CN117771349A - Neonatal meningitis escherichia coli glycoprotein conjugate vaccine and application - Google Patents
Neonatal meningitis escherichia coli glycoprotein conjugate vaccine and application Download PDFInfo
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- CN117771349A CN117771349A CN202311780737.5A CN202311780737A CN117771349A CN 117771349 A CN117771349 A CN 117771349A CN 202311780737 A CN202311780737 A CN 202311780737A CN 117771349 A CN117771349 A CN 117771349A
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- escherichia coli
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Landscapes
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Abstract
The invention relates to a neonatal meningitis escherichia coli glycoprotein conjugate vaccine and application thereof. Animal immunity experiments prove that the prepared vaccine can stimulate mice to generate protective antibodies, the protection rate can reach more than 90 percent, and the antibodies can be transferred to newborn mice through maternal and infant transfer, so that the newborn mice are protected from infection; the recombinant escherichia coli glycoprotein conjugate vaccine constructed by the invention provides a new choice for the immunotherapy of neonatal meningitis escherichia coli infection, and has good practical significance and application prospect.
Description
Technical Field
The invention belongs to the technical field of synthetic biology, and particularly relates to a neonatal meningitis escherichia coli glycoprotein conjugate vaccine and application thereof.
Background
Meningococcal escherichia coli (Neonetal meningitis Escherichia coli, NMEC) is one of the most common pathogenic bacteria responsible for neonatal bacterial meningitis, with a higher mortality rate. NMEC is capable of mutating, acquiring and transmitting plasmids and other mobile genetic elements encoding resistance genes, which enable it to develop resistance to almost all common antibacterial drugs. In recent years, NMEC has increased year by year resistance to the traditional drug compound Sulfamethoxazole (SXT) for the treatment of simple urinary tract infections, and many clinical NMEC isolates have also obtained genes encoding the ultra-broad spectrum lactamase (ESBL) which confer multiple resistance to the ultra-broad spectrum cephalosporins, aztreonam, aminoglycosides and tetracyclines. In premature infants, the incidence of early-onset neonatal sepsis caused by multi-drug resistant NMEC is on the rise. In addition, the development difficulty of novel antibiotics aiming at NMEC is greater and more serious, and the development speed is slower, so that the treatment of the antibiotics of NMEC is more difficult, and the development of novel NMEC treatment schemes becomes a problem to be solved urgently.
An effective NMEC vaccine can prevent infection and reduce the prevalence of multi-resistant NMEC strains. The bacterial surface polysaccharide (O antigen and K antigen) can be used as protective antigen of bacteria, and can stimulate mature B lymphocyte after immunization, and excite immune system to generate antibody to protect organism from subsequent invasion of homologous bacteria. Polysaccharide conjugate vaccines designed from these surface polysaccharides primarily induce immune responses without T cell involvement, yielding low affinity antibody IgM + And cannot form effective immunological memory, and cannot play an immune role in infants under 2 years old. The bacterial surface polysaccharide can be covalently coupled with carrier protein to form polysaccharide protein combined vaccine, and the vaccine can be used as T cell dependent antigen to induce high affinity immunityThe reaction is carried out, so that the proliferation and differentiation of B cells into plasma cells and memory B cells are induced, high-affinity antibody IgG+ is generated, and lasting immune memory is formed, thereby realizing the immune function on infants, and simultaneously providing long-time continuous immune response for adults.
Glycoprotein vaccines make up for the deficiencies of polysaccharide vaccines and are among the safest and most effective vaccines since the history. In recent years, in vivo bioconjugate techniques using protoribosylation systems have made significant advances in glycoconjugation techniques. In vivo bioconjugate technology refers to the biosynthesis of polysaccharides and carrier proteins in engineered bacterial cells and their subsequent in vivo conjugation by use of oligosaccharyl transferases. Compared with the traditional chemical coupling method, the biological coupling technology provides a more efficient and cost-saving strategy for developing and producing the sugar-coupled vaccine.
Disclosure of Invention
In order to solve the technical problems, the invention provides a neonatal meningitis escherichia coli glycoprotein conjugate vaccine and application thereof.
The technical scheme adopted by the invention is as follows: a neonatal meningitis escherichia coli glycoprotein conjugate vaccine comprising one or more immunoconjugates formed from a neonatal meningitis escherichia coli surface polysaccharide coupled to a carrier protein.
Preferably, the serotype neonatal meningitis escherichia coli surface polysaccharide is O1, O5, O6, O7, O18 or O45;
the carrier protein is a haemophilus surface protein HiD, a Streptococcus pneumoniae PspA protein, a diphtheria toxin carrier protein CRM197 or a cholera toxin B subunit CTB, preferably a cholera toxin B subunit CTB.
Preferably, the recombinant strain is prepared by mixing serotype neonatal meningitis escherichia coli surface polysaccharides O6, O18 and O45 respectively with glycoprotein coupled with cholera toxin B subunit CTB;
or hexavalent vaccine prepared by mixing serotype neonatal meningitis escherichia coli surface polysaccharides O1, O5, O6, O7, O18 and O45 respectively with glycoprotein coupled with cholera toxin B subunit CTB.
The preparation method of the neonatal meningitis escherichia coli glycoprotein conjugate vaccine constructs a recombinant engineering bacterium containing a surface polysaccharide plasmid capable of being expressed to obtain the neonatal meningitis escherichia coli and a carrier protein plasmid capable of being expressed to obtain the carrier protein plasmid, generates an immunoconjugate of the coupling of the neonatal meningitis escherichia coli surface polysaccharide and the carrier protein after culture, and can be used for preparing vaccine stock solution after extraction and purification.
Preferably, the recombinant engineering bacteria delete zwf, pfkB, glgC, wecG, wecC, ushA, wcaJ, wcaA-I and waaL genes.
Preferably, the E.coli surface polysaccharide plasmid capable of expression to give neonatal meningitis is obtained by cloning the galF-wzz region of E.coli O1, O5, O6, O7, O18 or O45 into the yeast E.coli shuttle vector pCRG 16;
the expression vector protein plasmid was obtained by cloning HiD, pspA, CRM197 or CTB, respectively, from the E.coli codon optimized O-glycosyltransferase encoding gene into the pCDFDuet-1 plasmid.
Preferably, the original T7 promoter and terminator can be replaced by two pairs of P15 promoter and BBa_B1004 terminator in the expression vector protein plasmid;
preferably, the O-glycosyltransferase gene is derived from Neisseria meningitidis, acinetobacter bailii, neisseria gonorrhoeae, pseudomonas aeruginosa, neisseria lactose or Neisseria polysaccharea, preferably pglL from Neisseria meningitidis.
A recombinant engineering bacterium can produce glycoprotein required for preparing a neonatal meningitis escherichia coli glycoprotein conjugate vaccine.
Preferably, the recombinant engineering bacteria deleted zwf, pfkB, glgC, wecG, wecC, ushA, wcaJ, wcaA-I and waaL genes.
The application of the neonatal meningitis escherichia coli glycoprotein conjugate vaccine in preparing a medicament for preventing NMEC infection.
The invention has the advantages and positive effects that: the neonatal meningitis escherichia coli glycoprotein conjugate vaccine related by the invention can stimulate mice to generate protective antibodies, the protection rate can reach more than 90%, and the antibodies can be transferred to neonatal mice through maternal and infant transfer, so that the neonatal mice are protected from infection.
