CN110240636A - Mycobacterium bovis molecular chaperone protein GroEL1 and its encoding gene and application - Google Patents
Mycobacterium bovis molecular chaperone protein GroEL1 and its encoding gene and application Download PDFInfo
- Publication number
- CN110240636A CN110240636A CN201910480091.6A CN201910480091A CN110240636A CN 110240636 A CN110240636 A CN 110240636A CN 201910480091 A CN201910480091 A CN 201910480091A CN 110240636 A CN110240636 A CN 110240636A
- Authority
- CN
- China
- Prior art keywords
- groel1
- gene
- mycobacterium bovis
- albumen
- mycobacterium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
- A61P31/06—Antibacterial agents for tuberculosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Communicable Diseases (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- General Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pulmonology (AREA)
- Food Science & Technology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Gastroenterology & Hepatology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Oncology (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
Abstract
The invention discloses a kind of Mycobacterium bovis molecular chaperone protein GroEL1 and its encoding genes, belong to biology and technical field of vaccines.Applicant clones from mycobacterium bovis bcg genome and obtains GroEL1 gene, the genetic recombination is expressed into mycobacterium smegmatis, determined its height stick and invasive ability after, it is final to obtain recombinant protein GroEL1 then by the gene in expression in escherichia coli.The albumen can improve the 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 roEL1, the albumen have high Adhering capacity and high invasive ability, can be improved the IgG of body, IgA antibody it is horizontal and
Th2 type cellular immune level shows that the albumen can cause high-level humoral immunity and cellular immunity, has certain anti-ox point
Branch bacillus infection ability, there is the potential as perlsucht vaccine.
Background technique
Perlsucht is a kind of chronic debilitating zoonosis, and bovine tuberculosis is listed in the animal that China preferentially prevents and treats
One of disease.Mycobacterium bovis (Mycobacterium bovis, M.bovis) is the main pathogen for causing perlsucht, together
When people and other animals can be also transmitted to from ox, bring huge economic loss and Industry losses to society.
Vaccine is one of mostly important and effective means of Field of Animal Epidemic Disease Control.But it since milk cow is that cream uses animal, examines
Consider public health factor, various countries are especially careful to the vaccine immunity of perlsucht especially bovine tuberculosis.With part state
The sternness increasingly of family's perlsucht prevention and control situation, more and more countries start selection and carry out vaccine immunity to ox to prevent tuberculosis
Disease.BCG vaccine is the preferred vaccine when perlsucht is immunized, but there is also biggish gaps for its immune effect and expection.To have
Prevention and control perlsucht is imitated, vaccine research and development increasingly receive significant attention.Effective vaccine targets are to develop the basis of efficient vaccine,
The albumen for finding high immunogenicity is the most important thing for developing efficient vaccine.
Summary of the invention
It is an object of that present invention to provide a kind of Mycobacterium bovis immune-related protein GroEL1, 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 GroEL1 gene and in mycobacterium smegmatis
It is cloned, analyzes and verify through Western Blot, the albumen successful expression, cell model demonstrates the recombinant bacterium with non-
It is often high to stick and invasive ability.Further GroEL1 is connected in prokaryotic expression carrier and is expressed in Escherichia coli
Purifying is analyzed through Western Blot and is verified, it was confirmed that the albumen has good immunogenicity.By the recombinant protein of the purifying
GroEL1 carries out mouse immune after mixing with adjuvant, discovery albumen can significantly improve mouse IgG, IgA antibody level and Th2 type
Cell immune response is horizontal, has important anti-bovine mycobacterium infection potential, is the important potential target for researching and developing new drug and vaccine
Point.
The immune-related molecular chaperone protein of Mycobacterium bovis provided by the present invention, entitled GroEL1 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 539 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 GroEL1 can be by by the DNA sequence dna shown in the 5 ' end 1-1620 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 GroEL1 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 GroEL1 albumen
DNA molecular；
3) there is 90% or more homology with the gene 1) limited, and encodes the DNA molecular of above-mentioned GroEL1 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 1620 base-pairs, is the open reading frame of GroEL1 gene
(ORF), encoding amino acid sequence is GroEL1 albumen shown in SEQ ID NO:6.
