CN108774628A

CN108774628A - Synthesis causes the colibacillus engineering and purposes of neonatal meningitis Escherichia coli Glycoprotein binding vaccine

Info

Publication number: CN108774628A
Application number: CN201810705416.1A
Authority: CN
Inventors: 王磊; 黄笛; 江小龙
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2018-07-02
Filing date: 2018-07-02
Publication date: 2018-11-09
Anticipated expiration: 2038-07-02
Also published as: CN108774628B

Abstract

The invention discloses colibacillus engineerings and purposes that a kind of synthesis causes neonatal meningitis Escherichia coli Glycoprotein binding vaccine.It is related to the method that structure synthesis causes ewborn infant Meningitic E. coil O1 serotype Glycoprotein binding vaccine bebcells factory.It is to utilize the efficient homologous recombination efficiency of saccharomyces cerevisiae, based on the method for DNA Assembler, builds O1 antigen synthetic gene cluster plasmids.O1 antigen synthetic gene cluster shuttle plasmids are converted in e. coli jm109, are extracted by lipopolysaccharides, and gel electrophoresis and silver staining identify plasmid；It is deleted under FLP-FRT auxiliary in JM109waaLWithwecA, exclude the interference of original imperfect O antigens.PET28a (+) plasmid is transformed, through induction, synthesizes Glycoprotein binding vaccine, by means of AKTA Primeplus protein purification work station purified glycoproteins and the glycoprotein is identified by western-blotting.The recombination bacillus coli that the present invention is built synthesizes Glycoprotein binding vaccine for bioanalysis and provides new thinking.

Description

Synthesis causes the Escherichia coli work of neonatal meningitis Escherichia coli Glycoprotein binding vaccine Journey bacterium and purposes

Technical field

The invention belongs to synthetic biology technical fields, are related to a kind of synthesized using recombination bacillus coli and cause newborn's meninx The method of scorching Escherichia coli Glycoprotein binding vaccine.More specifically a kind of synthesis causes neonatal meningitis Escherichia coli sugar egg The colibacillus engineering and purposes of white combined vaccine.

Background technology

Newborn causes Meningitic E. coil（NMEC）It is the big monoid for being under the jurisdiction of parenteral enteropathogenic E. Coli, mainly The crowd that object is newborn infants and hypoimmunity is infected, conditioned pathogen is belonged to.Via gastrointestinal tract or respiratory mucosa After field planting, NMEC can invade blood circulation system and mass propagation, form the bacteremia of high concentration.Bacteremia concentration reaches certain After threshold value, NMEC will infect link into brain.Host immune system attacks are resisted by expressing a variety of special virulence factors, NMEC can pass through human body blood-brain barrier, into cerebrospinal fluid and brain tissue flourish, and cause serious meningitis and can not The sequelae of recovery from illness.Compared with the disease that fowl enteropathogenic E. Coli and urinary tract enteropathogenic E. Coli cause, NMEC infection High lethality and high treatment difficulty are always one of problem of clinic study.NMEC tenable environment pressure, it is widely distributed, add The multi-drug resistant occurred in recent years so that the prevention and treatment of NMEC face huge difficulty.It is pair to be inoculated with corresponding vaccine The effective means of anti-NMEC infection.Currently, causing the vaccine development of Meningitic E. coil pathogenic bacteria less newborn.The present invention It provides a kind of for special pathogen more simply and easily production of vaccine strategy, is provided for production polysaccharide protein combined vaccine New approach.

Invention content

To achieve the above object, the invention discloses a kind of synthesis to cause neonatal meningitis Escherichia coli Glycoprotein binding epidemic disease The Recombinant organism of seedling, which is characterized in that contain NMEC different serotypes O antigens synthetic gene cluster plasmid and glycosyl Change system plasmid, deletes simultaneouslywaaLWithwecAGene is named as engineered strain JM109waaL。The Escherichia coli Genetic engineering bacterium, which is characterized in that it is described containing NMEC serotype O1 antigen synthetic gene cluster plasmids, refer to by BSPdb numbers Its O antigen synthetic gene clusters region is oriented according to library, and is connected to saccharomyces cerevisiae-shuttle vehicle pCRG16 On.The carrier, which is able to stablize in Escherichia coli and saccharomyces cerevisiae, replicates expression, contains synthesis O1 repetitive units simultaneously Gene, and polymerase and flippase that repetitive unit is aggregating.Its identification method is as follows：By vector introduction JM109 In, JM109/pCRG16-O1 is obtained, LPS is extracted, passes through the correctness of gel electrophoresis and silver staining map identification plasmid.The plasmid figure Spectrum is shown in Fig. 1.

It is of the present invention containing glycosylation system plasmid, refer to：PET28a (+) is transformed, two couples of tac for being inserted into synthesis are opened Mover and rrnB terminators replace original T7 promoters and terminator, which, which contains, comes from pseudomonas aeruginosa （Pseudomonas aeruginosa）The exotoxin A gene optimized by e. coli codon（Exotoxin A, GI： 877850）；With from campylobacter jejuni（Campylobacter jejuni）And by the N- of e. coli codon optimization Glycosyl transferase encoding gene（undecaprenyl-diphosphooligosaccharide--protein Glycotransferase, GI：905417）, two genes in the entry are respectively at a pair of of tac promoters and rrnB is terminated Under son control, which names pET28M-epa-pglB.Plasmid map is shown in Fig. 2.

The present invention further discloses the Escherichia coli that synthesis causes neonatal meningitis Escherichia coli Glycoprotein binding vaccine The construction method of genetic engineering bacterium, which is characterized in that steps are as follows：

1）Plasmid pSim importings are transformed into host strain JM109, the host strain for carrying plasmid is obtained；

2）Using pKD3 as template, amplification respectively carrieswaaLThe resistance of gene, wecA homology arms knocks out segment；

3) resistance that the same gene is first converted into the host strain of the carrying plasmid pSim obtained by step 1 knocks out segment, obtainswaaLThe recombinant bacterium replaced by FRT-cat-FRT sequences；

4) pCP20 plasmids are imported in the recombinant bacterium obtained by step 3, are relied on FLP recombinases identification FRT sequences progress resistance and are disappeared It removes；

5) using the recombinant bacterium of one gene of deletion obtained by step 4 as host strain, step 3 is repeated）Operation, deletedwaaLWithwecAThe recombinant bacterium of gene；

6) the structure waaL and wecA deletes primer and identifies the nucleotide sequence such as SEQ ID NO of primer:Shown in 1-8.

