CN110272910A - A kind of outer membrane vesicles and its application of the recombinant vector including JEV antigen gene, recombinant bacterial strain and submission JEV antigen - Google Patents

A kind of outer membrane vesicles and its application of the recombinant vector including JEV antigen gene, recombinant bacterial strain and submission JEV antigen Download PDF

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CN110272910A
CN110272910A CN201910552006.2A CN201910552006A CN110272910A CN 110272910 A CN110272910 A CN 110272910A CN 201910552006 A CN201910552006 A CN 201910552006A CN 110272910 A CN110272910 A CN 110272910A
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jev antigen
outer membrane
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membrane vesicles
jev
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曹三杰
何博
彭彦杰
梁伟
易强
赵勤
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Sichuan Agricultural University
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Abstract

The present invention provides a kind of recombinant vector including JEV antigen gene, the outer membrane vesicles of recombinant bacterial strain and submission JEV antigen and its applications, belong to immunological technique field, the recombinant vector including JEV antigen gene, including JEV antigen gene and pBAD-18-ClyA starting vector；The recombinant bacterial strain includes above-mentioned recombinant vector, and is able to produce the outer membrane vesicles of submission JEV antigen；Method is as follows: 1) recombinant bacterial strain being carried out Liquid Culture；2) preculture bacterium solution is transferred and carries out Fiber differentiation in induced fluid culture medium, be centrifuged；3) supernatant is successively filtered except thallus, ultrafiltration concentration and centrifugation, collects solid phase components, obtain the outer membrane vesicles of submission JEV antigen.The outer membrane vesicles of the submission JEV antigen can excite efficient specific immune response, have outstanding antigenicity, can generate higher specific antibody, can cause humoral immunity but also cause cellular immunity.

Description

A kind of recombinant vector including JEV antigen gene, recombinant bacterial strain and submission JEV antigen Outer membrane vesicles and its application
Technical field
The invention belongs to immunological technique field more particularly to a kind of recombinant vectors including JEV antigen gene, recombinant bacterium The outer membrane vesicles and its application of strain and submission JEV antigen.
Background technique
Japanese B encephalitis (Japanese encephalitis, JE) is also known as Japanese Type-B encephalitis, and abbreviation encephalitis is By Japanese B encephalitis virus (JEV, the Japanese of flaviviridae (Flaviviridae) Flavivirus (Flavivirus) Encephalitis virus) caused by threaten the acute central nervous system infectious disease of human and livestock health, it is total to be important people and animals Illness and arbovirus diseases.In clinic, human infection this fall ill after being ill relatively rapidly, it may appear that it is hyperpyrexia, the disturbance of consciousness, frightened It faints, characteristic symptoms, the infection serious person such as tonic spasm and meningeal irritation sign can often leave the disturbance of consciousness, dementia, mistake more afterwards The sequelae such as language and quadriplegia, epilepsy.It is the most universal with the infection of pig in animal, develop into the infection of this disease after infection Source, but its clinical symptoms is unobvious, and the death rate is lower.Pregnant sow may happen suddenly after infection miscarries, output stillborn foetus, mummy With weak tire, also see with tire it is positive produce fetus, replacement gilt Non Apparent Abnormality performance, and boar is in addition to having general symptom, Chang Fasheng mono- The enlargement of side property testis and inflammation, it is also even to have two sides lesion simultaneously.Since the disease subclinical infection chance is more, so japanese encephalitis virus It so far is still the pathogen with important clinical significance and global meaning.
Genetic engineering subunit vaccine (Sub-unit vaccine) is also referred to as biosynthesis subunit vaccine/recombinant subunit Vaccine.Studies have reported that proving, the JEV subunit vaccine based on E protein or NS1 albumen development can generate preferable protection effect Fruit.The safety with higher of such vaccine, will not infection animal generate side effect, be Vaccinum Encephalitidis Epidemicae development new direction, generation Numerous research institutions are all committed to the exploration of the direction within the scope of boundary.
Outer membrane vesicles (Outer Membrane Vesicles, OMV) are mainly by Gram-negative bacteria outer membrane in certain machine System, which issues, bears bud and in a kind of diameter comprising biological active agents of bacterium surface formation about between 20~250nm Capsule balloon-shaped structure.Ingredient mainly contains bacterial outer membrane and pericentral siphon, including nonprotein antigen lipopolysaccharides (LPS), phosphatide (PL), film egg It is white, toxin, the virulence factors such as invasion.Its biological function includes: 1. formation for facilitating biomembrane；2. excretory system replaces Generation；3. mass transfer；4. transfer of genetic material；5. virulence correlation factor etc. is protected, and most of all, it is as bacterial derivation Product can induce body and generate effective immune response.
Outer membrane vesicles still without efficient submission JEV antigen at present.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of recombinant vector including JEV antigen gene, recombinant bacterial strain and The outer membrane vesicles of submission JEV antigen；The recombinant bacterial strain can successful expression JEV antigen, and be able to produce submission JEV antigen Outer membrane vesicles；The outer membrane vesicles of the submission JEV antigen can excite efficient specific immune response, have outstanding antigen Property, higher specific antibody can be generated, humoral immunity can be caused but also cause cellular immunity.
It is another object of the present invention to provide the outer membrane vesicles of above-mentioned submission JEV antigen in the popular B-mode brain of preparation Application in scorching vaccine.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical schemes:
A kind of recombinant vector including JEV antigen gene, including JEV antigen gene and pBAD-18-ClyA starting vector； The JEV antigen gene be inserted in pBAD-18-ClyA starting vector Xba I restriction enzyme site and Hind III digestion site it Between.
Preferably, the nucleotide sequence of the JEV antigen gene is as shown in SEQ ID NO:1 or SEQ ID NO:2.
The present invention provides a kind of recombinant bacterial strain including JEV antigen gene, the recombinant bacterial strain includes above-mentioned recombination Carrier.
The present invention provides a kind of outer membrane vesicles of submission JEV antigen produced by the recombinant bacterial strain.
The present invention provides the preparation methods of the outer membrane vesicles of the submission JEV antigen, comprising the following steps:
1) recombinant bacterial strain is inoculated in the OD cultivated in fluid nutrient medium to bacterium solution600It is obtained for 0.4~0.6 Obtain preculture bacterium solution；
2) preculture bacterium solution described in step 1) is transferred and carries out 15~25h of Fiber differentiation in induced fluid culture medium Afterwards, it is centrifuged, collects supernatant；
3) supernatant described in step 2) is successively filtered except thallus, ultrafiltration concentration and centrifugation, collects solid phase group Point, obtain the outer membrane vesicles of submission JEV antigen.
Preferably, fluid nutrient medium described in step 1) is chlorampenicol resistant LB liquid medium；Described in step 2) Induced fluid culture medium be add 20wt% L-Arabinose solution chlorampenicol resistant LB liquid medium.
Preferably, the molecular cut off of ultrafiltration concentration described in step 3) is 100kDa.
Preferably, after step 4) the collection solid phase components, further include the solid phase components of collection are resuspended, density level bands Degree centrifugation and the outer membrane vesicles for collecting acquisition submission JEV antigen.
Preferably, the density gradient centrifugation uses OptiPrep-iodixanol gradient cell separating liquid, the density The centrifugal force of gradient centrifugation is 140000~160000g, and the time of the density gradient centrifugation is 10~14h.
The present invention provides the outer membrane vesicles of the submission JEV antigen to prepare answering in Japanese Encephalitis Vaccine With.
Beneficial effects of the present invention
Recombinant vector provided by the invention including JEV antigen gene, the JEV antigen gene of carrying can successful expression, Preparation for the outer membrane vesicles of subsequent submission JEV antigen provides condition.JEV mesh expressed by recombinant bacterial strain provided by the invention Antigen protein can be positioned on the outer membrane vesicles of bacterium excretion, make the outer membrane vesicles body can be stimulated to generate as antigen All kinds of specific immune responses and nonspecific immune reaction；The outer membrane vesicles of the submission JEV antigen of the recombinant bacterial strain secretion Structural integrity；The outer membrane vesicles of the submission JEV antigen can excite efficient specific immune response, have outstanding antigen Property, higher specific antibody can be generated, humoral immunity can be caused but also cause cellular immunity.
Mouse Immunization Protection according to embodiments of the present invention the result shows that, submission JEV provided by the invention is anti- Former outer membrane vesicles are 50%~90% to the protective rate of mouse, and it is good to attack malicious protecting effect；Indirect elisa method measures mice serum JE neutralizing antibody is the positive, and antibody titer is significantly higher than negative control group and unloaded control group；The outer membrane vesicle of the submission JEV antigen Bubble can stimulate body secretes IgG1 and IgG2A, while 3 kinds of cell factors IL-2, IL-4 and IFN-γ be in mice serum Increase, it is extremely significant with control group difference；Mouse spleen T lymphocyte subgroup sorting the experimental results showed that, the submission JEV antigen Outer membrane vesicles be immunized mouse after, mouse T cell subtype distribution CD3+, CD3+CD4+ be all remarkably higher than control group (P < 0.05), extremely extremely significant in CD3+CD8+ subgroup to be higher than PBS control group.
