CN108774628B - Escherichia coli engineering bacterium for synthesizing neonatal meningitis escherichia coli glycoprotein conjugate vaccine and application - Google Patents

Abstract

The invention discloses an escherichia coli engineering bacterium for synthesizing an escherichia coli glycoprotein conjugate vaccine for neonatal meningitis and application thereof. Relates to a method for constructing a cell factory for synthesizing Escherichia coli O1 serotype glycoprotein conjugate vaccine for causing neonatal meningitis. The O1 antigen synthetic gene cluster plasmid is constructed by utilizing the efficient homologous recombination efficiency of saccharomyces cerevisiae and based on a DNA Assembler method. Transforming O1 antigen into shuttle plasmid of synthetic gene cluster in Escherichia coli JM109, extracting lipopolysaccharide, performing gel electrophoresis and identifying plasmid by silver staining; deletion in JM109 with the aid of FLP-FRTwaaLAndwecAand eliminating the interference of original incomplete O antigen. The pET28a (+) plasmid was engineered, induced, glycoprotein-binding vaccines were synthesized, glycoproteins were purified by means of the AKTA Primeplus protein purification workstation and identified by western-blotting. The recombinant escherichia coli constructed by the invention provides a new idea for synthesizing glycoprotein conjugate vaccine by a biological method.

Description

Escherichia coli engineering bacterium for synthesizing neonatal meningitis escherichia coli glycoprotein conjugate vaccine and application

Technical Field

The invention belongs to the technical field of synthetic biology, and relates to a method for synthesizing a neonatal meningitis escherichia coli glycoprotein conjugate vaccine by utilizing recombinant escherichia coli. More specifically an engineered Escherichia coli bacterium for synthesizing Escherichia coli glycoprotein conjugate vaccine causing neonatal meningitis and its application.

Background

Meningitis-causing escherichia coli (NMEC) of newborns is a large group belonging to enteropathogenic escherichia coli, and main infection objects are newborns and infants and people with low immunity and belong to conditional pathogenic bacteria. After colonization through the gastrointestinal or respiratory mucosa, NMEC can invade the blood circulation system and multiply in large quantities to form high-concentration bacteremia. After the bacteremia concentration reaches a certain threshold, the NMEC will enter the brain infection link. By expressing various specific virulence factors to resist the attack of a host immune system, NMEC can cross the blood brain barrier of a human body, enter cerebrospinal fluid and brain tissues for reproduction and growth, and cause serious meningitis and sequelae which cannot be cured. Compared with diseases caused by avian pathogenic escherichia coli and uropathogenic escherichia coli, the high mortality rate and the high treatment difficulty of NMEC infection are one of the problems in clinical medical research. NMEC is resistant to environmental stresses, is widely distributed, and in addition to the multiple drug resistance that has emerged in recent years, has made the prevention and treatment of NMEC a great difficulty. Vaccination with the corresponding vaccine is an effective means to combat NMEC infection. At present, the development of vaccines for the Escherichia coli pathogenic bacteria causing meningitis of newborn is less. The invention provides a vaccine production strategy which is simpler and more convenient for special pathogenic bacteria, and provides a new way for producing polysaccharide protein conjugate vaccine.

Disclosure of Invention

In order to achieve the purpose, the invention discloses an escherichia coli genetic engineering bacterium for synthesizing an escherichia coli glycoprotein conjugate vaccine causing neonatal meningitis, which is characterized by containing different serotype O antigen synthetic gene cluster plasmids of NMEC and glycosylation system plasmids, and deleting simultaneouslywaaLAndwecAgene, named engineering strain JM109waaL。The escherichia coli genetic engineering bacteria are characterized in that the plasmid containing the NMEC serotype O1 antigen synthetic gene cluster is used for positioning an O antigen synthetic gene cluster region through a BSPdb database and connecting the O antigen synthetic gene cluster region to a saccharomyces cerevisiae-escherichia coli shuttle vector pCRG 16. The vector can be stably replicated and expressed in escherichia coli and saccharomyces cerevisiae respectively, and simultaneously contains a gene for synthesizing an O1 repeating unit, and polymerase and invertase for polymerizing the repeating unit. The identification method comprises the following steps: the vector is introduced into JM109 to obtain JM109/pCRG16-O1, LPS is extracted, and the correctness of the plasmid is identified by gel electrophoresis and silver staining. The plasmid map is shown in FIG. 1.

The plasmid containing the glycosylation system in the invention refers to: improvement ofpET28a (+) was constructed, and two synthetic pairs of tac promoter and rrnB terminator were inserted to replace the original T7 promoter and terminator, the plasmid containing the plasmid from P.aeruginosa (P.aeruginosa)Pseudomonas aeruginosa) Exotoxin a gene optimized by escherichia coli codon (exotoxin a, GI: 877850); and from Campylobacter jejuni: (Campylobacter jejuni) And codon-optimized N-glycosyltransferase coding gene of escherichia coli (undecaprenyl-diphosphologoccharide-protein glycosyltransferase, GI: 905417) under the control of a pair of tac promoter and rrnB terminator, respectively, the plasmid being designated pET 28M-epa-pglB. The plasmid map is shown in FIG. 2.