The present invention also discloses that carrying out the synthesis of glucoprotein vaccine cell fermentation using recombinant bacterium causes neonatal meningitis simultaneously The method of Escherichia coli O1 serotype Glycoprotein binding vaccines, it is characterised in that carried out by following step：

（1）Culture medium and fermentation process：

LB culture mediums（1L）:Tryptone (tryptone)：10g, Yeast Extract（Yeast extract）：5g, NaCl（Chlorine Change sodium）：5g, if configuration solid medium, adds 15g Agar（Agar）, by gained cell factory 20ml contain card that 50 μ g/ml of mycin, 37 DEG C in the LB culture mediums of 10 μ g/ml of chloramphenicol, 220rpm is activated overnight, and activation bacterium is transferred to 1L containing card 50 μ g/ml of that mycin, the LB culture mediums of 10 μ g/ml of chloramphenicol, 37 DEG C, 220rpm cultivate toOD ₆₀₀IPTG is added in ≈ 0.6 （1mM）, and 30 DEG C are gone to, 180rpm is cultivated, and after about 12h, thalline were collected by centrifugation, sonicated cells, by means of AKTA Primeplus protein purification work station large-scale purification glycoprotein；

（2）By the NMEC K1 of activation:O1 strain culturings are extremelyOD ₆₀₀≈ 0.6 takes suitable after being injected intraperitoneally three times in rabbit Blood centrifuges to obtain serum, and with JM109/pCRG16-O1 bacterial strains agglutinating reaction occurs for serum, and reflects by western-blotting It is special to determine band.By building one plant of recombination bacillus coli, realize from glucose to NMECO1 serotype Glycoprotein binding epidemic diseases The biosynthesis of seedling.The technology path taken is as shown in Figure 3.

The present invention further discloses Escherichia coli in the cause neonatal meningitis Escherichia coli for synthesizing different serotypes The application of Glycoprotein binding vaccine.Meningitis is that non-neonate central nervous system is most common, infection of most serious Property disease.Since neonatal immune system is still unsound, lack specific immunity antibody, susceptible chance is more；And barrier function Undeveloped mature (skin, enteron aisle and blood-brain barrier), therefore meningitis is for newborn, incidence, the death rate Relatively high with disability rate, mostly there are central nervous system complications not in time for rescue, are brought to patient and society huge negative Load.Although its case fatality rate is declined in developed country in recent years, drop to recent 10% hereinafter, sending out from the 50% of the seventies National its case fatality rate decline is but not obvious in exhibition.Escherichia coli are cause sepsis of the newborn and meningitis most common, most Main Gram-negative bacteria, caused average mortality reach 17%-38%, and the incidence of survivor's nervous system sequelae More up to 58%.Although for causing neonatal meningitis Escherichia coli to carry out many researchs, its specific pathogenic mechanism Still there are many unknown places, and treatment or drug development to neonatal meningitis cause many difficulties.Cause neonatal meningitis Escherichia coli have capsular polysaccharide, corresponding O antigens（O- polysaccharide）Type is mainly including O1, O18, O5, O7, O6, O11 etc..

In the treatment of directed toward bacteria property meningitis, the use of antibiotic easily causes human pathogenic's bacterolysis, in turn Local inflammation reaction is aggravated, causes blood-brain barrier further to damage, leads to the exacerbation of brain edema, intracranial hypertension；On the other hand, various Extensive, the long-term application of antibiotic, makes antibody-resistant bacterium increase, and drug-resistance factor is spread rapidly, and lethality increases, and treats difficulty and branch Go out further increasing.Vaccine can provide effective immunoprotection as a kind of biological agent to specific disease.Usual vaccine Be divided into the pathogenic bacteria of attenuation or inactivation, two class of specified microorganisms ingredient such as surface protein itself and other parts, when these at Divide after entering body and can be identified and be eliminated by immune system, while generating immunological memory, to receive again together when body Sample can play the role of being effectively protected when threatening.Vaccine plays an important role in human history, is that human infectious disease is pre- Anti- maximally efficient method.Due to vaccine inoculation, some serious infectious diseases have obtained thorough elimination, such as smallpox.In addition, also There are some diseases such as polio, measles, lockjaw etc., since the use of vaccine has also obtained very effective containment.Generation Boundary's Health Organisation Report has the vaccine that 25 kinds of infectious diseases can be had been approved by listing effectively to prevent and control at present.

In vaccine development, the antigenic component for finding its specificity is needed, and the vaccine based on bacterial surface polysaccharides is more next It is valued by people.Bacterial surface polysaccharides antigen（O antigens, K antigens etc.）It is present in nearly all Gram-negative bacteria thalline Outermost layer is the most important immunogenic components of bacterium surface, pathogenic closely related with bacterium, can evoke host's generation Immune response.Surface polysaccharide antigen is generally by oligosaccharides recurring unit（≤50）Composition, each recurring unit is usually by three to eight Monosaccharide forms.Bacterial surface polysaccharides antigenic structure is due to constituting the monosaccharide type of recurring unit, putting in order, combination and more The space structure of sugar chain is different and different, thus the diversity with height.According to the diversity of surface polysaccharide antigen, bacterium can It is divided into different serotype.Such as Escherichia coli carry the O antigens of 166 kinds of different structures, Shigella carries 34 kinds of differences The O antigens of structure.The gene for participating in the synthesis of surface polysaccharide antigen is generally present in a spy on chromosome in the form of gene cluster Anchor point, for example, in Escherichia coli, Shigella and salmonella, O antigen synthetic gene clusters are respectively positioned ongalFWithgndBase Because between.