Detailed description of the invention
Fig. 1 is pBAD-18-ClyA-E and pBAD-18-ClyA-Ez recombinant plasmid cleavage map；
Fig. 2 is plasmid order-checking DNAMAN comparison diagram, is followed successively by pBAD-18-ClyA-E plasmid and pBAD-18- from top to bottom ClyA-Ez plasmid；
Fig. 3 is recombination bacterioprotein expression figure；
Fig. 4 is outer membrane vesicles Electronic Speculum effect picture；
Fig. 5 is that outer membrane vesicles total protein measures standard curve；
Fig. 6 is outer membrane vesicles destination protein Wstern-blot figure；
Fig. 7 is that mouse attacks malicious Protection result；
Fig. 8 is that mouse attacks poison protection survivorship curve；
Fig. 9 is the immune anti-JEV serum IgG antibody indirect ELISA testing result of mouse；
Figure 10 is the standard curve of IgG1 and IgG2A；
Figure 11 is that IgG1 and IgG2A is horizontal in immunized mice serum；
Figure 12 is the standard curve of cell factor IL-2, IL-4 and IFN-γ；
Figure 13 is the level of cell factor in Post-immunisation serum, is followed successively by IL-2, IL-4 and the IFN-γ factor from top to bottom Level；
Figure 14 is the OMV-Ez dosage group plaque figure of 5 μ g；
Figure 15 is mouse spleen T lymphocyte subgroup variation diagram, and respectively CD3+T cell, CD3+CD4+T are thin from top to bottom Born of the same parents and CD3+CD8+T cell.
Specific embodiment
The present invention provides a kind of recombinant vectors including JEV antigen gene, including JEV antigen gene and pBAD-18- ClyA starting vector；The JEV antigen gene is inserted in the Xba I restriction enzyme site and Hind of pBAD-18-ClyA starting vector Between III digestion site.
In the present invention, the nucleotide sequence of the JEV antigen gene is preferably such as SEQ ID NO:1 or SEQ ID NO: Shown in 2.In the present invention, sequence shown in SEQ ID NO:1 is with SA14-14-2 plants of III type Japanese B encephalitis virus of gene Gene order be standard (GenBank:AF315119.1), select in open reading frame that (present invention is named as complete E gene E), sequence length 1539bp.In the present invention, sequence shown in SEQ ID NO:2 is that antigenic highest is selected from E gene Genetic fragment tandem compound obtain, the present invention is named as Ez, sequence length 855bp.In the present invention, the Ez includes: (1) Complete 3rd domain sequence of E protein, amino acid residue are located at 292-402；(2) 7 are able to guide body and generate neutralizing antibody B cell epitope sequences, amino acid residue are located at 75-92,149-163,258-285,307-316,356-362,373- 399,397-403；(3) CTL epitope sequences and a Th epitope sequences, amino acid residue are located at 60-68 and 436- 445；(4) epitope sequences on NS1 albumen, amino acid residue are located at 116-125.With a glycine and one between each epitope Serine separates, and keeps each epitope relatively independent and is conducive to the submission of antigen.In the present invention, sequence shown in SEQ ID NO:2 It is that the optimal gene order after expression codon optimization is carried out according to Escherichia coli inclined preferendum codon principle.The present invention is to described The preparation method of E gene and Ez gene is not particularly limited, using the preparation method of this field routine；Of the invention specific It in implementation process, is prepared using the method for gene chemical synthesis, gene chemical synthesis preferably entrusts Wuhan Jin Kairui bioengineering to have Limit company completes.
In the present invention, the pBAD-18-ClyA starting vector includes PBAD18 original plasmid and ClyA genetic fragment, The ClyA genetic fragment is inserted into PBAD18 original plasmid by digestion and is obtained；The pBAD18 original plasmid is commercially available Product.
In the present invention, the preparation method of the recombinant vector including JEV antigen gene, comprising the following steps: S1) by institute It states E gene or after Ez gene is connected to pMD19-T plasmid, imports in competent escherichia coli cell and cloned；S2) digestion institute Plasmid, recovery purifying E genetic fragment or Ez genetic fragment after stating clone；S3) by after purification E genetic fragment or Ez gene piece Section is connect with pBAD-18-ClyA starting vector obtains the recombinant vector including JEV antigen gene.In the present invention, the step S1) preferred that biological scientific & technical corporation is entrusted to complete.Plasmid of the present invention after being cloned carries out digestion；The body of the digestion System is preferred as follows:
The program of the digestion is preferably as follows: 37 DEG C of incubation 3.5h.The present invention after the endonuclease reaction preferably into Row agarose gel electrophoresis verifying is confirmed whether to be consistent with expected size, and whether the brightness of target fragment, which can be used for DNA fragmentation, is returned It receives.In the present invention, the recovery purifying E genetic fragment or Ez genetic fragment preferably use DNA purification kit to carry out, in detail Step is referring to kit specification.The present invention is after obtaining E genetic fragment or Ez genetic fragment after purification, by described in after purification E genetic fragment or Ez genetic fragment connect with pBAD-18-ClyA starting vector obtain include JEV antigen gene recombination carry Body.The linked system of the connection is preferably as follows:
The linker of the connection is preferably as follows: 16 DEG C connect overnight (10~14h), the connection product of the connection It is preferably disposed at 0~5 DEG C of preservation, is more preferably placed in 4 DEG C of preservations.
The present invention provides a kind of recombinant bacterial strain including JEV antigen gene, the recombinant bacterial strain includes above-mentioned recombination Carrier.In the present invention, the recombinant bacterial strain is prepared by the way that the recombinant vector to be transferred in competent escherichia coli cell It obtains；In the present invention, the competent escherichia coli cell is preferably α plants of competent escherichia coli cells of DH5 or JC8031 plants Competent escherichia coli cell；The present invention does not have the preparation method of the DH5 α plants or JC8031 plants competent escherichia coli cells There is particular determination, using the preparation method of this field routine.In the present invention, the recombinant vector is transferred to α plants of large intestines of DH5 The recombinant bacterial strain that bacillus competent cell obtains is clone strain；The recombinant vector is transferred to JC8031 plants of Escherichia coli impressions The recombinant bacterial strain that state cell obtains is expression bacterial strain.In the present invention, the method being transferred to preferably uses chemical method.The present invention It preferably further include the screening and identification of recombinant bacterial strain after described be transferred to；The present invention is not special to the screening and identification It is required that screening and identification method using this field routine.
The present invention provides a kind of outer membrane vesicles of submission JEV antigen produced by the recombinant bacterial strain.In the present invention In, the recombinant bacterial strain is preferably above-mentioned expression bacterial strain.
The present invention also provides the preparation methods of the outer membrane vesicles of the submission JEV antigen, comprising the following steps: 1) will The recombinant bacterial strain is inoculated in the OD cultivated in fluid nutrient medium to bacterium solution600Preculture bacterium solution is obtained for 0.4~0.6； 2) preculture bacterium solution described in step 1) is transferred after carrying out 15~25h of Fiber differentiation in induced fluid culture medium, centrifugation, Collect supernatant；3) supernatant described in step 2) is successively filtered except thallus, ultrafiltration concentration and centrifugation, collects solid phase Component obtains the outer membrane vesicles of submission JEV antigen.
In the present invention, the recombinant bacterial strain is tapped into row culture and obtains preculture bacterium solution.In the present invention, described heavy Group bacterial strain preferably carries out recovery culture, and the recovery culture preferably carries out on the LB solid medium of chlorampenicol resistant, The time of the recovery culture is preferably 8~12h, and the temperature of the recovery culture is preferably 37 DEG C.The present invention is in the recovery After culture, the thallus after the culture is inoculated in fluid nutrient medium and is cultivated；The fluid nutrient medium is preferably chlorine Chloramphenicol resistance LB liquid medium, wherein final concentration of 20~30 μ g/mL of chloramphenicol, more preferably 25 μ g/mL；The liquid The temperature of culture is preferably 36~38 DEG C, and more preferably 37 DEG C；The time of the Liquid Culture is preferably 5~7h, more preferably 6h；The Liquid Culture is preferably with concussion, and the revolving speed of the concussion is preferably 200~250rpm, more preferably 220rpm； The OD of the pre-culture solution600Preferably 0.5.
The present invention after obtaining the pre-culture solution, by the preculture bacterium solution transfer in induced fluid culture medium into After 15~25h of row Fiber differentiation, supernatant is collected in centrifugation.In the present invention, the induced fluid culture medium preferably adds The chlorampenicol resistant LB liquid medium of the L-Arabinose solution of 20wt%；The L-Arabinose solution of the 20wt% with The volume ratio of chlorampenicol resistant LB liquid medium is preferably 1:950~1050, more preferably 1:1000.It is heretofore described to lure Temperature and the revolving speed for leading culture are consistent with the temperature of aforesaid liquid culture and revolving speed, and details are not described herein.It is described to lure in the present invention The time for leading culture is preferably 18~22h, more preferably 20h.In the present invention, the centrifugal force of the centrifugation is preferably 8000~ 12000g, more preferably 9000~11000g, most preferably 10000g；The time of the centrifugation is preferably 8~12min, more excellent It is selected as 10min；The temperature of the centrifugation is preferably 0~5 DEG C, and more preferably 4 DEG C.The present invention collects supernatant after the centrifugation Liquid.
The present invention after obtaining the supernatant, the supernatant is successively filtered except thallus, be concentrated by ultrafiltration and from The heart collects solid phase components, obtains the outer membrane vesicles of submission JEV antigen.In the present invention, the filtration sterilization is preferably successively wrapped Include the film filtering in 0.45 μm of aperture and the film filtering in 22 μm of apertures；The film filtering in 0.45 μm of aperture is preferred to carry out 1~3 It is secondary, it is preferred to carry out 2 times；The film filtering in 0.22 μm of aperture is preferred to be carried out 1~3 time, preferred to carry out 2 times.This Invention collects filtrate, the filtrate is concentrated by ultrafiltration after the filtration sterilization；The molecular cut off of the ultrafiltration concentration Preferably 100kDa, the present invention in, the cycles of concentration of the ultrafiltration concentration is preferably 5~7 times, more preferably 6 times.In the present invention In, the centrifugal force of the centrifugation is preferably 38000~40000g, more preferably 39000g；The time of the centrifugation is preferably 50 ~70min, more preferably 60min.The present invention preferably collects solid phase components after the centrifugation, as submission JEV antigen The crude extract of outer membrane vesicles.