The invention further discloses a construction method of the Escherichia coli genetic engineering bacteria for synthesizing the Escherichia coli glycoprotein conjugate vaccine causing neonatal meningitis, which is characterized by comprising the following steps:

1) introducing the plasmid pSim into a host bacterium JM109 to obtain a host bacterium carrying the plasmid;

2) the respective bands were amplified using pKD3 as a templatewaaLGene, resistance knock-out fragment of wecA homology arm;

3) firstly, transforming a resistance knockout fragment of the same gene into the host bacterium carrying the plasmid pSim obtained in the step 1 to obtainwaaLRecombinant bacteria substituted by FRT-cat-FRT sequences;

4) introducing a pCP20 plasmid into the recombinant bacteria obtained in the step 3, and recognizing an FRT sequence by means of an FLP recombinase to eliminate resistance;

5) repeating the operation of the step 3) by taking the recombinant bacterium obtained in the step 4 and deleting one gene as a host bacterium to obtain a deletionwaaLAndwecArecombinant bacteria of the gene;

6) the nucleotide sequences of the constructed waaL and wecA deletion primers and the identification primers are shown in SEQ ID NO. 1-8.

The invention also discloses a method for synthesizing the neonatal meningitis escherichia coli O1 serotype glycoprotein conjugate vaccine by adopting recombinant bacteria to perform glycoprotein vaccine cell fermentation, which is characterized by comprising the following steps:

(1) culture medium and fermentation method:

LB medium (1L) Tryptone (Tryptone): 10g, Yeast Extract (Yeast Extract): 5g, NaCl (sodium chloride): 5g, if the solid medium, then 15g Agar, will be added, the cell factory in 20ml containing kanamycin 50 u g/ml, chloramphenicol 10 u g/ml LB medium 37 degrees C, 220rpm overnight activation, activation bacteria into 1L containing kanamycin 50 u g/ml, chloramphenicol 10 u g/ml LB medium, 37 degrees C, 220rpm culture toOD ₆₀₀About.0.6, adding IPTG (1 mM), culturing at 30 ℃ and 180rpm, centrifuging to collect thalli after about 12h, carrying out ultrasonic cell disruption, and purifying the glycoprotein in a large amount by means of an AKTA Primeplus protein purification workstation;

(2) activated strain NMEC K1: O1 was cultured toOD ₆₀₀Approximatively closing to 0.6, taking a proper amount of blood after three times of intraperitoneal injection to rabbits, centrifuging to obtain serum, carrying out agglutination reaction on the serum and JM109/pCRG16-O1 strains, and identifying the band specificity through western-blotting. Biosynthesis from glucose to the NMECO1 serotype glycoprotein conjugate vaccine was achieved by constructing a recombinant E.coli strain. The technical route taken is shown in fig. 3.

The invention further discloses application of the Escherichia coli in synthesizing different serotype neomeningitis Escherichia coli glycoprotein conjugate vaccines. Bacterial meningitis is the most common and serious infectious disease of the central nervous system in the neonatal period. Because the immune system of the newborn is not healthy and lacks specific immune antibodies, the newborn is easy to feel more frequently; and the barrier function is not developed and matured (skin, intestinal tract and blood brain barrier), so that the morbidity, mortality and disability rate of the bacterial meningitis are high for the newborn, and central nervous system complications are remained due to untimely rescue, which brings huge burden to patients and society. Although the fatality rate has been reduced in developed countries in recent years from 50% in the 70 s to less than 10% in the near future, the fatality rate has not been significantly reduced in developing countries. Coli is the most common, leading gram-negative bacterium causing neonatal sepsis and meningitis, with an average mortality rate of 17% -38% and a higher incidence of neurological sequelae of survivors of 58%. Although many studies have been conducted on Escherichia coli causing neonatal meningitis, the specific pathogenic mechanism thereof remains unknown, causing many difficulties in the treatment or drug development of neonatal meningitis. The Escherichia coli causing neonatal meningitis has capsular polysaccharide, and the corresponding O antigen (O-polysaccharide) types mainly comprise O1, O18, O5, O7, O6, O11 and the like.

In the treatment of bacterial meningitis, the use of antibiotics easily causes the dissolution of human pathogenic bacteria, further aggravates local inflammatory reaction, causes further injury to blood brain barrier, and leads to aggravation of cerebral edema and cranial hypertension; on the other hand, the wide and long-term application of various antibiotics increases drug-resistant strains, drug-resistant factors quickly diffuse, the fatality rate is increased, and the treatment difficulty and expenditure are further increased. The vaccine as a biological agent can provide effective immune protection against specific diseases. Vaccines are generally divided into attenuated or inactivated pathogens, specific microbial components such as surface proteins and other parts, which are recognized and destroyed by the immune system when they enter the body, and which simultaneously generate immunological memory, thus providing effective protection when the body is again threatened. Vaccines play an important role in human history and are the most effective method for the prevention of infectious diseases in humans. Due to vaccination, some serious infectious diseases, such as smallpox, are eradicated completely. In addition, there are some diseases such as polio, measles, tetanus, etc. which are very effectively suppressed due to the use of the vaccine. The world health organization reports that 25 infectious diseases can be effectively prevented and controlled by vaccines that have been approved for marketing.

In vaccine development, specific antigen components are required to be searched, and vaccines based on bacterial surface polysaccharides are increasingly regarded. Polysaccharide antigens (O antigen, K antigen and the like) on the surface of bacteria exist in the outermost layer of almost all gram-negative bacteria, are the most main immunogenic components on the surface of the bacteria, are closely related to the pathogenicity of the bacteria, and can stimulate a host to generate an immune response. Surface polysaccharide antigens generally consist of oligosaccharide repeats (< 50), each repeat usually consisting of three to eight monosaccharide groupsAnd (4) obtaining. Bacterial surface polysaccharide antigen structures vary in the type, order, manner of attachment, and spatial structure of the polysaccharide chain of the monosaccharide constituting the repeating unit, and thus are highly diverse. Bacteria can be classified into different serotypes according to the diversity of surface polysaccharide antigens. For example, Escherichia coli has 166 different structures of O antigen, Shigella has 34 different structures of O antigen. The genes involved in surface polysaccharide antigen synthesis are generally present at a specific site on the chromosome in the form of gene clusters, e.g., in Escherichia coli, Shigella and Salmonella, where the O antigen synthesis gene cluster is locatedgalFAndgndbetween the genes.