Recombinant vector, transgenic cell line and recombinant bacterium containing above-mentioned GroEL1 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 GroEL1 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-GroEL1, by the recombinant plasmid transformed to mycobacterium smegmatis
In, recombinant Mycobacterium smegmatis is obtained, Ms-pMV-G1 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 GroEL1 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-GroEL1
Coli pET-30a-GroEL1 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 2019333.The bacterial strain under IPTG induction, compile by expression GroEL1 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 GroEL1 of purifying with adjuvant, as a result shows
Show, GroEL1 albumen can induce high-caliber IgG, IgA antibody and Th2 type cellular immunity, have and resist Mycobacterium bovis, in advance
The potential of anti-perlsucht.
GroEL1 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, GroEL1 recombinant protein of the invention is encoded by Mycobacterium bovis GroEL1 gene, confirms there is height by inventor
Adhering capacity and invasive ability.
2, GroEL1 recombinant protein of the invention confirms there is immunogenicity by inventor.
3, GroEL1 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, GroEL1 recombinant protein of the invention is confirmed that body can be caused to generate Gao Shui in Mice Body by inventor
Flat Th2 type cellular immunity.
More detailed technical solution refers to " specific embodiment ".
Sequence table explanation:
Sequence table SEQ ID NO:1 is the sequence for expanding the primer GroEL1-F1 of GroEL1 genetic fragment, for constructing
pMV261-GroEL1。
Sequence table SEQ ID NO:2 is the sequence for expanding the primer GroEL1-R1 of GroEL1 genetic fragment, for constructing
pMV261-GroEL1。
Sequence table SEQ ID NO:3 is the sequence for expanding the primer GroEL1-F2 of GroEL1 genetic fragment, for constructing
pET-30a-GroEL1。
Sequence table SEQ ID NO:4 is the sequence for expanding the primer GroEL1-R2 of GroEL1 genetic fragment, for constructing
pET-30a-GroEL1。
Sequence table SEQ ID NO:5 is the sequence of the nucleotide of GroEL1 gene, and the length is 1620bp.
Sequence table SEQ ID NO:6 is the amino acid sequence of GroEL1 albumen, is made of altogether 539 amino acid.
Detailed description of the invention
Fig. 1: being the plasmid map of pMV216.
Fig. 2: being the map for the recombinant plasmid pMV261-GroEL1 that the present invention constructs.By pMV261 plasmid and GroEL1 base
It is recombinated because overall length connects after digestion with restriction enzyme.
Fig. 3: Western blot verifies the expression of GroEL1 in Ms-pMV-G1.Swimming lane M: high molecule mass protein mark
It is quasi-；The detection of GroEL1 expression in swimming lane 1:Ms-pMV-G1；Swimming lane 2: mycobacterium smegmatis empty bacterium control.
Fig. 4: is sticked and invasive ability quantitative detecting analysis figure after Ms-pMV-G1 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-GroEL1 that the present invention constructs.Be by pET-30a plasmid and
GroEL1 full length gene is connected after digestion with restriction enzyme and is recombinated.
Fig. 7: being the Mycobacterium bovis GroEL1 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-GroEL1 plasmid not induced；Containing after swimming lane 2:IPTG induction
The BL21 recombinant bacterium of pET-30a-GroEL1 plasmid；Expression in precipitating after swimming lane 3:IPTG induction；Swimming lane 4:IPTG induction
Expression in supernatant afterwards；Swimming lane 5-6: Protein Detection after elution；Swimming lane 7: GroEL1 albumen after purification.
The immunogenicity of Fig. 8: Western blot analysis rGroEL1 albumen.Swimming lane M: high molecule mass protein standards；
Swimming lane 1: the BL21 recombinant bacterium containing pET-30a-GroEL1 plasmid.
Fig. 9: being the changes of weight after each group mouse immune.Figure 10: being that total IgG after each group mouse immune and total IgA are anti-
Body variation.A: total IgG antibody test result；B: total IgA antibody testing result；*, P < 0.05；*, 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 that IL-6 after each group mouse immune and IFN-γ are horizontal.The detection of A:IL-6 expression；B:IFN- γ table
It is detected up to level；*, P < 0.05；* *, P < 0.001.