Bacterial surface polysaccharides can stimulate body to generate protective antibody, however there is no T cells in immunologic process Participate in, therefore the antibody and immunological memory of high-affinity can not be generated, simultaneously because infant immunisation system not yet develop it is perfect, Therefore 2 years old Infants Below of this vaccine pair is nearly unavailable.And bacterial surface polysaccharides are connected to form sugared egg with carrier protein appropriate In vain, the immunogenicity of polysaccharide can be greatly improved.Glycoprotein binding vaccine is that polysaccharide covalent is connected to carrier protein, the bottom of due to The presence of object makes individual polysaccharide（The non-dependent antigen of T cell）Conversion relies on antigen for T cell, excites humoral immunity, generates The antibody and immunological memory of high-affinity.Currently, glucoprotein vaccine occupies very high share, the vaccine product of listing on the market Mainly by after extraction purification albumen and polysaccharide be crosslinked by chemical method, obtain combined vaccine using multiple purifying.The party Method repeatedly purifies in process of production, causes that yield is relatively low, production cost is high.

Most of bacterium surfaces all have polysaccharide component, are the protective antigens of bacterium, have very strong specificity, simultaneously Also body immune system can be stimulated to generate specific antibody.In bacterium, glycoconjugate wide variety, including lipopolysaccharides （Lipopolysaccharide, LPS）, peptide glycan, glycoprotein, capsular polysaccharide（capsular polysaccharide, CPS）, the bacterial surface polysaccharides such as teichoic acid and exocellular polysaccharide body immune system can be stimulated to generate specific antibody, therefore can To be used for preparing polysaccharide vaccine.However bacterial surface polysaccharides molecular weight is small, is not belonging to T cell and relies on antigen, in immunologic process There is no T cell participations, can not generate the antibody and immunological memory of high-affinity.Bacterial surface polysaccharides and suitable attenuated carrier Albumen, which is covalently attached to the glycoprotein to be formed, can stimulate body to generate the immune response that T cell relies on, and greatly improve polysaccharide Immunogenicity.In the whole process, glycoprotein needs sugar chain extensively cross-linked with antibody therefore shorter unlike polysaccharide Effective immune response can still be generated.Immunologic mechanism caused by glycoprotein and polysaccharide are entirely different, it can cause T, B thin The joint of born of the same parents identifies, hence it is evident that improves immune effect.Glycoprotein can also generate immunological memory, therefore Glycoprotein binding vaccine is recognized To be the most successful vaccine of the mankind.Glycoprotein binding vaccine prevention of disease during Clinical practice produces highly significant Effect.This kind of vaccine has been successfully applied at present there are three types of clinics, is for Type B haemophilus influenzae, pneumonia chain respectively The Glycoprotein binding vaccine of coccus and Neisseria meningitidis.In addition, be in clinical test there are many more vaccine, it is such as golden yellow In color staphylococcus, Song and shigella flexneri etc..

For the genetic engineering bacterium of this patent structure through phenol water-swollen squid lipopolysaccharides, gel electrophoresis and silver staining display JM109 can The LPS of NMEC O1 serotypes is synthesized, and western-blotting results show ladder shape bands, it was demonstrated that synthesis Correct glycoprotein.

More detailed description of the present invention is as follows：

First of the present invention is designed to provide one plant of glucose fermentation and synthesizes NMEC O1 serotype Glycoprotein binding vaccines Cell factory.The colibacillus engineering deletes O antigens link enzyme gene from JM109（O-antigen ligase,waaL）With initial glycosyltransferase gene（wecA）；The bacterium contains the glycosylation plasmid pET28M-epa- of IPTG inductions simultaneously The shuttle plasmid pCRG16-O1 of pglB and expression O1 antigens.PET28M-epa-pglB is adapted from pET28a (+), is inserted into synthesis Two pairs of tac promoters and rrnB terminators replace original T7 promoters and terminator wherein,epaTo come from P. aeruginosa Bacterium（Pseudomonas aeruginosa）The exotoxin A gene optimized by e. coli codon（Exotoxin A, GI： 877850）；pglBTo come from campylobacter jejuni（Campylobacter jejuni）And optimize by e. coli codon N- glycosyl transferase encoding genes（undecaprenyl-diphosphooligosaccharide--protein Glycotransferase, GI：905417）PCRG16-O1 is containing cause neonatal meningitis Escherichia coli O1 antigen gene clusters Expression plasmid, contains the gene of synthesis O1 repetitive units simultaneously, and the polymerase and flippase that repetitive unit is aggregating.

Second object of the present invention is to provide a kind of one plant of glucose fermentation synthesis NMEC O1 serotype glycoprotein The step of construction method of combined vaccine, this method, is as follows：

1. deleting in JM109 bacterial strainswaaLWithwecAGene：

The structure of 1.1 JM109/pSim bacterial strains

1.1.1 extracting pSim plasmids

PSim plasmids are extracted with raw work pillar small amount plasmid extraction agent box, specific steps are shown in kit specification.

1.1.2 preparing JM109 competent cells

1.1.3 the acquisition of JM109/pSim bacterial strains

The pSim plasmids of extraction are converted by electroporation apparatus into JM109 competent cells, are then gone in recovery medium Or so recovery half an hour is coated with blasticidin-S resistance 2YT tablets, culture is protected from light overnight in 30 DEG C of incubators.Next day can see flat The single bacterium colony grown on plate is the JM109 bacterial strains of the plasmid containing pSim.

The amplification of 1.2 resistance gene fragments

Using pKD3 as template, using waaL-FRT-chl-FRT-F/R as primer, carried with pfu DNA polymerase amplificationswaaLThe chlorampenicol resistant segment of gene upstream and downstream homology arm.PCR product is into row agarose gel electrophoresis blend compounds reclaim reagent Box recycles target fragment.PCR programs and agarose gel electrophoresis step are shown in specification.

1.3 resistance fragments are converted to JM109/pSim competence bacterial strains

1.3.1 preparing JM109/pSim competent cells.

Substantially it is prepared with JM109 competent cells in 1.1.2, the difference is that need to be hot in 42 DEG C of shaking baths before ice bath 30min is swashed, with inductionThe expression of RED homologous recombination enzymes.It will carrywaaLThe chlorampenicol resistant segment electrotransformation of homology arm is extremely In JM109/pSim competent cells, recovery 1-2h is coated with chlorampenicol resistant 2YT tablets, is incubated overnight in 37 DEG C of incubators, secondary Day picking monoclonal carries out boiling bacterium PCR identifications.Both ends identify that primer is S-waaL-F/ R, and intersection identifies that primer is S-waaL-F/ inner-chl-R。

Identify that correct monoclonal bacterial strain has as inactivatedwaaLGene and the bacterial strain for carrying chloramphenicol resistance gene, Labeled as JM109 waaL::Cm preserves correct bacterial strain.