The bacterial surface polysaccharide can stimulate the body to generate protective antibodies, but T cells do not participate in the immune process, so that high-affinity antibodies and immune memory cannot be generated, and the vaccine is almost ineffective for infants under 2 years old because the immune system of the infants is not well developed. And the polysaccharide on the surface of the bacteria is connected with proper carrier protein to form glycoprotein, so that the immunogenicity of the polysaccharide can be greatly improved. Glycoprotein-binding vaccines are vaccines in which polysaccharides are covalently linked to a carrier protein, and the individual polysaccharides (T cell-independent antigens) are converted to T cell-dependent antigens by the presence of a substrate, triggering humoral immunity, generating high affinity antibodies and immunological memory. At present, glycoprotein vaccines have a high share in the market, and vaccine products on the market mainly comprise protein and polysaccharide which are extracted and purified and are crosslinked by a chemical method, and the combined vaccine is obtained by multiple purification. The method has the advantages of low yield and high production cost due to multiple purification in the production process.

Most bacteria have polysaccharide components on the surface, are protective antigens of the bacteria, have strong specificity and can stimulate the immune system of the body to produce specific antibodies. In bacteria, glycoconjugates are diverse in type, and bacterial surface polysaccharides including Lipopolysaccharide (LPS), peptidoglycan, glycoprotein, Capsular Polysaccharide (CPS), teichoic acid, and exopolysaccharide can stimulate the body's immune system to produce specific antibodies, and thus can be used to prepare polysaccharide vaccines. However, the molecular weight of the polysaccharide on the surface of the bacteria is small, the polysaccharide does not belong to T cell-dependent antigens, T cells do not participate in the immune process, and high-affinity antibodies and immune memory cannot be generated. The glycoprotein formed by covalently linking the bacterial surface polysaccharide with a suitable attenuated carrier protein can stimulate the body to generate T cell-dependent immune response, and greatly improves the immunogenicity of the polysaccharide. During the whole process, glycoproteins do not need to be extensively cross-linked with antibodies like polysaccharides, so that shorter sugar chains can still produce an effective immune response. The immune mechanism caused by glycoprotein is completely different from that of polysaccharide, and the glycoprotein can cause the combined recognition of T, B cells, thereby obviously improving the immune effect. Glycoproteins can also contribute to immunological memory, and thus glycoprotein-conjugated vaccines are considered to be the most successful vaccines for humans. The glycoprotein conjugate vaccine has a very remarkable effect on the prevention of diseases in the clinical use process. Three types of vaccines are currently successfully used in the clinic, namely glycoprotein-binding vaccines against Haemophilus influenzae type B, Streptococcus pneumoniae and Neisseria meningitidis. In addition, there are many vaccines in clinical trials, such as Staphylococcus aureus, Shigella sonnei and Shigella flexneri.

Lipopolysaccharide is extracted from the gene engineering bacteria constructed by the method through a phenol-water method, gel electrophoresis and silver staining show that JM109 can synthesize the LPS of the NMEC O1 serotype, and a western-blotting result shows a ladder-shaped strip, so that correct glycoprotein is proved to be synthesized.

The invention is described in more detail below:

the first objective of the invention is to provide a cell factory for synthesizing a glycoprotein conjugate vaccine of the NMEC O1 serotype by fermenting glucose. The Escherichia coli engineering bacterium starts from JM109, deletes O-antigen ligase gene (O-antigen ligase,waaL) And an initial glycosyltransferase gene(s) ((ii))wecA) (ii) a Meanwhile, the strain contains IPTG-induced glycosylation plasmid pET28M-epa-pglB and shuttle plasmid pCRG16-O1 for expressing O1 antigen. pET28M-epa-pglB was engineered from pET28a (+), inserted into two synthetic pairs of tac promoter and rrnB terminator replacing the original T7 promoter and terminator,epais derived from Pseudomonas aeruginosaPseudomonas aeruginosa) Through the large intestineBacillus codon-optimized exotoxin A gene (exotoxin A, GI: 877850);pglBis derived from Campylobacter jejuni: (Campylobacter jejuni) And codon-optimized N-glycosyltransferase coding gene of escherichia coli (undecaprenyl-diphosphologoccharide-protein glycosyltransferase, GI: 905417) pCRG16-O1 is expression plasmid containing O1 antigen gene cluster of Escherichia coli causing neonatal meningitis, and contains genes synthesizing O1 repeating units, polymerase and invertase for polymerizing the repeating units.

The second purpose of the invention is to provide a method for constructing the strain of fermented glucose synthesized NMEC O1 serotype glycoprotein conjugate vaccine, which comprises the following steps:

1. deletion in JM109 StrainwaaLAndwecAgene:

1.1 construction of JM109/pSim Strain

1.1.1 extraction of pSim plasmid

The pSim plasmid is extracted by using a small-scale extraction kit of the crude column type plasmid, and the specific steps are shown in the kit specification.

1.1.2 preparation of JM109 competent cells

1.1.3 acquisition of JM109/pSim Strain

Transforming the extracted pSim plasmid into JM109 competent cells by an electroporator, then transferring the JM109 competent cells into a recovery culture medium for recovery for about half an hour, coating a blasticidin resistant 2YT plate, and culturing the plasmid in an incubator at 30 ℃ overnight in the dark. The next day, a single colony on the plate was seen as strain JM109 harboring the pSim plasmid.

