next day, room temperature
Stirring dialysis 8-10h；Bag filter is transferred in 2L renaturation buffer 3, dialysed overnight is stirred at room temperature；Bag filter is transferred to 2L by next day
In renaturation buffer 4, dialysis 8-10h is stirred at room temperature；Bag filter is transferred in 2L renaturation buffer 5, dialysed overnight is stirred at room temperature；
Bag filter is transferred in 2L stock buffer by next day, and dialysis 8-10h is stirred at room temperature；It is secondary to replace stock buffer, room temperature dialysis
Overnight；1ml dialyzate is taken, room temperature 12000rpm is centrifuged 10min, collects supernatant, surveys protein concentration；Extremely by protein solution loading
The DEAE rubber column gel column of pre-balance with Gradient elution, and collects destination protein peak；Then loading is further to Phenyl drainage column
Eluting peak is collected in purifying；Last loading SP agarose Gel column, collects eluting peak；The purifying destination protein obtained will be collected to turn
It into bag filter, dialyses in the sodium chloride of 0.15M, is transferred at 4 DEG C and is stored for use after the completion；
Second, the preparation of 13 kinds of Pneumococcal serotype capsular polysaccharides
By fermenting respectively to 13 kinds of Pneumococcal serotypes, after bacterium solution separation, centrifuged supernatant is taken, carries out pod membrane
The purifying of polysaccharide obtains corresponding Pneumococcal serotype capsular polysaccharide；
Third, 13 kinds of Pneumococcal serotype capsular polysaccharide-protein conjugate synthesis
The diphtheria toxin that the pneumococcal capsular polysaccharide for 13 kinds of purifying that second obtains is obtained with first step respectively is dashed forward
The A catenin of variant CRM197 is covalently keyed, and obtains 13 kinds of pneumococcal capsular polysaccharide-protein conjugates；
1) 1 capsular polysaccharide-CRM197 albumin A catenin conjugate of Pneumococcus serotypes synthesizes
The Pn1 degradation of polysaccharide of 5mg is weighed in reaction flask, the 1mol/L NaCl for measuring 0.5ml is added in reaction flask；Magnetic
Power stirring is completely dissolved polysaccharide.The initial pH for recording polysaccharide solution measures suitable CDAP solution respectively, and reaction flask is added
In, it is stirred to react 1.5min at room temperature, when 30s measures the pH of solution.After 1.5min, solution is adjusted with 0.2mol/L NaOH
PH to 9.5, is stirred to react 3min at room temperature, maintains pH 9.5 with 0.2mol/L NaOH, crosses after 3min immediately into reaction flask
The CRM197 albumin A chain of 5mg is added, (25 DEG C) are stirred to react 1h at room temperature, measure 37.5 μ l2mol/L lysines and are added instead
It answers in bottle, adjusts pH value of solution to 9.0 with 0.1N HCl.It is stirred to react 30min at room temperature, reaction flask is transferred at 4 DEG C and is reacted
Overnight, reaction mixture is transferred in bag filter (MWCO6-8000), at 4 DEG C, is dialysed 3 times to 0.85%NaCl solution,
6L/ times, by reaction mixture 10000rpm after dialysis, supernatant is taken after being centrifuged 10min, using Sepharose CL-4B
Polysaccharide conjugate after the dialysis of gel column purification, and conjugate peak is collected, sample inspection；
2) 3 capsular polysaccharide-CRM197 albumin A chain combination object of Pneumococcus serotypes synthesizes
The Pn3 degradation of polysaccharide of 5mg is weighed in reaction flask, 0.68ml1mol/L NaCl is measured and is added in reaction flask；Magnetic force
Stirring is completely dissolved polysaccharide, records the initial pH of polysaccharide solution, measures suitable CDAP solution respectively, is added in reaction flask,
It is stirred to react 1.5min at room temperature, when 30s measures the pH of solution.After 1.5min, with 0.2mol/L NaOH adjust solution pH to
9.5, it is stirred to react 3min at room temperature, maintains pH 9.5 with 0.2mol/L NaOH, crosses after 3min immediately to addition 3mg
CRM197 albumin A chain, (25 DEG C) are stirred to react 1h at room temperature, measure 37.5 μ l2mol/L lysines and are added in reaction flask, use