1.4 the elimination of resistant gene

1.4.1 plasmid extraction kit is utilized to extract pCP20 plasmids.

1.4.2 JM109 is preparedwaaL::The competence of Cm, and electricity turns in pCP20 plasmids to the competence, recovers, and applies Cloth amicillin resistance 2YT tablets, are incubated overnight and are screened by 30 DEG C.

1.4.3 in the monoclonal access 5mL2YT culture mediums on second day picking amicillin resistance 2YT tablet, 37 DEG C It is incubated overnight.Third day is with 1：100 ratio is transferred in the 2YT culture mediums of 20mL, 42 DEG C, 180rpm shaken cultivations 8h.So Bacterium solution is diluted into 10-5 and 10-6 afterwards and is coated with non-resistant 2YT tablets.4th day, by the monoclonal colonies on nonreactive plate respectively at Photocopy is carried out in nonreactive and chlorampenicol resistant tablet pricks plate.Being chosen on nonreactive tablet within 5th day can grow, but mould in chlorine The monoclonal that cannot be grown in plain resistant panel carries out boiling bacterium PCR identifications, identifies that primer is S-waaL-F/R, identification electrophoretogram is shown in Fig. 4.Correct bacterial strain is required missing engineered strain JM109waaL。

1.4.4 for the correct bacterial strain of gained in 37 DEG C, 220rpm presses 1：100 continuous passage cultures 2 times, scribing line are flat with LB Plate, panel photocopy method identify the bacterial strain for having lost pCP20 plasmids.

wecAThe deletion of gene is samewaaLGene elmination method identifies that primer is S-wecA-F/R, and electrophoretogram is shown in Fig. 5.

The structure of 2.pET28M-pglB-EPA carriers

The transformation of (2.1pET28a+) plasmid

Synthesize the segment containing two pairs of tac promoters and rrnB terminators, by digestion connection replace original T7 promoters and T7 terminators

2.2pET28M-pglB-EPA

PglB the and EPA genes for synthesizing codon optimization, are connected to by digestion on Pet28M carriers

The structure of 3.pCRG16-O plasmids, identification and sequencing

The preparation of 3.1 competent yeast cells

The acquisition of the linear cloning vectors of 3.2 pCRG16

（1）PCRG16 plasmids are extracted with small amount plasmid extraction agent box.

（2）Plasmid is usedNotThe fast enzyme cuttings of I carry out single endonuclease digestion

The acquisition of 3.3 DNA Assembler segments

By taking NMEC O1 as an example, the genome of the corresponding serotypes of extraction NMEC passes through BSPdb databases（http:// bspdb.nankai.edu.cn/index.html）Corresponding O antigens synthetic gene cluster is positioned, by entire O antigens synthetic gene Cluster is expanded at 5 parts, per segment about 5K.First segment is 5 ' containing same at the linear cloning vector NotI restriction enzyme sites of pCRG16 Source sequence 40mer bases, second segment contain first segment 3 ' homologous sequence about 100-200mer, and so on, until Contain another NotI restriction enzyme site homologous sequence 40mer base of the linear cloning vectors of pCRG16 in the 3 ' of the last one segment.In detail Thin principle is as shown in Figure 6

3.4 saccharomyces cerevisiaein vivoRecombination to construct O antigen presentation plasmids

Segment that above-mentioned amplification obtains and the linear cloning vector common-batteries of pCRG16 are converted to CRY1-2 saccharomyces cerevisiaes, recovery in 1mlSD uracil-deficient fluid nutrient mediums take the 100 μ L of culture medium of recovery to be coated on SD uracil-deficient tablets, 30 DEG C of trainings 48-72h is supported, 3-5 single bacterium colony of random picking extracts yeast plasmid, PCR with the yeast plasmid extracts kit of Suo Laibao companies Method identifies plasmid, judges whether to recombinate successfully by size.The recombinant plasmid transformed extracted from yeast is extracted to DH10B Plasmid in DH10B send sequencing.

4. lipopolysaccharides extracts, gel electrophoresis and silver staining

Correct O Antigen Expression Vectors will be sequenced first to convert to in JM109 competent cells, coating chlorampenicol resistant 2YT is flat Plate, identified correct monoclonal with 20mL liquid 2YT medium cultures extremelyODLPS is extracted when 600 ≈ 0.6.Configuration 12% The LPS of extraction is carried out gel electrophoresis, then silver staining by SDS-PAGE running gels.

The present invention provides described in a kind of concrete application colibacillus engineering synthesize Glycoprotein binding vaccine method and Corresponding purification step and detection method, this method are as follows：

5. the fermentation synthesizing and purifying of Glycoprotein binding vaccine and detection：

PCRG16-O and pET28M-pglB-EPA corotation is entered into JM109waaL∆wecAIn competent cell, recovery 1-2h, It is coated with chloramphenicol and the dual anti-tablet of kanamycins, 37 DEG C of culture 48-60h.The monoclonal grown is carried out to boil bacterium PCR identifications.Mirror Fixed correctly clone carries out guarantor bacterium.

Glycoprotein binding vaccine synthesizes and purifying：

（1）Above-mentioned bacterial strains are inoculated in the LB culture mediums of 20mL and kanamycins is added（50μg/mL）And chloramphenicol（10μg/ mL）, 37 DEG C, 180rpm is incubated overnight.

（2）The next morning, by the bacterium solution being incubated overnight according to 1:100 ratio is forwarded in the LB culture mediums of 1L, 37 DEG C, 200rpm cultivates 10-12h.

（3）The expression of IPTG inducible proteins is added（Final concentration of 1mM）, 30 DEG C, 180rpm induces about 10-12h.

（4）With supercentrifuge 12000rpm, 10min receives bacterium, and thalline is gone in 50mL centrifuge tubes.

（5）Thalline washed once with 0.9%NaCl, then adds Binding buffer and thalline is resuspended to OD600 ≈ 200。

（6）Protease inhibitors is added（Final concentration of 1mM）And lysozyme（Final concentration of 1mg/mL）.Ice bath 30min.

