CN115851477B - Escherichia coli JC83 and application thereof - Google Patents
Escherichia coli JC83 and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides escherichia coli JC83 and application thereof, and belongs to the technical fields of bioengineering and immunology. The escherichia coli JC83 provided by the invention is preserved in China center for type culture collection, and the preservation number is CCTCCNO: m20211289. The lipopolysaccharide secreted by the outer membrane vesicle of the escherichia coli JC83 provided by the invention has obviously reduced pyrogenicity. The main immunogen protein VP2 of the infectious bursal disease virus is expressed by using the outer membrane vesicle secreted by the escherichia coli JC83 strain, and the VP2 protein can be directly expressed on the surface of the outer membrane vesicle and secreted to bacterial culture supernatant along with the outer membrane vesicle. The outer membrane vesicles expressing VP2 have better immunogenicity, and can stimulate animals to produce higher levels of antibodies after immunization.
Description
Technical Field
The invention belongs to the technical field of bioengineering and immunology, and particularly relates to escherichia coli JC83 and application thereof.
Background
Membrane proteins and virulence factors of Outer Membrane Vesicles (OMVs) induce an immune response in host cells, so OMVs can act as immunogens to stimulate cellular immune responses, preventing pathogen invasion. Therefore, OMVs with high immunogenicity and non-replicability are one of the good candidates for vaccines. Neisseria meningitidis OMVs of type B have been developed as a vaccine under the trade name Bexsero, developed by the nowa company, approved by the european commission for marketing in europe in month 1 of 2013, and are currently widely used in countries such as europe, copa, brazil, new zealand, etc.
Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria, and can cause a strong inflammatory response and regulate the immune response, stimulating the production of antibodies against various antigens by cells. OMVs are a mimetic of the ability to recognize pathogen recognition receptors, which are similar in character to adjuvants, and contain LPS, and thus have the potential to develop as adjuvants. In neisseria meningitidis mutants there is a mutant gene LpxL 1 homologous to the classical pathogen LpxL, which gene is also involved in the acylation of lipid a, resulting in 5 fatty acyl chains instead of 6 for mutant lipid a. Thus, researchers have constructed neisseria meningitidis LpxL 1 mutants by genetic engineering and found neisseria meningitidis mutants lacking the lipid a biosynthesis gene LpxL to be less toxic than wild-type strains while retaining good adjuvant activity. The introduction of exogenous LPS-modifying enzymes can also alter LPS structure. Helicobacter pylori Hp 0021 (structural gene of lipid a-1-phosphatase) as expressed in e.coli can synthesize monophosphoryl lipid a therein instead of the natural biphosphorylated form, which is likely to reduce the stimulation of host innate immune responses.
At present, various researches show that OMVs can be used as subunit vaccines developed by antigen carriers to provide protection for animals. Coli genetic engineering OMVs express exon domain matrix protein 2 (23 amino acid sequences conserved by influenza a virus), and animals can be protected from attack of influenza a H1N1 virus by using the OMVs as vaccines for vaccination; nicole et al presented GFP on OMVs of non-pathogenic and pathogenic strains of E.coli, respectively, with successful intracellular observation of green fluorescence; schroederJ fuses the leishmania antigen to the C-terminus of the E.coli autotransporter, ultimately presented at the OMVs of Salmonella; muralinath M fused the pneumococcal protein PspA to the N-terminus of beta lactamase, successfully presented on OMVs of Salmonella against infection by pneumococci; huang et al presented Omp22 protein on E.coli OMVs to combat Acinetobacter baumannii infection; chen et al present O antigen polysaccharides on lipopolysaccharides on escherichia coli OMVs to combat infection by franciscensis.
Infectious bursal disease (Infectious bursal disease, IBD) is an important immunosuppressive disease in chickens, severely threatening the health development of the poultry industry. The pathogen is Infectious Bursal Disease Virus (IBDV), which belongs to a member of the genus avian Birna virus of the family Birna virus, the genome comprises two double-stranded RNAs, i.e., A, B segments. Segment a encodes 4 proteins, of which VP2 is the capsid protein and the major protective antigen of IBDV, associated with viral virulence, cell tropism and antigenic variation. There is no research on the preparation of avian IBDV vaccines using the OMVs system to express the IBDV VVP2 protein.