0.1mol/L HCl adjusts pH value of solution to 9.0, is stirred to react 30min at room temperature, and reaction flask is transferred at 4 DEG C and is reacted overnight,
Reaction mixture is transferred in bag filter (MWCO6-8000), at 4 DEG C, is dialysed 3 times to 0.85%NaCl solution, 6L/ times,
By reaction mixture 10000rpm after dialysis, supernatant is taken after being centrifuged 10min, using Sepharose CL-4B gel column
Polysaccharide conjugate after purifying dialysis, and conjugate peak is collected, sample inspection；
3) 4 capsular polysaccharide-CRM197 albumin A chain combination object of Pneumococcus serotypes synthesizes
5mg activated polysaccharide is weighed into reaction flask, measures the 0.5M sodium phosphate buffer of 100 μ l, is added in reaction flask, amount
Take the CRM197 albumin A chain of 5mg into reaction flask, magnetic agitation is completely dissolved polysaccharide；It measures 0.5ml pure water and reaction flask is added
In, magnetic agitation mixes；Weigh the NaBH of 5.0mg3(CN), it is added in reaction flask, reaction system is placed in 30 DEG C of dry bath device
Middle reaction 12h, after reaction, the 0.15MNaCl solution for measuring 1.5ml are added in reaction flask, weigh the sodium of 2.5mg
Borohydride is added in reaction flask, and reaction system, which is placed at 22 DEG C, reacts 5h；Reaction mixture is transferred to bag filter
(MWCO12-14Kd), it at 4 DEG C, dialyses 3 times to 0.15M sodium chloride solution, dialyse liquid measure 6L every time；
By reaction mixture 10000rpm after dialysis, supernatant is taken after being centrifuged 10min, using Sepharose CL-4B gel column
Polysaccharide conjugate after purifying dialysis, and conjugate peak is collected, sample inspection；
4) 5 capsular polysaccharide-CRM197 albumin A chain combination object of Pneumococcus serotypes synthesizes
5.0mg activated polysaccharide is weighed, is added into reaction flask, the 0.5M sodium phosphate buffer of 100 μ l is added into reaction flask
Liquid；The CRM197 albumin A chain of 4.0mg is weighed, is added in reaction flask, 0.5ml pure water is measured and is added in reaction flask, magnetic agitation makes
Reactant dissolution, measures the pH of reaction system；Weigh 5.0mgNaBH3(CN), it is added in reaction flask；Reaction system is placed in room
Temperature lower reaction 48 hours；The NaBH for weighing 2.5mg is dissolved in 10 μ l pure water, after mixing dissolution completely with liquid-transfering gun, is added anti-
It answers in bottle；Reaction system is placed at 23 DEG C and is stirred to react 5 hours；Reaction mixture is transferred to bag filter (MWCO6-8Kd)
In, at 4 DEG C, dialyses 3 times, change the liquid once to 0.15M sodium chloride solution within every 5 hours；By reaction mixture after dialysis
10000rpm takes supernatant after being centrifuged 10min, the polysaccharide conjugate after being dialysed using Sepharose CL-4B gel column purification,
And conjugate peak is collected, sample inspection；
5) Pneumococcus serotypes 6A capsular polysaccharide-CRM197 albumin A chain combination object synthesizes
6.0mg activation Pn6A polysaccharide is weighed, is dissolved in 1mL purified water, stirring surveys initial pH value to after being completely dissolved；With
0.1M NaOH adjusts reacting liquid pH value to 7.0；The CRM197 albumin A chain of 4mg is added into reaction system, stirs and evenly mixs；It weighs
The NaBH of 5.0mg3(CN), it is added in above-mentioned reaction flask, in room temperature reaction 18 hours；Inspection is sampled after reaction；It weighs
The NaBH of 2.7mg was added in above-mentioned reaction flask, in room temperature reaction 5 hours；Inspection is sampled after reaction；By reaction mixture
It is transferred to bag filter, at 4 DEG C, is dialysed 5 times to 0.15M sodium chloride solution, 6L/ times, by reaction mixture after dialysis
10000rpm takes supernatant after being centrifuged 10min, the polysaccharide conjugate after being dialysed using Sepharose CL-4B gel column purification,
And conjugate peak is collected, sample inspection；
6) Pneumococcus serotypes 6B capsular polysaccharide-CRM197 albumin A chain combination object synthesizes
The Pn6B for weighing 5.0mg is dissolved in 1mL purified water, and stirring surveys initial pH value to after being completely dissolved；Use 0.1M