Drawings
FIG. 1 shows the structure of pCRG16-O1/O5/O6/O7/O18/O45 plasmid;
FIG. 2 construction of pCDF-pglL-HiD/PspA/CRM197/CTB plasmids;
FIG. 3pCRG16-O1/O5/O6/O7/O18/O45 plasmid identification;
FIG. 4 glycoprotein conjugate vaccine western blot detection;
FIG. 5 glycoprotein vaccine immunization of mice with methods and challenge experimental procedures;
FIG. 6 is a graph of survival of immunized adult mice after challenge;
FIG. 7 antibody titers in serum of immunized adult mice;
FIG. 8 is a diagram showing a method for transferring a mother and infant to an animal experiment mouse and a toxicity attack experiment flow;
FIG. 9 antibody titers in offspring young mouse serum after immunization of adult mice;
FIG. 10 level of bacteremia in offspring produced after immunization of a female mouse.
Detailed Description
Embodiments of the present invention are described below with reference to the accompanying drawings.
The invention relates to a neonatal meningitis escherichia coli glycoprotein conjugate vaccine, which comprises one or more immunoconjugates, wherein the immunoconjugates are formed by coupling neonatal meningitis escherichia coli surface polysaccharide and carrier protein. Wherein, the surface polysaccharide of the serovars neonatal meningitis escherichia coli is O1, O5, O6, O7, O18 or O45; the carrier protein is a haemophilus surface protein HiD, a Streptococcus pneumoniae PspA protein, a diphtheria toxin carrier protein CRM197 or a cholera toxin B subunit CTB, preferably a cholera toxin B subunit CTB.
The immune composition containing multivalent neonatal meningitis escherichia coli conjugate is formed by coupling, separating and purifying surface polysaccharides of multiple serotypes neonatal meningitis escherichia coli with proteins, and mixing, wherein different polysaccharides can be coupled with the same proteins to form glycoproteins and then mixing, or different polysaccharides can be coupled with different proteins to form glycoproteins and then mixing. Respectively preparing plasmid systems containing NMEC different serotypes O antigen synthesis gene clusters and glycosylated plasmid systems capable of expressing and obtaining carrier proteins, transferring the plasmid systems and the glycosylated plasmid systems into engineering bacteria prepared by the same host bacteria, and preparing the synthesized lipopolysaccharide which can be used for preparing neonatal meningitis escherichia coli glycoprotein conjugate vaccine.
Serotypes of E.coli with neonatal meningitis include O1, O5, O6, O7, O18, O45, the gene cluster being of specific origin GenBank accession No. GU299791, AB811596, AB811597, AF125322, AB811603, AY771223. An immune composition may be prepared that also includes a multivalent neonatal meningitis escherichia coli conjugate. Plasmids containing NMEC different serotypes of O antigen synthesis gene cluster refer to cloning the galF-wzz region of E.coli O1, O5, O6, O7, O18, O45 into the yeast E.coli shuttle vector pCRG 16. In E.coli, almost all genes responsible for the synthesis of O-polysaccharide are located in the gene cluster between galF and gnd, while wzz responsible for the regulation of O antigen chain length is located downstream of gnd. The recombinant vector can be stably replicated and expressed in escherichia coli and saccharomyces cerevisiae, and contains genes for synthesizing the O antigen repeating units, and polymerase and invertase for polymerizing the repeating units.
In certain embodiments of the invention, the carrier protein coupled to the neonatal meningitis E.coli surface polysaccharide may be the Haemophilus influenzae surface protein HiD, the Streptococcus pneumoniae PspA protein, the diphtheria toxin carrier protein CRM197, or the cholera toxin B subunit CTB. The glycosylation plasmid system is based on pCDFDuet-1 plasmid, the original T7 promoter and terminator in pCDFDuet-1 plasmid are replaced by two pairs of synthesized P15 promoter and BBa_B1004 terminator, and then the O-glycosyltransferase coding gene and the gene capable of synthesizing expression carrier protein are integrated under the control of the pair of P15 promoter and BBa_B1004 terminator, so that the glycosylation plasmid system can be obtained. By modifying and replacing the T7 promoter in the original plasmid, the phenomenon that expression cannot be identified in the escherichia coli is avoided, the replaced P15 promoter and BBa_B1004 terminator can stably express under a non-induction condition, meanwhile, the use of IPTG is avoided, and the production cost is greatly reduced. In certain embodiments of the invention, the plasmid pCDF-pglL-HiD/PspA/CRM197/CTB may be prepared.
Wherein the O-glycosyltransferase may be an O-glycosyltransferase-encoding gene derived from Neisseria meningitidis, acinetobacter bailii, neisseria gonorrhoeae, pseudomonas aeruginosa, neisseria lactose or Neisseria polysaccharea, and which has been codon-optimized by E.coli. The acquisition numbers of the genes encoding oligosaccharyl transferase and the synthesized carrier protein are respectively as follows: neisseria meningitidis (GenBank accession No.: JN 200826.1), acinetobacter besseyi (GenBank accession No.: WP 004923783.1), neisseria gonorrhoeae (GenBank accession No.: UWT 15409.1), pseudomonas aeruginosa (GenBank accession No.: wp_ 058150674.1), neisseria lactose (GenBank accession No.: CP 031253.1), neisseria polysaccharose (GenBank accession No.: CP 031325.1); haemophilus influenzae surface protein HiD (GenBank accession No.: ACV 30033.1), streptococcus pneumoniae PspA protein (GenBank accession No.: M74122.1), diphtheria toxin carrier protein CRM197 (GenBank accession No.: AMV 91693.1) or cholera toxin B subunit CTB (GenBank accession No.: M23050.1). Among them, O-glycosyltransferase preferably uses pglL from Neisseria meningitidis (Campylobacter jejuni) and codon optimized by E.coli.
The preparation method of the neonatal meningitis escherichia coli glycoprotein conjugate vaccine constructs a recombinant engineering bacterium containing a surface polysaccharide plasmid capable of being expressed to obtain the neonatal meningitis escherichia coli and a carrier protein plasmid capable of being expressed to obtain the carrier protein plasmid, generates an immunoconjugate of the coupling of the neonatal meningitis escherichia coli surface polysaccharide and the carrier protein after culture, and can be used for preparing vaccine stock solution after extraction and purification. One or more plasmids containing NMEC different serotypes O antigen synthesis gene clusters and one or more glycosylation plasmids are transferred into the same host cell, and expressed glycoprotein is the main component of glycoprotein vaccine. Purifying and mixing one or more expressed glycoproteins to prepare the neonatal meningitis escherichia coli glycoprotein conjugate vaccine stock solution.
In certain embodiments of the invention, to avoid competitive formation of LPS, waaL encoding an O-polysaccharide ligase enzyme capable of transferring lipid-linked polysaccharide to lipid A of LPS was deleted in these recombinant strains. Meanwhile, zwf and pfkB are deleted, so that the most key intermediate metabolites of the synthesized sugar precursor, namely glucose-6-phosphate and fructose-6-phosphate, can flow to NDP-sugar to the greatest extent, and an important synthetic substrate is provided for synthesizing O antigen. Knocking out ushA avoids hydrolysis of synthetic NDP-sugar into 1-phosphate sugar and NMP, and reduces decomposition thereof. Blocking wecG, wecC can prevent the generated UDP-ManNAc from synthesizing UDP-ManNAca and entering the ECA synthesis path, thereby improving accumulation of UDP-ManNAc. Knockout of glgC can accumulate more of the intermediary metabolite glucose 1-phosphate, preventing its breakdown to form ADP-glucose. Deletion of wcaJ, wcaA-I blocks UDP-glucose, GDP-fucose, UDP-galactose, UDP-GlcA from entering the clara acid synthesis pathway to reduce the competing pathway flux of nucleoside sugar precursor synthesis. The above knock-out of these genes ultimately allows for the maximal synthesis of strain O antigen, and thus the highest yield of glycoprotein.