Disclosure of Invention
In view of the above, the present invention aims to provide an E.coli JC83 with significantly reduced immunogenicity of outer membrane vesicle lipopolysaccharide, which E.coli JC83 can be used for preparing highly immunogenic IBDV subunit vaccines.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an escherichia coli JC83, wherein the escherichia coli JC83 is preserved in China center for type culture collection, and the preservation number is CCTCC NO: m20211289.
Preferably, the lipopolysaccharide secreted by the outer membrane vesicles by E.coli JC83 is significantly reduced in pyrogenicity.
The invention also provides an outer membrane vesicle secreted by the escherichia coli JC 83.
The invention also provides an application of the escherichia coli JC83 or the outer membrane vesicle in preparing an outer membrane vesicle vaccine.
The invention also provides an application of the escherichia coli JC83 or the outer membrane vesicle in preparing an IBDV subunit vaccine.
Preferably, the immunogen of the infectious bursal disease virus is the viral protein VP2.
Preferably, the method comprises the following steps: constructing an immunogen gene vp2 recombinant expression vector, transferring the recombinant expression vector into JC83 competent cells to obtain engineering bacteria, performing amplification culture, and separating and purifying.
Preferably, the method for constructing the immunogen gene vp2 recombinant expression vector comprises the following steps: the vp2 gene and the ClyA gene are cloned in series into a pBAD-His expression plasmid to construct a pBAD-ClyA-vp2 recombinant expression vector.
Preferably, the nucleotide sequence of the vp2 gene is shown as SEQ ID NO. 1.
The invention has the beneficial effects that:
the lipopolysaccharide secreted by the outer membrane vesicle of the escherichia coli JC83 provided by the invention has obviously reduced pyrogenicity.
According to the invention, the main immunogen protein VP2 of the infectious bursal disease virus is expressed by using OMVs secreted by Escherichia coli JC83, VP2 protein can be directly expressed on the surface of the OMVs, the OMVs are secreted into bacterial culture supernatant, and the high-concentration protein is obtained by collecting and purifying the OMVs. The OMV-VP2 constructed by the invention has better immunogenicity, and can stimulate animals to produce higher level antibodies after immunization.
The invention utilizes OMVs system to express IBDV VVP2 protein, provides basis for developing novel vaccine of poultry IBDV, provides product support for the prevention and control of poultry IBDV, improves the integral prevention and control level of IBDV, and promotes the sustainable healthy development of poultry farming industry.
Preservation description
The escherichia coli JC83 (Escherichia coli JC 83) is preserved in China center for type culture collection, the preservation time is 2021, 10 months and 18 days, the preservation address is in eight-path 299-number university of Wuhan in Wuhan, the city of Hubei province, and the preservation number is CCTCC NO: m20211289.
Drawings
FIG. 1 is a graph showing the results of an OMVs pyrogenicity analysis;
FIG. 2 shows the quantitative detection results of OMVs proteins;
FIG. 3 shows the result of immunoblotting experiments to determine whether the VP2 of the target protein is expressed;
FIG. 4 is a graph showing the results of the particle size analyzer analysis;
FIG. 5 is a graph showing the morphology of OMVs observed by transmission electron microscopy;
FIG. 6 shows the results of antibody levels induced by OMVs-VP2 in animals.
Detailed Description
The invention provides an escherichia coli JC83, wherein the escherichia coli JC83 is preserved in China center for type culture collection, and the preservation number is CCTCC NO: m20211289.
The preservation time of the escherichia coli JC83 is 2021, 10 months and 18 days, and the preservation address is in the university of Wuhan, eight-path 299 of Wuhan in Wuhan, the city of Wuhan, hubei province. The escherichia coli JC83 provided by the invention is obtained by modifying a Nissle 1917 strain, and compared with the Nissle 1917 strain, the lipopolysaccharide pyrogenicity of the escherichia coli JC83 secreting outer membrane vesicles is obviously reduced.