1.2 amplification of resistance Gene fragments

pKD3 as template and waaL-FRT-chl-FRT-F/R as primer are used to amplify the band with pfu DNA polymerasewaaLChloramphenicol resistant fragments of the upstream and downstream homology arms of the gene. The PCR product was subjected to agarose gel electrophoresis and the target fragment was recovered using a gel recovery kit. The PCR procedure and the agarose gel electrophoresis procedure are described in the specification.

1.3 transformation of resistant fragments into JM109/pSim competent strains

1.3.1 preparation of JM109/pSim competent cells.

Substantially the same JM109 competent cells as in 1.1.2 were prepared, except that heat shock was carried out in a 42 ℃ water bath shaker for 30min before ice bath to induce

Expression of RED homologous recombinase. Will be provided withwaaLThe chloramphenicol resistant fragment of the homologous arm was electrically transformed into JM109/pSim competent cells, revived for 1-2h, coated with a chloramphenicol resistant 2YT plate, cultured overnight in an incubator at 37 ℃, and single clones were picked the next day for the identification by boiling PCR. The two-end identification primer is S-waaL-F/R, and the cross identification primer is S-waaL-F/inner-chl-R.

Identifying the correct monoclonal strain as having been inactivatedwaaLThe strain, whose gene is and which carries the chloramphenicol resistance gene, is labeled as Δ waaL:: Cm for JM109, and the correct strain is preserved.

1.4 Elimination of resistance genes

1.4.1 extraction of pCP20 plasmid using plasmid extraction kit.

1.4.2 preparation of JM109waaLCm, and the pCP20 plasmid was electrotransferred into the competence, recovered, spread on ampicillin-resistant 2YT plates, cultured overnight at 30 ℃ and screened.

1.4.3 on the following day, single clones on ampicillin-resistant 2YT plates were picked up and inoculated into 5mL of 2YT medium and cultured overnight at 37 ℃. The third day was measured at 1:100 percent of the culture medium is transferred into 20mL of 2YT culture medium, and the culture is carried out for 8h at 42 ℃ and 180rpm by shaking. Then, 10-5 and 10-6 of the diluted bacterial liquid are coated on non-resistant 2YT plates. On the fourth day, the monoclonal colonies on the non-resistant plates were replica-plated on non-resistant and chloramphenicol-resistant plates, respectively. And selecting a monoclonal which can grow on a non-resistant plate but cannot grow on a chloramphenicol resistant plate on the fifth day, performing bacteria boiling PCR identification, identifying a primer as S-waaL-F/R, and identifying an electrophoretogram as shown in figure 4. The correct strain is the desired deletion engineering strain JM109 ΔwaaL。

1.4.4 the correct strain obtained was grown at 37 ℃ at 220rpm in a 1:100 serial subcultures were performed 2 times, streaked onto LB plates, and the plates were photocopied to identify strains that had lost the pCP20 plasmid.

wecADeletion of geneswaaLGene deletion method, identifying primer as S-wecA-F/R, electrophoretogram as figure 5.

Construction of pET28M-pglB-EPA vector

2.1 transformation of pET28a (+) plasmid

Synthesizing a fragment containing two pairs of tac promoters and rrnB terminators, and replacing the original T7 promoter and T7 terminator by enzyme digestion connection

2.2pET28M-pglB-EPA

Synthesizing codon-optimized pglB and EPA genes, and connecting the genes to a Pet28M vector through enzyme digestion

Construction, identification and sequencing of pCRG16-O plasmid

3.1 preparation of Yeast competent cells

3.2 obtaining of pCRG16 Linear cloning vector

(1) The pCRG16 plasmid was extracted using a plasmid mini-extraction kit.

(2) Use of plasmidsNotI fast-cutting enzyme for Single enzyme digestion

3.3 obtaining of DNA Assembler fragments

Taking NMEC O1 as an example, extracting the genome of the corresponding serotype of NMEC, positioning the corresponding O antigen synthetic gene cluster through a BSPdb database (http:// bsspdb. nankai. edu. cn/index. html), and clustering the whole O antigen synthetic gene into 5 parts for amplification, wherein each fragment is about 5K. The first fragment contains the base of the 40mer homologous sequence at the NotI cleavage site of the pCRG16 linear cloning vector at 5 ', the second fragment contains the about 100-200mer homologous sequence of the first fragment at 3 ', and the like, until the 3 ' of the last fragment contains the base of the 40mer homologous sequence at the other NotI cleavage site of the pCRG16 linear cloning vector. The detailed principle is shown in FIG. 6

3.4 Saccharomyces cerevisiaein vivoRecombinant construction of O antigen expression plasmid

And (2) carrying out cotelecation transformation on the amplified fragment and a pCRG16 linear cloning vector to CRY1-2 Saccharomyces cerevisiae, recovering the fragment in 1ml of SD uracil-deficient liquid culture medium, coating 100 mu L of the recovered culture medium on an SD uracil-deficient plate, culturing for 48-72h at 30 ℃, randomly picking 3-5 single colonies, extracting yeast plasmids by using a yeast plasmid extraction kit of Solebao company, identifying the plasmids by a PCR method, and judging whether the recombination is successful or not according to the size. The recombinant plasmid extracted from yeast was transformed into DH10B, and the plasmid in DH10B was extracted and sequenced.

4. Lipopolysaccharide extraction, gel electrophoresis and silver staining

Firstly transforming the correctly sequenced O antigen expression vector into JM109 competent cells, coating chloramphenicol resistant 2YT plates, and culturing the correctly identified monoclonals with 20mL liquid 2YT culture mediumODLPS was extracted at 600 ≈ 0.6. Preparing 12% SDS-PAGE electrophoresis gel, performing gel electrophoresis on the extracted LPS, and performing silver staining.

