CN110240637A - The immune-related molecular chaperone protein GroEL2 of Mycobacterium bovis and its encoding gene and application - Google Patents
The immune-related molecular chaperone protein GroEL2 of Mycobacterium bovis and its encoding gene and application Download PDFInfo
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Abstract
The invention discloses a kind of Mycobacterium bovis molecular chaperone protein GroEL2 and its encoding genes, belong to biology and technical field of vaccines.Applicant clones from the genome of mycobacterium bovis bcg and obtains GroEL2 gene, the genetic recombination is expressed into mycobacterium smegmatis, determined its height stick and invasive ability after, it is final to obtain recombinant protein GroEL2 then by the gene in expression in escherichia coli.The albumen can improve the Th1 type and Th2 type cellular immune level of the IgG of body, IgA antibody level and body, have certain anti-bovine mycobacterium infection ability, have application prospect in the research and development of the research of Mycobacterium bovis pathogenic mechanism, vaccine and diagnostic reagent.
Description
Technical field
The invention belongs to biology and technical field of vaccines, and in particular to a kind of immune-related molecular chaperones of Mycobacterium bovis
Protein G roEL2, the albumen have high Adhering capacity and high invasive ability, can improve IgG, IgA antibody level and the machine of body
The Th1 type and Th2 type cellular immune level of body, show that the albumen can cause high-level humoral immunity and cellular immunity, have
Certain anti-bovine mycobacterium infection ability.
Background technique
Mycobacterium bovis (Mycobacterium bovis, M.bovis) is a kind of important pathogenic bacteria of ox, can be caused
The gradual syntexis of ox, output of milk decline, usage ability reduction etc., and people and other animals can be transmitted to by ox, it causes huge
Big social danger and economic loss.Endemic conditions situation is presented in China in perlsucht, earliest related perlsucht epidemic situation
Written report the Xinjiang cattle farm positive rate lungy reported at that time by 1956 can be traced reach 11%.
" quarantine+slaughter " is the effective ways for perlsucht control general in the world, but due to prapes patient and his family
Bottom is unclear, and the economic loss caused by slaughtering is huge, and after the assessment of a large amount of economics, more and more scholars think vaccine
The immune important means that can be used as perlsucht prevention and control.BCG vaccine is official's vaccine currently used for human tuberculosis, but in portion
Country is divided also to have the application in perlsucht prevention and control.In terms of the prevention of perlsucht, since milk cow is cream with animal, card is situated between
The essence of seedling is the Attenuation strain of Mycobacterium bovis again, it is possible to endanger human health by dairy products, therefore in application side
It is very careful that face needs.Subunit vaccine have the characteristics that it is safe and efficient, undoubtedly and the important development direction of perlsucht vaccine,
And the albumen for finding high immunogenicity is the most important thing for developing efficient subunit vaccine.
Summary of the invention
It is an object of that present invention to provide a kind of Mycobacterium bovis immune-related protein GroEL2, which is molecule companion
Companion's albumen has the ability for improving immunity of organism level.
In order to achieve the object of the present invention, Hua Zhong Agriculture University's agromicrobiology state key experiment where applicant
Room ruminant aetology locellus is from amplification in mycobacterium bovis bcg to GroEL2 gene and in mycobacterium smegmatis
It is cloned, analyzes and verify through Western Blot, which confirms that the recombinant bacterium has by cell model
It is very high to stick and invasive ability.Further GroEL2 is connected in prokaryotic expression carrier and the carry out table in Escherichia coli
It up to purifying, analyzes and verifies through Western Blot, it was confirmed that the albumen has good immunogenicity.By the recombination egg of the purifying
White GroEL2 carries out mouse immune after mixing with adjuvant, discovery albumen can significantly improve mouse IgG, IgA antibody level and Th1
Type cell immune response is horizontal, has important anti-bovine mycobacterium infection potential, is research and develop new drug and vaccine important potential
Target spot.
The immune-related molecular chaperone protein of Mycobacterium bovis provided by the present invention, entitled GroEL2 albumen, source
It is following protein 1) or 2) in Mycobacterium bovis (Mycobacterium bovis) BCG vaccine:
1) protein that the amino acid sequence shown in SEQ ID NO:6 forms；
2) by the amino acid sequence of SEQ ID NO:6 by one or several amino acid residues substitution and/or missing and/
Or addition and the protein as derived from 1).
SEQ ID NO:6 is made of 540 amino acid residues.