The invention also provides an outer membrane vesicle secreted by the escherichia coli JC 83. Compared with outer membrane vesicles secreted by other strains of escherichia coli, lipopolysaccharide secreted by escherichia coli JC83 into the outer membrane vesicles obviously reduces pyrogenicity, and when the outer membrane vesicles are used for outer membrane vesicle subunit vaccines, nonspecific immunity is greatly reduced, and specific immunity is better.
The invention also provides an application of the escherichia coli JC83 or the outer membrane vesicle in preparing an outer membrane vesicle vaccine. The invention has no special limitation on the type of vaccine, and the escherichia coli JC83 or outer membrane vesicle can be used for preparing any type of outer membrane vesicle vaccine, and has strong immunity.
The invention also provides an application of the escherichia coli JC83 or the outer membrane vesicle in preparing an IBDV subunit vaccine.
In the present invention, the IBDV immunogen is preferably VP2 protein, and the nucleotide sequence of the VP2 protein is preferably shown in SEQ ID NO. 1. When the escherichia coli JC83 of the invention is used for preparing an IBDV subunit vaccine, the method preferably comprises the following steps: constructing an immunogen gene vp2 recombinant expression vector, transferring the recombinant expression vector into JC83 competent cells to obtain engineering bacteria, performing amplification culture, and separating and purifying.
In the present invention, the method for constructing the recombinant expression vector of the immunogen gene vp2 preferably comprises the following steps: the vp2 gene and the ClyA gene are cloned in series into a pBAD-His expression plasmid to construct a pBAD-ClyA-vp2 recombinant expression vector. The ClyA gene of the invention can present and express VP2 protein on the surface of OMVs, and the tandem is preferably that the VP2 gene and the ClyA gene are connected in series through glycine peptide chain (GGTGGTGGCGGCGGT). The specific source of the pBAD-His expression plasmid is not particularly limited, and the present invention can be carried out by using products conventionally and commercially available in the art. The specific method for transferring the recombinant expression vector into JC83 competent cells is not particularly limited, and the conventional transfer method in the field can be adopted. The invention has no special limitation on the steps of amplifying culture and separating purification of engineering bacteria, and the engineering bacteria can be amplified culture and separated purification by adopting the conventional steps in the field.
The outer membrane vesicle subunit vaccine obtained by the method is of a spherical and double-layer membrane structure, the particle size is about 20-250 nm, the main components of the subunit vaccine comprise VP2 protein and lipid, the immunogenicity is good, humoral immunity can be induced after animals are immunized, and high antibodies are generated.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Escherichia coli JC83 and secretory OMVs pyrogenicity detection thereof
The JC83 strain is obtained by modifying an escherichia coli Nissle 1917 strain. Coli Nissle 1917, BL21 and JC83 strains were cultured in large amounts, and OMVs secreted by the three strains were enriched and purified by ultracentrifugation methods for use in determining the pyrogenicity of OMVs. Fresh blood was collected from mice and added to 96-well plates (200. Mu.L/well), and 20. Mu.L ofrOMVs, 200. Mu.L lipid Iva (Carbosynth), or endotoxin standard (Sigma-Aldrich) were added, respectively. After 18h incubation at 37 ℃, the supernatant was centrifuged and assayed for IL-1β content by double sandwich ELISA, and the pyrogenicity of the secreted OMVs of Nissle 1917, BL21 and JC83 strains was determined by making a curve with endotoxin standards. The results are shown in FIG. 1. As can be seen from FIG. 1, the lipopolysaccharide of the outer membrane vesicles secreted by the Escherichia coli JC83 provided by the invention has obviously reduced pyrogenicity.
Example 2
Cloning of vp2 Gene
Extracting genome of IBDV mutant strain by TRIzol reagent, designing specific primer (F: 5'-ATGCAGATTGTTCCGTTCAT-3'; R: 5'-TCGAGCAGTTCCTGAAGCAG-3') according to the vp2 gene sequence of the mutant strain, cloning vp2 gene to pMD19T vector by high-fidelity polymerase, and sequencing and verifying to obtain vp2 gene sequence with nucleotide sequence shown in SEQ ID NO. 1.