（7）Centrifuge tube is placed on ice, somatic cells are crushed with Ultrasonic Cell Disruptor.Super 3S, stops 2S, adds up ultrasonic time about 40min, until liquid improves in sepia and liquid fluidity.The protease inhibitors is bought in thermo fisher Scientific companies.

（8）A small amount of DNA enzymatic and RNA enzyme is added（Final concentration of 5 μ g/mL）, ice bath 15min.

（9）4 DEG C, 8000rpm centrifuges 15min, and it is spare that supernatant is gone to another 50mL centrifuge tubes（- 80 DEG C of preservations can be placed in 2-3 days）.

Protein purification is carried out with AKTA Primeplus systems, steps are as follows：

（1）By 2.1.6.2 preparation of reagents requirements prepare IMAC buffer A, IMAC buffer B, go endotoxin buffer, AEC bufferA, AEC bufferB and 20% absolute ethyl alcohol.The above reagent was both needed to moisture film processing and ultrasound 30min.

（2）Open instrument, the cleaning robot pipeline under load patterns loads onto nickel ion after screen upper curve to be shown is all steady Chelate chroma-tography column.

（3）It uses IMAC buffer A instead to be cleaned again, until curve is steady again.

（4）Supernatant after ultrasonication is crossed into 0.22 μm of filter membrane, and in the supreme sample ring of sample introduction.At this time if curve Fluctuation continues to be rinsed to steady with IMAC buffer A in such a mode.

（5）Instrument is adjusted to inject patterns, the sample in loading ring is made to be combined with nickel column.Until the sample in loading ring It is combined completely with nickel column.

（6）Again load patterns are adjusted to, endotoxin buffer [61] 40 column volumes of flushing is first spent and uses IMAC again Buffer A continue to rinse, until curve is steady again.

（7）Be adjusted to gradient elution mode, make IMAC buffer B concentration in 6 column volumes from 0% rise to 100% into Row gradient elution destination protein.When indicating that apparent absorption peak occurs in the curve of UV on display screen, collect corresponding when appearance Receipts sample pipe in sample be destination protein.

（8）Sample, which is put into bag filter and is placed in 4 DEG C of refrigerators in dialyzate, dialyses 2h to remove imidazoles.

（9）The albumen purified with nickel ion affinity chromatograph is purified again with ion-exchange chromatography.By nickel ion Chelate chroma-tography column changes ion-exchange chromatography into, with AEC buffer A flushing lines.

（10）By albumen loading to loading ring, inject patterns are adjusted to after curve is steady makes albumen and ion-exchange chromatography Column fully combines.

（11）10 column volumes are rinsed with AEC buffer A.

（12）Gradient elution destination protein is carried out with AEC buffer A and AEC buffer B.When indicating UV on display screen Curve when there is apparent absorption peak, sample when collecting appearance in corresponding receipts sample pipe is our destination protein （Such as SEQ ID NO:Shown in 9）, add 10% -80 DEG C of preservations of glycerine.

Glycoprotein binding vaccine detects -- western blot detections：

（1）Sample is put into gel in 1 × transferring film Buffer after SDS-PAGE is detached, and is placed on horizontal shaker and shakes 15min。

（2）Prepare the pvdf membrane and filter paper of suitable size, pvdf membrane need to impregnate 5min before use in absolute methanol, filter Paper need to infiltrate in 1 × transferring film Buffer.Sponge, filter paper, gel, pvdf membrane, filter paper, sponge are clipped successively in this order, It is put into transferring film instrument.4 DEG C, constant pressure 70V turns 1h.

（3）After the completion of transferring film, pvdf membrane is put into and is incubated in container, sealed with skimmed milk power solution room temperature on horizontal shaker Close 1h.

（4）Pvdf membrane is washed three times with the TBST solution prepared, every time 10 min, and the primary antibody diluted, room temperature water is added It is incubated 1h on yawing bed（Or 4 DEG C be incubated overnight）.

（5）It is washed three times with TBST, every time 10 min, the secondary antibody diluted is added, 1h is incubated on room temperature horizontal shaker.

（6）It is washed three times with TBST, every time 10 min, be washed once with TBS again.Use highly sensitive ECL luminescence reagents box In AI600 hypersensitive Multifunctional imaging instrument exposure images.Testing result is shown：Negative control does not have band at 72KD, experimental group There is apparent ladder shapes band at 72KD, it was demonstrated that synthesized Glycoprotein binding vaccine correct.The Glycoprotein binding vaccine Body immune system can be stimulated to generate the immune response that T cell relies on, the immunogenicity of polysaccharide is greatly improved, have simultaneously There is very strong specificity.In entire immunologic process, glycoprotein needed unlike polysaccharide it is extensively cross-linked with antibody, therefore compared with Short sugar chain can still generate effective immune response.Immunologic mechanism caused by glycoprotein and polysaccharide are entirely different, its energy Cause the joint identification of T, B cell, hence it is evident that improve immune effect.Glycoprotein can also generate immunological memory, therefore glycoprotein knot It is considered as the most successful vaccine of the mankind to close vaccine.Glycoprotein binding vaccine prevention of disease during Clinical practice generates The effect of highly significant.This kind of vaccine has been successfully applied at present there are three types of clinics, is to be directed to the bloodthirsty bar of Type B influenza respectively The Glycoprotein binding vaccine of bacterium, streptococcus pneumonia and Neisseria meningitidis.In addition, being in clinical test there are many more vaccine In, such as in staphylococcus aureus, Song and shigella flexneri.

Description of the drawings

Fig. 1 is plasmid pCRG16-O1；Meningitic E. coil O1 antigen synthetic gene clusters are caused for expressing；

Fig. 2 is the collection of illustrative plates of plasmid pET28M-epa-pglB, is used for induced expression and glycosylates system；

Fig. 3 is the engineer of cell factory and the Technology Roadmap of synthesis；

Fig. 4 iswaaLBy the electrophoretogram that FRT-chl-FRT is replaced and waaL genes are deleted；

（In figure, M:Marker；Swimming lane 1-2 is respectively：Control,waaLDelete bacterial strain；

Fig. 5 iswecABy the electrophoretogram that FRT-chl-FRT is replaced and waaL genes are deleted；In figure, M:Marker；Swimming lane 1- 2 are respectively：Control,wecADelete bacterial strain；

Fig. 6 is the construction method of pCRG16-O1 plasmids.