Above-mentioned sequence can be artificial synthesized, can also first synthesize its encoding gene, then carries out biological expression and obtain.It is above-mentioned 2) in
The encoding gene of GroEL2 can be by by the DNA sequence dna shown in the 5 ' end 1-1623 bit bases shown in SEQ ID NO:5
The codon of middle one or several amino acid residues of missing, and/or the missense mutation of one or several base-pairs of progress obtain.
The encoding gene of above-mentioned albumen also belongs to protection scope of the present invention.
The encoding gene of GroEL2 albumen concretely following 1) -3) any one of described in gene:
1) its nucleotide sequence is sequence shown in SEQ ID NO:5；
2) it can hybridize under strict conditions with the SEQ ID NO:5 DNA sequence dna limited and encode above-mentioned GroEL2 albumen
DNA molecular；
3) there is 90% or more homology with the gene 1) limited, and encodes the DNA molecular of above-mentioned GroEL2 albumen.
It is described 3) in gene, 1) or 2) 95% or more homology is preferably formed with the gene limited.
Sequence shown in SEQ ID NO:5 is made of 1623 base-pairs, is the open reading frame of GroEL2 gene
(ORF), encoding amino acid sequence is GroEL2 albumen shown in SEQ ID NO:6.
Recombinant vector, transgenic cell line and recombinant bacterium containing above-mentioned GroEL2 protein coding gene also belong to the present invention
Protection scope.
The specific technical solution of the present invention is as described below:
Mycobacterium bovis bcg Pasteur strain BCG[American Type Culture Collection (ATCC) used in applicant:
It 35734] is the state university Luiz professor Bermudez present of Oregon, America.Previous work of the invention includes to preservation
The extraction of mycobacterium bovis bcg Pasteur pnca gene group.
The present invention clones GroEL2 gene by template of mycobacterium bovis bcg Pasteur strain BCG full-length genome, and even
It is connected in pMV261 carrier, constructs recombinant plasmid pMV261-GroEL2, by the recombinant plasmid transformed to mycobacterium smegmatis
In, recombinant Mycobacterium smegmatis is obtained, Ms-pMV-G2 is named as.Western Blot result confirms the albumen successful expression
And there is reactionogenicity, stick and the testing result of invasive ability shows that the recombinant Mycobacterium smegmatis compares wild strain and has height
Stick and invasive ability.Further the GroEL2 gene of clone is connected in pET-30a carrier, constructs recombinant plasmid pET-
The recombinant plasmid transformed bacillus coli DH 5 alpha is obtained recombinant escherichia coli strain, is named as Escherichia by 30a-GroEL2
Coli pET-30a-GroEL2 delivers the Chinese Typical Representative culture positioned at Wuhan City, Hubei Province Wuhan University on May 7th, 2019
Object collection preservation, deposit number are CCTCC M 2019334.The bacterial strain under IPTG induction, compile by expression GroEL2 gene
The recombinant protein of code, the Western Blot result verification high immunogenicity and reactionogenicity of the recombinant protein.It should for verifying
The immunological role of albumen in vivo carries out mouse immune after mixing the recombinant protein GroEL2 of purifying with adjuvant, as a result shows
Show, GroEL2 albumen can induce high-caliber IgG, IgA antibody and Th1 type, Th2 type cellular immunity, have and resist ox branch
Bacillus infection prevents the potential of perlsucht.
GroEL2 albumen provided by the present invention can also be used to prepare diagnosing bovine tuberculosis kit, such as Mycobacterium bovis
IgG or IgA antibody enzyme-linked immunologic detecting kit.
The invention has the following advantages that
1, GroEL2 recombinant protein of the invention is encoded by Mycobacterium bovis GroEL2 gene, confirms there is height by inventor
Adhering capacity and invasive ability.
2, GroEL2 recombinant protein of the invention confirms there is immunogenicity by inventor.
3, GroEL2 recombinant protein of the invention is confirmed that body can be caused to generate Gao Shui in Mice Body by inventor
Flat IgA, IgG antibody.
4, GroEL2 recombinant protein of the invention is confirmed that body can be caused to generate Gao Shui in Mice Body by inventor
Flat Th1 type cellular immune level and Th2 type humoral immunity level.
More detailed technical solution refers to " specific embodiment ".