Specific embodiment
Embodiment 1: the building and verifying of recombinant Mycobacterium smegmatis Ms-pMV-G1
The building of 1.1 recombinant Mycobacterium smegmatis Ms-pMV-G1
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 GroEL1 gene, obtain the complete sequence of GroEL1 gene, sequence length is
1620bp。
The primer sequence for expanding GroEL1 gene is as follows:
1, forward primer GroEL1-F1:GCCAAGACAATTGCGGATCCATGAGCAAGCTGATCGAATACGACG.Under
Scribing line show the restriction enzyme site of BamH I.Sequence shown in corresponding sequence table SEQ ID NO:1.
2, reverse primer GroEL1-R1:TGGTGGTGGTGGTGGATATCGTGCGCGTGCCCGTGGT.Shown in underscore
For the restriction enzyme site of EcoRV.Sequence shown in corresponding sequence table SEQ ID NO:2.
PCR reaction system is as follows:
Template DNA 2L, 2 × plantaTM Master Mix 25L, primer each 2L, ultrapure water 19L.
PCR amplification condition is as follows:
98 DEG C of initial denaturations 5min, 98 DEG C of denaturation 30sec, 62 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 GroEL1 gene and pMV261 plasmid TreliefTM SoSoo Cloning Kit after digestion
(TSINGKE company) is attached, and obtains recombinant plasmid pMV261-GroEL1 (Fig. 2).By recombinant plasmid pMV261-
GroEL1 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.
1.2 Western blot identify the expression of GroEL1 gene in recombinant Mycobacterium smegmatis Ms-pMV-G1
The recombinant Mycobacterium smegmatis Ms-pMV-G1 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-G1 can react with His antibody, and specific reaction band can be detected, and molecular weight is about 60kDa (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-
GroEL1 gene successful expression in pMV-G1.
Embodiment 2: recombinant Mycobacterium smegmatis Ms-pMV-G1 sticks, the detection of invasive ability
The detection of 2.1 recombinant Mycobacterium smegmatis Ms-pMV-G1 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
G1 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-G1 Adhering capacity is significantly higher than wild strain Ms, P by count of bacteria
< 0.001 (Fig. 4 A) is 2.28 times of wild strain.Show that GroEL1 has the function of improving bacterial adhesion power.
The detection of 2.2 recombinant Mycobacterium smegmatis Ms-pMV-G1 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-G1 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-G1 invasive ability is significantly higher than
Wild strain, P < 0.001 (Fig. 4 B) are 3.19 times of wild strain.Show that GroEL1 has the function of improving bacteria attack power.
Clone, expression and purifying of the embodiment 3:GroEL1 gene in Escherichia coli
The clone of 3.1 recombination pET-30a-GroEL1 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 GroEL1 gene, obtains the complete sequence of GroEL1 gene, sequence length is
1620bp。
The primer sequence for expanding GroEL1 gene is as follows:
1, forward primer GroEL1-F2:CGGGATCCATGAGCAAGCTGATCGAATACGA.Underscore show BamHI
Restriction enzyme site.Sequence shown in corresponding sequence table SEQ ID NO:3.
2, reverse primer GroEL1-R2:CGGAATTCGTGCGCGTGCCCGTGGT.Underscore show the digestion of EcoRI
Site.Sequence shown in corresponding sequence table SEQ ID NO:4.
PCR reaction system is as follows: template DNA 2L, 2 × plantaTM Master Mix 25L, each 2L of primer, ultrapure water
19L。
PCR amplification condition is as follows: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30sec, 62 DEG C of annealing 30sec, 72 DEG C extend
45s carries out 30 circular responses；72 DEG C thoroughly extend 10min.
Pcr amplification product is recycled, with EcoR I and BamHI digestion, while by pET-30a (+) plasmid (Fig. 5) with identical
Enzyme carries out double digestion.By after digestion GroEL1 gene and pET-30a (+) plasmid be attached with T4DNA ligase, obtain weight
Group plasmid pET-30a-GroEL1 (Fig. 6).After recombinant plasmid pET-30a-GroEL1 is converted 5 α of Escherichia coli DH, it is placed in
37 DEG C of shaking tables cultivate 12h at 180r/min, extract plasmid (nucleotide sequence corresponding sequence table SEQ ID after being sequenced correctly
Sequence shown in NO:5) conversion e. coli bl21 bacterium, which is cultivated in LB liquid medium to OD=
It takes 1mL bacterium solution as control before induction when 0.6, while isopropylthiogalactoside (IPTG) extremely final concentration of 0.8mM is added,
37 DEG C of shaking table inducing expression 4h.Sample 12000r/min is then centrifuged 1min, discards supernatant, 12000r/min after PBS is resuspended
It is centrifuged 1min, abandons supernatant, 600 μ L PBS are added and are resuspended, carries out bacterial cell disruption, cracking pressure 1000kar by hydraulic crushing instrument
It is 3 times broken, until thallus is clarified；Then 12000r/min is centrifuged 10min, separation supernatant precipitating, the isometric 600 μ L of precipitating
PBS is resuspended, and separately sampled to carry out that suitable sample-loading buffer is added, boiling water boiling 10min, 4000r/min are centrifuged 2min.Take supernatant
And precipitating carries out SDS-PAGE, the existence form and expression of object observing albumen respectively.The results show that GroEL1 success exists
(Fig. 7, swimming lane 4) is expressed 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-GroEL1 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 2019333.