The present invention preferably further includes that will collect after obtaining the crude extract of outer membrane vesicles of the submission JEV antigen Solid phase components are resuspended, density gradient centrifugation and collect the outer membrane vesicles for obtaining submission JEV antigen.In the present invention, described Resuspension is preferably carried out with PBS buffer solution；The density gradient centrifugation preferably uses OptiPrep-iodixanol gradient thin Born of the same parents' separating liquid；The density gradient of the density gradient centrifugation is preferably as follows: 1.5ml 50%, 1.5ml 45%, 1.5ml 40%, 1.5ml 35%, 1.5ml 30% and 1.5ml 25%；The cell separating liquid original liquid concentration is preferably 60%, no Separating liquid with concentration is preferably formulated with 50mM HEPES-150mM NaCl dilution stoste.In the present invention, the density The centrifugal force of gradient centrifugation is preferably 140000~160000g, more preferably 15000g；The time of the density gradient centrifugation is excellent It is selected as 10~14h, more preferably 11~13h.The present invention is preferred to collect enrichment outer membrane vesicles after the density gradient centrifugation The component of layer the outer membrane vesicles that solid phase components are submission JEV antigen after purification are collected by centrifugation again.In the present invention, The centrifugal force being centrifuged again is preferably 140000~160000g, more preferably 15000g, and the time being centrifuged again is excellent It is selected as 50~70min, more preferably 60min.
In the present invention, the outer membrane vesicles of the submission JEV antigen after purification, which are preferably resuspended in PBS buffer solution, to be protected It deposits；The temperature of the preservation is preferably -20 DEG C, and the time of the preservation is preferably within 4 weeks.The present invention is excellent before the preservation Choosing further includes no dientification of bacteria, and the outer membrane vesicles of the submission JEV antigen after resuspension are preferably applied to nonreactive by the no dientification of bacteria Property LB agar plate, seen whether that bacterium colony is formed after culture, regarded as sterile if being formed without bacterium colony, carried out subsequent preservation Step.
The present invention provides the outer membrane vesicles of the submission JEV antigen to prepare answering in Japanese Encephalitis Vaccine With.The outer membrane vesicles of submission JEV antigen provided by the invention can also be helped separately as subunit vaccine application with vaccine Agent combination prepares vaccine application.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood For limiting the scope of the present invention.
Material and reagent used in the embodiment of the present invention are as follows:
1 material
1.1 biomaterial
Bacterial strain and plasmid: bacillus coli DH 5 alpha and JC8031 plants of competent cells are by Sichuan Agricultural University's swine disease research center It prepares and saves；Bacillus coli DH 5 alpha is from commercially available, and JC8031 plant by Cornell University Yung-fu professor Chang present.
Prokaryotic expression plasmid pBAD-18-ClyA carrier is constructed and is saved by Sichuan Agricultural University's swine disease research center, described PBAD-18-ClyA starting vector includes PBAD18 original plasmid and ClyA genetic fragment, by digestion by the ClyA gene piece Section, which is inserted into PBAD18 original plasmid, to be obtained.
1.2 main agents
DNA T4 ligase, DNA purification kit,RT reagent Kit、DL10000、 DL5000 gel electrophoresis Maker and Xba I, III restriction enzyme of Hind are purchased from precious bioengineering (Dalian) Co., Ltd, Japan； The reagents such as tryptone and yeast extract, glycerol, sodium chloride, sodium hydroxide, coomassie brilliant blue R250, methanol are huge purchased from Guangzhou Blue Chemical Industry Science Co., Ltd；Chloramphenicol (Chl) is purchased from U.S. AMERSCO company；Mini-scale plasmid extraction agent box is purchased from the U.S. OMEGA company；PAGE gel reagent preparation box is purchased from Beijing Ku Laibo Science and Technology Ltd.；Chemiluminescence developing solution reagent The sheep anti-mouse igg ELIAS secondary antibody of box and HRP label is purchased from Beijing Bo Aosen company；Primary antibody dilution and BCA determination of protein concentration Kit is purchased from Beijing Suo Laibao biotechnology company.
1.3 preparation of reagents
(1) chloramphenicol: weighing chloramphenicol dry powder 5.0g, is dissolved in 100ml sterilizing ultrapure water, being configured to concentration is 50mg/ml Kanamycins solution be placed in -20 DEG C of preservations with 0.22 μm of membrane filtration after dissolution.
(2) LB liquid medium: weighing tryptone 10g, yeast extract 5g, and it is ultrapure to be dissolved in 950ml by sodium chloride 10g Water is slowly added to a small amount of NaOH and adjusts PH=7.0, is settled to 1L.115 DEG C of high pressure steam sterilization 30min, 4 after being cooled to room temperature DEG C save.
(3) LB solid medium: weighing 15g agar powder and be mixed into 1L LB Liquid Culture, 115 DEG C of high pressure steam sterilizations 30min needs to be added chloramphenicol (25mg/ml) by test in suitable temperature, pours into sterilizes culture dish rapidly, after cooling 4 DEG C of refrigerators are inverted in save.
(4) 50 × TAE buffers: weighing Tris 242g and EDTA 18.612g, and 800ml deionized water is added and sufficiently stirs 51.1ml glacial acetic acid is added after mixing uniformly, NaOH adjusts PH=8.3 and deionized water is added to be settled to 1L, room temperature preservation.
(5) sodium chloride 8g, potassium chloride 0.2g, disodium hydrogen phosphate 1.42g, potassium dihydrogen phosphate 0.27g PBS buffer solution: are weighed In beaker, appropriate sterilizing ultrapure water is added, dissolution is sufficiently stirred, concentrated hydrochloric acid adjusting PH=7.4 is added dropwise, is settled to 1L, 115 DEG C 4 DEG C of preservations after sterilizing.
(6) 50% glycerites: 25ml glycerol and 25ml ultrapure water, 115 DEG C of high steams are added into 100ml conical flask Sterilize 30min, 4 DEG C of preservations.
(7) SDS-PAGE gel electrophoresis buffer: Tris-bass 15.1g is weighed, Gly glycine 94g, SDS 5g are in beaker In, it is last repeatedly to add water that dissolution is sufficiently stirred, it is settled to 1L.
1.4 key instrument equipment
Embodiment 1
The building of recombinant vector, recombinant bacterial strain including JEV antigen gene
The nucleotide sequence of JEV antigen gene is as shown in SEQ ID NO:1；What is wherein underlined is respectively upstream and downstream enzyme Enzyme site；Thickened portion is His label, and italicized item is terminator.
The amino acid sequence (SEQ ID NO:3) of coding:
CSRFNCLGMGNRDFIEGASGATWVDLVLEGDSCLTIMANDKPTLDVRMINIEASQLAEVRSYCYHASV TDISTVTRCPTTGEAHNEKRADSSYVCKQGFTDRGWGNGCGLFGKGSIDTCAKFSCTSKAIGRMIQPENIKYEVGI FVHGTTTSENHGNYSAQVGASQAAKFTVTPNAPSITLKLGDYGEVTLDCEPRSGLNTEAFYVMTVGSKSFLVHREW FHDLSLPWTSPSSTAWRNRELLMEFEEAHATKQYVVALGSQEGGLHQALAGAIVVEYSSSVKLTSGHLKCRLKMDK LALKGTTYGMCTEKFSFAKNPADTGHGTVVIELTYSGSDGPCKIPIVSVASLNDMTPVGRLVTVNPFVATSSSNSK VLVEMEPPFGDSYIVVGRGDKQINHHWHKAGSTLGKAFSTTLKGAQRLAALGDTAWDFGSIGGVFNSIGKAVHQVF GGAFRTLFGGMSWITQGLMGALLLWMGVNARDRSIALAFLATGGVLVFLATNVHAHHHHHH。
It entrusts Wuhan Jin Kairui bioengineering Co., Ltd to complete the synthesis of E gene, and E gene is connected to plasmid The cloned plasmids pMD19-T-E containing target gene fragment is obtained on pMD19-T.
The cloned plasmids pMD19-T-E that company is provided carries out digestion, and digestion system is as follows with reaction condition:
Program: 37 DEG C of incubation 3.5h.Product is added in 2% Ago-Gel after endonuclease reaction, in 90 DEG C of voltages Lower electrophoresis 30min.Gel after electrophoresis is placed in gel imaging system, in the UV lamp observe target fragment whether with It is expected that size (1539bp) is consistent, whether brightness can be used for DNA fragmentation recycling.
The purifying of target fragment
It will be cut at purpose band place using scalpel in Ago-Gel, recycled using DNA Purification Kit, Specific steps are as follows:
(1) gel cut is placed in centrifuge tube and is weighed, the Buffer DC of 3 times of gel amounts is added, sets after evenly mixing In 55 DEG C of baking ovens, Ago-Gel is waited to melt.
(2) mixed solution after melting upper step is transferred in filter column, and 12000rpm is centrifuged 1min, and repeated centrifugation once mentions High-recovery.
(3) the WB Buffer, 12000rpm that 700 μ l are added into filter column are centrifuged 30s, are repeated once and abandon filtrate.
(4) filter column is arranged on the centrifuge tube of 1.5ml, 25~30 μ lElution is added in the centre of filter membrane Buffer is stored at room temperature 1min.Note: should be placed in the heating of 55 DEG C of baking ovens for Elution Buffer in advance, and when use is conducive to mention High elution efficiency.
(5) last room temperature 12000rpm centrifugation 1min elution, centrifuge tube is placed in -20 DEG C of preservations.
The connection of target fragment and pBAD-18-ClyA
Above-mentioned DNA fragmentation after purification is connect with pBAD-18-ClyAVector, linked system and program are as follows:
Program: overnight, 4 DEG C of product save for 16 DEG C of connections.