Sequence table explanation:
Sequence table SEQ ID NO:1 is the sequence for expanding the primer GroEL2-F1 of GroEL2 genetic fragment, for constructing
pMV261-GroEL2。
Sequence table SEQ ID NO:2 is the sequence for expanding the primer GroEL2-R1 of GroEL2 genetic fragment, for constructing
pMV261-GroEL2。
Sequence table SEQ ID NO:3 is the sequence for expanding the primer GroEL2-F2 of GroEL2 genetic fragment, for constructing
pET-30a-GroEL2。
Sequence table SEQ ID NO:4 is the sequence for expanding the primer GroEL2-R2 of GroEL2 genetic fragment, for constructing
pET-30a-GroEL2。
Sequence table SEQ ID NO:5 is the sequence of the nucleotide of GroEL2 gene, and the length is 1623bp.
Sequence table SEQ ID NO:6 is the sequence of the amino acid of GroEL2 albumen, is made of 540 amino acid residues.
Detailed description of the invention
Fig. 1: being the plasmid map of pMV216.
Fig. 2: being the map for the recombinant plasmid pMV261-GroEL2 that the present invention constructs.By pMV261 plasmid and GroEL2 base
It is recombinated because overall length connects after digestion with restriction enzyme.
Fig. 3: Western blot verifies the expression of GroEL2 in Ms-pMV-G2.Swimming lane M: high molecule mass protein mark
It is quasi-；GroEL2 expression in swimming lane 1:Ms-pMV-G2；Swimming lane 2: mycobacterium smegmatis empty bacterium control.
Fig. 4: is sticked and invasive ability quantitative detecting analysis figure after Ms-pMV-G2 infection A549 cell." * * * " expression P <
0.001；A: Adhering capacity quantitative detection result；B: invasive ability quantitative detection result.
Fig. 5: being the plasmid map of pET-30a.PET-30a is commercialization plasmid, is purchased from Novagen company.
Fig. 6: being the map for the recombinant plasmid pET-30a-GroEL2 that the present invention constructs.Be by pET-30a plasmid and
GroEL2 full length gene is connected after digestion with restriction enzyme and is recombinated.
Fig. 7: being the Mycobacterium bovis GroEL2 protein SDS-PAGE figure of purifying.Swimming lane M: high molecule mass protein mark
It is quasi-；Swimming lane 1: the BL21 recombinant bacterium containing pET-30a-GroEL2 plasmid not induced；Containing after swimming lane 2:IPTG induction
The BL21 recombinant bacterium of pET-30a-GroEL2 plasmid；Expression in precipitating after swimming lane 3:IPTG induction；Swimming lane 4:IPTG induction
Expression in supernatant afterwards；Swimming lane 5-8: Protein Detection after elution；Swimming lane 9: GroEL2 albumen after purification.
The immunogenicity of Fig. 8: Western blot analysis GroEL2 albumen.Swimming lane M: high molecule mass protein standards；
Swimming lane 1: the BL21 recombinant bacterium containing pET-30a-GroEL2 plasmid.
Fig. 9: being the changes of weight after each group mouse immune.
Figure 10: being the total IgG after each group mouse immune and the variation of total IgA antibody.A: total IgG antibody test result；B: total
IgA antibody testing result；*, P < 0.01；* *, P < 0.001.
Figure 11: being the specific IgG after each group mouse immune and Specific IgA antibody variation.A: specific IgG antibodies inspection
Survey result；B: Specific IgA antibody testing result；* *, P < 0.001.
Figure 12: being the IFN-γ and IL-6 level after each group mouse immune.The detection of A:IFN- γ expression；B:IL-6 table
It is detected up to level；*, P < 0.01；* *, P < 0.001.
Specific embodiment
Embodiment 1: the building and verifying of recombinant Mycobacterium smegmatis Ms-pMV-G2
The building of 1.1 recombinant Mycobacterium smegmatis Ms-pMV-G2
Using mycobacterium bovis bcg Pasteur strain BCG (ATCC:35734) full-length genome as template, following design is utilized
Primer (sequence table SEQ ID NO:1-2) clone GroEL2 gene, obtain the complete sequence of GroEL2 gene, sequence length is
1623bp。
The primer sequence for expanding GroEL2 gene is as follows:
1, forward primer GroEL2-F1:GCCAAGACAATTGCGGATCCATGGCCAAGACAATTGCGTA.Underscore institute
It is shown as the restriction enzyme site of BamH I.Sequence shown in corresponding sequence table SEQ ID NO:1.
2, reverse primer GroEL2-R1:TGGTGGTGGTGGTGGATATCGAAATCCATGCCACCCATGT.Underscore institute
It is shown as the restriction enzyme site of EcoRV.Sequence shown in corresponding sequence table SEQ ID NO:2.
PCR reaction system is as follows:
Template DNA 2 μ L, 2 × plantaTM25 μ L of Master Mix, each 2 μ L of primer, 19 μ L of ultrapure water.