NaOH adjusts reacting liquid pH value to 7.0；The CRM197 albumin A chain of 2.5mg is added into reaction system, stirs and evenly mixs；It weighs
The NaBH of 5.0mg3(CN), it is added in above-mentioned reaction flask, in room temperature reaction 20 hours；The NaBH of 2.5mg is weighed, is added above-mentioned anti-
It answers in bottle, in room temperature reaction 6 hours；Reaction mixture is transferred to bag filter, at 4 DEG C, to 0.15M NaCl solution dialysis 5
It is secondary, 6L/ times；By reaction mixture 10000rpm after dialysis, supernatant is taken after being centrifuged 10min, using Sepharose CL-
Polysaccharide conjugate after the dialysis of 4B gel column purification, and conjugate peak is collected, sample inspection；
7) Pneumococcus serotypes 7F capsular polysaccharide-CRM197 albumin A chain combination object synthesizes
The Pn7F polysaccharide for weighing 10.0mg, is dissolved in 1mL purified water, and stirring is to being completely dissolved；0.1M is added dropwise respectively
NaOH solution adjusts pH to 7.0 into polysaccharide solution；The CRM197 albumin A chain of 3.5mg is added into reaction system, stirring is mixed
It is even；Weigh the NaBH of 5.0mg3(CN), it is added in above-mentioned reaction flask, in room temperature reaction 20 hours；990 μ l are added into reaction flask
Pure water stirs and evenly mixs；The NaBH for weighing 2.5mg was added in above-mentioned reaction flask, in room temperature reaction 6 hours；Reaction mixture is turned
Bag filter (MWCO6000-8000) is moved to, at 4 DEG C, is dialysed 5 times to 5mM succinate/0.9% sodium chloride buffer, 6L/
It is secondary；By reaction mixture 10000rpm after dialysis, supernatant is taken after being centrifuged 10min, using Sepharose CL-4B gel
Polysaccharide conjugate after column purification dialysis, and conjugate peak is collected, sample inspection.
8) Pneumococcus serotypes 9V capsular polysaccharide-CRM197 albumin A chain combination object synthesizes
The Pn9V activated polysaccharide of 10mg is weighed, the sodium phosphate buffer of 125 μ l is measured, 125 μ l pure water is measured and reaction is added
In bottle, magnetic agitation is completely dissolved polysaccharide；The CRM197 albumin A chain of 15mg is measured to being added in reaction flask, stirring and dissolving is complete
Entirely；Weigh the NaBH of 10mg3(CN), it is added in reaction flask；Reaction system is placed at 22 DEG C and is reacted 48 hours；Weigh 2.5mg
NaBH4, it is added in reaction flask, is reacted 5 hours at 22 DEG C；By reaction mixture 10000rpm, supernatant is taken after being centrifuged 10min
Liquid, using AKTA system, polysaccharide conjugate after the dialysis of Sepharose CL-4B gel column purification, and collect in conjunction with peak；
9) 14 capsular polysaccharide-CRM197 albumin A chain combination object of Pneumococcus serotypes synthesizes
The Pn14 activated polysaccharide for weighing 5mg measures the CRM197 albumin A chain of 1ml3.9mg, is added in reaction flask, magnetic force stirs
Mixing is completely dissolved polysaccharide；Weak reductant NaBH is added3(CN) it after 5mg, is reacted 48 hours at 22 DEG C；Strong reductant is added
NaBH42.5mg reacts 4 hours at room temperature；Reaction mixture is transferred in bag filter (MWCO12-14Kd), it is saturating with 2ml
Analyse liquid rinse reaction flask；At 4 DEG C, dialyses 3 times, 6L/ times, change the liquid once within every 5 hours to 0.15M sodium chloride solution.Dialysis knot
Shu Yihou collects dialyzate, and 10000rpm takes supernatant after being centrifuged 10min, is dialysed using Sepharose CL-4B gel column purification
Polysaccharide conjugate afterwards, and collect conjugate peak；
10) Pneumococcus serotypes 18C capsular polysaccharide-CRM197 albumin A chain combination object synthesizes
The Pn18C degradation of polysaccharide for weighing 5mg is dissolved with 1mL1M sodium chloride solution；Its initial pH value is surveyed after dissolution completely；
Suitable CDAP solution is added, stirs 1.5min at room temperature, the pH to 9.0, Zhi Houyu that 0.2M NaOH solution adjusts solution is added
React at room temperature 3min；The CRM197 albumin A chain of 10mg is added, reacts 45min at 25 DEG C；After reaction, 37.5 μ are added
L2M lysine solution；30min is reacted at 25 DEG C is placed on 4 DEG C of reactions overnight；Reaction mixture is gone into bag filter