The preparation of α plants of E. coli competents of DH5
(1) α plants of Escherichia coli of non-resistant LB flat lining out recovery DH5, picking when growing up to single bacterium colony are inoculated in dress Have in 3~5ml non-resistant LB liquid medium test tube, is placed in shaking table, 220r/min, 37 DEG C of culture 12h or so, until logarithm Phase.
(2) bacterium solution in logarithmic phase is inoculated in without in the LB Liquid Culture of antibody by 50ml with the ratio of 1:100,37 DEG C, 220r/min cultivates 3~4h, until bacterium solution OD600nmValue is equal to 0.3~0.5.
(3) after taking out bacterium solution ice bath 30min, 4 DEG C are placed in a centrifuge, 10min is centrifuged under the conditions of 4000rpm, it is heavy to collect It forms sediment.
(4) CaCl of the 10ml 0.1mol/L concentration after ice bath is added2Solution is softly resuspended, again ice bath 30min, from 4 DEG C of scheming, 4000rpm is centrifuged 10min, and repeats this step.
(5) glycerol-CaCl of 2ml pre-cooling is added2Solution (contains 15% glycerol), after precipitating is resuspended, with 1.5ml centrifuge tube point Dress, every 100 μ l of pipe are placed in -70 DEG C and save backup.
Connect the conversion of plasmid
Using chemical transformation, above-mentioned connection product is transferred to DH5 α E. coli competent, specific steps are as follows:
(1) E. coli competent prepared in advance is taken out, is placed on ice chest and it is waited slowly to melt.Then by 10 μ l connection product is transferred in the competent cell of 100 μ l, is mixed, -20 DEG C of ice bath 30min.
(2) after the completion of ice bath, centrifuge tube is placed in heat shock 90s in 42 DEG C of water-bath immediately.Then ice bath 3min again, The not antibiotic LB liquid medium of 600 μ l is added after the completion.37 DEG C are placed in air table, is converted under the conditions of 180r/min 1h。
(3) after the completion of cultivating, 4000rpm, is centrifuged 5min at room temperature.The LB culture solution of 500 μ l is removed, it is remaining After the piping and druming of 200 μ l culture solution tendernesses mixes, drawn with liquid-transfering gun to chlorampenicol resistant culture dish, with spreading rod even spread.
(4) culture dish is placed in 30min in 37 DEG C of incubators, then back-off is placed, and overnight incubation is obvious to single bacterium colony.
The screening and identification of positive plasmid
Plasmid extracts
The smooth round single bacterium colony of milky and surface on culture dish is selected, is chosen with oese single to mould equipped with 3ml chlorine It in the test tube of plain resistance LB liquid medium, is placed in the air table of 220r/min, expands 10~12h of culture under the conditions of 37 DEG C After take 2ml bacterium solution extract plasmid, specific steps are as follows:
Plasmid enzyme restriction identification
Above extracted plasmid is subjected to digestion identification, digestion system and program are as follows:
Program: 37 DEG C of digestion 30min, after by product carry out 1% Ago-Gel run glue, condition be 85V voltage under Electrophoresis 30min.Gel after electrophoresis is taken pictures imaging, according to Marker band observe in the UV lamp target fragment whether with It is expected that size is consistent.
Plasmid order-checking identification
Digestion is identified that successful plasmid send biotech firm to carry out determined dna sequence, splices sequencing knot with DNAMAN software Fruit, and be compared with implementation sequence.Remaining 1ml bacterium solution is expanded into 3~4h of culture after identifying successfully, it is sweet with 50% later Cryopreservation tube, -80 DEG C of preservations are added in 1:1 ratio in oily glycerite.
Recombinantly express the building and identification of bacterium
Recombinantly express the building of bacterium
JC8031 plants of competence is prepared referring to the preparation method of above-mentioned DH5 α competence.
Recombinant plasmid and empty plasmid are transferred in JC8031 competent cell referring to above-mentioned chemical transformation.
Recombinantly express the expression identification of bacterium
Plasmid enzyme restriction identification is carried out to recombinant expression bacterium referring to the above method, sequencing is identified.Furthermore SDS-PAGE method is used Identification carries out Protein Separation identification to recombinant bacteria, it is ensured that and plasmid is correctly expressed, specific steps are as follows:
(1) sample preparation: expand culture 5ml bacterium solution about 6h in test tube, in OD600nmValue is added between 0.4~0.6 20%L-Arabinose overnight induction.0.5ml bacterium solution is taken after the completion of induction, and centrifuge 3000 × g, 4 DEG C of centrifugation 3min give up Supernatant.The 20% 50 μ L of albumen loading Buffer containing beta -mercaptoethanol is added, gently piping and druming is resuspended.Boiling water bath 5-10min is cold But spare to room temperature.
(2) offset plate preparation refers to kit operation instruction: being 1. ready to experiment mould needed for glue, with washing powder by glue Plate and penicillin bottle, which clean up, is placed in baking oven drying, assembles glass plate and checks whether leak.2. small to penicillin Distilled water 2.08ml, 30%Acr-Bis1.67ml, separation gel buffer 1.25ml, 10%APS0.05ml are sequentially added into bottle The SDS-PAGE separation gel as 10% is prepared with TEMED0.002ml, quickly mixes and moves in offset plate gap, be slowly added to Distilled water flattens, and waits 30~40min.3. pouring out upper layer distilled water, distilled water is sequentially added into penicillin bottle 1.14ml, 30%Acr-Bis0.34ml, concentration glue buffer 0.5ml, 10%APS0.02ml and TEMED0.002ml are configured to Glue is concentrated for 5%SDS-PAGE, quickly mixes and moves in separation gel upper gap, is inserted into comb, waits 30min.
(3) protein electrophoresis: offset plate is installed into electrophoresis tank, injects electrophoretic buffer, sample is added into glue hole, is started Run glue, program 80V, 30min, 120V, 1h.
(4) it dyes: the glue run is taken out, cut off redundance, be placed in culture dish, 0.25% Coomassie brilliant blue is added Dye liquor did not had glue surface, was protected from light overnight dyeing.
(5) it decolourizes: the glue for completing dyeing is placed in culture dish, Coomassie brilliant blue destainer is added and did not had glue surface, is put into It is clear to band to rock decoloration under appropriate speed for shaking table.
(6) it is imaged: the glue for completing decoloration being placed in gel imager, selection mode is taken pictures.With albumen Maker band pair Than analyzing band correctness.It is named as pBAD-18-ClyA-E and pBAD-18-ClyA-Ez after identifying successfully, and bacterium solution is expanded 3~4h is cultivated, cryopreservation tube, -80 DEG C of preservations are added in 1:1 ratio with 50% glycerol glycerite later.
Experimental result
Plasmid enzyme restriction result and analysis
Plasmid is identified using double digestion, as shown in Figure 1, comparison DL10000Marker size, it is seen that band obtained by electrophoresis is big It is small to be consistent with desired value, wherein E gene 1539bp, pBAD-18-ClyA plasmid about 7000bp.
According to two groups of plasmid double digestions as a result, preliminary judgement pBAD-18-ClyA-E recombined pronucleus expression plasmid construction at Function.
Plasmid order-checking result
Shanghai Sheng Gong Biotechnology Co., Ltd compares plasmid nucleotide sequencing, sequencing result with DNAMAN software Analysis, it is as a result as shown in Figure 2, completely the same with expected sequence.
Sequencing result is consistent with the gene order of design synthesis, shows that gene loci is not mutated, it is ensured that express albumen Accuracy.Determine that expression plasmid pBAD-18-ClyA-E is constructed successfully according to sequencing result.Recombinantly express the expression of results of bacterium
Recombinant bacterium after inducing expression carries out full bacterium sds polyacrylamide gel electrophoresis experiment, as a result as shown in the right side Fig. 3, Using unloaded bacterium as control, it is seen that apparent protein band, and albumen size and expection are consistent.According to recombinant bacteria protein expression The case where illustrate, pBAD-18-ClyA-E plasmid can express in JC8031 plants of competence of Escherichia coli, and the obvious table of band It is high up to amount.This illustrates that recombinantly expressing bacterium constructs successfully.By building, successfully recombinant expression bacterium is named as JC8031-E, unloaded bacterium note For JC8031-P.
Embodiment 2
Outer membrane vesicles are prepared with the recombinant expression bacterium JC8031-E in embodiment 1
The recombinant bacterial strain for being stored in -80 DEG C is taken out, 4 DEG C of defrostings are placed in, culture bacterium prepares OMV, concrete operation step It is as follows:
(1) two groups of recombination bacterium solutions after thawing are dipped with oese, triangular in shape point on the culture dish of chlorampenicol resistant Grade scribing line recovery bacterium.
(2) oese picking single bacterium colony is used, is inoculated in the test tube for filling 10ml chlorampenicol resistant LB liquid medium, It is placed in 37 DEG C of shaking tables that condition is 220rpm, cultivates about 6h, make OD600nmValue is between 0.4~0.6.
(3) again by the dilution proportion of 1:100 into 1000mL LB chlorampenicol resistant fluid nutrient medium, by the ratio of 1:1000 Inducer 20%L-Arabinose solution is added in example.It is equally placed in 37 DEG C of shaking tables that condition is 220rpm, cultivates about 20h.
(4) supernatant is collected by centrifugation, centrifugal condition is 4 DEG C, 10000 × g, 10min, as far as possible removal precipitating.
(5) under condition of ice bath, the filter in 0.45 μm of aperture of the supernatant being collected into is filtered 2 times, then with 0.22 μm The filter in aperture filters 2 times, sufficiently removal thallus.
(6) the ultra-filtration centrifuge tube filter liquor again for the use of retention relative molecular mass being 100kDa, is concentrated into original volume 1/6, collect filtrate.