PCR amplification condition is as follows:
98 DEG C of initial denaturation 5min；98 DEG C of denaturation 30sec, 58 DEG C of annealing 30sec, 72 DEG C of extension 2min carry out 30 circulations
Reaction；72 DEG C thoroughly extend 10min.
Recycle pcr amplification product, with BamH I and EcoRV digestion, while by pMV261 plasmid (Fig. 1) with identical enzyme into
Row double digestion.By the GroEL2 gene and pMV261 plasmid TreliefTM SoSoo Cloning Kit after digestion
(TSINGKE company) is attached, and obtains recombinant plasmid pMV261-GroEL2 (Fig. 2).By recombinant plasmid pMV261-
GroEL2 converts mycobacterium smegmatis mc2After 155, it is placed in 37 DEG C of shaking tables and cultivates at 110r/min to logarithmic phase (OD600=0.8
~1.0).Expression is measured using the correct rear Western blot method that carries out of bacterium solution PCR identification.
1.2Western blot identifies the expression of GroEL2 gene in recombinant Mycobacterium smegmatis Ms-pMV-G2
The recombinant Mycobacterium smegmatis Ms-pMV-G2 of 1.1 preparations is collected into bacterium solution after 42 DEG C of heat-inducible 3-4h, 4 DEG C,
L0000rpm/min is used PBS washing thalline 3 times after being centrifuged 5min, and sonicated cells carry out SDS-PAGE electricity after thallus is resuspended
Swimming, glue is transferred on pvdf membrane, after film washing, film is put into in the diluted 5%BSA of TBST, room temperature closes 3h；Sufficiently wash
Pvdf membrane is put into 2000 times of diluted HIS antibody after washing, 4 DEG C of overnight incubations；Pvdf membrane is sufficiently put into 10000 after washing
Again in the sheep anti-mouse igg of diluted horseradish peroxidase-labeled, room temperature 1h；TBST washing three times afterwards sends out the ECL mixed
Light liquid drops evenly on film, detects signal using Kodak Image Station chemiluminescence detector.As the result is shown: Ms-
PMV-G2 can react with His antibody, and specific reaction band can be detected, and molecular weight is about 62kDa (Fig. 3, swimming lane 1),
Meanwhile mycobacterium smegmatis control cannot react with HIS antibody, be not detected signal (Fig. 3, swimming lane 2), therefore Ms-
GroEL2 gene successful expression in pMV-G2.
Embodiment 2: recombinant Mycobacterium smegmatis Ms-pMV-G2 sticks, the detection of invasive ability
The detection of 2.1 recombinant Mycobacterium smegmatis Ms-pMV-G2 Adhering capacities
By A549 cell (the state university Luiz professor Bermudez present of Oregon, America) in 12 porocyte culture plates
Culture, single layer to be grown up to simultaneously reach 2 × 105Behind a/hole, recombinant Mycobacterium smegmatis Ms-pMV- is added than 10:1 according to infection
G2 sets up blank mycobacterium smegmatis as control, is sufficiently washed after 4 DEG C of effect 30min with PBS, Triton X- is then added
100 (Bio-Rad company) lytic cells collect bacterium coating 7H11 solid (purchased from BD company) plate intracellular, in 37 DEG C, 5%CO2
It is cultivated 5-7 days in incubator.Find that recombinant bacterium Ms-pMV-G2 Adhering capacity is significantly higher than wild strain Ms, P by count of bacteria
< 0.001 (Fig. 4 A) is 2.51 times of wild strain.Show that GroEL2 has the function of improving bacterial adhesion ability.
The detection of 2.2 recombinant Mycobacterium smegmatis Ms-pMV-G2 invasive abilities
By A549 cell according to 2 × 105A/12 porocyte culture plates of Kong Puzhi, using infection than being that 10:1 will recombinate shame
Dirty mycobacteria Ms-pMV-G2 is seeded in A549 cell, and setting wild strain mycobacterium smegmatis Ms is control.By bacterium with
A549 cell is in 37 DEG C, 5%CO2Triton X-100 (Bio-Rad company) lytic cell, benefit is added after acting on 1h in incubator
Clump count is quantitative determined with colony counting method.The results show that recombinant Mycobacterium smegmatis Ms-pMV-G2 invasive ability is significantly higher than
Wild strain, P < 0.001 (Fig. 4 B) are 4.21 times of wild strain.Show that GroEL2 has the function of improving bacteria attack ability.