The invention provides a method for synthesizing glycoprotein combined vaccine by specifically applying escherichia coli engineering bacteria, and a corresponding purification step and detection method, wherein the method specifically comprises the following steps:

5. fermentation, synthesis, purification and detection of glycoprotein-binding vaccines:

co-transformation of pCRG16-O and pET28M-pglB-EPA into JM109 ΔwaaL∆wecARecovering from competent cells for 1-2h, coating chloramphenicol and kanamycin double-resistant plate, and culturing at 37 deg.C for 48-60 h. And (4) performing bacteria boiling PCR identification on the grown single clone. And identifying the correct clone for bacterium preservation.

Glycoprotein-binding vaccine synthesis and purification:

(1) the above strain was inoculated into 20mL of LB medium and added with kanamycin (50. mu.g/mL) and chloramphenicol (10. mu.g/mL), cultured at 37 ℃ overnight at 180 rpm.

(2) The next morning, the overnight-cultured broth was transferred to 1L LB medium at a ratio of 1:100, and cultured at 37 ℃ and 200rpm for 10-12 h.

(3) Protein expression was induced by addition of IPTG (final concentration of 1 mM) and induced at 30 ℃ for about 10-12h at 180 rpm.

(4) The bacteria are collected by a high-speed centrifuge at 12000rpm for 10min, and the bacteria are transferred to a 50mL centrifuge tube.

(5) The cells were washed once with 0.9% NaCl and then resuspended in Binding buffer to OD 600. apprxeq.200.

(6) Protease inhibitor (final concentration of 1 mM) and lysozyme (final concentration of 1 mg/mL) were added. Ice-cooling for 30 min.

(7) The tube was placed on ice and the cells were disrupted by ultrasonication. And (4) stopping the ultrasonic treatment for 2 seconds after the ultrasonic treatment is carried out for 3 seconds, and accumulating the ultrasonic treatment time for about 40 minutes until the liquid is brownish and the liquid mobility is good. The protease inhibitor was purchased from thermo fisher scientific.

(8) Small amounts of DNase and RNase (final concentration 5. mu.g/mL) were added and the mixture was incubated in ice for 15 min.

(9) Centrifuging at 8000rpm for 15min at 4 deg.C, transferring the supernatant to another 50mL centrifuge tube (which can be stored at-80 deg.C for 2-3 days).

Protein purification was performed using the AKTA Primeplus system, as follows:

(1) IMAC buffer A, IMAC buffer B, endotoxin-removing buffer, AEC buffer A, AEC buffer B and 20% absolute ethanol are prepared according to the preparation requirement of 2.1.6.2 reagent. All the above reagents need to be processed by water film and treated by ultrasound for 30 min.

(2) And opening the instrument, cleaning the machine pipeline in a load mode, and installing the nickel ion chelating chromatographic column after the curves on the display screen are stable.

(3) Wash again with IMAC buffer a until the curve again plateaus.

(4) The sonicated supernatant was passed through a 0.22 μm filter and loaded into the loading loop. At this point the flushing with IMAC buffer A in this mode continues to plateau if the curve fluctuates.

(5) The instrument was set to inject mode and the sample in the loading loop was allowed to bind to the nickel column. Until the sample in the loading ring is completely combined with the nickel column.

(6) Readjust to load mode, first use endotoxin buffer [61] washing 40 column volume and then IMAC buffer A to continue washing, until the curve again stable.

(7) The gradient elution mode is adjusted to allow the IMAC buffer B concentration to rise from 0% to 100% in 6 column volumes for gradient elution of the protein of interest. When the curve indicating UV on the display screen has obvious absorption peak, the sample in the corresponding sampling tube is the target protein when the peak is collected.

(8) The sample was placed in a dialysis bag and dialyzed in the dialysate at 4 ℃ for 2h in a refrigerator to remove imidazole.

(9) The protein purified by nickel ion affinity chromatography was purified again by ion exchange chromatography. The nickel ion chelating column was replaced with an ion exchange column, and the line was flushed with AEC buffer A.

(10) And (3) loading the protein to a loading ring, and adjusting to an inject mode after the curve is stable so that the protein is fully combined with the ion exchange chromatographic column.

(11) Wash 10 column volumes with AEC buffer A.

(12) The target protein was eluted with a gradient of AEC buffer A and AEC buffer B. When the curve indicating UV on the display screen shows obvious absorption peak, the sample in the corresponding sampling tube when the peak is collected is our target protein (shown as SEQ ID NO: 9), and 10% glycerol is added for preservation at-80 ℃.

Glycoprotein binding vaccine assay-western blot assay:

(1) after SDS-PAGE separation, the gel was placed in a 1 × rotating membrane Buffer and shaken for 15min on a horizontal shaker.

(2) PVDF membrane and filter paper with proper sizes are prepared, the PVDF membrane is soaked in absolute methanol for 5min before use, and the filter paper is soaked in a 1 × rotating membrane Buffer. Sequentially clamping the sponge, the filter paper, the gel, the PVDF membrane, the filter paper and the sponge in this order, and placing the clamped sponge, the filter paper, the gel, the PVDF membrane, the filter paper and the sponge into a membrane rotating instrument. 4 ℃, constant pressure of 70V and rotation for 1 h.

(3) After the membrane transfer was completed, the PVDF membrane was placed in an incubation container and sealed with a skimmed milk powder solution on a horizontal shaker at room temperature for 1 hour.

(4) The PVDF membrane was washed three times with the prepared TBST solution, 10min each time, the diluted primary antibody was added, and incubated for 1h on a room temperature water flat shaker (or overnight incubation at 4 ℃).

(5) Washing with TBST for 10min three times, adding diluted secondary antibody, and incubating for 1h on a room temperature water flat shaking bed.

