CN105567660B - A kind of method and its application of Recombinant protein expression mycobacterium tuberculosis Rv2837c activated protein - Google Patents
A kind of method and its application of Recombinant protein expression mycobacterium tuberculosis Rv2837c activated protein Download PDFInfo
- Publication number
- CN105567660B CN105567660B CN201610011743.8A CN201610011743A CN105567660B CN 105567660 B CN105567660 B CN 105567660B CN 201610011743 A CN201610011743 A CN 201610011743A CN 105567660 B CN105567660 B CN 105567660B
- Authority
- CN
- China
- Prior art keywords
- rv2837c
- tuberculosis
- mycobacterium tuberculosis
- protein
- albumen
- 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.)
- Active
Links
Classifications
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- 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
-
- 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
-
- 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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G01N2333/922—Ribonucleases (RNAses); Deoxyribonucleases (DNAses)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Pulmonology (AREA)
- Communicable Diseases (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a kind of method and its application of Recombinant protein expression mycobacterium tuberculosis Rv2837c activated protein.Mycobacterium tuberculosis Rv2837c protein gene cloning is entered into prokaryotic expression carrier pET28a+, is named as pET-CnpB.The Rv2837c activated protein of purifying is obtained using Ni ion affinity chromatography method.Recombination Rv2837c albumen can be used for detecting mycobacterium tuberculosis infection animal and patients serum.Recombinating Rv2837c albumen can induce the high-caliber humoral and cellular immune response response of mouse, can be used for the development of tuberculosis novel subunit vaccine.Therefore, Rv2837c albumen has a good application prospect.The present invention relates to the active recombinant proteins to prepare the application in diagnosis of tuberculosis and preventing preparation or drug.
Description
Technical field
The invention belongs to Diagnosis of Tuberculosis reagent and vaccines arts.The present invention relates to a kind of Recombinant protein expression and obtain
The method of mycobacterium tuberculosis Rv2837c activated protein.The invention further relates to the recombinant proteins to prepare diagnosis of tuberculosis and pre-
Application in anti-preparation or drug.
Background technique
TB endemic status
Tuberculosis (Tuberculosis, TB) be by mycobacterium tuberculosis (Mycobacterium tuberculosis,
Mtb chronic infectious disease caused by) infecting, is still the infectious disease that most serious is endangered in worldwide at present.According to WHO report,
The whole world has about 1/3 population infection mycobacterium tuberculosis, new hair patient about 9,000,000 in 2013, the tuberculosis in all infectious diseases
The sick death rate is only second to AIDS.TB is classified as the most important infectiousness killer of the mankind by WHO together with AIDS, malaria.China
Belong to one of 22 TB high burden countries in the whole world, ranked first position in 27 kinds of China notifiable infectious diseases.The Ministry of Public Health of China will
TB is classified as one of the major disease of national priority control, and national Eleventh Five-Year Plan and " 12th Five-Year Plan " serious infectious diseases special project project will
Tuberculosis is classified as one of primary study object.
Diagnosis of tuberculosis present Research
Sediments microscope inspection, Sputum culturing are the main means of pathogen detection, but the diagnosis based on the detection of pathogen
The disadvantages of method is long there are experimental period, susceptibility is low, specificity is undesirable and time-consuming.Molecular biology method such as PCR,
Hybridization and gene and protein chip have also been applied to quick diagnosis lungy, but because its recall rate is undesirable, and there are technologies
It is required that the reasons such as high, expensive, are not suitable for clinical Large-scale Screening.Novel diagnostic method and reagent is actively studied to help
In control lungy.
The fast development of immunological technique promotes the development of diagnostic techniques.After human infection's mycobacterium tuberculosis, generate
Delayed allergy occurs while immunity.Tuberculin test is that mycobacterium tuberculosis, whether there is or not super by measurement body
Quick reaction, determines whether subject has against mycobacterium tuberculosis cellular immunity, to judge whether subject infects the cause of disease
A kind of method of bacterium.It is still widely used at present in clinic with the tuberculin test (PPD) of this principle development, but since card is situated between
The extensive inoculation of seedling, the results of tuberculin test positive can not be diagnosed as tuberculosis, and more monitor for vaccine effect.
After tuberculosis infection, the memory t cell of internal long-term existence antigentic specificity, when encountering antigenic stimulus again,
Energy prompt activation proliferation, generates cytokine profiles, and wherein IFN-γ is crucial cell factor.IFN-γ release test
(interferon gamma release assay, IGRAs) is a kind of ion vitro immunization inspection for mycobacterium tuberculosis infection
The new method of survey has definite meaning in terms of identifying active tuberculosis and latent tuberculosis infection, prediction tuberculosis onset risk,
But limitation is compared to clinical Diagnosis of Tuberculosis value, in addition, T-SPOT.TB experimental technique high operation requirements, kit is expensive,
It is also limited in the application of the not high countries and regions of economic level.