Specific implementation mode

With reference to specific embodiment, the present invention is described further, but the present invention should not be limited by the examples；

Material used in following embodiment, reagent, instrument and method without specified otherwise be conventional material in this field, Reagent, instrument and method can be obtained by commercial channel；

Plasmid extraction is using raw work bioengineering in the present invention（Shanghai）The SanPrep pillar Plasmid DNA of Co., Ltd is taken out in a small amount Extraction reagent kit（Catalog NO.：B518191）, gel extraction is using raw work bioengineering（Shanghai）Co., Ltd SanPrep gel extraction kits（Catalog NO.：B518131）, the connection of DNA fragmentation is to use fermentas companies T4 DNA Ligase（Catalog NO.：EL0014）, the pfu DNA of DNA fragmentation expanded using fermentas companies polymerase（Catalog NO.：EP0571）, the Fast of PCR plasmid templates digested using fermentas companies Digest KpnI（Catalog NO.：FD0524）, BamHI（Catalog NO.：FD0054） NcoI（Catalog NO.： FD0574）BglII（Catalog NO.：FD0083）, NotI（Catalog NO.：FD0593）

The electroporated experiments of E.coli use the electroporation of Bio-Rad（Catalog NO.：165-2100）.Bacterial genomes carry It takes using the bacterial genomes extracts kit that Beijing health is century biochemical technology Co., Ltd（Catalog NO.： CW0552S）；

Table 1 is deletedwaaLWithwecAThe primer sequence

The source for the bacterial strain and raw material that 2 present invention of table uses

The amino acid sequence of Exotoxin A in 3 Glycoprotein binding vaccine of table

mkkiwlalaglvlafsasaaeeafdlwnecakacvldlkdgvrssrmsvdpaiadtngqgvlhysmvleggnd alklaidnalsitsdgltirleggvepnkpvrysytrqargswslnwlvpighekpsnikvfihelnagnqlshmsp iytiemgdellaklardatffvrahesnemqptlaishagvsvvmaqaqprrekrwsewasgkvlclldpldgvyny laqqrcnlddtwegkiyrvlagnpakhdldikdnnnstptvishrlhfpeggslaaltahqachlpletftrhrqpr gweqleqcgypvqrlvalylaarlswnqvdqvirnalaspgsggdlgeaireqpeqarlaltlaaaeserfvrqgtg ndeagaasadvvsltcpvakdqnrtkgecagpadsgdallernyptgaeflgdggdvsfstrgtqnwtverllqahr qleergyvfvgyhgtfleaaqsivfggvrarsqdldaiwrgfyiagdpalaygyaqdqepdargrirngallrvyvp rwslpgfyrtgltlaapeaageverlighplplrldaitgpeeeggrvtilgwplaertvvipsaiptdprnvggdl dpssipdkeqaisalpdyasqpgkppredlkhhhhhh*

* it is terminator codon

Embodiment 1

The acquisition of gene

In the present embodiment, obtains and derive from pseudomonas aeruginosa（Pseudomonas aeruginosa）By Escherichia coli password The exotoxin A gene of son optimization（Exotoxin A, GI：877850）；With from campylobacter jejuni（Campylobacter jejuni）And by the N- glycosyl transferase encoding genes of e. coli codon optimization（undecaprenyl- Diphosphooligosaccharide--protein glycotransferase, GI：905417）.

Embodiment 2

The design of gene elmination primer

The present embodiment knocks out two genes of JM109 using λ Red recombination systems, and this method often knocks out a gene and all resisted The elimination of property.Below withwaaLFor gene, the step of elaborating gene knockout,wecAGene elmination design of primers and this phase Together.

JM109waaL nucleotide sequences are searched, the deletion primer and identification primer of waaL are designed.The deletion primer of waaL is WaaL-FRT-chl-FRT-F/R identifies that primer is S-waaL-F/R.Nucleotide sequence is as shown in table 1-8.

Embodiment 3

JM109ΔwaaLStructure

The conversion of 3.1 plasmid pSim

In the flat lining outs of LB of non-resistant, 37 DEG C are incubated overnight the wild type JM109 that -80 DEG C of picking freezes.Second day picking Monoclonal is seeded in 5mL LB culture mediums, 37 DEG C, and 220rpm is incubated overnight.Second day inoculum concentration for pressing 1%, is forwarded to In the LB culture mediums of 200ml.37 DEG C, 220rpm, culture to OD₆₀₀About 0.6-0.8, ice bath 20min, 5500rpm, 5min are received Collect thalline in sterilized 50ml centrifuge tubes, 4 DEG C, 5500rpm, centrifuges 5min, abandon supernatant, crossed with 50ml ice baths sterile 10% glycerine resuspension thalline, 4 DEG C, 5500rpm, then 5min is centrifuged, repeat aforesaid operations 3 times, last time, when using abandoning supernatant Residual liquid thalline is resuspended, take 80 μ L in in new sterile EP tube.- 80 DEG C freeze.

The competent cell that -80 DEG C freeze is placed in ice and melts 10min, the pSim plasmids of 1 μ L are added, are added after mixing It shocks by electricity in cup, ice bath 2min, 1.8KV is electroporated, the LB culture mediums of 1ml is added after electric shock immediately, 30 DEG C, 220rpm/min is multiple Revive 20min, and appropriate bacterium solution is taken to be coated on blasticidin-S tablet（200 μ g/ml of blasticidin-S concentration）, 30 DEG C of inversions are incubated overnight, and The monoclonal grown for two days is the JM109 for carrying pSim plasmids.