Clone, expression and purifying of the embodiment 3:GroEL2 gene in Escherichia coli
The clone of 3.1 recombination pET-30a-GroEL2 plasmids and expression
Using mycobacterium bovis bcg Pasteur strain (ATCC:35734) full-length genome as template, drawn using what is designed as follows
Object (sequence table SEQ ID NO:3-4) clones GroEL2 gene, obtains the complete sequence of GroEL2 gene, sequence length is
1623bp。
The primer sequence for expanding GroEL2 gene is as follows:
1, forward primer GroEL2-F2:CGGAATTCATGGCCAAGACAATTGCGTA.Underscore show EcoR's I
Restriction enzyme site.Sequence shown in corresponding sequence table SEQ ID NO:3.
2, reverse primer GroEL2-R2:CCAAGCTTGAAATCCATGCCACCCATGT.Underscore show Hind's III
Restriction enzyme site.Sequence shown in corresponding sequence table SEQ ID NO:4.
PCR reaction system is as follows:
Template DNA 2 μ L, 2 × plantaTM25 μ L of Master Mix, each 2 μ L of primer, 19 μ L of ultrapure water.
PCR amplification condition is as follows:
94 DEG C of initial denaturation 5min；It is anti-to carry out 30 circulations by 94 DEG C of denaturation 30sec, 62 DEG C of annealing 30sec, 72 DEG C of extension 45s
It answers；72 DEG C thoroughly extend 10min.
Pcr amplification product is recycled, with III digestion of EcoR I and Hind, while by pET30a (+) plasmid (Fig. 5) with identical
Enzyme carries out double digestion.By after digestion GroEL2 gene and pET-30a (+) plasmid be attached with T4DNA ligase, obtain weight
Group plasmid pET-30a-GroEL2 (Fig. 6).After recombinant plasmid pET-30a-GroEL2 is converted 5 α of Escherichia coli DH, it is placed in
37 DEG C of shaking tables are cultivated 12 hours at 180r/min, extract plasmid (nucleotide sequence corresponding sequence table SEQ after being sequenced correctly
Sequence shown in ID NO:5) conversion e. coli bl21 bacterium, which is cultivated in LB liquid medium to OD
Take 1mL bacterium solution as control before induction when=0.6, while it is extremely final concentration of that isopropylthiogalactoside (IPTG) is added
0.8mM, 37 DEG C of shaking table inducing expression 4h.Sample 12000r/min is then centrifuged 1min, supernatant is discarded, after PBS is resuspended
12000r/min is centrifuged 1min, abandons supernatant, and 600 μ L PBS are added and are resuspended, and carries out bacterial cell disruption by hydraulic crushing instrument, is crushed pressure
Power 1000kar is 3 times broken, until thallus is clarified；The bodies such as then 12000r/min is centrifuged 10min, and separation supernatant precipitates, and precipitating is used
600 μ L PBS of product are resuspended, separately sampled to carry out that suitable sample-loading buffer, boiling water boiling 10min, 4000r/min centrifugation is added
2min.Supernatant and precipitating is taken to carry out SDS-PAGE, the existence form and expression of object observing albumen respectively.The results show that
GroEL2 success expresses (Fig. 7, swimming lane 4) in supernatant.Sequence shown in amino acid sequence corresponding sequence table SEQ ID NO:6.
The Escherichia coli recombinant strain is named as Escherichia coli pET-30a-GroEL2 by applicant；In 2019
On May 7, in delivers China typical culture collection center (CCTCC) preservation positioned at Wuhan City, Hubei Province Wuhan University, preservation
Number is CCTCC M 2019334.
The purifying of 3.2GroEL2 albumen
After the e. coli bl21 of recombination according to the above method inducing expression cracking, after PAGE gel electrophoresis,
Determine GroEL2 protein expression in supernatant.It is purified using nickel column:
(1) ethyl alcohol for closing pillar is flowed out through pillar；
(2) washing of 12mL sterile deionized water is added into affinity column；
(3) the Binding buffer that 50mL is added balances pillar；
(4) albumen supernatant by the filtering of 0.45 μm of aperture filter is added, collects former drops and filters out liquid, number 1.
(5) the Binding buffer that 30mL is added balances pillar, collects former drop filter liquors, number 2.
(6) the Washing buffer that 50mL is added washes away foreign protein, collects former drops, number 3.
(7) the Elution buffer that 6mL is added elutes destination protein, collects former drops, number 4.
(8) it will number in the pipe for 1-4 and 50 μ L sample-loading buffers are respectively added boil 10min.