Serodiagnosis has spy by detection infected person anteserum's tuberculosis specific antibody level, indirect diagnosis of tuberculosis
Anisotropic good, time-consuming significant advantage that is short and being easy to batch detection.Mycobacterium tuberculosis and BCG vaccine genome difference are selected at present
Area's antigen is carried out the research of auxiliary diagnosis using specific antibody in the methods of ELISA detection patients serum, studied more deep
Such as Early insulin secretion antigen target 6kD albumen (early secretary antigenic target 6ku protein, ESAT-
6), culturing filtrate albumen (culture filtrate protein, CFP-10), fat arabian mannan antigen, Ag85 are multiple
Close object, 38KD albumen, A60 antigen, 16KD albumen, MPT64, MTB48 etc..These antigens differ from one another, although a variety of both at home and abroad
The listing of antitubercle sera diagnostic kit, but its clinical report result is different, therefore it is relevant to disease special to continue sieve series
Antigen combines existing dominant antigen, helps to develop more effective serological diagnostic method.
Tuberculosis subunit vaccine present Research
BCG vaccine (Bacillus Calmette-Gu é rin vaccine, BCG) be currently used for tuberculosis prophylaxis only
One vaccine, but there is the problems such as protection period is short, and protective immune response is weaker, in addition, as a kind of attenuated live vaccine, no
It can be used for the inoculation of immunocompromised crowd.The TB new generation vaccine being currently being deployed mainly include subunit vaccine, gene vaccine,
Recombinant BCG vaccine, attenuation or the whole cell of enhancing live vaccine, auxotroph live vaccine, in conjunction with vaccine of dendritic cells etc..Its
Middle subunit vaccine by one or more can induce protective immunity at being grouped as, since constituent is clear, structure is simple,
Safety and the specifically concern by researcher.So far, in tuberculosis candidate vaccine, about 50% is subunit's epidemic disease
Seedling makees vaccine component using the drive member (such as albumen, polypeptide, mycolic acid, glycolipid) of mycobacterium tuberculosis, can induce machine
Body generates immunoprotection or achievees the effect that immunization therapy.
Research by many years to mycobacterium tuberculosis protective antigens has identified a variety of with stronger immunogene
Property albumen, mainly include secretory protein, such as ESAT-6, CFP10, Ag85, TB10.4；The T cell antigen of mouse and the mankind
Target point protein (such as MTB39 and MTB32 combine the fusion protein MTB72f formed)；The specific antigen of latent infection expression is such as
Hsp65, Hsp70, HspX etc..Wherein the Ag85B in Ag85 family studies more early, Horwitz et al (rotective
immunity against tuberculosis induced by vaccination with major extracellular
proteins of Mycobacterium tuberculosis.Proceedings of the National Academy of
Sciences of the United States of America,1995,92(5):1530-4.) use injected s.c.
To be attacked with strain after Ag85B protein immunization cavy 3 times, compared with the control group, it is immune after cavy can generate stronger cell
Immune response simultaneously shows certain immunity to strain, and the time-to-live increases.The rBCG of expression Ag85B, which has gone through to enter, to be faced
Bed test.Ag85B the and TB10.4 amalgamation protein vaccine of AERAS, SSI, Sanofi and Intercell joint research and development, at present just
Carrying out Phase I clinical trial.Therefore, multinomial research shows that Ag85B albumen is good anti-TB vaccine component, has good
Development volue.
Although above-mentioned protective antigens all shows certain vaccine potential, so far, not obtaining also can be used for replacing
For the tuberculosis new generation vaccine of BCG, show that the tuberculosis antigen effect based on secretory protein is limited.To mycobacterium tuberculosis physiology
The rapid development of the characteristic study provides more candidate molecules.Multipath, multimachine antigen collective effect, just having can
More effective immune effect can be obtained, therefore is also the research direction of the following novel vaccines against tuberculosis.