(6) The plates were washed three times with TBST, 10min each, once with TBS. And (3) exposing and imaging by using a high-sensitivity ECL luminescence kit in an AI600 ultra-sensitive multifunctional imager. And (3) displaying a detection result: the negative control has no band at 72KD, and the experimental group has obvious ladder-shaped bands at 72KD, which proves that the glycoprotein binding vaccine is synthesized correctly. The glycoprotein combined vaccine can stimulate an immune system of an organism to generate T cell-dependent immune response, greatly improves the immunogenicity of polysaccharide and has strong specificity. During the whole immunization process, the glycoprotein does not need to be cross-linked with the antibody as extensively as the polysaccharide, so that the short sugar chain can still generate effective immune reaction. The immune mechanism caused by glycoprotein is completely different from that of polysaccharide, and the glycoprotein can cause the combined recognition of T, B cells, thereby obviously improving the immune effect. Glycoproteins can also contribute to immunological memory, and thus glycoprotein-conjugated vaccines are considered to be the most successful vaccines for humans. The glycoprotein conjugate vaccine has a very remarkable effect on the prevention of diseases in the clinical use process. Three types of vaccines are currently successfully used in the clinic, namely glycoprotein-binding vaccines against Haemophilus influenzae type B, Streptococcus pneumoniae and Neisseria meningitidis. In addition, there are many vaccines in clinical trials, such as Staphylococcus aureus, Shigella sonnei and Shigella flexneri.

Drawings

FIG. 1 is plasmid pCRG 16-O1; the gene cluster is used for expressing the meningitis Escherichia coli O1 antigen synthesis;

FIG. 2 is a map of plasmid pET28M-epa-pglB, used for the induction of expression of the glycosylation system;

FIG. 3 is a technical roadmap for the artificial design and synthesis of cell factories;

FIG. 4 iswaaLElectropherograms with FRT-chl-FRT replacement and waaL gene deletion;

(in the figure, M: Marker; lanes 1-2 are respectively: control,waaLdeleting the strain;

FIG. 5 iswecAElectropherograms with FRT-chl-FRT replacement and waaL gene deletion; in the figure, M is Marker; lanes 1-2 are: in contrast to this, the present inventors have found that,wecAdeleting the strain;

FIG. 6 shows the construction of pCRG16-O1 plasmid.

Detailed Description

The present invention will be further described with reference to the following specific examples, but the present invention is not limited by the examples;

the materials, reagents, apparatus and methods used in the following examples are all conventional in the art and are commercially available without specific recitation;

in the present invention, the plasmid extraction was carried out using a small amount of plasmid DNA extraction kit of the SanPrep column type (Catalog NO: B518191) from Biotechnology (Shanghai) Co., Ltd., gel cutting recovery was carried out using a Small amount of plasmid DNA extraction kit of the SanPrep column type (Catalog NO: B518131) from Biotechnology (Shanghai) Co., Ltd., ligation of DNA fragment was carried out using T4 DNA library (Catalog NO: EL 0014) from cementias, amplification of DNA fragment was carried out using pfu DNA polymerase (Catalog NO: EP 0571) from cementias, digestion of PCR plasmid template was carried out using Fast Digest KpnI (Catalog NO: FD 0524) from cementias, BamHI (Catalog NO: FD 0054) NcoI (Catalog NO: FD 0574) BglII (Catalog NO: 0083) and FD NO: 0593) from cementias

Coli electroporation experiments used a Bio-Rad electrotransformation apparatus (Catalog No.: 165-2100). Bacterial genome extraction a bacterial genome extraction kit (Catalog No.: CW 0552S) of beijing kang, a century biochemical technology limited was used;

table 1 deletionwaaLAndwecAprimer sequences used

TABLE 2 sources of strains and raw materials used in the present invention

TABLE 3 amino acid sequence of Exotoxin A in glycoprotein conjugate vaccines

mkkiwlalaglvlafsasaaeeafdlwnecakacvldlkdgvrssrmsvdpaiadtngqgvlhysmvleggndalklaidnalsitsdgltirleggvepnkpvrysytrqargswslnwlvpighekpsnikvfihelnagnqlshmspiytiemgdellaklardatffvrahesnemqptlaishagvsvvmaqaqprrekrwsewasgkvlclldpldgvynylaqqrcnlddtwegkiyrvlagnpakhdldikdnnnstptvishrlhfpeggslaaltahqachlpletftrhrqprgweqleqcgypvqrlvalylaarlswnqvdqvirnalaspgsggdlgeaireqpeqarlaltlaaaeserfvrqgtgndeagaasadvvsltcpvakdqnrtkgecagpadsgdallernyptgaeflgdggdvsfstrgtqnwtverllqahrqleergyvfvgyhgtfleaaqsivfggvrarsqdldaiwrgfyiagdpalaygyaqdqepdargrirngallrvyvprwslpgfyrtgltlaapeaageverlighplplrldaitgpeeeggrvtilgwplaertvvipsaiptdprnvggdldpssipdkeqaisalpdyasqpgkppredlkhhhhhh*

Is a stop codon

Example 1

Acquisition of genes

In this example, the extract was obtained from Pseudomonas aeruginosaPseudomonas aeruginosa) Exotoxin a gene optimized by escherichia coli codon (exotoxin a, GI: 877850); and from Campylobacter jejuni: (Campylobacter jejuni) And codon-optimized N-glycosyltransferase coding gene of escherichia coli (undecaprenyl-diphosphologoccharide-protein glycosyltransferase, GI: 905417).

Example 2

Design of Gene deletion primers

This example uses the lambda Red recombination system to knock out two genes of JM109, which is performed to eliminate resistance for each gene knocked out. The following arewaaLThe gene knockout step is described in detail by taking an example as a gene,wecAthe gene deletion primers were designed in the same way.