(7) upper step filtrate is injected in super filter tube, is centrifuged 1h under conditions of 39000 × g, 4 DEG C using ultracentrifuge. Supernatant is abandoned, the precipitating for being attached to tube wall is rinsed with PBS buffer solution and is resuspended.Slightly to mention OMV.
(8) 2ml is slightly mentioned by several times in OMV suspension injection ultracentrifugation pipe, is separately added into from bottom to up with lower density OptiPrep-iodixanol gradient cell separating liquid: 1.5ml 50%, 1.5ml 45%, 1.5ml 40%, 1.5ml 35%, (original liquid concentration 60%, separating liquid dilute stoste with 50mM HEPES-150mM NaCl by 1.5ml 30% and 1.5ml 25% It is formulated).High speed centrifugation is carried out, condition is 150000 × g, 4 DEG C, is stayed overnight.
(9) after the completion of being centrifuged, each component centrifugate is successively collected respectively from top to bottom.With 10 × 50mMHEPES-150mM NaCl solution dilution carries out polyacrylamide gel electrophoresis experimental identification OMV and is enriched in which layer separating liquid.Carry out again from The heart precipitating, condition be 150000 × g, 4 DEG C, 1h.
(10) after the completion of being centrifuged, the OMV of precipitating is resuspended in 2ml PBS buffer solution, then re-suspension liquid is applied to by reject supernatant Non-resistant LB agar plate, after carrying out without the dientification of bacteria, -20 DEG C of storages, and use within 4 weeks.
The Electronic Speculum of outer membrane vesicles form is observed
The OMV sample for taking purifying, is added drop-wise to respectively on carbon coating copper mesh, and 2% uranium acetate is added dropwise, and drying at room temperature is sent Wuhan Biotechnology Co., Ltd, Google detect under 80KV Hitachi h-7650 electron microscope.
BCA method measures outer membrane vesicles total protein concentration
According to kit specification, prepare BSA titer and BCA working solution in advance, then with micropore board detecting method into Row detection, concrete operations are as follows:
(1) dilution standard product: standard items is taken to be diluted to final concentration of 0.5mg/mL with PBS.Standard items are pressed 0,2,4,6, 8,12,16,20 μ L are added in protein standard sample wells, and PBS is added to make 20 μ L of total volume.
(2) appropriate dilute sample (selecting multiple gradients), in the sample well for adding 20 μ L to 96 orifice plates.
(3) 200 μ LBCA working solutions are injected in every hole, are protected from light 15~30min of standing in 37 DEG C of incubators.It is measured with microplate reader A562nm calculates protein concentration according to standard curve.
The identification of target protein in outer membrane vesicles
OMV is identified using Western-blot, it is ensured that sample OMV can present target protein.Concrete operations are such as Under:
10% sds polyacrylamide gel electrophoresis experiment is first carried out according to above-mentioned steps.Reagent needed for preparing simultaneously:
Transferring film liquid: glycine 2.9g, Tris5.8g, SDS0.37g are weighed, adds distilled water constant volume after completely dissolution in beaker To 1L.
TBSTbuffer: NaCl40g, Tris12.1g, HCl4.2ml are weighed, adds distilled water fixed after completely dissolution in beaker Hold and to 500ml and adjust PH=7.5, when use is diluted to 1 × TBS, and the Tween 20 of 500 μ l/L is added.
Block buffer: weighing 5% skimmed milk power 5g, TBST buffer 100ml is added, cannot save for a long time need to show With current.
Secondary antibody diluent: 6ml TBST buffer is added into 9ml Block buffer.
(1) transferring film: shearing the pvdf membrane of suitable size, and mark, be placed in formaldehyde and activate 30s, ultrapure water rinse 1 It is secondary, it is put into transferring film liquid and impregnates balance 15min, the glue run through and filter paper are also placed in transferring film liquid and impregnated.
(2) 3 pieces of filter paper being layered on transfer instrument, drives bubble away as far as possible, the wetting of transferring film liquid puts pvdf membrane, drives bubble away, It soaks again, puts PAGE glue, spread other 3 pieces of filter paper, test tube drives bubble away again with losing no time, and covers transfer instrument lid, adjusts Section program is 25V, transfers 25min.
(3) transfer finishes, and takes out pvdf membrane, is placed in methanol and impregnates 5s, after ultrapure water rinse is primary, be placed in confining liquid In, it is incubated for 1.5h.
(4) after the completion of being incubated for, film 3 times are washed at room temperature in shaking table with TBSTbuffer, each 4min.By anti-His label Primary antibody and primary antibody dilution press the dilution proportion of 1:5000, submerge film, 4 DEG C of overnight incubations.
(5) after the completion of being incubated for, film 3 times are washed at room temperature in shaking table with TBSTbuffer, each 4min.By HRP sheep anti mouse two Anti- and secondary antibody diluent presses the dilution proportion of 1:5000, submerges film, while mixing colour developing bottom liquid, and shaking table room temperature is protected from light incubation 30min~60min.
(6) after the completion of being incubated for, film 3 times are washed at room temperature in shaking table with TBSTbuffer, each 4min.Guarantee albumen towards On, according to PVEF membrane area size according to 0.1ml/cm2Chemiluminescence detection liquid westarworking needed for calculating Solution submerges film after then mixing AB liquid according to the ratio of 1:1, is protected from light and is incubated for 1min.
(7) in gel imager middle berth layer of transparent gloves, the PVDF albumen completed will be incubated for and be placed on gloves up Chemiluminescent substance polluting platform is prevented, immunoblotting program is selected, the time for exposure is set, last exposed photographic saves result.
It will identify that successful two groups of recombination outer membrane vesicles are named as OMV-E.
Experimental result
It is shown according to outer membrane vesicles electron microscope (Fig. 4), OMV structural integrity meets expected size.Illustrate Escherichia coli JC8031 plants have efficient OMV production capacity.
Outer membrane vesicles total protein measurement result
Standard curve (Fig. 5) is drawn according to the operating procedure of BCA method kit, measures the total protein concentration of OMV, it is as a result outer Membrane vesicle total protein concentration is 4.4 μ g/ μ l of OMV-E；OMV-P 0.8μg/μl.Antigen protein testing result contained by outer membrane vesicles
According to Wstern-blot result figure (Fig. 6) as it can be seen that JEV purpose antigen albumen expressed by recombinant expression bacterium can It is positioned on the OMV of bacterium excretion, it is made body can be stimulated to generate all kinds of specific immune responses and non-specificity as antigen Immune response.
Embodiment 3
The building of recombinant vector, recombinant bacterial strain including JEV antigen gene；In addition to the nucleotide sequence of JEV antigen gene Other than as shown in SEQ ID NO:2, other methods and step are same as Example 1.
The nucleotide sequence of JEV antigen gene is as shown in SEQ ID NO:2；What is wherein underlined is respectively upstream and downstream enzyme Enzyme site；Thickened portion is His label, and italicized item is terminator.
The amino acid sequence of coding is following (SEQ ID NO:4):
CSRDKLALKGTTYGMCTEKFSFAKNPADTGHGTVVIELTYSGSDGPCKIPIVSVASLNDMTPVGRLVTV NPFVATSSSNSKVLVEMEPPFGDSYIVVGRGDKQINHHWHKAGSTGSCYHASVTDIGSPTTGEAHNEKRADSSYVCG SSENHGNYSAQVGASQGSQEGGLHQALAGAIVVEYSSSVKLTSGHLGSKFSFAKNPADGSTVNPFVAGSEMEPPFGD SYIVVGRGDKQINHHWHKAGSHKAGSTLGSSIGKAVHQVFGSKAWGKSILFAHHHHHH
Experimental result
Plasmid enzyme restriction result and analysis
Plasmid is identified using double digestion, as shown in Figure 1, comparison DL10000Marker size, it is seen that band obtained by electrophoresis is big It is small to be consistent with desired value, wherein Ez gene 855bp, pBAD-18-ClyA plasmid about 7000bp.
According to two groups of plasmid double digestions as a result, preliminary judgement pBAD-18-ClyA-Ez recombined pronucleus expression plasmid construction at Function.
Plasmid order-checking result
Shanghai Sheng Gong Biotechnology Co., Ltd compares plasmid nucleotide sequencing, sequencing result with DNAMAN software Analysis, it is completely the same with expected sequence as a result as shown under Fig. 2.
Sequencing result is consistent with the gene order of design synthesis, shows that gene loci is not mutated, it is ensured that express albumen Accuracy.Determine that expression plasmid pBAD-18-ClyA-Ez is constructed successfully according to sequencing result.
Recombinantly express the expression of results of bacterium
Recombinant bacterium after inducing expression carries out full bacterium sds polyacrylamide gel electrophoresis experiment, as a result as shown in the left side Fig. 3, Using unloaded bacterium as control, it is seen that apparent protein band, and albumen size and expection are consistent.According to recombinant bacteria protein expression The case where illustrate, pBAD-18-ClyA-Ez plasmid can express in JC8031 plants of competence of Escherichia coli, and the obvious table of band It is high up to amount.This illustrates that recombinantly expressing bacterium constructs successfully.By building, successfully recombinant expression bacterium is named as JC8031-Ez, unloaded bacterium Remember JC8031-P.
Embodiment 4
Outer membrane vesicles are prepared with the recombinant expression bacterium JC8031-Ez in embodiment 3
Other than recombinant expression bacterium is JC8031-Ez, remaining method and step is same as Example 2.