Mycobacterium tuberculosis Rv2837c progress
2008, a kind of new two adenosine (Cyclic di-adenosine of micromolecule nucleotide class signaling molecule ring
Monophosphate, c-di-AMP) it is found, it is primarily involved in bacterial spore formation, cell wall is formed, cell wall feeling of stress
The physiology courses such as bacterium size are answered and control, it is horizontal related to bacterial virulence, thus cause the extensive interest of researcher.Jun
Yang, et al. (Deletion of the cyclic di-AMP phosphodiesterase gene (cnpB) in
Mycobacterium tuberculosis leads to reduced virulence in a mouse model of
infection.Mol Microbiol.2014 93(1):65-79.) discovery Rv2837c be c-di-AMP degrading enzyme, and by its
It is named as CnpB.Rv2837c contains DHH-DHHAI structural domain, and the albumen containing the structural domain is widely present in bacterium, such as golden yellow
Color staphylococcus, hay bacillus etc..Manikandan Ket al.(Two-step synthesis and hydrolysis of
cyclic di-AMP in Mycobacterium tuberculosis.PLoS One.2014 23；9(1):E86096. it) sends out
C-di-AMP first can be degraded to pApA by existing Rv2837c, further be degraded to 5'-AMP.Postic G et al
(Characterization of NrnA homologs from Mycobacterium tuberculosis and
Mycoplasma pneumoniae.RNA.201218(1):155-65.) discovery Rv2837c has RNA enzyme activity, degradable
NanoRNA, and dephosphorylation pAp, thus it is speculated that there is important adjustment effect in bacterium pathogenic course.Cron LE et al.
(Two DHH subfamily 1proteins contribute to pneumococcal virulence and confer
protection against pneumococcal disease.Infect Immun.2011 79(9):3697-710.) hair
Albumen now containing DHH structural domain is related with the virulence of bacterium, after the protein immunization mouse, can resist the sense of a certain amount of bacterium
Dye, thus there is immune protective efficiency, prompt the Rv2837c of DHH structural domain family that there is similar biology and immunological characteristic.
Summary of the invention
The object of the present invention is to provide a kind of sides of Recombinant protein expression mycobacterium tuberculosis Rv2837c activated protein
Method.The recombination Rv2837c activated protein that this method obtains can be used for the development of tuberculosis new diagnostic and/or vaccine (preparation).
The invention is realized in this way：
A kind of method of Recombinant protein expression mycobacterium tuberculosis Rv2837c activated protein, according to tuberculosis branch bar
H37Rv plants of bacterium of genome Rv2837c gene order design primer：
P1：The site 5 '-CGGCATATGGTGACGACGATCGACCCAAG-3 ', NdeI
P2：The site 5 '-TTTAAGCTTGCCAGCTGCGCGATCTGACG-3 ', HindIII
PCR amplification c-di-AMP catabolic enzyme Rv2837c gene, reaction ginseng are used from Mycobacterium tuberculosis H37Rv genome
Number：95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 30s, 57 DEG C of renaturation 30s, 72 DEG C of extension 100s, 35 recycle, 72 after last circulation
DEG C extend 5min；The 1023bp target fragment that PCR amplification goes out, with NdeI and HindIII double digestion, the connection of T4DNA ligase is same
Sample double digestion and the pET28a+ carrier recycled, Transformed E .coli DH5 α competent cell, with kalamycin resistance plate screening,
Picking positive colony is inoculated in 37 DEG C of shaken cultivations of fluid nutrient medium and stays overnight, and kit extracts Plasmid DNA, and double digestion identification is positive
Plasmid carries out sequencing, and sequencing shows successfully to construct Rv2837c prokaryotic expression carrier, is named as pET-CnpB, complete
Gene order such as SEQ ID NO：Shown in 1, pET-CnpB Transformed E .coli BL21, IPTG induce destination gene expression, using anti-
His tag monoclonal antibody Western-blot method identifies destination protein, which is：CCTCC NO：
M2015703；Inducible protein is expressed by the way of 0.05mmoL/L low concentration IPTG and 16 DEG C of Low- temperature cultures, using Ni ion
Affinity chromatography purifying obtains solvable, high yield destination protein, and purifying protein is detected through HPLC, has degradation c-di-
The activity of AMP, amino acid complete genome sequence such as SEQ ID NO：Shown in 2.
The recombination mycobacterium tuberculosis Rv2837c albumen is for diagnosing and preventing the application in preparation lungy.
The result of the recombinant protein detection mycobacterium tuberculosis infection serum：The Rv2837c albumen that the present invention purifies
10 μ g/mL are coated with elisa plate, detect different infection period mice serums and clinical tubercular's serum.Testing result shows
In infecting mouse body, Rv2837c antibody continues with infection time and is increased, and its level is higher than Ag85B；According to tuberculosis patient
Serum Rv2837c antibody level draws ROC curve, and area under the curve is greater than 0.5, shows with diagnostic.