Finding JM109waaL nucleotide sequence, and designing a deletion primer and an identification primer of waaL. The deletion primer of waaL is waaL-FRT-chl-FRT-F/R, and the identification primer is S-waaL-F/R. The nucleotide sequences are shown in tables 1-8.

Example 3

JM109ΔwaaLConstruction of

3.1 transformation of plasmid pSim

Wild type JM109 frozen at-80 ℃ was streaked on non-resistant LB plates and cultured overnight at 37 ℃. Picking the next dayThe single clone was inoculated into 5mL of LB medium and cultured overnight at 37 ℃ and 220 rpm. The following day, the cells were inoculated into 200ml of LB medium at an inoculum size of 1%. Incubated at 37 ℃ and 220rpm to OD₆₀₀About 0.6-0.8, ice-cooling for 20min, 5500rpm, 5min, collecting the thallus in a sterilized 50ml centrifuge tube, 4 ℃, 5500rpm, centrifuging for 5min, discarding the supernatant, resuspending the thallus with 50ml of ice-cooled sterile 10% glycerol, 4 ℃, 5500rpm, centrifuging for 5min, repeating the above operation for 3 times, finally, resuspending the thallus with the residual liquid when discarding the supernatant, and taking 80 μ L to be put in a new sterile EP tube. Freezing and storing at minus 80 ℃.

Thawing the competent cells frozen at-80 ℃ in ice for 10min, adding 1 μ L of pSim plasmid, mixing uniformly, adding the mixture into an electric shock cup, carrying out ice bath for 2min, carrying out electric shock transformation at 1.8KV, immediately adding 1ml of LB culture medium after electric shock, recovering for 20min at 30 ℃ and 220rpm, taking a proper amount of bacterial solution, coating the bacterial solution on an blasticidin plate (with the concentration of blasticidin of 200 μ g/ml), carrying out inversion overnight culture at 30 ℃, and obtaining a monoclonal JM109 carrying the pSim plasmid after the next day.

3.2 deletion of the Gene of interest

3.2.1 preparation of homologous recombination fragments

Using pKD3 plasmid (purchased from Wuhan vast Ling Biotech Co., Ltd.) as template, using primer waal-FRT-chl-FRT-F/R to perform PCR amplification, cutting gel, purifying and recovering to obtain a plasmid containing two endswaaLKnock-out fragment of homology arm

3.2.2 first step homologous recombination

A single clone JM109 harboring the pSim plasmid was picked and inoculated into 5ml of LB medium, incubated at 37 ℃ and 220rpm overnight. The following day, the cells were inoculated into 200ml of LB medium at an inoculum size of 1%. Incubated at 37 ℃ and 220rpm to OD₆₀₀About 0.6-0.8, transferring the bacterial liquid to a 42 ℃ water bath shaking table, 150rpm, 20min, ice-cooling for 20min, 4 ℃, 5500rpm, centrifuging for 5min, collecting the thallus in a sterilized 50ml centrifuge tube, 4 ℃, 5500rpm, centrifuging for 5min, discarding the supernatant, resuspending the thallus with 50ml ice-cooled sterile 10% glycerol, 4 ℃, 5500rpm, centrifuging for 5min, repeating the above operation for 3 times, finally, resuspending the thallus with the residual liquid when discarding the supernatant, taking 80 mu L of the thallus in a new sterile EP tube, adding the thallus in the new sterile EP tube, and performing centrifugation for 5minAdding 4 mu L of homologous fragments, uniformly mixing, adding into an electric shock cup, carrying out ice bath for 2min, carrying out 1.8KV electric shock transformation, immediately adding 1ml of LB culture medium after electric shock, culturing for 2h at 37 ℃ and 180rpm/min, taking a proper amount of bacterial liquid, coating on a resistant plate, and carrying out overnight culture at 37 ℃. The next day, single clones were picked and identified by PCRwaaLThe correct clone with the gene replaced by waaL-FRT-chl-FRT, that is, JM109waaL::FRT-chl-FRT

3.3 Elimination of resistance after Gene deletion

3.3.1JM109 ∆waaLComprises the preparation of competent cells of the FRT-chl-FRT strain

PickingE.coli BL21 (DE3) ∆lacZThe single clone was inoculated into 5ml LB medium, competent according to the above procedure, pCP20 plasmid was electrotransferred, 1ml LB medium was added immediately after electric shock, cultured at 30 ℃ and 180rpm/min for 20min, and an appropriate amount of the bacterial solution was spread on ampicillin plates (ampicillin concentration 100. mu.g/ml) and cultured overnight at 30 ℃. The next day, the single clone was picked up into 5ml LB medium (chloramphenicol concentration 25. mu.g/ml), cultured at 30 ℃ and 180rpm/min for 10h, transferred to a new liquid medium, added with no antibiotics, cultured at 42 ℃ for 6h, diluted and plated, inverted overnight, and picked up the next day, and then photocopied on a nonreactive plate and a chloramphenicol plate, respectively. The monoclonal antibody which does not grow on the chloramphenicol plate but grows on the position corresponding to the non-resistant plate, namely the positive clone with successfully eliminated resistance, is further verified by PCR.

3.4 wecADeletion of

wecADeletion principles and procedures andwaaLis identical to deletewaaLBy repeating 3.2 and 3.3 of the experimental steps on the basis of the strain of (2)wecAGene deletion, and finally JM 109. delta. is constructedwaaLΔwecA

FIGS. 4 and 5 are JM109 and deletewaaL/wecAThe PCR verification result of (a) shows that the band in lane 1 of lane 2 in fig. 4 is significantly reduced, indicating that the corresponding target gene has been successfully knocked out.

The pSim plasmid is a temperature sensitive plasmid, and the plasmid is lost when the culture temperature is higher than 30 ℃, so that the strain is always cultured at 30 ℃ after the pSim plasmid is transferred into JM109, so as to prevent the loss of the pSim plasmid.