Experimental result
It is shown according to outer membrane vesicles electron microscope (Fig. 4), OMV structural integrity meets expected size.Illustrate Escherichia coli JC8031 plants have efficient OMV production capacity,
Outer membrane vesicles total protein measurement result
Standard curve (Fig. 5) is drawn according to the operating procedure of BCA method kit, measures the total protein concentration of OMV, it is as a result outer Membrane vesicle total protein concentration is 2.8 μ g/ μ l of OMV-Ez；OMV-P 0.8μg/μl.Antigen protein testing result contained by outer membrane vesicles
According to Wstern-blot result figure (Fig. 6) as it can be seen that JEV purpose antigen albumen expressed by recombinant expression bacterium can It is positioned on the OMV of bacterium excretion, it is made body can be stimulated to generate all kinds of specific immune responses and non-specificity as antigen Immune response.
Embodiment 5
Mouse immune attacks malicious Protection
3 week old cleaning grade female KM mouse 70 is bought from Chengdu up to large experimental animal company, is randomly divided into 7 groups, often Group 10.OMV vaccine injection dosage in reference literature report, by tri- groups of OMV-P, OMV-E and OMV-Ez respectively with 50 μ g/ only and The dose inoculation dorsal sc of 75 μ g/ only is immune.Three groups of OMV are diluted to 50 μ by external mesaxon vesica total protein content measured value G/200 μ l and 75 μ g/200 μ l.If 1 group of blank control group, immune using the identical approach of PBS buffer solution.It is identical after 7 days immune Mode booster immunization 1 time.Abdominal cavity is carried out with the III virulent SC2013-SN5 of type encephalitis of gene to every group of mouse respectively after two epidemic diseases 7 days to attack Poison, every 600 μ l (75LD50).It is observed continuously 14 days after attacking poison, records mouse invasion and death condition on time.
1 mouse immune protest test of table and grouping
The detection of mice serum JE neutralizing antibody level
Mice serum acquisition
Mouse blood sampling time is to attack before poison 3 days the 5th day after booster immunization.Blood collection be using blood sampling capillary into The blood sampling of row orbital venous plexus.By blood in being stored at room temperature 30min after blood sampling, goes to 4 DEG C of refrigerators and stand 1h, 1000 × g of centrifuge, 4 DEG C of centrifugation 10min, serum are separated in sterile super-clean bench and is dispensed are stored in -20 DEG C.
The detection of mice serum JE neutralizing antibody
Select the pig Japanese B encephalitis virus of Shenzhen Combined Biotech Co., Ltd.'s production of market sale anti- It is horizontal that body ELISA detection kit detects clear middle JE neutralizing antibody of taking a blood sample.Specific steps are as follows:
(1) 10 × cleaning solution is diluted with distilled water by 1:10, is formulated as working concentration washing lotion.
(2) recommended method sample diluting liquid is diluted serum to be checked by 1:100 to specifications.Negative and positive mice The dilution of serum same procedure.100 μ l sample diluting liquids are added in 2 hole of blank control, every hole；Sealing plate film is posted, is kept away under the conditions of 37 DEG C Light reaction 30min.
(3) liquid in hole is got rid of rapidly, and every hole is injected 300 μ l cleaning solutions and washed 3 times, every time washing concussion 1min, finally Net rear blotting paper is once got rid of to pat dry.
(4) in addition to blank control wells, remaining every hole adds the diluted 50 μ l of sheep anti-mouse igg of 1:5000, covers sealing plate film, and 37 DEG C It is protected from light 30min.Repeat board-washing step.
(5) institute's expense is calculated in advance, takes isometric substrate solution and developing solution piping and druming to mix, 100 μ l, lid is added in every hole Film is covered well, and 37 DEG C are protected from light 10min.
(6) 50 μ l of reaction terminating liquid is added, is returned to zero with blank control, is read with microplate reader in 450nm wavelength and record OD Value.
The detection of mice serum neutralizing antibody level
The OMV-Ez dosage group mice serum of the best 75 μ g of choice experiment effect is reduced using plaque and neutralizes experiment (PRNT) It is horizontal to detect neutralizing antibody, specific steps are as follows:
(1) shift to an earlier date 2 days and be paved with 6 orifice plates with hamster kidney cell (BHK-21) in good condition, it is properly spare to its state.
(2) mice serum of packing 56 DEG C of inactivation 30min in PCR pipe are taken, are made after aseptic process using viral maintaining liquid 2-1、2-2、2-3、2-4、2-5Serial doubling dilution.The JEV-SC2013 strain cell toxicant of known titre is diluted to viral dilution 100PFU/100μl.By in the virus liquid and 100 μ l serum addition centrifuge tube after 100 μ l dilution, it is sufficiently mixed and is placed on 37 DEG C Cell incubator carries out neutralization reaction, and reaction time 90min is primary every the concussion of 15min tenderness.Cell controls are set simultaneously Hole and virus control wells.
(3) 6 orifice plates 2 times for covering with BHK-21 cell monolayer are cleaned with PBS buffer solution, and upper step is neutralized to the virus liquid finished It is inoculated in hole, every 200 μ l of hole.It marks and is placed in 37 DEG C of cell incubator culture 90min, shaken every 15min fixed-direction Shake it is several under.
(4) 6 orifice plates finished will be colonized to take out, culture solution in hole is quickly sucked out, every hole is added 2ml and contains 1% Methyl cellulose Plain maintaining liquid is cultivated 2~3 days in 37 DEG C of incubators.Observe and record Cytopathic effect.
(5) 6 orifice plates for finishing culture are taken out, and liquid in hole is quickly sucked out, and the dyeing of 1ml crystal violet solution is added in every hole 20min.6 orifice plates are finally rinsed, plaque quantity is calculated, 50% is reduced as judgement than control group using the plaque number of experimental group and marks Standard, serum dilution are exactly its neutralization titer.
The detection of mice serum cytokine levels
Select the mice serum interleukin 2 (IL-2), white of the Hangzhou Lian Ke biotechnology company production of market sale 3 kinds of ELISA detection kits of cytokine 4 (IL-4) and interferon gamma (IFN-γ) detect adopted Cytokine of Serum water It is flat.Laboratory operating procedures are as follows:
(1) reagent needed for preparing washing lotion, buffer etc. in advance, blood serum sample are balanced to room temperature.By standard items by recommendation Method dilutes to obtain each group normal concentration, using standard dilutions as the zero-dose of standard curve.
(2) 300 μ 1 × washing lotions of l are added into hole and impregnate 30s, pat dry later.
(3) standard items of 100 μ l doubling dilutions are added in standard sample wells.100 μ l standard dilutions are added in blank well.Sample 80 μ l 1 × detection buffers and 20 μ l blood serum samples are added in hole.The diluted detection antibody of 50 μ l is added in every hole.Sealing plate film sealing plate, 300r/min concussion, is incubated at room temperature 1.5h.
(4) liquid in hole is thrown away after being incubated for, 300 μ l washing lotion board-washings are added in each hole, wash 6 times.Every hole adds after the completion Enter the Streptavidin of the 100 diluted horseradish peroxidase-labeleds of μ l.Sealing plate film sealing plate, 300r/min concussion, incubation at room temperature 1.5h。
(5) board-washing, every hole are protected from light 100 μ l chromogenic substrate TMB of addition, are incubated at room temperature 5~30min again.
(6) 100 μ l terminate liquids are added in every hole.It is read using microplate reader and records the OD value under 450nm.Mouse spleen T leaching The sorting of bar cell subsets
The grouping of mouse and immunization ways, immunizing dose attack malicious Protection referring to above-mentioned mouse immune.Two exempt from nothing after 7 days Bacterium separating mouse spleen lymphocyte is thin by CD3+, CD4+ and CD8+ three classes T lymph in selected by flow cytometry apoptosis mouse spleen Born of the same parents.Specific steps are as follows:
(1) get out required reagent in advance, by with the mouse after 75% alcohol soaking disinfection in sterile in superclean bench Separating spleen is drawn in 1ml Hank ' s buffer piercing spleen with 1ml syringe and is blown and beaten repeatedly, until spleen is at white clear Shape.
(2) lymphocyte blown and beaten out transfer is set in 15ml centrifuge tube, supplements 11ml Hank ' s liquid, at room temperature 300 × G is centrifuged 5min, abandons supernatant.
(3) it is slowly injected into erythrocyte cracked liquid 2ml, 300 × g is centrifuged 5min after soft piping and druming mixes, and abandons supernatant.
(4) Hank ' s liquid 12ml is added again, 300 × g is centrifuged 5min after soft piping and druming mixes, and abandons supernatant.
(5) 3ml contains the abundant suspension cell of RPMI-1640 culture medium of 10% fetal calf serum, counts to cell, adjusts Ganglion cell's concentration is to 2~4 × 105A/ml.
(6) by 100 μ L (1 × 105It is a) cell suspension is transferred in another centrifuge tube, and with rat anti-mouse CD3- FITC SPRD, rat anti-mouse CD4-PE and rat anti-mouse the CD8a-PerCP 15min at 4 DEG C in the dark.By dyeing Cell is washed in PBS and is resuspended in 450 μ l PBS.Cell is resuspended by BD FACS Calibur flow cytometry analysis T cell subgroup.
The classification of mice serum antibody subtype
IgG1 the and IgG2a antibody water in ELISA kit detection each group mice serum produced using R&D company, the U.S. Flat, specific experiment operating procedure is as follows:
(1) it is loaded: all solution equilibrias to room temperature.100 μ 1 × dilutions of l are added in blank well, and 100 μ l are added in sample aperture 100 μ l of various concentration standard items is added in sample liquid, gauge orifice.Reacting hole, equilibrium at room temperature stationary incubation 60 are sealed with sealing plate gummed paper Minute.
(2) it dilutes cleaning solution: 20 times of cleaning solutions is diluted with 20 times of ultrapure water.
(3) it washs: liquid in the ELISA Plate after incubation is dried, shaken after 250 μ l cleaning solutions are added with micro oscillator 3min is washed repeatedly 3 times.