The purifying of 3.2 GroEL1 albumen
After the e. coli bl21 of recombination according to the above method inducing expression cracking, after PAGE gel electrophoresis,
Determine GroEL1 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 50L sample-loading buffer is respectively added boils 10min.
(9) SDS-PAGE polyacrylamide gel is configured, by (the every hole 20L/), 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 7) purified.
The detection of 3.3 GroEL1 protein immunogenics
The identification of rGroEL1 protein immunogenic, key step are as follows: by the weight of purifying are carried out using Western blot method
Histone GroEL1 (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: GroEL1 albumen can be sent out with ox positive serum
Specific reaction band can be detected in raw reaction, and molecular weight is about 60kDa (Fig. 8), therefore GroEL1 albumen has good exempt from
Epidemic focus.
The detection that embodiment 4:GroEL1 albumen causes mouse immune to be reacted
The immune and Avoirdupois monitoring of 4.1 mouse
GroEL1 albumen is carried out 1:1 with adjuvant (MONTANIDETM IMS 1313VG N) 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, rGroEL1 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 GroEL1+ adjuvant immunity group mouse is with regard to significant after immune 14th day
Higher than simple immunologic adjuvant and PBS group, P < 0.05.21st 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 (Figure 10 B).Total IgG and total IgA antibody level are presented rapid increase and become
Gesture.Show that GroEL1 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:
GroEL1 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
It is significant that duration is presented in GroEL1+ adjuvant immunity group mouse specific IgG (Figure 11 A) and specificity IgA (Figure 11 B) antibody level
Increase.Extremely significant difference (P < 0.001) just is presented with simple immunologic adjuvant group and PBS group 7th day after immune.Show
GroEL1 albumen can just cause mouse to generate high-caliber specific IgG and Specific IgA antibody in Initial stage of immunization.
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: from the 0th day to the 21st day, each group mouse IFN-γ level has a degree of rising, and the 28th day
Downward trend is presented, but GroEL1+ adjuvant immunity group and adjuvant control group show GroEL1 albumen without significant difference (Figure 12 B)
Mouse is not caused to generate high-caliber IFN-γ.After immune 7th day, IL- in GroEL1+ adjuvant immunity group mice serum
6 levels are just significantly higher than adjuvant control group and PBS immune group (P < 0.001) (Figure 12 A), show that rGroEL1 albumen can exempt from
Epidemic disease early stage just causes mouse to generate high-caliber IL-6, i.e. Th2 type cellular immune level.
To sum up, GroEL1 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 significantly rising for cell factor IL-6 expression demonstrates GroEL1
Albumen can cause high-caliber Th2 type cellular immune level, show that the albumen has certain anti-bovine mycobacterium infection latent
Can, it can be researched and developed as vaccine, the important target of diagnostic preparation.