Construction of vp2 recombinant expression vector by homologous recombination method
Cloning vp2 gene and ClyA gene in series to pBAD-His expression plasmid, constructing pBAD-ClyA-vp2 recombinant expression vector, converting into DH5 alpha engineering bacteria (DH 5 alpha engineering bacteria are used for vector construction and screening), screening resistance of recombinant engineering bacteria by using chloramphenicol, and identifying by using a PCR method.
Construction of engineering bacteria
By CaCl 2 Preparing competent cells of JC83 escherichia coli by the method; the recombinant plasmid pBAD-ClyA-vp2 was transferred into JC83 competent cells.
Identification of engineering bacteria
The expression engineering strain is painted on a chloramphenicol resistant solid culture medium (chloramphenicol concentration 20 ng/mL), cultured overnight at 37 ℃, and the monoclonal colony is picked up into 200 mu L of chloramphenicol resistant culture solution (chloramphenicol concentration 10 ng/mL), and cultured for 4-6h at 37 ℃ with a shaking table at 200 rpm. Identification was performed using PCR methods.
Identification primers of pBAD-His expressing bacteria:
F1:CACCCTGTCTAACACGGTTA
R1:CAGTGATGGTGATGGTGATG
amplifying the fragment: 276bp
pBAD-ClyA-vp2 expression bacterium identification primer
F2:ATGCAGATTGTTCCGTTCAT
R2:ATGATGTGGGTAAGCTGAGG
Amplifying the fragment: 493bp
PCR reaction conditions:
pre-denaturation 94 ℃; denaturation 94 ℃, annealing 55 ℃, extension 72 ℃ and 34 cycles; the termination was at 72 ℃.
Engineering bacteria expansion culture
Adding 40 mu L of the identified expression strain into 4mL of chloramphenicol resistance culture solution, and culturing at 200rpm with a shaking table at 37 ℃ for overnight; the bacterial solution was transferred to 300mL of chloramphenicol-resistant medium (chloramphenicol concentration 10 ng/mL), and cultured at 37℃to OD with shaking at 200rpm 600 The value reaches 0.5 to 0.7; 3 mLL-arabinose was added as inducer (final concentration 0.2%) to the above bacterial liquid and induced overnight (about 16 h) at 37℃with shaking table 200 rpm; centrifuging the collected bacterial liquid at 7000rpm at 4 ℃ for 15min, and collecting a supernatant; the supernatant was filtered through a 0.65 μm filter and then filtered again through a 0.45 μm filter.
Transferring the filtered bacterial culture supernatant to a precooled sterile overspeed separation tube, balancing (three positions are completely consistent after decimal point is accurate), centrifuging for 2 hours at 4 ℃ and 40000rpm, discarding the supernatant, re-suspending and precipitating by using sterile PBS, and transferring the re-suspension to a clean EP tube to obtain the escherichia coli outer membrane vesicle for expressing VP2 protein.
Example 3
Identification of whether the outer membrane vesicles obtained in example 1 express VP2 protein
0.2% Triton-100 was added to the purified OMVs for cleavage, 1:1 mixture, and the cleaved outer membrane vesicle proteins were used for protein quantification and immunoblotting experiments by cleavage on ice for 30 min.
Quantitative detection of OMVs proteins: BCA protein quantification
The concentrations of the purified OMVs were determined by BCA method, and the concentrations of the three samples were 1342.318, 2190.34 and 3038.67. Mu.g/mL. Dilution of BSA standard: the BSA standard was diluted with PBS diluent consistent with the protein sample to be tested. The absorbance of the background value (i.e., absorbance at a protein concentration of 0. Mu.g/. Mu.L) is subtracted from the average value of the absorbance of each standard as an abscissa, the final concentration of each standard as an ordinate, a standard curve is drawn, and then the concentration of the protein sample to be measured is calculated. The results are shown in FIG. 2.
Immunoblotting experiment to detect whether VP2 of target protein is expressed or not
Adding a protein loading buffer into the outer membrane vesicle protein after cleavage, boiling in boiling water for 8min for denaturation, and detecting the expression of the protein by using a WesternBlot method. The results are shown in FIG. 3.