The deletion of 3.2 target gene

3.2.1 the preparation of homologous recombination segment

With pKD3 plasmids（It buys from the Wuhan bio tech ltd Miao Ling）For template, primer waal-FRT-chl- is used FRT-F/R carries out PCR amplification, cuts glue purification recycling, obtains both ends and containswaaLThe knockout segment of homology arm

3.2.2 first step homologous recombination

The JM109 monoclonals that picking carries pSim plasmids are seeded in 5ml LB culture mediums, and 37 DEG C, 220rpm is incubated overnight.The Two days inoculum concentrations for pressing 1%, are forwarded in the LB culture mediums of 200ml.37 DEG C, 220rpm, culture to OD₆₀₀About 0.6-0.8, will Bacterium solution is gone in 42 DEG C of shaking baths, 150rpm, 20min, then ice bath 20min, 4 DEG C, 5500rpm, centrifuges 5min, collects bacterium Body is in sterilized 50ml centrifuge tubes, 4 DEG C, 5500rpm, centrifuges 5min, abandons supernatant, sterile 10% crossed with 50ml ice baths Thalline is resuspended in glycerine, 4 DEG C, 5500rpm, then centrifuge 5min, repeats aforesaid operations 3 times, last time, using residual when abandoning supernatant Extraction raffinate weight hangs thalline, takes 80 μ L in 4 μ L homologous fragments in new sterile EP tube, are added, is added after mixing in electric shock cup, ice 2min is bathed, 1.8KV is electroporated, and the LB culture mediums of 1ml are added after electric shock immediately, 37 DEG C, 180rpm/min, cultivates 2h, takes suitable Amount bacterium solution is coated in resistant panel, and 37 DEG C are incubated overnight.Second day picking monoclonal, PCR are identifiedwaaLGene is by waaL- The correct clone that FRT-chl-FRT is replaced, i.e. JM109waaL::FRT-chl-FRT

The elimination of resistance after 3.3 gene delections

3.3.1JM109 ∆waaL::The preparation of FRT-chl-FRT bacterial strain competent cells

PickingE.coli BL21 (DE3) ∆lacZMonoclonal is seeded in the LB culture mediums of 5ml, is prepared according to previous step Competence, electricity turn pCP20 plasmids, and the LB culture mediums of 1ml are added after electric shock immediately, 30 DEG C, 180rpm/min, cultivates 20min, takes Appropriate bacterium solution is coated on ampicillin plate（100 μ g/ml of ampicillin concentration）, 30 DEG C are incubated overnight.It chooses within second day It takes in monoclonal to the LB culture mediums of 5ml（25 μ g/ml of chloramphenicol concentration）, 30 DEG C, 180rpm/min, 10h is cultivated, is transferred to New fluid nutrient medium, added with antibiotic, after cultivating 6h, does not dilute coated plate, 37 DEG C, inversion is incubated overnight, and chooses list within second day by 42 DEG C Clone, carries out photocopy on nonreactive plate and chloramphenicol plate respectively.It does not grow on chloramphenicol plate, but is grown on nonreactive plate corresponding position Monoclonal, as resistance eliminates successful positive colony, further verified by PCR.

3.4 wecADeletion

wecADelete principle and step withwaaLIt is identical, to deletewaaLBacterial strain based on, by repeat experimental procedure in 3.2 and 3.3, can willwecAJM109 Δs are finally constructed in gene elminationwaaLΔwecA

Fig. 4 and Fig. 5 is JM109 and deletionwaaL/wecAPCR verification results, the band phase of the band swimming lane 1 of swimming lane 2 in Fig. 4 Than there is significant reduction, showing corresponding target gene by successful knockout.

PSim plasmids are temperature-sensitive plasmid, and cultivation temperature is higher than under conditions of 30 DEG C, and plasmid will be lost, therefore, After pSim plasmids are transferred to JM109, bacterial strain will be cultivated under the conditions of 30 DEG C always, to prevent the loss of pSim plasmids.

Embodiment 4

Immunization experiment and antibacterial experiment：

Select 6 ~ 8 weeks Balb/c female mices（Tie up experimental animal Co., Ltd of tonneau China）Carry out immunization experiment and antibacterial experiment, tool Body process is as follows：

（1）Mouse is randomly divided into two groups, respectively glycosylation group and negative control group, every group 6.

（2）It was immunized respectively at the 0th, 14,28 day.Every mouse per injection amount total volume is 100 μ L, including 2 μ g Glycoprotein binding vaccine and isometric Freund's adjuvant, be subcutaneously injected.Wherein first time immune Freund's complete adjuvant, after It is incomplete Freund's adjuvant twice.Negative control group injects isometric PBS.

（3）After final immunization terminates 35 days, blood is taken to every mouse tail vein in every group, 3000rpm, 4 °C centrifuge To serum be placed in -80 °C it is spare.

（4）Carry out antibacterial experiment.The O1 serotypes of 1 × PBS solution are dissolved in every mouse injection 100CFU of three groups of mouse Cause Meningitic E. coil, tail vein injection.Eyeball is plucked after four hours and takes blood, and a part of blood 3000rpm, 4 °C centrifuge To serum, another part is placed in dilution spread LB solid plates in anticoagulant tube, 37 °C, static gas wave refrigerator in carbon dioxide incubator. It is counted after tablet grows single bacterium colony.

（5）Western blot detections are carried out respectively to the serum of collection：

Western blot detections, which are done, with the serum obtained after immune finds that glycosylation group can detect the purpose item of ladder shapes Band, it was confirmed that the Glycoprotein binding vaccine can stimulate mouse immune system to generate corresponding antibody, while analyze glycosylation group It is found with the testing result after injection bacterium solution before injecting bacterium solution, internal antibody level has apparent rising after injection bacterium solution, injects Bacterium solution at secondary immunity original, stimulation mouse generates second set response, and it is multiple also to further illustrate that polysaccharide protein combined vaccine passes through Miscellaneous immunologic mechanism stimulation body produces memory cell.

It is counted and is shown by antibacterial experiment coated plate, the experimental group flat-plate bacterial colony number vaccinated is significantly lower than injection PBS's Negative control group, it is extremely significantly, to illustrate that polysaccharide protein combined vaccine stimulation mouse is exempted from that clump count, which reduces 100 times of statistic analysis results, Epidemic disease system generates memory cell and antibody, plays significant antibacterial action.

The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited, although with reference to above-mentioned reality Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation Technical solution recorded in example is modified or equivalent replacement of some of the technical features.And these are changed or replace It changes, the scope and spirit for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.