The preparation method comprises the following steps:
step one: taking E.coli K-12MG1655 (ATCC: 25404), deleting the waaL gene encoding O-polysaccharide ligase, and deleting zwf, pfkB, glgC, wecG, wecC, ushA, wcaJ, wcaA-I and waaL genes simultaneously to block the competing pathways for the synthesis of nucleoside sugar precursors;
1.1, transforming plasmid pCas into host bacterium escherichia coli K12 MG1655 to obtain host bacterium carrying the plasmid;
1.2 designing sgRNA and a homology arm of a gene to be deleted by taking a K12 MG1655 genome as a template, constructing pTarget plasmid in vitro by a Gibson assembly method, and converting competent cells DH5 alpha;
1.3 extracting pTarget plasmid with correct sequencing;
1.4 transferring pTarget plasmid into the host bacterium carrying plasmid pCas obtained in the step 1.1, and coating the plasmid on a flat plate of LB+spectinomycin (50 ug/ml) +L- (+) -arabinose (10 mM) after electric shock recovery to obtain recombinant bacterium with target genes deleted;
1.5 the recombinant bacterium deleted of one gene obtained in the step 1.4 is used as a host bacterium, and the steps 1.2 are repeated in sequence,
1.3, 1.4, and finally obtaining the recombinant bacteria deleted from zwf, pfkB, glgC, wecG, wecC, ushA, wcaJ, wcaA-I and waaL.
Step two: construction of glycosylation plasmid
2.1 taking pCDFDuet-1 plasmid, synthesizing a fragment containing two pairs of P15 promoter and BBa_B1004 terminator, and replacing the original T7 promoter and T7 terminator by enzyme digestion connection;
2.2PCR amplified codon optimized pglL and HiD/PspA/CRM197/CTB genes were ligated to pCDFDuet-1 vector by cleavage to give pCDF-pglL-HiD/PspA/CRM197/CTB with plasmid structure shown in FIG. 2.
Step three: construction of pCRG16-O plasmid
3.1 preparation of Yeast competent cells;
3.2 obtaining pCRG16 linear cloning vector, extracting pCRG16 plasmid and digesting pCRG16 by NotI restriction endonuclease.
3.3 obtaining DNA Assembler fragments; taking NMEC O6: K1 as an example, extracting genomes of corresponding serotypes of NMEC, locating corresponding O antigen synthesis gene clusters through a BSPdb database (http:// BSPdb. Nankai. Edu. Cn/index. Html), and amplifying the whole O antigen synthesis gene clusters into 5 parts, wherein each part is about 5K; the first fragment contains 40mer bases of homologous sequence at the NotI cleavage site of pCRG16 linear cloning vector at 5', the second fragment contains about 100-200mer of homologous sequence at the 3' of the first fragment, and so on until the 3' of the last fragment contains 40mer bases of homologous sequence at the NotI cleavage site of pCRG16 linear cloning vector;
3.4 recombinant construction of O antigen expression plasmid by Saccharomyces cerevisiae in vivo;
co-electrotransformation of the pCRG16 linear cloning vector prepared in the step 3.2 of the fragment obtained by the amplification in the step 3.3 to CRY1-2 saccharomyces cerevisiae, resuscitating in 1ml SD uracil deficiency liquid culture medium, taking 100 mu L of resuscitated culture medium, coating on SD uracil deficiency flat plates, culturing for 48-72 hours at 30 ℃, randomly picking up 3-5 single colonies, extracting yeast plasmids by using a yeast plasmid extraction kit, identifying plasmids by a PCR method, and judging whether recombination is successful or not by size; the plasmid structure is shown in FIG. 1, recombinant plasmid extracted from yeast is transformed into NEB-10β, and plasmid in NEB-10β is extracted and sequenced. Finally, pCRG16-O1/O5/O6/O7/O18/O45 is obtained.
Step four: construction of recombinant strain of E.coli glycoprotein from meningitis
Transferring pCRG16-O1/O5/O6/O7/O18/O45 and pCDF-pglL-HiD/PspA/CRM197/CTB into the competent cells of the recombinant strain MG1655 prepared in the first step, resuscitating for 1-2h, coating chloramphenicol and streptomycin double-antibody plates, and culturing at 37 ℃ for 48-60h; and carrying out PCR identification and screening on the monoclonal to obtain the recombinant engineering bacteria.
Step five: fermenting and synthesizing glycoprotein-binding vaccine; culturing one or more recombinant engineering bacteria prepared in the fourth step, extracting protein, purifying to obtain target glycoprotein, and mixing one or more glycoproteins to obtain the neonatal meningitis escherichia coli glycoprotein conjugate vaccine stock solution.
The glycoprotein conjugate vaccine can stimulate the immune system of the organism to generate T cell dependent immune response, greatly improves the immunogenicity of polysaccharide and has strong specificity. In the whole immunization process, glycoprotein is not required to be widely crosslinked with antibody like polysaccharide, so that shorter sugar chains can still generate effective immune reaction, and glycoprotein vaccine prepared by coupling polysaccharide and protein has better effect than simple glycoprotein vaccine. Binding of bacterial surface polysaccharides to carrier proteins can elicit T cell-dependent immune responses that elicit high affinity and long lasting memory antibody responses. Glycoprotein conjugate vaccines have very remarkable effects on disease prevention during clinical use.
The prepared neonatal meningitis escherichia coli glycoprotein conjugate vaccine can be used for NMEC infection, and particularly has outstanding performance when the covalent products of bacterial surface polysaccharide O6/O18/O45 and PspA and the covalent products of bacterial surface polysaccharide O1/O5/O6/O7/O18/O45 and PspA are purified and mixed to form the hexavalent vaccine. Animal immunization proves that the protective antibody can be stimulated to generate protective antibodies by the mice, the mice are induced to generate specific antibodies, the mice are protected from NMEC infection, the protection rate reaches 90-100%, and the occurrence and the development of meningitis can be prevented; compared to glycoprotein vaccines, glycoprotein vaccines are able to produce long lasting antibodies in the mother, and can also deliver antibodies to neonates via maternal and infant delivery, and specific antibodies passively transferred from vaccinated mice have been shown to be effective in protecting neonate mice against pathogens in the first month after birth, and can deliver antibodies to neonates via maternal and infant delivery, protecting them from infection.
The following description of the present invention is made with reference to the accompanying drawings, wherein the experimental methods without specific description of the operation steps are performed according to the corresponding commodity specifications, and the instruments, reagents and consumables used in the embodiments can be purchased from commercial companies without specific description.