(9) SDS-PAGE polyacrylamide gel is configured, by (the 20 every hole μ L/), electrophoresis in the sample adding hole handled well
(it is 80 volts that concentration gel electrophoresis condition, which is DC voltage, and separation gel deposition condition is that DC voltage is 120 volts), electrophoresis is completed
Afterwards, gel coomassie brilliant blue staining is removed to stay overnight.Then it decolourizes, determines the destination protein (Fig. 7, swimming lane 9) purified.
The detection of 3.3GroEL2 protein immunogenic
The identification of GroEL2 protein immunogenic, key step are as follows: by the weight of purifying are carried out using Western blot method
Histone GroEL2 (2.0 μ g/ swimming lane) carries out SDS-PAGE electrophoresis, and albumen on glue is transferred on pvdf membrane, will after film washing
Film is put into in the diluted 5%BSA of TBST, and room temperature closes 3h；Pvdf membrane is put into 5000 times of diluted ox sun sufficiently after washing
Property serum antibody in, 4 DEG C overnight incubation；Pvdf membrane is sufficiently put into 5000 times of diluted horseradish peroxidase-labeleds after washing
Sheep anti-mouse igg in, room temperature 1h；TBST washing three times afterwards drops evenly the ECL luminescent solution mixed on film, applies
Kodak Image Station chemiluminescence detector detects signal.As the result is shown: GroEL2 albumen can be sent out with ox positive serum
Specific reaction band can be detected in raw reaction, and molecular weight is about 62kDa (Fig. 8), therefore GroEL2 albumen has good exempt from
Epidemic focus.
The detection that embodiment 4:GroEL2 albumen causes mouse immune to be reacted
The immune and Avoirdupois monitoring of 4.1 mouse
GroEL2 albumen is carried out 1:1 with adjuvant (1313 VG N of MONTANIDETM IMS) to mix, is dripped after emulsification completely
Female Balb/C mouse 3 of 6-8 week old 16-18g, every 25 μ g are immunized in nose.Independent immunologic adjuvant group is set up simultaneously and is immunized
PBS group is control.Weighing in 0th day is primary, then weighs weekly 1 time and makes a record, changes of weight curve table is made.As a result
It has been shown that, with the extension of time, GroEL2 protein immunization group, adjuvant immunity group, the weight of PBS group mouse are slowly increased from 18g
To 22g, for changes of weight without significant difference (P > 0.05) (Fig. 9), mouse mental status is good between each group.
The detection of 4.2 immunized mice total IgGs, IgA antibody
With the detection of the mouse IgG ELISA kit and IgA ELISA kit of Xin Bosheng company it is immune after the 0th, 7,14,
21,28 days mouse total IgGs, IgA antibody.It is detected according to specification, step is summarized as follows:
Serum after dilution is added in the reaction plate after rising again, 37 DEG C of incubation 90min.Biotin sufficiently is added after washing
Change antibody working solution.37 DEG C of incubation 60min.Enzyme conjugates working solution is sufficiently added after washing, 37 DEG C are protected from light incubation 30min.It fills
Addition chromogenic substrate (TMB) after washing is divided to be protected from light 37 DEG C of incubation 15min.OD450 value is measured at once after terminate liquid is added.
The results show that the total IgG antibody level of GroEL2+ adjuvant immunity group mouse is with regard to significant after immune 14th day
Higher than simple immunologic adjuvant and PBS group, P < 0.01.21st and 28 day difference and its significant, P < 0.001 (Figure 10 A).Total IgA is anti-
Body then just continued significantly to increase from the 7th day, P < 0.001.Rapid increase trend (figure is presented in total IgG and total IgA antibody level
10B).Show that GroEL2 albumen can just cause mouse to generate high-caliber total IgG and total IgA antibody in Initial stage of immunization.
The detection of 4.3 immunized mice specific IgGs, IgA antibody
Docking blood sampling was carried out to mouse in 0th, 7,14,21,28 day after immune respectively, carries out IgA, IgG after collecting serum
Antibody test and cytokines measurement.Specific step is as follows:
GroEL2 albumen (final concentration of 2 μ g/mL) is used as antigen coat ELISA Plate, 4 DEG C of sufficiently washings rear overnight are added
37 DEG C of closing 1h after confining liquid；The diluted serum to be checked of 100 μ L100 times, 37 DEG C of incubation 2h sufficiently are added after washing；Sufficiently washing
The sheep anti-mouse igg or IgA of horseradish peroxidase-labeled are added afterwards, 37 DEG C of incubation 2h, sufficiently after washing plus substrate TMB develops the color
10min；Terminate liquid 2M sulfuric acid is added, is read with enzyme-linked immunosorbent assay instrument, measurement wavelength is the OD value at 450nm.