The size of OMVs particle size was detected using a Zetasizer instrument according to Stokes-Einstein principle using dynamic light scattering to detect the variation over time of fluctuations in scattered light due to brownian motion of particles. The particle size analysis was performed by the re-suspended outer membrane vesicles from febrile company after completion of super-isolation, and the results are shown in fig. 4. As can be seen from fig. 4, the main particle size peak of OMVs sample analyzed by using a zetasizer ultra instrument is within the particle size range of Exosome, and the particle distribution coefficient (PDI) obtained by detection is 0.2223, which proves that the system dispersity is moderate and the confidence of the detection result is high. The percentage of the particle size of OMVs in the range of 20-200nm is 90.7613 percent respectively.
Observation of OMVs morphology by Transmission Electron microscopy
The outer membrane vesicles resuspended after completion of the super-ionization were subjected to electron microscopy morphological analysis, and the fields of view were selected to be 200, 100 and 50nm in view of the particle size analysis performed, and the results are shown in FIG. 5.
Example 4
35 SPF chicks were randomly divided into 7 groups, 5/group, group I immunized OMVs-VP2 (50 ng) by nasal drip, group II immunized OMVs-VP2 (50 ng) by subcutaneous injection, group III immunized attenuated vaccine 2512 (1-feather, shihua) by water drinking, group IV immunized inactivated vaccine (1-feather, dahua farmer) by intramuscular injection, group V immunized OMVs (50 ng) by nasal drip and eye drip, group VI immunized OMVs (50 ng) by subcutaneous injection, and group VII served as blank. After 21 days of first immunization, the immunization was boosted once in the same manner, blood was collected 7 days after second immunization, and the titers of the anti-VP 2 antibodies in the serum were detected by ELISA kit. The results are shown in figure 6, after secondary immunization, higher levels of anti-VP 2 antibodies were produced in chickens immunized both subcutaneously and nasally.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> agricultural university of south China
<120> an Escherichia coli JC83 and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1455
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
atgcagattg ttccgttcat acggagcctt ctgatgccaa caaccggacc ggcgtccatt 60
ccggacgaca ccctagagaa gcatactctc aggtcagaga cctcgaccta caatttgact 120
gtgggggaca cagggtcagg gctaattgtc tttttccctg gtttccctgg ctcaattgtg 180
ggtgctcact acacactgca gagcaatggg aactacaagt tcgatcagat gctcctgact 240
gcccagaacc taccggccag ctacaactac tgcaggctag tgagtcggag tctcacagtg 300
aggtcaagca cactccctgg tggcgtttat gcactaaatg gcaccataaa cgccgtgacc 360
ttccaaggaa gcctgagtga actgacagat gttagctaca atgggttgat gtctgcaaca 420
gccaacatca acgacaaaat cgggaacgtc ctagtagggg aaggggtaac cgtcctcagc 480
ttacccacat catatgatct tgggtatgtg agactcggtg accccattcc cgctataggg 540
ctcgacccaa aaatggtggc aacatgtgac agcagtgaca ggcccagagt ctacaccata 600
actgcagccg atgattacca attctcatca cagtaccaag caggtggggt aacaatcaca 660
ctgttctcag ctaatatcga tgccatcaca agcctcagca tcgggggaga actcgtgttt 720
caaacaagcg tccaaggcct tatactgggt gctaccatct accttatagg ctttgatggg 780
actgcggtaa tcaccagagc tgtggccgca gacaatgggc taacggccgg cactgacaac 840
cttatgccat tcaatattgt gattccaacc agcgagataa cccagccaat cacatccatc 900
aaactggaga tagtgacctc caaaagtggc ggtcaggcgg gagatcagat gtcatggtca 960
gcaagtggga gcctagcagt gacgatccac ggtggcaact atccaggagc cctccgtccc 1020