Example 4

Immune experiment and antibacterial experiment:

a Balb/c female mouse (Witonglihua laboratory animals Co., Ltd.) with the period of 6-8 weeks is selected for immune experiments and antibacterial experiments, and the specific process is as follows:

(1) mice were randomly divided into two groups, a glycosylation group and a negative control group, 6 mice per group.

(2) Immunizations were performed on days 0, 14, and 28, respectively. Each mouse was injected subcutaneously in a total volume of 100. mu.L per injection, containing 2. mu.g of glycoprotein-binding vaccine and an equal volume of Freund's adjuvant. Wherein the first immunization is carried out with Freund's complete adjuvant, and the last two immunizations are both Freund's incomplete adjuvant. The negative control groups were injected with an equal volume of PBS.

(3) 35 days after the end of the last immunization, the tail vein of each mouse in each group was bled and centrifuged at 3000rpm at 4 ℃ to obtain serum, which was placed at-80 ℃ for further use.

(4) Antibacterial experiments were performed. Three groups of mice were injected with 100CFU of O1 serotype of meningitidis E.coli in 1 XPBS solution per mouse, tail vein injection. After four hours, the eyeball is picked to draw blood, one part of the blood is centrifuged at 3000rpm and 4 ℃ to obtain serum, and the other part of the blood is placed in an anticoagulation tube to be diluted and coated with an LB solid plate and is statically cultured in a carbon dioxide incubator at 37 ℃. And counting after the single fungus grows out of the flat plate.

(5) And (3) performing western blot detection on the collected serum respectively:

the serum obtained after immunization is used for western blot detection, and the glycosylation group can detect a ladder-shaped target band, so that the glycoprotein-conjugated vaccine can stimulate a mouse immune system to generate corresponding antibodies, and meanwhile, the detection results before and after the glycosylation group is injected with bacterial liquid are analyzed, so that the level of the antibodies in vivo is obviously increased after the injection of the bacterial liquid, the injected bacterial liquid becomes secondary immunogen, the mouse is stimulated to generate secondary response, and the glycoprotein-conjugated vaccine is further proved to stimulate an organism to generate memory cells through a complex immune mechanism.

The bacterial colony number of the experimental group plate injected with the vaccine is obviously lower than that of the negative control group injected with PBS (phosphate buffer solution), and the bacterial colony number is reduced by 100 times, so that the statistical analysis result is extremely remarkable, and the polysaccharide protein combined vaccine stimulates a mouse immune system to generate memory cells and antibodies, thereby playing a remarkable antibacterial role.

Although the present invention has been described in detail with reference to the above embodiments, it will be apparent to one skilled in the art that modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for some of the features thereof. And such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

SEQUENCE LISTING

<110> university of southern kayak

<120> Escherichia coli engineering bacteria for synthesizing Escherichia coli glycoprotein conjugate vaccine causing neonatal meningitis and application thereof

<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

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<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

gtgggtatgg gaagaatcag 20

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<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

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gccccatgcc aataatccat ag 22

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<211> 649

<212> PRT

<213> 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 (3)

1. An Escherichia coli genetic engineering bacterium for synthesizing an Escherichia coli glycoprotein conjugate vaccine causing neonatal meningitis, which is characterized by containing NMEC (neuronal peptides)Escherichia coliNMEC) O1 antigen Synthesis Gene Cluster plasmid and glycosylation System plasmid, with deletion of the samewaaLAndwecAgene, named engineering strain JM109waaL ∆wecAThe carrier is/pCRG 16-O1 pET 28M-epa-pglB; the plasmid containing the NMEC O1 antigen synthetic gene cluster refers to: the O1 antigen synthetic gene cluster region is located through a BSPdb database and is connected to a saccharomyces cerevisiae-escherichia coli shuttle vector pCRG 16;

the glycosylation system-containing plasmid refers to: pET28a (+) was engineered to insert two synthetic pairs of tac promoter and rrnB terminator in place of the original T7 promoter and terminator, and this plasmid contained a plasmid from Pseudomonas aeruginosa (Pseudomonas aeruginosa)Pseudomonas aeruginosa) Exotoxin A gene optimized by escherichia coli codon; and from Campylobacter jejuni: (Campylobacter jejuni) And the two genes in the entry are respectively under the control of a pair of tac promoter and rrnB terminator, and the plasmid is named as pET 28M-epa-pglB.

2. The use of the engineered Escherichia coli strain of claim 1 in preparing O1 serotype neomeningitis Escherichia coli glycoprotein conjugate vaccine.

3. A method for constructing Escherichia coli genetic engineering bacteria of the newborn meningitis Escherichia coli glycoprotein conjugate vaccine of claim 1, which comprises the following steps:

1) introducing the plasmid pSim into a host bacterium JM109 to obtain a host bacterium carrying the plasmid;

2) the respective bands were amplified using pKD3 as a templatewaaLGene, resistance knock-out fragment of wecA homology arm;

3) firstly, transforming a resistance knockout fragment of the same gene into the host bacterium carrying the plasmid pSim obtained in the step 1) to obtainwaaLRecombinant bacteria substituted by FRT-cat-FRT sequences;

4) introducing a pCP20 plasmid into the recombinant bacteria obtained in the step 3), and recognizing an FRT sequence by means of an FLP recombinase to eliminate resistance;

5) repeating the operation of the step 3) by taking the recombinant bacterium obtained in the step 4) and deleted with one gene as a host bacterium to obtain a deletionwaaLAndwecArecombinant bacteria of the gene;

6) the nucleotide sequences of the constructed waaL and wecA deletion primers and the identification primers are shown in SEQ ID NO. 1-8.

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