The results show that GroEL2+ adjuvant immunity group mouse specific IgG (Figure 11 A) and specificity IgA (Figure 11 B) antibody
Level is presented duration and significantly increases.After immune 7th day just with simple immunologic adjuvant group and PBS group difference extremely significantly (P
<0.001).Show that GroEL2 albumen can just cause mouse to generate high-caliber specific IgG and specificity in Initial stage of immunization
IgA antibody.
The detection of 4.4 immunized mice cell factors
With the detection of the mouse IFN-γ ELISA kit and IL-6ELISA kit of Xin Bosheng company it is immune after the 0th, 7,
14,21,28 days mouse IFN-γ and IL-6 are horizontal.It is detected according to specification, step is summarized as follows:
Serum after dilution is added in the reaction plate after rising again, 37 DEG C of incubation 90min.Biotin sufficiently is added after washing
Change antibody working solution.37 DEG C of incubation 60min.Enzyme conjugates working solution is sufficiently added after washing, 37 DEG C are protected from light incubation 30min.It fills
Addition chromogenic substrate (TMB) after washing is divided to be protected from light 37 DEG C of incubation 15min.OD450 value is measured at once after terminate liquid is added.
As the result is shown: respectively after immune 14th day and the 7th day, total IFN-γ of GroEL2+ adjuvant immunity group mouse
And IL-6 level is just significantly higher than control group (P < 0.001) (Figure 12).Show that GroEL2 albumen can just cause in Initial stage of immunization
Mouse generates high-caliber IFN-γ and IL-6, i.e. Th1 type and Th2 type cellular immune level.
To sum up, GroEL2 albumen has the function of that raising mycobacteria is sticked and invasive ability, the indication albumen can mention
The virulence of high bacterium；Its prokaryotic expression protein can cause mouse and generate high level IgA and IgG antibody.Although current antibody pair
There is also certain arguements for resistant function lungy, but IFN-γ and significantly rising for IL-6 expression demonstrate GroEL2
Albumen can either cause high-caliber Th1 type cellular immune level, while can also cause high-level Th2 type humoral immunity water
It is flat, it is determined that the albumen has certain anti-mycobacterial infections potential, can research and develop as vaccine, the important target of diagnostic preparation
Mark.
Sequence table
<110>Hua Zhong Agriculture University
<120>the immune-related molecular chaperone protein GroEL2 of Mycobacterium bovis and its encoding gene and application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 40
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 1
gccaagacaa ttgcggatcc atggccaaga caattgcgta 40
<210> 2
<211> 40
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 2
tggtggtggt ggtggatatc gaaatccatg ccacccatgt 40
<210> 3
<211> 28
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 3
cggaattcat ggccaagaca attgcgta 28
<210> 4
<211> 28
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 4
ccaagcttga aatccatgcc acccatgt 28
<210> 5
<211> 1623
<212> DNA
<213>Mycobacterium bovis (Mycobacterium bovis)
<400> 5
atggccaaga caattgcgta cgacgaagag gcccgtcgcg gcctcgagcg gggcttgaac 60
gccctcgccg atgcggtaaa ggtgacattg ggccccaagg gccgcaacgt cgtcctggaa 120
aagaagtggg gtgcccccac gatcaccaac gatggtgtgt ccatcgccaa ggagatcgag 180
ctggaggatc cgtacgagaa gatcggcgcc gagctggtca aagaggtagc caagaagacc 240
gatgacgtcg ccggtgacgg caccacgacg gccaccgtgc tggcccaggc gttggttcgc 300
gagggcctgc gcaacgtcgc ggccggcgcc aacccgctcg gtctcaaacg cggcatcgaa 360
aaggccgtgg agaaggtcac cgagaccctg ctcaagggcg ccaaggaggt cgagaccaag 420
gagcagattg cggccaccgc agcgatttcg gcgggtgacc agtccatcgg tgacctgatc 480
gccgaggcga tggacaaggt gggcaacgag ggcgtcatca ccgtcgagga gtccaacacc 540