(4) enzyme: the 100 diluted IgG2A/IgG1 secondary antibodies of μ l 1:100 are added in every hole.
(5) it is incubated for: incubation at room temperature 20min.It repeats step (4).
(6) 100ul developing solution is added in every hole；Room temperature is protected from light colour developing 10min.
(7) 100 μ l terminate liquids are added in every hole.
It is returned to zero with blank well, microplate reader measures the OD value in each hole in 450nm, and keeps a record.
Mouse immune attacks malicious Protection result
Mouse is carried out to attack poison within the 7th day after booster immunization.As a result as shown in Figure 7 and Figure 8, display: (1) PBS control group 10 are all dead, the 5th day and the 7th day each dead two, the 6th day dead 4, the 8th day and the 10th day each dead 1, guarantor Shield rate 0%；The OMV-E group of (2) 50 μ g dead 5, the 5th, 7,9,10 day each dead 1, then the 6th day dead 2, protective rate 50%；The OMV-Ez group of (3) 50 μ g dead 4, the 6th day dead 2, the 7th, 8 day each dead 1, protective rate 60%；(4) The OMV-P group of 50 μ g dead 9, the 5th, 6,8,10 day each dead 1, the 7th day dead 3, the 9th day dead 2, protection Rate 10%；The OMV-E group of (5) 75 μ g dead 4, the 5th day dead 2, the 6th, 9 day dead 1, protective rate 60%；(6) The OMV-Ez group of 75 μ g is only 1 dead, in death in the 10th day, protective rate 90%；The OMV-P group of (7) 75 μ g is all dead, the 5,6,7 days each dead 2, the 8th day dead 4, protective rate 0%.All mouse diing times concentrate on attacking 5~10 after poison It.The 3.2 horizontal testing results of mice serum JE neutralizing antibody
It is horizontal using indirect elisa method measurement mice serum JE neutralizing antibody, as a result as Fig. 9 is shown: 5 after booster immunization It, experimental comparison group PBS, 50 μ g OMV-P and 75 μ g OMV-P are less than cutoff value, are determined as feminine gender.In addition to control group, 4 groups of recombination outer membrane vesicles groups of remaininging all produce JE neutralizing antibody, and serum detection is all the positive, and the OMV-Ez dosage group of immune 75 μ g Potency highest, OD value reading is 1.1725 at microplate reader 450nm wavelength, but is not significantly higher than other groups (P > 0.05).
Antibody subtype classification experiments result
As shown in Figure 10, select immunizing dose for the group of 75 μ g, using ELISA kit probe into antibody subtype IgG1 and IgG2A situation of change, the coefficient R of two category directrix curves2Value be respectively 0.9996 and 0.9933, measurement result conforms to It asks.Measurement result such as Figure 11 shows that each immune group can stimulate body secretes IgG1 and IgG2A.OMV-P experimental comparison group and The content of OMV-E experimental group IgG2A is significantly higher than IgG1 (P < 0.05), and the content of OMV-Ez group IgG2A is extremely significant is higher than IgG1(P<0.01)。
Mice serum cytokines measurement result
In order to which to induce the specific cellular immunity for JEV in Mice Body anti-for immune group each when probing into 75 μ g dosage It answers, cell factor IL-2, IL-4 and IFN-γ growth pattern in 3 in mice serum, standard is measured using indirect ELISA reagent kit Curve is as shown in figure 12, and as a result as shown in figure 13,3 type cytokines of three immune groups increased, with control group phase Than, the amount difference pole of OMV-Ez group IL-2 is extremely extremely significant (P < 0.0001), and the amount difference of IFN-γ is extremely significant (P < 0.01), The amount difference of IL-4 is extremely significant (P < 0.01), is increment and maximum one group of growth rate.Remaining two group's variation is lower than OMV-Ez group.
Mice serum neutralizing antibody experimental result
According to experimental principle it is found that the number of Plaque Formation is equal to number viral in every hole in each dilution hole, Compared with the control group, the viral load of reduction is the quantity neutralized by antibody, is subtracted with the plaque quantity of test group than control group Few 50% is used as criterion, this group of serum dilution ratio is exactly its plaque reduction assay potency.As shown in Figure 14 and table 2,75 μ The OMV-Ez dosage group of g is 2-3The virus of half can be neutralized when dilution, therefore generated neutralizing antibody average titer is 1: 8。
2 mice serum neutralizing antibody testing result of table
Mouse spleen T lymphocyte subgroup sorts experimental result
By fluidic cell method (FCM) detect the CD3+ with T cell subtype representative scatter diagram, CD3+CD4+ with The average measurement percentage of CD3+CD8+T cell assesses CD3+ in splenocyte, the distribution of CD4+ and CD8+T cell by FACS. Counting the detection average value with standard deviation and arranging is bar chart 15, represents CD3+, CD3+CD4+ and the CD3+CD8 of spleen The percentage of+T cell.OMV-Ez group is all remarkably higher than control group (P < 0.05) in CD3+, CD3+CD4+, and in the Asia CD3+CD8+ It is extremely extremely significant in group to be higher than PBS control group.
As can be seen from the above embodiments, the recombinant vector provided by the invention including JEV antigen gene, takes The JEV antigen gene of band can successful expression, the preparation for the outer membrane vesicles of subsequent submission JEV antigen provides condition.This hair JEV purpose antigen albumen expressed by the recombinant bacterial strain of bright offer can be positioned on the outer membrane vesicles of bacterium excretion, be made described Outer membrane vesicles can stimulate body to generate all kinds of specific immune responses and nonspecific immune reaction as antigen；Height can be excited The specific immune response of effect has outstanding antigenicity, can generate higher specific antibody, can cause humoral immunity Cellular immunity can be caused again.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.
Sequence table
<110>Sichuan Agricultural University
<120>a kind of recombinant vector including JEV antigen gene, recombinant bacterial strain and submission JEV antigen outer membrane vesicles and its answer With
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1539
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tgctctagat ttaactgctt aggtatgggc aaccgcgatt ttattgaagg tgcaagcggt 60
gcaacctggg ttgatctggt tctggaaggt gatagctgtc tgaccattat ggcaaatgat 120
aaaccgacac tggatgtgcg catgattaac attgaagcaa gtcagctggc agaagttcgt 180
agctattgtt atcatgcaag cgttaccgat attagcaccg ttacacgttg tccgaccacc 240
ggtgaagcac ataatgaaaa acgtgcagat agcagctatg tgtgcaaaca gggttttacc 300
gatcgtggtt ggggtaatgg ttgtggtctg tttggtaaag gtagcattga tacctgtgcc 360
aaatttagct gtaccagcaa agcaattggt cgtatgattc agccggaaaa catcaaatat 420
gaagtgggca tttttgtgca tggcaccacc accagtgaaa atcatggtaa ttatagcgca 480
caggttggtg caagccaggc agcaaaattt acagttaccc cgaatgcacc gagcattacc 540
ctgaaactgg gtgattatgg tgaagttacc ctggattgtg aaccgcgtag cggtctgaat 600
accgaagcat tttatgttat gaccgttggc agcaaatcat ttctggttca tcgtgaatgg 660
tttcatgatc tgagcctgcc gtggaccagt ccgagcagca ccgcatggcg taatcgtgaa 720
ctgctgatgg aatttgaaga agcacacgca accaaacagt atgttgttgc actgggtagc 780
caagaaggtg gtctgcatca ggcactggca ggcgcaattg ttgttgaata tagcagcagc 840
gttaaactga ccagcggtca tctgaaatgt cgtctgaaaa tggataaact ggcactgaaa 900
ggcaccacct atggtatgtg taccgaaaaa ttcagctttg ccaaaaatcc ggcagatacc 960
ggtcatggca ccgttgttat tgaactgacc tatagcggta gtgatggtcc gtgtaaaatt 1020
ccgattgtta gcgttgcaag cctgaatgat atgacaccgg ttggtcgtct ggttaccgtt 1080
aatccgtttg ttgcaaccag cagcagcaat agcaaagttc tggttgaaat ggaaccgcct 1140
tttggcgata gctatattgt ggttggtcgc ggtgataagc agattaatca tcattggcat 1200
aaagcaggta gcaccctggg taaagcattt agcacaaccc tgaaaggtgc acagcgtctg 1260
gcagccctgg gtgataccgc atgggatttt ggtagtattg gtggtgtgtt taacagcatt 1320
ggtaaagcag ttcatcaggt ttttggtggt gcatttcgta cgctgtttgg tggtatgagc 1380
tggattacac agggtctgat gggtgcactg ctgctgtgga tgggtgttaa tgcacgtgat 1440
cgtagcattg cactggcatt tctggcaacc ggtggtgttc tggtttttct ggccaccaat 1500
gttcatgcac atcatcacca tcaccactaa aagcttggg 1539
<210> 2
<211> 855