Sequence table
<110>Hua Zhong Agriculture University
<120>Mycobacterium bovis molecular chaperone protein GroEL1 and its encoding gene and application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 45
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 1
gccaagacaa ttgcggatcc atgagcaagc tgatcgaata cgacg 45
<210> 2
<211> 37
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 2
tggtggtggt ggtggatatc gtgcgcgtgc ccgtggt 37
<210> 3
<211> 31
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 3
cgggatccat gagcaagctg atcgaatacg a 31
<210> 4
<211> 25
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 4
cggaattcgt gcgcgtgccc gtggt 25
<210> 5
<211> 1620
<212> DNA
<213>Mycobacterium bovis (Mycobacterium bovis)
<400> 5
atgagcaagc tgatcgaata cgacgaaacc gcgcgtcgcg ccatggaggt cggcatggac 60
aagctggccg acaccgtgcg ggtgacgctg gggccgcgcg gccggcatgt ggtgctggcc 120
aaggcgtttg gcggacccac ggttaccaac gacggcgtca cggtggcacg tgagatcgag 180
ctggaagatc cgtttgaaga cttgggcgcc cagctggtga agtcggtggc caccaagacc 240
aacgatgtgg ccggtgacgg caccaccacc gcaaccatct tggcgcaggc actgatcaag 300
ggcggcctga ggctagtggc cgccggcgtc aacccgatcg cgctcggcgt gggaatcggc 360
aaggccgccg acgcggtatc cgaggcgctg ctggcatcgg ccacgccggt gtccggcaag 420
accggcatcg cgcaggtggc gacggtgtcc tcgcgcgacg agcagatcgg tgacctggtt 480
ggcgaagcga tgagcaaggt cggccacgac ggcgtggtca gcgtcgaaga atcctcgacg 540
ctgggcaccg agttggagtt caccgagggt atcggcttcg acaagggctt cttgtcggca 600
tacttcgtta ccgacttcga taaccagcag gcggtgctcg aggacgcgtt gatcctgctg 660
caccaagaca agatcagctc gcttcccgat ctgttgccat tgctggaaaa ggttgcagga 720
acgggtaagc cactactgat cgtggctgaa gacgtggagg gcgaagcgtt ggcgacgctg 780
gtcgtcaacg cgattcgcaa gacgttgaaa gcggtcgcgg tcaaggggcc gtacttcggt 840
gaccgccgta aggcgttcct tgaggacctg gcggtggtga cgggtggcca ggtggtcaac 900
cccgacgccg gcatggtgct gcgcgaggtg ggcttggagg tgctgggctc ggcccgacgc 960
gtggtggtca gcaaggacga cacggtcatt gtcgacggcg gcggcaccgc agaagcggtg 1020
gccaaccggg cgaagcactt gcgtgccgag atcgacaaga gcgattcgga ttgggatcgg 1080
gaaaagcttg gcgagcggct ggccaaactg gccggcgggg ttgctgtcat caaggtgggt 1140
gccgccaccg agaccgcact caaggagcgc aaggaaagcg tcgaggatgc ggtcgcggcc 1200
gccaaggccg cggtcgagga gggcatcgtc cctggtgggg gagcctcgct catccaccag 1260
gcccgcaagg cgctgaccga actgcgtgcg tcgctgaccg gtgacgaggt cctcggtgtc 1320
gacgtgttct ccgaagccct tgccgcgccg ttgttctgga tcgccgccaa cgctggcttg 1380
gacggctcgg tggtggtcaa caaggtcagc gagctacccg ccgggcatgg gctgaacgtg 1440
aacaccctga gctatggtga cttggccgct gacggcgtca tcgacccggt caaggtgact 1500
aggtcggcgg tgttgaacgc gtcatcggtt gcccggatgg tactcaccac cgagacggtc 1560
gtggtcgaca agccggccaa ggcagaagat cacgaccatc accacgggca cgcgcactga 1620
<210> 6
<211> 539
<212> RNA
<213>Mycobacterium bovis (Mycobacterium bovis)
<400> 6
Claims (9)
1. Mycobacterium bovis molecular chaperone protein GroEL1 has the amino acid sequence as shown in sequence table SEQ ID NO:6.
2. encoding the gene of Mycobacterium bovis molecular chaperone protein GroEL1 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 GroEL1 described in claim 1 in preparation perlsucht vaccine.