(MWCO6000-8000) it in, at 4 DEG C, dialyses to 0.85% sodium chloride solution, changes liquid 3 times, 6L/ times, change liquid one within every 5 hours
Secondary, dialyzate is collected in dialysis after terminating, and 10000rpm is centrifuged 10min, takes supernatant, and using CL-4B gel-purified, this is combined more
Sugar, and collect conjugate peak；Detect the albumen and polyoses content in conjugate solution；
11) Pneumococcus serotypes 19A capsular polysaccharide-CRM197 albumin A chain combination object synthesizes
10.0mg oxidation Pn19A polysaccharide is weighed, is dissolved in the buffer of 0.5mL, sets bar magnet in reaction flask, stirring is extremely
Polysaccharide is completely dissolved；The CRM197 albumin A chain of 10mg is added；It stirs and evenly mixs, weighs the NaBH of 5.0mg3(CN), it is added to reaction
In bottle, react 20 hours at room temperature；Weigh the NaBH of 2.5mg4, it is added in reaction flask, reacts 5 hours at room temperature；It will be anti-
It answers mixed liquor to go in bag filter (MWCO6000-8000), at 4 DEG C, dialyses to 0.85% sodium chloride solution, change liquid 3 times, 6L/
It is secondary, it changes the liquid once within every 5 hours；Dialyzate is collected in dialysis after terminating, 10000rpm is centrifuged 10min, supernatant is taken, using CL-4B
This combines polysaccharide to gel-purified, and collects conjugate peak；Detect the albumen and polyoses content in conjugate solution；
12) Pneumococcus serotypes 19F capsular polysaccharide-CRM197 albumin A chain combination object synthesizes
5.2mg oxidation Pn19F polysaccharide is weighed, 1ml pure water is dissolved in, bar magnet is set in reaction flask, on magnetic stirring apparatus
It stirs to polysaccharide and is completely dissolved at room temperature；The CRM197 albumin A chain of 3.0mg is added；After stirring and evenly mixing, the NaBH of 4.9mg is weighed3
(CN), it is added and sets in reaction flask, stirring is always maintained on magnetic stirring apparatus；It is reacted 24 hours at 18 DEG C of room temperature；It weighs
The NaBH of 2.5mg4, it is added to reaction flask；It is reacted 5 hours at 18 DEG C of insulating box；Reaction mixture is transferred to bag filter
(MWCO12-14000), it at 4 DEG C, dialyses 5 times to buffer, dialyse liquid measure 6L every time, changes the liquid once within every 5 hours；Dialysis knot
Shu Yihou collects dialyzate, and 10000rpm is centrifuged 10min, takes supernatant, and using CL-4B gel-purified, this combines polysaccharide, and collects
Conjugate peak；Detect the albumen and polyoses content in conjugate solution；
13) Pneumococcus serotypes 23F capsular polysaccharide-CRM197 albumin A chain combination object synthesizes
4.9mg oxidation Pn23F polysaccharide is weighed, 1ml pure water is dissolved in, bar magnet is set in reaction flask, on magnetic stirring apparatus
It stirs to polysaccharide and is completely dissolved at room temperature；The CRM197 albumin A chain of 5.0mg is added；Weigh the NaBH of 5.1mg3(CN), addition is set
In reaction flask, stirring is always maintained on magnetic stirring apparatus；It is reacted 17 hours at 18 DEG C of insulating box；Weigh 2.5mg's
NaBH4, it is added to reaction flask；It is reacted 5 hours at 18 DEG C of insulating box；Reaction mixture is transferred to bag filter (MWCO12-
14000) it, at 4 DEG C, dialyses to 0.15M sodium chloride solution, dialyse liquid measure 6L every time, changes the liquid once within every 5 hours, totally 5 times；Thoroughly
Dialyzate is collected in analysis after terminating, 10000rpm is centrifuged 10min, takes supernatant, and using CL-4B gel-purified, this combines polysaccharide, and
Collect conjugate peak；Detect the albumen and polyoses content in conjugate solution；
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 blood
It is about 40mg/ml that clear type conjugate solution, which is concentrated into polysaccharide concentration,；It is dense that 6B serotype conjugate solution concentration is concentrated into polysaccharide
Degree is about 80mg/ml；The monovalent serum type conjugate solution that respective volume is added is calculated into office preparation bottle according to following table:
By the 0.22 μm of film aseptic filtration of conjugate mixed liquor；Aluminum phosphate colloid, ultimate density 125mg/ml is added；With slow
Fliud flushing is settled to final volume；It is filling, 0.5ml/ bottles；
5th, the mixture immunogenicity experiments of polynary pneumococcal capsular polysaccharide-protein conjugate:
(1) injection mouse detects the A catenin conjugate mixture preparation of 13 valence pneumococal polysaccharide-CRM197:
Take 5-6 weeks CM57 system mouse 70, the polynary pneumococal polysaccharide-CRM197 albumen of every mouse injection preparation
A chain combination vaccine, injection capacity be 0.1ml/ mouse/time, injection mouse be divided into three groups, one group of injection present invention prepares more
First pneumococal polysaccharide-CRM197 albumin A chain vaccine, the PVC13(Pfizer of another group of injection clinical use in the market) epidemic disease
Seedling is blank control as control, third group, and it is as follows specifically to vaccinate and acquire immune serum program list:
Mouse blood is acquired to centrifuge tube, static 2 hours at room temperature, is centrifuged 10 minutes, uses under the conditions of 10000rpm
Liquid-transfering gun draws the supernatant serum of centrifugation, is stored in 4 DEG C of refrigerators, to be checked；
(2) polysaccharide antibody titre in ELISA method detection mice serum
Prepare in 13 kinds of different serotypes pneumococal polysaccharide stock solution 1mg/ml(1 × PBS solutions respectively), it is stored in ice
Case dilutes Pneumococcal serotype polysaccharide to be checked storage liquid to coating buffer 2-4 μ g/ml, adds 100 μ l coating solution to each
It is coated with elisa plate in a hole, is incubated at room temperature overnight, is washed 4 times with board-washing buffer, 100 μ l Block buffers are added,
It is incubated for 2 hours at room temperature, is washed 4 times, can be saved one week at 4 DEG C with board-washing buffer；Mouse is injected into combined vaccine and control
The corresponding test serum 1:10 that sample obtains is diluted to working prototype serum, dilutes suitable multiple, is added to elisa plate first row
In hole, 200 μ l of total volume carries out two times downwards since first row and is serially diluted, is incubated at room temperature 2 hours, slow with board-washing
Fliud flushing is washed 4 times, and 100 μ l alkali phosphatase enzyme mark sheep anti-mouse antibodies (1:2000 dilution) are added, are incubated at room temperature 4 hours, is used
Board-washing buffer is washed 4 times, 100 μ l phosphoric acid -4- nitro phenyl ester disodium salt substrate solutions is added, in 405nm disk-read；
The results show that the immunogenicity of combined vaccine of the present invention is good, anti-specificity in the mice serum after injecting three needles
IgG antibody titre in the significant serum for being higher than one needle of injection and two needles of the IgG antibody titre of pneumococal polysaccharide；After injecting three needles
Each serotype antibody titer be higher than injection 4 times or more of one needle, meet the standard that WHO improves combined vaccine titre；
Compared with clinical use PVC13 vaccine in the market, the titre of each serotype antibody, after injecting three needles, mouse resisting anteserum
Titre is close to even better.
(3) opsono-cytophagic test (Opsonophagocytic Assay, OPA)
Opsono-cytophagic test is evaluation pneumococcal Polysaccharide Conjugate Vaccine fungicidal effectiveness method, according to " the lung of UAB-MOPA
The scorching many types of opsonophagocytosis sterilization test method of streptococcal capsular polysaccharide specific antibody " tries the mouse immune serum of acquisition
It tests；
OPA titre results are as follows:
| Serotype | Control group | Test group (serum after three needles of injection) |
| 1 | 16 | 2048 |
| 3 | 16 | 1024 |
| 4 | 32 | 2048 |
| 5 | 16 | 2048 |
| 6A | 16 | 1024 |
| 6B | 8 | 1024 |
| 7F | 32 | 2048 |
| 9V | 16 | 1024 |
| 14 | 16 | 4096 |
| 18C | 8 | 2048 |
| 19A | 8 | 1024 |
| 19F | 16 | 2048 |
| 23F | 16 | 2048 |
Test result as it can be seen that injection three needles after mice serum and do not inject polynary polysaccharide conjugate vaccine group mice serum ratio
Compared with the OPA titre of antibody is significantly improved.