gtcacactag tagcctacga aagagtggca acaggatctg tcgttacggt cgccggggtg 1080
agcaacttcg agctgatccc aaatcctgaa ctagcaaaga acctggtcac agaatacggc 1140
cgatttgacc caggagctat gaactacaca aaattgatac tgagtgagag ggaccgtctt 1200
ggcatcaaga ccgtatggcc aacaagggag tacactgact tccgcgagta cttcatggag 1260
gtggccgacc tcaactctcc cctgaagatt gcaggagcat ttggcttcaa agacataatc 1320
cgggccctaa ggaggatagc tgtgccggtg gtctctacac tgttcccacc cgctgccccc 1380
ctagcccatg caattgggga aggtgtagac tacctgctgg gcgatgaggc acaggctgct 1440
tcaggaactg ctcga 1455
<210> 2
<211> 2373
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atgactgaaa tcgttgcaga taaaacggta gaagtagtta aaaacgcaat cgaaaccgca 60
gatggagcat tagatcttta taataaatat ctcgatcagg tcatcccctg gcagaccttt 120
gatgaaacca taaaagagtt aagtcgcttt aaacaggagt attcacaggc agcctccgtt 180
ttagtcggcg atattaaaac cttacttatg gatagccagg ataagtattt tgaagcaacc 240
caaacagtgt atgaatggtg tggtgttgcg acgcaattgc tcgcagcgta tattttgcta 300
tttgatgagt acaatgagaa gaaagcatcc gcccagaaag acattctcat taaggtactg 360
gatgacggca tcacgaagct gaatgaagcg caaaaatccc tgctggtaag ctcacaaagt 420
ttcaacaacg cttccgggaa actgctggcg ttagatagcc agttaaccaa tgatttttca 480
gaaaaaagca gctatttcca gtcacaggta gataaaatca ggaaggaagc atatgccggt 540
gccgcagccg gtgtcgtcgc cggtccattt ggattaatca tttcctattc tattgctgcg 600
ggcgtagttg aaggaaaact gattccagaa ttgaagaaca agttaaaatc tgtgcagaat 660
ttctttacca ccctgtctaa cacggttaaa caagcgaata aagatatcga tgccgccaaa 720
ttgaaattaa ccaccgaaat agccgccatc ggtgagataa aaacggaaac tgaaacaacc 780
agattctacg ttgattatga tgatttaatg ctttctttgc taaaagaagc ggccaaaaaa 840
atgattaaca cctgtaatga gtatcagaaa agacacggta aaaagacact ctttgaggta 900
cctggtggtg gcggcggtat gcagattgtt ccgttcatac ggagccttct gatgccaaca 960
accggaccgg cgtccattcc ggacgacacc ctagagaagc atactctcag gtcagagacc 1020
tcgacctaca atttgactgt gggggacaca gggtcagggc taattgtctt tttccctggt 1080
ttccctggct caattgtggg tgctcactac acactgcaga gcaatgggaa ctacaagttc 1140
gatcagatgc tcctgactgc ccagaaccta ccggccagct acaactactg caggctagtg 1200
agtcggagtc tcacagtgag gtcaagcaca ctccctggtg gcgtttatgc actaaatggc 1260
accataaacg ccgtgacctt ccaaggaagc ctgagtgaac tgacagatgt tagctacaat 1320
gggttgatgt ctgcaacagc caacatcaac gacaaaatcg ggaacgtcct agtaggggaa 1380
ggggtaaccg tcctcagctt acccacatca tatgatcttg ggtatgtgag actcggtgac 1440
cccattcccg ctatagggct cgacccaaaa atggtggcaa catgtgacag cagtgacagg 1500
cccagagtct acaccataac tgcagccgat gattaccaat tctcatcaca gtaccaagca 1560
ggtggggtaa caatcacact gttctcagct aatatcgatg ccatcacaag cctcagcatc 1620
gggggagaac tcgtgtttca aacaagcgtc caaggcctta tactgggtgc taccatctac 1680
cttataggct ttgatgggac tgcggtaatc accagagctg tggccgcaga caatgggcta 1740
acggccggca ctgacaacct tatgccattc aatattgtga ttccaaccag cgagataacc 1800
cagccaatca catccatcaa actggagata gtgacctcca aaagtggcgg tcaggcggga 1860
gatcagatgt catggtcagc aagtgggagc ctagcagtga cgatccacgg tggcaactat 1920
ccaggagccc tccgtcccgt cacactagta gcctacgaaa gagtggcaac aggatctgtc 1980
gttacggtcg ccggggtgag caacttcgag ctgatcccaa atcctgaact agcaaagaac 2040
ctggtcacag aatacggccg atttgaccca ggagctatga actacacaaa attgatactg 2100
agtgagaggg accgtcttgg catcaagacc gtatggccaa caagggagta cactgacttc 2160
cgcgagtact tcatggaggt ggccgacctc aactctcccc tgaagattgc aggagcattt 2220
ggcttcaaag acataatccg ggccctaagg aggatagctg tgccggtggt ctctacactg 2280
ttcccacccg ctgcccccct agcccatgca attggggaag gtgtagacta cctgctgggc 2340
gatgaggcac aggctgcttc aggaactgct cga 2373
Claims (7)
1. The Escherichia coli JC83 is characterized in that the Escherichia coli JC83 is preserved in China center for type culture collection, and the preservation number is CCTCC NO: m20211289.