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tgctctagag ataaactggc actgaaaggc accacctatg gtatgtgtac cgaaaaattc 60
agctttgcca aaaatccggc agataccggt catggcaccg ttgttattga actgacctat 120
agcggtagtg atggtccgtg taaaattccg attgttagcg ttgcaagcct gaatgatatg 180
acaccggttg gtcgtctggt taccgttaat ccgtttgttg caaccagcag cagcaatagc 240
aaagttctgg ttgaaatgga accgcctttt ggcgatagct atattgttgt tggtcgtggt 300
gataagcaga tcaatcatca ttggcataaa gcaggtagca ccggtagctg ttatcatgca 360
agcgttaccg atattggtag cccgaccacc ggtgaagcac ataatgaaaa acgtgcagat 420
agcagctatg tttgtggtag cagcgaaaat catggtaatt atagcgcaca ggttggtgca 480
agccagggta gccaagaagg tggtctgcat caggcactgg caggcgcaat tgttgtggaa 540
tatagcagca gcgttaaact gaccagcggt catctgggta gcaaattttc attcgcaaaa 600
aatcctgccg atggcagcac agtgaacccg tttgtggcag gtagcgagat ggaacctccg 660
tttggtgatt catatatcgt ggttggccgt ggcgacaaac aaattaacca tcactggcac 720
aaagccggtt cacataaagc gggttcaacc ctgggtagct caattggtaa agcagttcat 780
caggtttttg gtagcaaagc atggggtaaa agcattctgt ttgcacatca tcaccatcac 840
cactaaaagc ttggg 855
<210> 3
<211> 509
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Cys Ser Arg Phe Asn Cys Leu Gly Met Gly Asn Arg Asp Phe Ile Glu
1 5 10 15
Gly Ala Ser Gly Ala Thr Trp Val Asp Leu Val Leu Glu Gly Asp Ser
20 25 30
Cys Leu Thr Ile Met Ala Asn Asp Lys Pro Thr Leu Asp Val Arg Met
35 40 45
Ile Asn Ile Glu Ala Ser Gln Leu Ala Glu Val Arg Ser Tyr Cys Tyr
50 55 60
His Ala Ser Val Thr Asp Ile Ser Thr Val Thr Arg Cys Pro Thr Thr
65 70 75 80
Gly Glu Ala His Asn Glu Lys Arg Ala Asp Ser Ser Tyr Val Cys Lys
85 90 95
Gln Gly Phe Thr Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly
100 105 110
Lys Gly Ser Ile Asp Thr Cys Ala Lys Phe Ser Cys Thr Ser Lys Ala
115 120 125
Ile Gly Arg Met Ile Gln Pro Glu Asn Ile Lys Tyr Glu Val Gly Ile
130 135 140
Phe Val His Gly Thr Thr Thr Ser Glu Asn His Gly Asn Tyr Ser Ala
145 150 155 160
Gln Val Gly Ala Ser Gln Ala Ala Lys Phe Thr Val Thr Pro Asn Ala
165 170 175
Pro Ser Ile Thr Leu Lys Leu Gly Asp Tyr Gly Glu Val Thr Leu Asp
180 185 190
Cys Glu Pro Arg Ser Gly Leu Asn Thr Glu Ala Phe Tyr Val Met Thr
195 200 205
Val Gly Ser Lys Ser Phe Leu Val His Arg Glu Trp Phe His Asp Leu
210 215 220
Ser Leu Pro Trp Thr Ser Pro Ser Ser Thr Ala Trp Arg Asn Arg Glu
225 230 235 240
Leu Leu Met Glu Phe Glu Glu Ala His Ala Thr Lys Gln Tyr Val Val
245 250 255
Ala Leu Gly Ser Gln Glu Gly Gly Leu His Gln Ala Leu Ala Gly Ala
260 265 270
Ile Val Val Glu Tyr Ser Ser Ser Val Lys Leu Thr Ser Gly His Leu
275 280 285
Lys Cys Arg Leu Lys Met Asp Lys Leu Ala Leu Lys Gly Thr Thr Tyr
290 295 300
Gly Met Cys Thr Glu Lys Phe Ser Phe Ala Lys Asn Pro Ala Asp Thr
305 310 315 320
Gly His Gly Thr Val Val Ile Glu Leu Thr Tyr Ser Gly Ser Asp Gly
325 330 335
Pro Cys Lys Ile Pro Ile Val Ser Val Ala Ser Leu Asn Asp Met Thr
340 345 350
Pro Val Gly Arg Leu Val Thr Val Asn Pro Phe Val Ala Thr Ser Ser
355 360 365
Ser Asn Ser Lys Val Leu Val Glu Met Glu Pro Pro Phe Gly Asp Ser
370 375 380
Tyr Ile Val Val Gly Arg Gly Asp Lys Gln Ile Asn His His Trp His
385 390 395 400
Lys Ala Gly Ser Thr Leu Gly Lys Ala Phe Ser Thr Thr Leu Lys Gly
405 410 415
Ala Gln Arg Leu Ala Ala Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser
420 425 430
Ile Gly Gly Val Phe Asn Ser Ile Gly Lys Ala Val His Gln Val Phe
435 440 445
Gly Gly Ala Phe Arg Thr Leu Phe Gly Gly Met Ser Trp Ile Thr Gln
450 455 460
Gly Leu Met Gly Ala Leu Leu Leu Trp Met Gly Val Asn Ala Arg Asp
465 470 475 480
Arg Ser Ile Ala Leu Ala Phe Leu Ala Thr Gly Gly Val Leu Val Phe
485 490 495
Leu Ala Thr Asn Val His Ala His His His His His His
500 505
<210> 4
<211> 281
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Cys Ser Arg Asp Lys Leu Ala Leu Lys Gly Thr Thr Tyr Gly Met Cys
1 5 10 15
Thr Glu Lys Phe Ser Phe Ala Lys Asn Pro Ala Asp Thr Gly His Gly
20 25 30
Thr Val Val Ile Glu Leu Thr Tyr Ser Gly Ser Asp Gly Pro Cys Lys
35 40 45
Ile Pro Ile Val Ser Val Ala Ser Leu Asn Asp Met Thr Pro Val Gly
50 55 60
Arg Leu Val Thr Val Asn Pro Phe Val Ala Thr Ser Ser Ser Asn Ser
65 70 75 80
Lys Val Leu Val Glu Met Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val
85 90 95
Val Gly Arg Gly Asp Lys Gln Ile Asn His His Trp His Lys Ala Gly
100 105 110
Ser Thr Gly Ser Cys Tyr His Ala Ser Val Thr Asp Ile Gly Ser Pro
115 120 125
Thr Thr Gly Glu Ala His Asn Glu Lys Arg Ala Asp Ser Ser Tyr Val
130 135 140
Cys Gly Ser Ser Glu Asn His Gly Asn Tyr Ser Ala Gln Val Gly Ala
145 150 155 160
Ser Gln Gly Ser Gln Glu Gly Gly Leu His Gln Ala Leu Ala Gly Ala
165 170 175
Ile Val Val Glu Tyr Ser Ser Ser Val Lys Leu Thr Ser Gly His Leu
180 185 190
Gly Ser Lys Phe Ser Phe Ala Lys Asn Pro Ala Asp Gly Ser Thr Val
195 200 205
Asn Pro Phe Val Ala Gly Ser Glu Met Glu Pro Pro Phe Gly Asp Ser
210 215 220
Tyr Ile Val Val Gly Arg Gly Asp Lys Gln Ile Asn His His Trp His
225 230 235 240
Lys Ala Gly Ser His Lys Ala Gly Ser Thr Leu Gly Ser Ser Ile Gly
245 250 255
Lys Ala Val His Gln Val Phe Gly Ser Lys Ala Trp Gly Lys Ser Ile
260 265 270
Leu Phe Ala His His His His His His
275 280

Claims (10)

1. a kind of recombinant vector including JEV antigen gene, which is characterized in that including JEV antigen gene and pBAD-18-ClyA Starting vector；The JEV antigen gene is inserted in XbaI enzyme cutting site and the Hind III enzyme of pBAD-18-ClyA starting vector Between enzyme site.
2. recombinant vector according to claim 1, which is characterized in that the nucleotide sequence such as SEQ of the JEV antigen gene Shown in ID NO:1 or SEQ ID NO:2.
3. a kind of recombinant bacterial strain including JEV antigen gene, which is characterized in that the recombinant bacterial strain includes claims 1 or 2 institute The recombinant vector stated.
4. a kind of outer membrane vesicles of the submission JEV antigen produced by recombinant bacterial strain as claimed in claim 3.
5. the preparation method of the outer membrane vesicles of submission JEV antigen as claimed in claim 4, comprising the following steps:
1) recombinant bacterial strain as claimed in claim 4 is inoculated in the OD cultivated in fluid nutrient medium to bacterium solution600For 0.4~ 0.6 obtains preculture bacterium solution；
2) preculture bacterium solution described in step 1) is transferred after carrying out 15~25h of Fiber differentiation in induced fluid culture medium, Supernatant is collected in centrifugation；
3) supernatant described in step 2) is successively filtered except thallus, ultrafiltration concentration and centrifugation, collects solid phase components, obtains Obtain the outer membrane vesicles of submission JEV antigen.
6. preparation method according to claim 5, which is characterized in that fluid nutrient medium described in step 1) is chloramphenicol Resistance LB liquid medium；Induced fluid culture medium described in step 2) is the L-Arabinose solution for adding 20wt% Chlorampenicol resistant LB liquid medium.
7. preparation method according to claim 5, which is characterized in that the molecular cut off of ultrafiltration concentration described in step 3) For 100kDa.
8. preparation method according to claim 6 or 7, which is characterized in that after step 4) the collection solid phase components, also wrap Include the solid phase components of collection are resuspended, density gradient centrifugation and collect obtain submission JEV antigen outer membrane vesicles.
9. preparation method according to claim 8, which is characterized in that the density gradient centrifugation uses OptiPrep- Iodixanol gradient cell separating liquid, the centrifugal force of the density gradient centrifugation are 140000~160000g, the density level bands The time of degree centrifugation is 10~14h.
10. the outer membrane vesicles of submission JEV antigen as claimed in claim 4 are preparing the application in Japanese Encephalitis Vaccine.
CN201910552006.2A 2019-06-25 2019-06-25 A kind of outer membrane vesicles and its application of the recombinant vector including JEV antigen gene, recombinant bacterial strain and submission JEV antigen Pending CN110272910A (en)

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CN116478899A (en) * 2023-04-28 2023-07-25 华南农业大学 Preparation method and application of duck pasteurella multocida outer membrane vesicle vaccine

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Application publication date: 20190924