In the examples, the plasmid was extracted using the SanPrep column type plasmid DNA miniextraction kit (Catalog NO. B518191) from the Shanghai Co., ltd., and the cut gel was recovered using the SanPrep column type gel recovery kit (Catalog NO. B518131) from the Shanghai Co., ltd., and the DNA fragment was amplified using pfu DNA polymerase (Catalog NO. EP 0571) from the ferments Co., and the PCR plasmid template was digested using Fast Digest NotI (Catalog NO. FD 0593) from the ferments Co., ltd.). The experiment of the electrical shock transformation of the coll was performed using an electrotransport apparatus (Catalog NO.: 165-2100) of Bio-Rad. Bacterial genome extraction the bacterial genome extraction kit (Catalog NO.: CW 0552S) from Beijing, a century Biochemical technology Co., ltd.
Example 1: recombinant host bacterium E.coli K-12MG1655
Construction of ΔzwfΔpfkB ΔglgC ΔwecC ΔushA ΔwcaΔwcaJ ΔwcaA-I ΔwaaL
Construction of the coll K-12MG1655/pCas
(1) Extraction of pCas plasmid
The pCas plasmid is extracted by using a small amount of extraction kit of the engineering column type plasmid, and specific steps are shown in the specification of the kit. (2) Preparation of E.coli K-12MG1655 competent cells
(3) E.coli K-12MG1655/pCas Strain acquisition
The extracted pCas plasmid was transformed into E.coli K-12MG1655 competent cells by electroporation, and then transferred into resuscitating medium for about half an hour, plated with kanamycin resistance 2YT plate, and cultured overnight in a 30℃incubator. The following day it can be seen that the single colony grown on the plate is E.coli K-12MG1655 strain containing pCas plasmid.
1.2 construction of pTarget-waaL plasmid
(1) Design of the gRNA principle: the http:// www.rgenome.net/Cas-designer/PAM Type is opened to select SpCas9 from Streptococcus pyogenes:5' -NGG-3' (because Cas9 protein is derived from Streptococcus pyogenes), the other is selected from the Organism Type, the genome is selected to Escherichia coli (K-12, MG1655), the CDS sequence of waaL is taken to paste about 900bp (not more than 1000 bp) to the Target sequence, the submission is performed, the high scoring spacer is selected, the last three bases in spacer are PAM sequences, no replication is required, spacer+cas9BIRE+spy-tem constitutes gRNA, a ranging primer is designed according to the gRNA sequence, the spacer of the upstream primer is a non-binding region, the downstream primer 5' contains 25-30bp bases which are consistent with the sequence at the left end of the upstream homology arm, the pTarget is used as template for amplification, the high fidelity primer amplification is recommended to be added at the time of amplification, and a secondary structure of 0.5 mu L-1 mu L is favorable.
(2) The principle of amplifying the upstream homology arm and the downstream homology arm is that the left end of the downstream homology arm is consistent with the right end of the upstream homology arm by 25-30 base sequences, and the downstream homology arm can be obtained by connecting the upstream homology arm and the downstream homology arm through overlap extension PCR.
(3) The pTarget plasmid was reverse amplified except for the gRNA, which contained 25-30 homologous bases at the left end, consistent with the left end of the gRNA.
(4) According toHiFi DNA Assembly description, construction
pTarget-GwaaL-HwaaL。
(5) Preparation of E.coli K-12MG1655/pCas competence (cultured at 30 ℃ C.) (this competence may or may not be induced with 10mM-100mM L- (+) -arabinoside), transformation
The pTarget-GwaaL-HwaaL plasmid was recovered in the SOC medium and cultured at 30℃for 2 hours.
(7) Inoculating the resuscitated bacterial liquid to 20ml LB+Kan+SpeC+10mM L- (+) -arabinose, culturing at 30 ℃ for 48 hours, streaking the bacterial liquid on an LB non-resistance plate, and culturing at 37 ℃; or streaked on LB+Kan+SpeC+L- (+) -arabinose plates with both Kan and SpeC working concentrations of 25mg/L.
(8) And (3) carrying out monoclonal PCR verification, and carrying out sequencing deletion to obtain the monoclonal with the genes deleted correctly.
1.3 the recombinant bacterium deleted of one gene obtained in the step 1.2 is taken as a host bacterium, the operations of the steps (1) - (8) are sequentially repeated, and finally the recombinant bacterium deleted of zwf, pfkB, glgC, wecG, wecC, ushA, wcaJ, wcaA-I and waaL is obtained.
EXAMPLE 2 construction of pCRG16-O1/O5/O6/O7/O18/O45 plasmid
2.1 amplification of O antigen Gene clusters (O-AGC)
The O antigen fragment was amplified from the wcaM to hisI region on E.coli O1/O5/O6/O7/O18/O45 genome (accession No. CP054353.1; CP026641.1; CP068155.1; CP003034.1; CP041535.1; CP 031916.1). The PCR fragment is divided into fragments containing about 4-5 kb in each section, wherein the 5 '-end of the first PCR fragment and the 3' -end of the last PCR fragment respectively carry 40bp pCRG16 homologous sequences, and a PCR amplification system (taking KOD-neoplus as a high-fidelity enzyme for example) is as follows:
The PCR amplification procedure (three-step method) was as follows:
the annealing temperature was set to the Tm value of the primer.
And separating and purifying the PCR products of each section through gel electrophoresis, cutting the target fragment into gel, recovering and measuring the concentration.
2.2 preparation of Yeast competence
1. Single colonies were picked and placed in 20mL 2YT, incubator at 30℃at 180rpm, and incubated for 16h in the dark.
2. Transfer into 200mL 2YT, and culture. And when the OD is about 0.6-1.0, the ice bath is kept for 30min.
3. The pre-cooled cells were centrifuged at 3000rpm for 5min to collect the cells. The supernatant was discarded and 30mL of precooled ddH was added 2 O re-suspended cells, and once again 3000rpm,and (5) centrifuging for 5min to collect the thalli.
4. 25mL of pre-chilled transformation solution was added to each tube of cells and the cells were resuspended by repeated sliding on ice. After confirming that the cells were sufficiently suspended, they were left to stand at room temperature for 30 minutes.
The formula of the conversion solution is as follows:
5. the cells after standing were centrifuged at 3000rpm for 5min to collect the cells. 25mL of a 1M sorbitol solution was added to each tube to resuspend the cells, and centrifuged. This step was repeated once.
6. Removing supernatant, adding appropriate volume of sorbitol solution to resuspend thallus, subpackaging into 80 μl per tube, and placing in a refrigerator at-80deg.C for use.
2.3 in vivo recombination E.coli O antigen in Yeast
1. The target fragments purified in step 2.1 were premixed separately, and the digested linearized plasmid pCRG16 vector fragment (about 80 ng) was added to the system and thoroughly mixed.
2. All fragments in this system were added to freshly prepared yeast competence and placed on ice for 30min. Subsequently, all solutions were transferred to a 2mm cuvette for shock inversion. The conversion parameters were set as: fungi-SC2-1.5kv, after the conversion is completed, 1mL SD medium is rapidly added into the system and kept stand for 3min for resuscitation.
3. All solutions in the cuvette were transferred to a sterilized 1.5mL centrifuge tube and incubated at 30℃for 3-5h at 180 rpm.
4. The recovered cells were centrifuged at 3000rpm for 5min to collect the cells. 100 mu L of supernatant is reserved for resuspension precipitation, the whole supernatant is coated on an SD plate after resuspension, the culture is carried out for more than 48 hours at 30 ℃ in dark, and after single colony is grown, the monoclonal plasmid is selected for PCR sequencing and identification.