Still there are many embodiment, all technical sides formed using equivalents or equivalent transformation by the present invention
Case is within the scope of the present invention.
Claims (3)
1. a kind of mixture of polynary pneumococcal capsular polysaccharide-protein conjugate, characterized in that include 13 kinds of pneumococcus
The adjuvant that capsular polysaccharide-protein conjugate and enhancing are immunized, each pneumococcal capsular polysaccharide-protein conjugate
Pass through the combination of covalent bond and same protein carrier, 13 kinds of pneumonia balls by corresponding Pneumococcal serotype capsular polysaccharide
Bacterium capsular polysaccharide be pass through from Pneumococcus serotypes 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F bacterium it is pure
Change to extract and obtain, the protein carrier is the A of the diphtheria toxin muton CRM 197 obtained by gene recombined escherichia coli expression
Chain, the nucleotide sequence of the A chain gene of the diphtheria toxin muton CRM 197 are as follows:
2. the mixture of polynary pneumococcal capsular polysaccharide-protein conjugate according to claim 1, characterized in that institute
Stating adjuvant is aluminum phosphate or aluminium hydroxide.
3. the preparation method of the mixture of polynary pneumococcal capsular polysaccharide-protein conjugate described in claim 1:
First, the preparation of the A catenin of diphtheria toxin muton CRM 197
Step 1, the building of the A catenin genetic fragment of diphtheria toxin muton CRM 197
It include the restricted of Nco I digestion recognition site CCATGG with the A chain nucleotide sequence of PCR method synthesis CRM197
Endonuclease recognition sequence and restriction endonuclease recognition sequence containing Bam H digestion recognition site GGATCC, above-mentioned two limit
The identification sequence of property restriction endonuclease processed is connected with 5 ' ends of the A chain gene sequence of diphtheria toxin muton CRM 197 and 3 ' ends respectively,
Obtain the recombination segment of the A chain of diphtheria toxin muton CRM 197；
Step 2, the plasmid construction of the A catenin of diphtheria toxin muton CRM 197 is expressed
The A chain nucleotide gene segment of CRM197 is synthesized with Nco I/Bam H digestion with restriction enzyme PCR method, after purification,
The gene fragment clone that digestion is obtained obtains the A chain egg of expression diphtheria toxin muton CRM 197 into an expression vector
White plasmid；
Step 3, the preparation of the A catenin of diphtheria toxin muton CRM 197
The plasmid of the A catenin for the expression diphtheria toxin muton CRM 197 that step 2 obtains is transferred to Bacillus coli expression host
In BL21, the Bacillus coli expression bacterium of the A catenin of expression diphtheria toxin muton CRM 197 is obtained, by prominent to diphtheria toxin
It after the Escherichia coli of the A catenin of variant CRM197 are cultivated, induced, is separated from thallus, purifying obtains diphtheria toxin and dashes forward
The A catenin of variant CRM197；
Second, the preparation of 13 kinds of Pneumococcal serotype capsular polysaccharides
By fermenting respectively to 13 kinds of Pneumococcal serotypes, after bacterium solution separation, centrifuged supernatant is taken, carries out capsular polysaccharide
Purifying, obtain corresponding Pneumococcal serotype capsular polysaccharide；
Third, 13 Pneumococcal serotype capsular polysaccharide-protein conjugate synthesis
The diphtheria toxin mutation that the pneumococcal capsular polysaccharide for 13 kinds of purifying that second obtains is obtained with first step respectively
The A catenin of CRM197 is covalently keyed, and obtains 13 kinds of pneumococcal capsular polysaccharide-protein conjugates；
4th, the preparation of 13 valence pneumococcal capsular polysaccharides-protein conjugate mixture
The adjuvant of 13 kinds of pneumococcal capsular polysaccharide-protein conjugates and enhancing conjugate immunity that third is obtained carries out
It uniformly mixes up to object.
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