SEQUENCE LISTING

<110>Nankai University

<120>Synthesis causes the colibacillus engineering and purposes of neonatal meningitis Escherichia coli Glycoprotein binding vaccine

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 59

<212> DNA

<213>Artificial sequence

<400> 1

gcagttttgg aaaagttatc atcattataa aggtaaaacg tgtaggctgg agctgcttc 59

<210> 2

<211> 59

<212> DNA

<213>Artificial sequence

<400> 2

agaagtgagt tttaactcac ttcttaaact tgtttattca tgggaattag ccatggtcc 59

<210> 3

<211> 59

<212> DNA

<213>Artificial sequence

<400> 3

ttatacttct gctaataatt ttctctgaga gcatgcattg tgtaggctgg agctgcttc 59

<210> 4

<211> 59

<212> DNA

<213>Artificial sequence

<400> 4

ccggtttccc aggcattggt tgtgtcatca catcctcata tgggaattag ccatggtcc 59

<210> 5

<211> 20

<212> DNA

<213>Artificial sequence

<400> 5

gtgggtatgg gaagaatcag 20

<210> 6

<211> 20

<212> DNA

<213>Artificial sequence

<400> 6

accctaattc acgtactccg 20

<210> 7

<211> 22

<212> DNA

<213>Artificial sequence

<400> 7

ggtgggtttg gaacggactt tc 22

<210> 8

<211> 22

<212> DNA

<213>Artificial sequence

<400> 8

gccccatgcc aataatccat ag 22

<210> 9

<211> 649

<212> PRT

<213>The amino acid sequence of Exotoxin A

<400> 9

Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser

1 5 10 15

Ala Ser Ala Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys

20 25 30

Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser

35 40 45

Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr

50 55 60

Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp

65 70 75 80

Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly

85 90 95

Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala

100 105 110

Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys

115 120 125

Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln

130 135 140

Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu

145 150 155 160

Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu

165 170 175

Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser

180 185 190

Val Val Met Ala Gln Ala Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu

195 200 205

Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val

210 215 220

Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu

225 230 235 240

Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu

245 250 255

Asp Ile Lys Asp Asn Asn Asn Ser Thr Pro Thr Val Ile Ser His Arg

260 265 270

Leu His Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln

275 280 285

Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg

290 295 300

Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val

305 310 315 320

Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val

325 330 335

Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu

340 345 350

Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala

355 360 365

Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu

370 375 380

Ala Gly Ala Ala Ser Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala

385 390 395 400

Lys Asp Gln Asn Arg Thr Lys Gly Glu Cys Ala Gly Pro Ala Asp Ser

405 410 415

Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu

420 425 430

Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp

435 440 445

Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly

450 455 460

Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser

465 470 475 480

Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile

485 490 495

Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr

500 505 510

Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala

515 520 525

Leu Leu Arg Val Tyr Val Pro Arg Trp Ser Leu Pro Gly Phe Tyr Arg

530 535 540

Thr Gly Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg

545 550 555 560

Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro

565 570 575

Glu Glu Glu Gly Gly Arg Val Thr Ile Leu Gly Trp Pro Leu Ala Glu

580 585 590

Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val

595 600 605

Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile

610 615 620

Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu

625 630 635 640

Asp Leu Lys His His His His His His

645

Claims

1. a kind of synthesis causes the Recombinant organism of neonatal meningitis Escherichia coli Glycoprotein binding vaccine, feature It is, contains NMEC（neonatal meningitisEscherichia coli, NMEC）O1 antigen synthetic gene cluster plasmids With glycosylation system plasmid, it is named as engineered strain JM109waaL ∆wecA/pCRG16-O1 pET28M-epa-pglB。

2. Recombinant organism described in claim 1, it is characterised in that described to be closed containing NMEC serotype O1 antigens It is referred at gene cluster plasmid：Go out its O1 antigen synthetic gene clusters region by BSPdb database-locateds, and is connected to wine On brewer yeast-shuttle vehicle pCRG16.

3. Recombinant organism described in claim 1, it is characterised in that described to refer to containing glycosylation system plasmid It is：PET28a (+) is transformed, the two pairs of tac promoters and rrnB terminators for being inserted into synthesis replace original T7 promoters and termination Son, which, which contains, comes from pseudomonas aeruginosa（Pseudomonas aeruginosa）Optimize by e. coli codon Exotoxin A gene（Exotoxin A, GI：877850）；With from campylobacter jejuni（Campylobacter jejuni） And by the N- glycosyl transferase encoding genes of e. coli codon optimization（undecaprenyl- Diphosphooligosaccharide--protein glycotransferase, GI：905417）, two in the entry Gene is respectively under a pair of of tac promoters and the control of rrnB terminators, which is named as Pet28M-EPA-pglB.

4. any one of the claim 1-3 Escherichia coli are in the cause neonatal meningitis Escherichia coli sugar of synthesis different serotypes The application of protein conjugate vaccines.

5. synthesis described in a kind of claim 1 causes the bacillus coli gene of neonatal meningitis Escherichia coli Glycoprotein binding vaccine The construction method of engineering bacteria, which is characterized in that steps are as follows：

6. a kind of carrying out glucoprotein vaccine cell fermentation synthesis cause neonatal meningitis e. coli serotype sugar using recombinant bacterium The method of protein conjugate vaccines, it is characterised in that carried out by following step：

（1）Culture medium and fermentation process：

LB culture mediums（1L）:Tryptone (tryptone)：10g, Yeast Extract（Yeast extract）：5g, NaCl（Chlorine Change sodium）：5g, if configuration solid medium, adds 15g Agar（Agar）, by gained cell factory 20ml contain card that 50 μ g/ml of mycin, 37 DEG C in the LB culture mediums of 10 μ g/ml of chloramphenicol, 220rpm is activated overnight, and activation bacterium is transferred to 1L containing card 50 μ g/ml of that mycin, the LB culture mediums of 10 μ g/ml of chloramphenicol, 37 DEG C, 220rpm cultivate toOD ₆₀₀IPTG is added in ≈ 0.6 （1mM）, and 30 DEG C are gone to, 180rpm is cultivated, and after about 12h, thalline were collected by centrifugation, sonicated cells, by means of AKTA Primeplus protein purification work station large-scale purification glycoprotein；Such as SEQ ID NO:Shown in 9；

（2）By the NMEC K1 of activation:O1 strain culturings are extremelyOD ₆₀₀≈ 0.6 takes suitable after being injected intraperitoneally three times in rabbit Blood centrifuges to obtain serum, and with JM109/pCRG16-O1 bacterial strains agglutinating reaction occurs for serum, and reflects by western-blotting It is special to determine band.