The PCR identification system is as follows (PrimerSTAR max for example):
the PCR amplification procedure was as follows:
after amplification, the plasmid construct identified as correct was electrotransferred to E.coli competence, and the results of plasmid identification for pCRG16-O1/O5/O6/O7/O18/O45 are shown in FIG. 3.
2.4 extraction of Yeast plasmids
A plurality of recombinant microzyme monoclonals are selected and respectively inoculated into 20mL SD culture medium, and the culture is carried out for 48 hours at 30 ℃ and 180 rpm. And (5) centrifuging at 3000rpm for 5min to collect the bacterial cells and extracting plasmids. The extraction steps are as follows:
1. the above precipitate was added to 470. Mu.L of sorbitol solution, followed by 25. Mu.L of yeast wall breaking enzyme and 5. Mu.L of mercaptoethanol. After full shaking and uniform mixing, standing at 30 ℃ for 2 hours. The centrifuge tubes were inverted up and down several times every 30min interval to mix the solutions. After completion of the standing, the cells were collected by centrifugation at 12000rpm for 1 min. To the tube containing the pellet, 250. Mu.L of YP1 solution containing RNaseA was added and the pellet was suspended thoroughly with shaking.
2. Subsequently, 250. Mu.L of an RNaseA-containing YP2 solution was added to the tube and gently turned up and down several times to ensure the lysis effect.
3. 350. Mu.L of YP3 solution was added to the tube and the solution was immediately gently inverted several times to mix thoroughly. At this time, a somewhat white flocculent precipitate appears in the tube. The supernatant was transferred to an adsorption column by high-speed centrifugation at 12000rpm for 20min, and the column was loaded by high-speed centrifugation at 12000rpm for 1 min.
4. 600. Mu.L of the rinse solution I containing absolute ethanol was added to the column, and the mixture was centrifuged at 12000rpm for 1min to wash the column.
5. 600. Mu.L of rinse solution II containing absolute ethanol was added to the column, and the column was again washed by high-speed centrifugation at 12000rpm for 1 min. This step was repeated once, and the DNA was precipitated again.
And (5) centrifuging at a high speed at 6.12000rpm for 2min, and putting the mixture into an oven drying adsorption column to ensure complete volatilization of the absolute ethyl alcohol.
7. Adding 50-100 mu L ddH into adsorption column 2 O, after standing at room temperature for 2min, the plasmid was recovered by high-speed centrifugation at 12000rpm for 1 min.
Preparation of electrical conversion competence of 2.5E.coli
1. Single colonies were picked and placed in 20mL LB, 37℃incubator, 180rpm, and cultured for 16h.
2. Transfer into 200mL LB, and culture was performed. E.coli competence was prepared by ice bath for 30min when grown to an OD of about 0.6-1.0.
3. The ice-bath-completed culture broth was centrifuged at 3000rpm for 5min at 4℃and the supernatant was discarded to collect the precipitate.
4. 50mL of pre-chilled 10% glycerol was added to each tube, and the cells were suspended thoroughly by repeated blowing.
5. The suspended bacterial liquid is centrifuged at 3000rpm for 5min at 4 ℃ again, and the supernatant is discarded and the precipitate is collected.
6. 50mL of pre-chilled 10% glycerol was added to each tube, and the cells were suspended thoroughly by repeated blowing.
7. Steps 5 and 6 are repeated once.
8. Re-adding proper 10% glycerol to the centrifuged precipitate, re-suspending, sub-packaging into 100 μl of each tube, and placing in a refrigerator at-80deg.C for use.
Transformation enrichment plasmid concentration: the correct positive plasmid identified in 2.4 above was transformed into freshly prepared e.coli competence. The specific procedure of the electric conversion is as follows: about 15. Mu.L of plasmid was pipetted into DH10B competent cells first, after 15min on ice, all solutions were added to a 1mm cuvette and the transformation parameters of Ec-SC1-1.5V were selected for electric shock. 1mL of LB medium was then added and left to stand for 5min. The cells were incubated at 37℃in an incubator at 180rpm for 3 hours and coated with chloramphenicol plates. And finally, performing bacterial liquid PCR on the single bacterial colony to identify positive bacteria.
Conversion: after extracting plasmid from positive colonies identified in DH10B, 1. Mu.L of plasmid was extracted and transformed into E.coli MG1655 for competence, after ice bath for 5min, all the solutions were added to a 1mm cuvette, and transformation parameters of Ec-SC1-1.5V were selected for electric shock. 1mL of LB medium was then added and left to stand for 5min. The cells were incubated at 37℃in an incubator at 180rpm for 1 hour, and chloramphenicol plates were applied. And finally, performing bacterial liquid PCR on the single bacterial colony to identify positive bacteria. And the positive colonies were inoculated into 20mL LB medium, cultured at 37℃and 180rpm for 16 hours, and LPS was prepared for extraction to select transformants with the final correct heterologous expression of O antigen.
Example 3
Construction of recombinant plasmid pCDF-pglL-HiD/PspA/CRM197/CTB
3.1 modification of pCDFDuet-1
(1) Fragments containing two pairs of P15 promoter and BBa_B1004 terminator were synthesized from Gene company, with both EcoNI and XhoI cleavage sites.
(2) The pCDFDuet-1 plasmid was extracted using the kit and the plasmid pCDFDuet-1 and the synthetic fragment were digested with EcoNI and XhoI endonucleases, respectively.
And (3) enzyme cutting system:
plasmid 15. Mu.L (containing 3. Mu.g) EcoNI 1.5. Mu.L XhoI 1.5. Mu.L Buffer 3. Mu.L was filled with water to 30. Mu.L
30min at 37 ℃; inactivating at 65 ℃ for 20 min; preserving at 4 ℃.
The digested plasmid pCDFDuet-1 and synthetic fragment were purified using an engineered SanPrep column gel purification kit, and the concentration of the recovered product was determined using a NanoDrop2000 ultramicro spectrophotometer.
(3) The digested plasmid and fragment were mixed in a molar ratio of 1:3, and FD Ligase Buffer were added and ligated at 22℃for 2h. The enzyme-linked product was electrotransformed into DH 5. Alpha. Strain, plated with 50mg/L streptomycin-resistant LB plate, and incubated overnight at 37 ℃. The following day single colonies were picked in 5mL medium (50 mg/L streptomycin added) and plasmids were extracted. The recombinant plasmid extracted by double restriction enzymes EcoNI and XhoI is used for identifying whether the recombinant plasmid is inserted into a fragment containing two pairs of P15 promoters and BBa_B1004 terminators, the sequences are amplified by PCR and are transmitted, and the accuracy of cloning is determined by comparison with the original sequences, and the fragment is named pCDFm.
3.2 construction of pCDF-pglL
(1) Synthesis of Gene fragments
pglL was synthesized from Gene company and ligated into pUC57, and the restriction sites at both ends of pglL were EcoRI and HindIII.
(2) Extraction of pCDFm plasmid
DH5a with pCDFm plasmid was picked up and incubated overnight in 20mL medium at 37℃with shaking. The next day the pCDFm plasmid was extracted according to the bacterial plasmid extraction kit method and the plasmid concentration was determined using a spectrophotometer. Plasmid pUC57-pglL containing the pglL fragment was extracted by the same method.
(3) Cleavage and purification of plasmids and fragments
The plasmids pCDFm and pUC57-pglL were digested with EcoRI and HindIII enzymes, respectively.