2. The escherichia coli JC83 of claim 1, wherein the escherichia coli JC83 secretes lipopolysaccharide of the outer membrane vesicle with a substantial reduction in pyrogenicity.
3. An outer membrane vesicle secreted by the escherichia coli JC83 of claim 1.
4. Use of the outer membrane vesicle of e.coli JC83 of claim 1 or of claim 3 for the preparation of an infectious bursal disease virus subunit vaccine, characterized in that the immunogen of the infectious bursal disease virus is the viral protein VP2.
5. The use according to claim 4, characterized in that it comprises the following steps: constructing an immunogen gene vp2 recombinant expression vector, transferring the recombinant expression vector into JC83 competent cells to obtain engineering bacteria, performing amplification culture, and separating and purifying.
6. The use according to claim 5, wherein the method for constructing the recombinant expression vector of the immunogen gene vp2 comprises the steps of: the vp2 gene and the ClyA gene are cloned in series into a pBAD-His expression plasmid to construct a pBAD-ClyA-vp2 recombinant expression vector.
7. The use according to claim 6, wherein the nucleotide sequence of the vp2 gene is shown in SEQ ID No. 1.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2007248A1 (en) * | 1989-01-06 | 1990-07-06 | Peter R. Paradiso | Meningococcal class 1 outer-membrane protein vaccine |
| CN105861402A (en) * | 2015-07-20 | 2016-08-17 | 中国医学科学院医学生物学研究所 | Novel vaccine form for submitting heterologous protein to surface of escherichia coli outer membrane vesicles |
| CN107099496A (en) * | 2017-04-25 | 2017-08-29 | 中国农业科学院哈尔滨兽医研究所 | Recombinant strains of lactic acid bacteria of amalgamation and expression infections chicken cloacal bursa virus VP2 albumen and Salmonella outer membrane protein and application thereof |
| WO2021224439A1 (en) * | 2020-05-08 | 2021-11-11 | De Staat Der Nederlanden, Vert. Door De Minister Van Vws, Ministerie Van Volksgezondheid, Welzijn En Sport | CLICK OMVs |
-
2022
- 2022-05-12 CN CN202210519074.0A patent/CN115851477B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2007248A1 (en) * | 1989-01-06 | 1990-07-06 | Peter R. Paradiso | Meningococcal class 1 outer-membrane protein vaccine |
| CN105861402A (en) * | 2015-07-20 | 2016-08-17 | 中国医学科学院医学生物学研究所 | Novel vaccine form for submitting heterologous protein to surface of escherichia coli outer membrane vesicles |
| CN107099496A (en) * | 2017-04-25 | 2017-08-29 | 中国农业科学院哈尔滨兽医研究所 | Recombinant strains of lactic acid bacteria of amalgamation and expression infections chicken cloacal bursa virus VP2 albumen and Salmonella outer membrane protein and application thereof |
| WO2021224439A1 (en) * | 2020-05-08 | 2021-11-11 | De Staat Der Nederlanden, Vert. Door De Minister Van Vws, Ministerie Van Volksgezondheid, Welzijn En Sport | CLICK OMVs |
Non-Patent Citations (1)
| Title |
|---|
| 革兰氏阴性菌外膜囊泡作为亚单位疫苗的研究进展;易洁;刘青;孔庆科;;微生物学报(06);全文 * |
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