tttgggctgc agctcgagct caccgagggt atgcggttcg acaagggcta catctcgggg 600
tacttcgtga ccgacccgga gcgtcaggag gcggtcctgg aggaccccta catcctgctg 660
gtcagctcca aggtgtccac tgtcaaggat ctgctgccgc tgctcgagaa ggtcatcgga 720
gccggtaagc cgctgctgat catcgccgag gacgtcgagg gcgaggcgct gtccaccctg 780
gtcgtcaaca agatccgcgg caccttcaag tcggtggcgg tcaaggctcc cggcttcggc 840
gaccgccgca aggcgatgct gcaggatatg gccattctca ccggtggtca ggtgatcagc 900
gaagaggtcg gcctgacgct ggagaacgcc gacctgtcgc tgctaggcaa ggcccgcaag 960
gtcgtggtca ccaaggacga gaccaccatc gtcgagggcg ccggtgacac cgacgccatc 1020
gccggacgag tggcccagat ccgccaggag atcgagaaca gcgactccga ctacgaccgt 1080
gagaagctgc aggagcggct ggccaagctg gccggtggtg tcgcggtgat caaggccggt 1140
gccgccaccg aggtcgaact caaggagcgc aagcaccgca tcgaggatgc ggttcgcaat 1200
gccaaggccg ccgtcgagga gggcatcgtc gccggtgggg gtgtgacgct gttgcaagcg 1260
gccccgaccc tggacgagct gaagctcgaa ggcgacgagg cgaccggcgc caacatcgtg 1320
aaggtggcgc tggaggcccc gctgaagcag atcgccttca actccgggct ggagccgggc 1380
gtggtggccg agaaggtgcg caacctgccg gctggccacg gactgaacgc tcagaccggt 1440
gtctacgagg atctgctcgc tgccggcgtt gctgacccgg tcaaggtgac ccgttcggcg 1500
ctgcagaatg cggcgtccat cgcggggctg ttcctgacca ccgaggccgt cgttgccgac 1560
aagccggaaa aggagaaggc ttccgttccc ggtggcggcg acatgggtgg catggatttc 1620
tga 1623
<210> 6
<211> 540
<212> RNA
<213>Mycobacterium bovis (Mycobacterium bovis)
<400> 6
Claims (9)
1. the immune-related molecular chaperone protein GroEL2 of Mycobacterium bovis has the ammonia as shown in sequence table SEQ ID NO:6
Base acid sequence.
2. encoding the gene of the immune-related molecular chaperone protein GroEL2 of Mycobacterium bovis described in claim 1.
3. gene according to claim 2, it is characterised in that: the gene has as shown in sequence table SEQ ID NO:5
Nucleotide sequence.
4. the recombinant expression carrier containing gene described in Claims 2 or 3.
5. the recombinant bacterium containing gene described in Claims 2 or 3.
6. recombinant expression carrier according to claim 4, it is characterised in that: the recombinant expression carrier is in pET30a
The recombinant expression carrier that gene described in Claims 2 or 3 obtains is inserted between multiple cloning sites.
7. recombinant bacterium according to claim 5, it is characterised in that: the recombinant bacterium is to carry described in Claims 2 or 3
The Escherichia coli of gene.
8. purposes of the molecular chaperone protein GroEL2 described in claim 1 in preparation perlsucht vaccine.
9. molecular chaperone protein GroEL2 described in claim 1 is preparing the purposes in diagnosing bovine tuberculosis kit.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101969976A (en) * | 2008-01-11 | 2011-02-09 | 美国政府健康与人类服务部秘书处 | Polypeptide vaccine and vaccination strategy against mycobacterium |
| CN102174555A (en) * | 2010-12-29 | 2011-09-07 | 中国人民解放军第四军医大学 | Recombinant expression vector of Hsp65-hIL-2 fusion expression and recombinant strain |
| CN102666575A (en) * | 2009-10-16 | 2012-09-12 | 艾西斯创新有限公司 | Mycobacterial vaccines |
| CN107202882A (en) * | 2016-03-17 | 2017-09-26 | 广东体必康生物科技有限公司 | Purposes of the Rv0440 albumen in diagnosis latency/active tuberculosis |
-
2019
- 2019-06-04 CN CN201910480093.5A patent/CN110240637A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101969976A (en) * | 2008-01-11 | 2011-02-09 | 美国政府健康与人类服务部秘书处 | Polypeptide vaccine and vaccination strategy against mycobacterium |
| CN102666575A (en) * | 2009-10-16 | 2012-09-12 | 艾西斯创新有限公司 | Mycobacterial vaccines |
| CN102174555A (en) * | 2010-12-29 | 2011-09-07 | 中国人民解放军第四军医大学 | Recombinant expression vector of Hsp65-hIL-2 fusion expression and recombinant strain |
| CN107202882A (en) * | 2016-03-17 | 2017-09-26 | 广东体必康生物科技有限公司 | Purposes of the Rv0440 albumen in diagnosis latency/active tuberculosis |
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