And (3) enzyme cutting system:
plasmid 15. Mu.L (containing 3. Mu.g) EcoRI 1.5. Mu.L HindIII 1.5. Mu.L Buffer 3. Mu.L was filled with water to 30. Mu.L
30min at 37 ℃; inactivating at 65 ℃ for 20 min; preserving at 4 ℃.
The digested plasmid pCDFm and pglL fragments were purified using an engineered SanPrep column gel purification kit and the concentration of the recovered product was determined using a NanoDrop2000 ultramicro spectrophotometer.
(4) Enzymatic ligation and transformation of restriction enzymes
The digested plasmid and fragment were mixed in a molar ratio of 1:3, and added to FD Ligase and FD Ligase Buffer, followed by enzymatic ligation at 22℃for 2h. The enzyme-linked product was electrotransformed into DH 5. Alpha. Strain, plated with 50mg/L streptomycin-resistant LB plate, and incubated overnight at 37 ℃. The following day single colonies were picked in 5mL medium (50 mg/L streptomycin added) and plasmids were extracted. The recombinant plasmid extracted was digested with the restriction enzymes EcoRI and HindIII to identify whether it was inserted into the pglL fragment. And amplifying the sequences by PCR, carrying out transmission measurement, and comparing the sequences with the original sequences to determine the correctness of cloning.
3.3 construction of pCDF-pglL-HiD/PspA/CRM197/CTB
(1) Synthesis of Gene fragments
HiD, pspA, CRM197, CTB were synthesized from Gene Inc., each gene was ligated into pUC57, hiD, pspA, CRM, and the restriction sites at both ends of CTB were NdeI and NotI.
(2) Extraction of pCDFm-pglL plasmid
DH5a with pCDFm-pglL plasmid was picked up and incubated overnight in 20mL medium with shaking at 37 ℃. The next day the pCDFm-pglL plasmid was extracted according to the bacterial plasmid extraction kit method and the plasmid concentration was determined using a spectrophotometer. The plasmid pUC57-HiD/PspA/CRM197/CTB containing HiD, pspA, CRM, CTB fragment was extracted by the same method.
(3) Cleavage and purification of plasmids and fragments
The plasmids pCDFm-pglL and pUC57-HiD/PspA/CRM197/CTB were digested with NdeI and NotI enzymes, respectively.
And (3) enzyme cutting system:
plasmid 15. Mu.L (containing 3. Mu.g) NdeI 1.5. Mu.L NotI 1.5. Mu.L Buffer 3. Mu.L was supplied with water to 30. Mu.L
30min at 37 ℃; inactivating at 65 ℃ for 20 min; preserving at 4 ℃.
The digested plasmid pCDFm-pglL and fragment HiD/PspA/CRM197/CTB were purified using an engineered SanPrep column gel purification kit and the concentration of recovered product was determined using a NanoDrop2000 ultramicro spectrophotometer.
(4) Enzymatic ligation and transformation of restriction enzymes
The digested plasmid and fragment were mixed in a molar ratio of 1:3, and added to FD Ligase and FD Ligase Buffer, followed by enzymatic ligation at 22℃for 2h. The enzyme-linked product was electrotransformed into DH 5. Alpha. Strain, plated with 50mg/L streptomycin-resistant LB plate, and incubated overnight at 37 ℃. The following day single colonies were picked in 5mL medium (50 mg/L streptomycin added) and plasmids were extracted. The recombinant plasmid extracted by double cleavage with restriction enzymes NdeI and NotI was used to identify whether it was inserted into the HiD/PspA/CRM197/CTB fragment. And (3) amplifying the sequences by PCR, carrying out transmission measurement, comparing with the original sequences, and determining the cloning correctness to finally obtain the pCDF-pglL-HiD/PspA/CRM197/CTB.
Example 4
4.1 Synthesis of glycoprotein conjugate vaccines
(1) Preparation of recombinant host bacterium E.coli K-12
MG 1655. Delta. Zwf. DELTA. PfkB. DELTA. GlgC. DELTA.wecC. DELTA.ushA. DELTA.wcaJ. DELTA.wcaA-I.DELTA.waaL competent cells were transformed into competent cells by electroporation of the extracted pCRG16-O1/O5/O6/O7/O18/O45 and pCDF-pglL-HiD/PspA/CRM197/CTB plasmids, and then transferred to a resuscitating medium for about half an hour, coated with 50MG/L streptomycin and 10MG/L chloramphenicol resistant 2YT plates, and cultured overnight in a 37℃incubator. The following day it can be seen that the single colony growing on the plate is the production strain containing the double plasmids. The strain was inoculated into 20mL of LB medium and added with streptomycin (50. Mu.g/mL) and chloramphenicol (10. Mu.g/mL), and cultured overnight at 37℃at 180 rpm.
(2) The next morning, the overnight culture broth was transferred to 1L of NBSYT medium at a ratio of 1:100, and incubated at 37℃for 10-12h at 200 rpm.
(3) Adding IPTG to induce protein expression (final concentration is 1 mM), inducing at 30 ℃ and 180rpm for about 10-12h, and screening to obtain the target recombinant engineering bacteria.
4.2 purification of glycoprotein conjugate vaccines
(1) Bacteria were harvested by high speed centrifuge 12000rpm for 10min and transferred to a 50mL centrifuge tube.
(2) The cells were washed once with 0.9% NaCl and then resuspended to OD 600. Apprxeq.200 by adding Binding buffer.
(3) Protease inhibitor (final concentration 1 mM) and lysozyme (final concentration 1 mg/mL) were added. Ice bath for 30min.
(4) The centrifuge tube was placed on ice and the somatic cells were disrupted by an ultrasonic disrupter. And (3) exceeding 3S, stopping 2S, and accumulating the ultrasonic time for about 40min until the liquid is tan and the liquid fluidity is good. The protease inhibitors were purchased from thermo fisher scientific.
(5) Small amounts of dnase and rnase (final concentration 5 μg/mL) were added and the ice bath was kept for 15min.
(6) Centrifuge at 8000rpm for 15min at 4deg.C, transfer supernatant to another 50mL centrifuge tube (which can be kept at-80deg.C for 2-3 days).
Protein purification was performed using the AKTA primaplus system as follows:
(1) Preparing IMAC buffer A, IMAC buffer B, endotoxin removing buffer, AEC buffer A, AEC buffer B and 20% absolute ethyl alcohol. All the reagents need to be treated by a water film and are subjected to ultrasonic treatment for 30min.
(2) Opening the instrument, cleaning the machine pipeline in the load mode, and loading the nickel ion chelating chromatographic column after the curves on the display screen are all stable.
(3) And cleaning again by using the IMAC buffer A until the curve is stable again.
(4) The supernatant after ultrasonication was filtered through a 0.22 μm filter and was sampled into a loading ring. At this point, if the curve fluctuates, the IMAC buffer A is continuously flushed to a plateau in this mode.
(5) The instrument was set to the object mode and the samples in the loading ring were bound to the nickel column. Until the sample in the upper sample ring is fully bound to the nickel column.
(6) And (3) returning to the load mode, flushing 40 column volumes by using an endotoxin removing buffer, and continuing flushing by using an IMAC buffer A until the curve is stable again.
(7) And (3) adjusting to a gradient elution mode, so that the concentration of the IMAC buffer B is increased from 0% to 100% in 6 column volumes, and gradient elution of the target protein is performed. When the curve indicating UV on the display screen shows obvious absorption peak, collecting the sample in the corresponding sampling tube when the peak is detected, namely the target protein.