9. molecular chaperone protein GroEL1 described in claim 1 is preparing the purposes in diagnosing bovine tuberculosis kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910480091.6A CN110240636A (en) | 2019-06-04 | 2019-06-04 | Mycobacterium bovis molecular chaperone protein GroEL1 and its encoding gene and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910480091.6A CN110240636A (en) | 2019-06-04 | 2019-06-04 | Mycobacterium bovis molecular chaperone protein GroEL1 and its encoding gene and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110240636A true CN110240636A (en) | 2019-09-17 |
Family
ID=67886029
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910480091.6A Pending CN110240636A (en) | 2019-06-04 | 2019-06-04 | Mycobacterium bovis molecular chaperone protein GroEL1 and its encoding gene and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110240636A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1556856A (en) * | 1999-08-19 | 2004-12-22 | �Ϻ���ͨ��ѧ | Vaccines from infectious agents |
| CN102573896A (en) * | 2009-05-13 | 2012-07-11 | 因波特医疗股份有限公司 | Compositions and methods for immunodominant antigens of mycobacterium tuberculosis |
-
2019
- 2019-06-04 CN CN201910480091.6A patent/CN110240636A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1556856A (en) * | 1999-08-19 | 2004-12-22 | �Ϻ���ͨ��ѧ | Vaccines from infectious agents |
| CN102573896A (en) * | 2009-05-13 | 2012-07-11 | 因波特医疗股份有限公司 | Compositions and methods for immunodominant antigens of mycobacterium tuberculosis |
Non-Patent Citations (2)
| Title |
|---|
| 付凤云等: "结核分枝杆菌GroEL基因的克隆及真核表达质粒的构建", 《浙江实用医学》 * |
| 何磊等: "结核分枝杆菌热休克蛋白GroEL1的研究进展", 《微生物与感染》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110760006A (en) | African swine fever immune system targeted genetic engineering vaccine | |
| CN110684116B (en) | Mycobacterium tuberculosis EEC fusion protein, preparation method and application thereof | |
| CN113150084B (en) | Nanocrystallized coronavirus antigen and application thereof | |
| CN103059109B (en) | Mycoplasma pneumonia antigen, preparation method and immunodetection kit | |
| CN106220716A (en) | Infectious coryza of chicken subunit vaccine and preparation method thereof | |
| CN105884902A (en) | Fusion protein of mycoplasma pneumonia protein epitope and preparation and application thereof | |
| CN105567660B (en) | A kind of method and its application of Recombinant protein expression mycobacterium tuberculosis Rv2837c activated protein | |
| CN108774628B (en) | Escherichia coli engineering bacterium for synthesizing neonatal meningitis escherichia coli glycoprotein conjugate vaccine and application | |
| CN107936123B (en) | Swine transmissible gastroenteritis virus fusion protein and preparation method and application thereof | |
| CN110483625A (en) | A kind of Mycoplasma bovis imagination albumen MbovP732 and its application | |
| CN103323585A (en) | ELISA kit used for detecting fish streptococcus agalactiae IgM antibody, and preparation method thereof | |
| CN116003540B (en) | Preparation and application of mycobacterium tuberculosis antigen composition PFHP010 | |
| CN116023506B (en) | ASFV nonstructural protein dominant antigen epitope fusion protein, kit and application thereof | |
| CN110240636A (en) | Mycobacterium bovis molecular chaperone protein GroEL1 and its encoding gene and application | |
| CN106226520A (en) | Antigen of mycobacterium tuberculosis albumen Rv0865 and the application of B cell epitope peptide thereof | |
| CN105671016B (en) | Improve S- adenosine-L-cysteine hydrolase immunogenicity method and six sugar-modified ahcy albumen | |
| CN113512098B (en) | Indirect ELISA (enzyme-Linked immuno sorbent assay) method for identifying swine fever virus and bovine viral diarrhea virus serum antibodies and application thereof | |
| CN105906716B (en) | Preparation and application of echovirus 9 type VP1 protein specific epitope and fusion protein thereof | |
| CN110382519A (en) | The completely new recombinant protein antigen of Orientia Tsutsugamushi and the vaccine composition for using above-mentioned recombinant protein antigen | |
| CN110240637A (en) | The immune-related molecular chaperone protein GroEL2 of Mycobacterium bovis and its encoding gene and application | |
| CN112409495A (en) | Streptococcus suis recombinant ZSD recombinant subunit vaccine, recombinant epitope prokaryotic plasmid, preparation and application | |
| CN106397546A (en) | O type foot-and-mouth disease virus artificial recombinant antigen and preparation and application thereof | |
| CN117003888B (en) | Enterotoxin-producing escherichia coli antigen multi-epitope fusion protein and preparation method and application thereof | |
| CN116041541B (en) | Mycobacterium tuberculosis antigen EPPA011 and application thereof | |
| CN114377121B (en) | Recombinant African swine fever antigen cocktail vaccine containing intramolecular adjuvant and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190917 |
|
| RJ01 | Rejection of invention patent application after publication |