(8) The samples were placed in dialysis bags and dialyzed in a 4 ℃ refrigerator for 2h in dialysate to remove imidazole.
(9) The proteins purified by nickel ion affinity chromatography were purified again using ion exchange chromatography columns. The nickel ion chelating chromatographic column is replaced by an ion exchange chromatographic column, and the pipeline is flushed by AEC buffer A.
(10) And (3) loading the protein into a loading ring, and adjusting to an object mode after the curve is stable so that the protein is fully combined with the ion exchange chromatographic column.
(11) 10 column volumes were flushed with AEC buffer A.
(12) And performing gradient elution on the target protein by using an AEC buffer A and an AEC buffer B. When the curve indicating UV on the display screen shows obvious absorption peak, collecting the sample in the corresponding sampling tube when the peak is detected, namely the target protein, adding 10% glycerol and preserving at-80 ℃.
Example 5
Glycoprotein-bound vaccine detection-western blot detection:
(1) The supernatant was centrifuged at 1mL and 100. Mu.L ddH was used 2 O was resuspended, and an equal amount of 2 XSDS loading buffer was added, followed by a 10 minute boiling water bath.
(2) The sample prepared in (1) was centrifuged, and the supernatant was applied to SDS-PAGE gel loading wells, and the protein or lipopolysaccharide was separated by electrophoresis (80V, 20, 120V,1.5 hours).
(3) The proteins were transferred onto the PVDF membrane using a transfer membrane apparatus with a constant pressure of 70V for about 1 hour in the order of filter paper, gel, PVDF membrane, filter paper from top to bottom.
(4) The membrane was immersed in WB blocking solution and incubated at 37 ℃ for 1 hour.
(5) Primary antibodies (diluted in proportion with TBST) were incubated in a 37 ℃ incubator for 1 hour.
(6) The membranes were washed 3 times with TBST solution every 7 minutes.
(7) HRP-labeled secondary antibody (diluted in proportion with TBST) was incubated in a 37 ℃ incubator for 1 hour.
(8) The membrane was again washed 3 times with TBST solution every 7 minutes.
(9) The solution A and the solution B in the WB luminous reagent are uniformly mixed according to the volume of 1:1, incubated on the surface (protein) of the membrane, uniformly mixed according to the volume, incubated on the surface (protein) of the membrane, and detected by a WB imager, and the structure is shown in figure 4.
EXAMPLE 6LPS extraction and silver staining
(1) Bacteria were cultured in a 37℃Erlenmeyer flask at 220rpm for 12 hours, and then centrifuged at 8000rpm for 8 minutes to collect the bacteria, and the supernatant was discarded. The pellet was washed 3 times with pre-chilled PBS, and after the last centrifugation the supernatant was discarded and allowed to empty for 3 minutes.
(2) The thalli are resuspended according to the proportion of adding 3mL of distilled water into each gram of thalli with wet weight, the thalli are resuspended according to the proportion of distilled water placed on ice, the thalli are placed on ice for 3 minutes and then placed in a water bath kettle with the temperature of 68 ℃ for 30 minutes, and the process is repeated for 3 times.
(3) Adding an equal volume of 90% phenol solution, shaking vigorously in a water bath at 68℃for 30 minutes
(4) The mixture was centrifuged at 150000rpm for 20 minutes and the upper aqueous phase fraction was carefully aspirated. Equal amount of distilled water was added, and after shaking again vigorously in a water bath at 68℃for 30 minutes, the aqueous phase fraction was centrifuged.
(5) Adding absolute ethanol pre-cooled at-20deg.C to make the volume of absolute ethanol reach 75% (v/v), standing at-20deg.C for 20min, centrifuging at 15000rpm for 10min, and discarding supernatant. Volatilizing at room temperature.
(6) Adding cutmart buffer into ddH2O in proportion, adding 4 μl of RNase and DNase I each of 10mg/ml to 50 μl system, digesting at 37deg.C for 2 hr, adding 4 μl of proteinase K of 10mg/ml, and digesting at 56 deg.C for 2 hr
(7) Adding absolute ethanol pre-cooled at-20deg.C to make the volume of absolute ethanol reach 75% (v/v), standing at-20deg.C for 20min, centrifuging at 15000rpm for 10min, and discarding supernatant. Volatilizing at room temperature.
(8) Adding proper deionized water for redissolving and precipitating to obtain LPS water solution
(9) The sample to be tested is mixed with an equal volume of 2 Xsilver dye loading buffer solution, and the mixture is boiled in boiling water for 5 minutes and then loaded, and is separated by SDS-PAGE. After 20 minutes at a constant pressure of 80V, the 120V constant pressure was maintained for about 1.5 hours until bromophenol blue was run out of the lowest edge of the gel.
(10) Carefully taking down the gel, immersing in the fixing solution, slowly shaking for 15 minutes in a shaking table, and then replacing the fixing solution and shaking for 15 minutes.
(11) After fixation, the glue is placed in the sensitization liquid and slowly rocked for 30 minutes.
(12) Wash 3 times with ddH2O for 15 minutes each.
(13) After washing, the gel was put into silver nitrate solution and reacted for 20 minutes.
(14) The solution was washed twice with ddH2O for 1 minute.
(15) The gel was placed in a developer and the reaction was stopped depending on the color. After the occurrence of the banding, the developing solution was poured off, and a stop solution was added to stop for 10 minutes.
(16) The solution was further washed with ddH2O 3 times for 5 minutes. The scanner then scans.
The genetic engineering bacteria constructed by the invention extract lipopolysaccharide by a phenol water method, gel electrophoresis and silver staining show that the constructed bacterial strain can synthesize LPS of NMEC O1/O5/O6/O7/O18/O45 serotypes, but MG1655 which is not subjected to recombination modification cannot be synthesized, and a western-blotting result shows that a laber-shaped strip is provided, so that the recombinant engineering bacteria is proved to synthesize correct glycoprotein.
EXAMPLE 7 method for Mass preparation of neonatal meningitis E.coli serotype glycoprotein conjugate vaccine
Preparing a neonatal meningitis escherichia coli serotype glycoprotein conjugate vaccine by adopting recombinant bacteria to carry out glycoprotein vaccine cell fermentation;
fermentation medium (NBSYT) (1L) Tryptone (Tryptone): 5-10g,Yeast Extract (Yeast extract): 3-5g, (NH) 4 ) 2 HPO 4 (potassium dihydrogen phosphate): 2-4g, K 2 HPO 4 ：2-4g，KH 2 PO 4 ·3H 2 O:2-5g. If a solid medium is prepared, 10-15g of Agar (Agar) is added.
Activating each recombinant engineering bacterium prepared in the example 4 in 20ml of NBSYT culture medium containing 20-100g/L glucose and 20-100g/L glycerol at 37 ℃ and 220rpm overnight; transferring the activated bacteria into 1L NBSYT culture medium containing 20-100g/L glucose and 20-100g/L glycerol, culturing at 37deg.C and 220rpm to OD 600 And (3) carrying out culture at 180rpm at 30 ℃ for about 72 hours, centrifuging to collect bacterial cells, crushing the cells by an ultrahigh pressure crusher, and purifying the cells in a large quantity by means of an AKTA Primeplus protein purification workstation to obtain glycoprotein.
Example 8 use of a neonatal meningitis e coli glycoprotein conjugate vaccine:
immunization experiments and antibacterial experiments were performed on 5-week-old CD-1 female mice (Vetolihua laboratory animal Co., ltd.) and the flow of the glycoprotein vaccine immunization and challenge experiments are shown in FIG. 5.
The specific process is as follows:
(1) Mice were randomly divided into 3 groups, PBS group, positive control group and experimental group, respectively.
(2) Mixing the purified glycoprotein vaccine with Freund's complete adjuvant, and subcutaneously injecting into CD1 mice of 5 weeks of age three times; immunization was performed on days 0, 14, 28, respectively, with each mouse injected subcutaneously in a total volume of 100 μl, including 2.5 μg glycoprotein conjugate vaccine (calculated as polysaccharide) and equal volume of freund's adjuvant; wherein the first immunization uses Freund's complete adjuvant, and the second immunization uses Freund's incomplete adjuvant. The negative control groups were all injected with equal volumes of PBS. Positive control group 10 8 65 ℃ and 30 DEG Cmin inactivated dead bacteria.
(3) After 7 days from the end of the last immunization, blood was collected from each mouse tail vein in each group, and the serum was obtained by centrifugation at 3000rpm at 4℃and placed at-80℃for use.
(4) An antibacterial experiment was performed. A lethal dose of NMEC O1:K1, O5:K1, O6:K1, O7:K1, O18:K1 and O45:K1 was intravenously injected into the tail vein of each of three groups of mice, and after 24 hours, tail vein blood was taken out of an anticoagulant tube to dilute and coat LB solid plates, and the plates were counted after single colonies were grown. The survival of the mice was observed continuously for 4 days. As shown in figure 6, the survival rate of mice injected with glycoprotein conjugate vaccine is high, the protection rate reaches 90-100%, and the occurrence and development of meningitis can be prevented.
And then carrying out glycoprotein-bound vaccine-induced mice to generate specific antibodies, taking blood from tail veins of the mice immunized for the third time, centrifuging at 4 ℃ for 30min at 1000 Xg, and taking the supernatant to prepare serum. The results of measuring specific IgG antibodies in blood by ELISA are shown in FIG. 7, and the protein-bound vaccine can stimulate the organism to produce protective antibody IgG.
From the above experimental results, it can be seen that the purified glycoproteins of various serotypes of neonatal meningitis escherichia coli are mixed to form a hexavalent vaccine, which can induce mice to generate specific antibodies and protect the mice from NMEC infection.
Example 9 mother-infant transfer animal experiment
The steps of the method for transferring the mice of the animal experiment to the mother and infant and the toxicity attack experiment are shown in figure 8.
Adult female mice immunized three times were bred and fed individually with cages. Pregnant mice were bred after about 1 month of completion of the breeding, and young mice were born. Blood was taken from 16 day old mice and assayed for antibody titer, and tail vein injections NMEC O1: K1, O5: K1, O6: K1, O7: K1, O18: K1 and O45: K1 were performed, respectively. The blood and cerebrospinal fluid of the young mice are taken to determine the bacterial load.
The results are shown in fig. 9 and 10, where all neonatal mice born with the hexavalent vaccine immunized mice had significantly increased specific IgG titers detected in blood compared to neonatal mice of the control PBS vaccinated mice (fig. 9). Meanwhile, neonatal mice of these immunized mice showed low bacteremia level, inhibition of meningitis development and increased survival rate (fig. 10), indicating that maternal immunization with the glycoconjugate vaccine prepared by the invention can effectively protect neonatal mice from NMEC attack.
The foregoing describes the embodiments of the present invention in detail, but the description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. All equivalent changes and modifications within the scope of the present invention are intended to be covered by the present invention.
Claims (10)
1. A neonatal meningitis escherichia coli glycoprotein conjugate vaccine, characterized by: comprising one or more immunoconjugates formed by coupling a neonatal meningococcal escherichia coli surface polysaccharide with a carrier protein.
2. The neonatal meningitis escherichia coli glycoprotein conjugate vaccine according to claim 1, wherein: the surface polysaccharide of the serovars neonatal meningitis escherichia coli is O1, O5, O6, O7, O18 or O45;
the carrier protein is a haemophilus surface protein HiD, a Streptococcus pneumoniae PspA protein, a diphtheria toxin carrier protein CRM197 or a cholera toxin B subunit CTB, preferably a cholera toxin B subunit CTB protein.
3. The neonatal meningitis escherichia coli glycoprotein conjugate vaccine according to claim 1, wherein: is formed by mixing serotype neonatal meningitis escherichia coli surface polysaccharides O6, O18 and O45 with glycoprotein coupled with cholera toxin B subunit CTB respectively;
Or hexavalent vaccine prepared by mixing serotype neonatal meningitis escherichia coli surface polysaccharides O1, O5, O6, O7, O18 and O45 respectively with glycoprotein coupled with cholera toxin B subunit CTB.
4. A method for preparing a neonatal meningitis escherichia coli glycoprotein conjugate vaccine according to any one of claims 1-3, characterized by: constructing recombinant engineering bacteria containing surface polysaccharide plasmid capable of expressing and obtaining neonatal meningitis escherichia coli and carrier protein plasmid, culturing to generate immunoconjugate of coupling of the neonatal meningitis escherichia coli surface polysaccharide and the carrier protein, extracting and purifying to prepare vaccine stock solution.
5. The method for preparing the neonatal meningitis escherichia coli glycoprotein conjugate vaccine according to claim 4, wherein the method comprises the following steps of: the recombinant engineering bacteria delete zwf, pfkB, glgC, wecG, wecC, ushA, wcaJ, wcaA-I and waaL genes.
6. The method for preparing the neonatal meningitis escherichia coli glycoprotein conjugate vaccine according to claim 5, wherein the method comprises the following steps of: the polysaccharide plasmid on the surface of the escherichia coli capable of expressing and obtaining neonatal meningitis is obtained by cloning a galF-wzz region of escherichia coli O1, O5, O6, O7, O18 or O45 into a yeast-escherichia coli shuttle vector pCRG 16;
The expression vector protein plasmid was obtained by cloning HiD, pspA, CRM197 or CTB, respectively, from the E.coli codon optimized O-glycosyltransferase encoding gene into the pCDFDuet-1 plasmid.
7. The method for preparing the neonatal meningitis escherichia coli glycoprotein conjugate vaccine according to claim 4, wherein the method comprises the following steps of: the original T7 promoter and terminator can be replaced by two pairs of P15 promoters and BBa_B1004 terminators in the expression obtained carrier protein plasmid;
preferably, the O-glycosyltransferase gene is derived from Neisseria meningitidis, acinetobacter bailii, neisseria gonorrhoeae, pseudomonas aeruginosa, neisseria lactose or Neisseria polysaccharea, preferably pglL from Neisseria meningitidis.
8. The recombinant engineering bacterium is characterized in that: capable of producing the glycoprotein required for the preparation of the neonatal meningitis escherichia coli glycoprotein conjugate vaccine of any one of claims 1-3.
9. The recombinant engineering bacterium according to claim 8, wherein: the recombinant engineering bacteria delete zwf, pfkB, glgC, wecG, wecC, ushA, wcaJ, wcaA-I and waaL genes.
10. Use of a neonatal meningitis escherichia coli glycoprotein conjugate vaccine according to any one of claims 1-3 in the preparation of a medicament for preventing NMEC infection.
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