The result of the recombinant protein stimulation immune response：The Rv2837c albumen dorsal sc that the present invention purifies
Immune 50 μ g/ of mouse only, is spaced 2 weeks, is immunized three times；Detection humoral immune response level includes that antibody titer and Subclass of antibody are surveyed
It is fixed；It is horizontal to detect cellullar immunologic response, including spleen lymphocyte proliferation, CD4+/CD8+Ratio, RT-PCR detection cell factor turn
The expression of record level, ELISA detection cell factor.Testing result shows the Rv2837c protein immunization mouse of recombination, can
Induce higher humoral and cellular immune response response horizontal, partial immunity response level is higher than existing mycobacterium tuberculosis and is immunized by force
Antigen A g85B.The stronger immune response of body can be stimulated after recombination Rv2837c protein immunization, improves the immune guarantor of body
Tuberculosis novel subunit vaccine can be used for because its mechanism of action and existing tuberculosis antigen are all different alone or in combination by protecting power
Development, thus have a good application prospect.
Ours the study found that in expression in escherichia coli, and the degradable c- of Rv2837c purified by affinity chromatography
Di-AMP, therefore there is c-di-AMP to decompose enzymatic activity.In infection animal body, Rv2837c antibody titer is with infection time
Extension and gradually rise, and the antibody titer of the antigen is significantly higher than non-tuberculosis patient in tubercular's body, shows that it can
Development for tuberculosis serological diagnosis kit.After Rv2837c protein immunization mouse, detection discovery its can induce be immunized it is small
Mouse generates the titre of high antibody, and antibody titer is higher；Recombinant protein can significantly induce spleen lymphocyte proliferation, especially stimulate
CD8+Cell increases；The more cell factors of inducible lymphocyte release, the especially raising of key cytokines IFN-γ,
Be conducive to body and resist mycobacterium tuberculosis infection.Therefore, Rv2837c albumen can be used as the candidate set of tuberculosis subunit vaccine
Point.
In conclusion Rv2837c as newfound c-di-AMP catabolic enzyme, has existing with other in Bacterial Physiological
The different mechanism of action of proteantigen.Our research obtains active mycobacterium tuberculosis c-di-AMP catabolic enzyme
Rv2837c, and can be used as the component for developing diagnostic reagent of tuberculosis and vaccine, thus there is preferable market application value.
Detailed description of the invention：
Fig. 1：Rv2837c gene magnification PCR result figure.
Fig. 2：Rv2837c prokaryotic expression carrier digestion qualification result figure.
Fig. 3：Rv2837c recombinant protein expresses SDS-PAGE and analyzes result figure.
Fig. 4：Rv2837c recombinant protein expresses Western-blot and analyzes result figure
Fig. 5：Rv2837c recombinant protein purification SDS-PAGE analysis chart.
Fig. 6：Rv2837c recombinant protein enzymatic activity HPLC analyzes result schematic diagram.
Fig. 7：Rv2837c recombinant protein detects infecting mouse specific antibody result schematic diagram.
Fig. 8：Rv2837c recombinant protein detects tuberculosis patient serum specific antibody ROC curve.
Fig. 9：Rv2837c recombinant protein is immunized mouse specific antibody level and analyzes result schematic diagram.
Figure 10：Rv2837c recombinant protein is immunized mice spleen lymphocytes proliferation and analyzes result schematic diagram.
Figure 11：Mouse spleen lymphocyte flow cytomery result figure is immunized in Rv2837c recombinant protein.
Figure 12：Rv2837c recombinant protein is immunized mouse spleen lymphocyte levels of cytokine secretion and analyzes result schematic diagram.
Figure 13：Rv2837c recombinant protein is immunized mouse lung levels of cytokine secretion and analyzes result schematic diagram.
Depositary institution's title：China typical culture collection center, depositary institution address：Wuhan, China Wuhan University is protected
Hide the date：On November 26th, 2015, classification naming：E. coli bl21/pET-CnpB Escherichia coliBL21/
pET-CnpB。
Specific embodiment
The building of Rv2837c prokaryotic expression carrier：
According to the genome Rv2837c gene order design primer of Mycobacterium tuberculosis H37Rv strain：
P1：The site 5 '-CGGCATATGGTGACGACGATCGACCCAAG-3 ', NdeI
P2：The site 5 '-TTTAAGCTTGCCAGCTGCGCGATCTGACG-3 ', HindIII
By Shanghai Sani Biotechnology Co., Ltd synthetic primer.Using Mycobacterium tuberculosis H37Rv pnca gene group DNA as mould
Plate PCR amplification target gene, response parameter：95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 30s, 57 DEG C of renaturation 30s, 72 DEG C extend
100s, 35 circulations, 72 DEG C of extension 5min after last recycles.1% agarose gel electrophoresis is analysis shows amplify 1023bp mesh
Band (see Fig. 1).Gel reclaims kit recycles target fragment, with NdeI and HindIII double digestion, 1% Ago-Gel
Electrophoresis recycles digestion products.The pET28a+ carrier that T4DNA ligase connects same double digestion and recycles, 16 DEG C overnight, conversion
E.coli DH5 α competent cell, with kalamycin resistance plate screening.Picking positive colony is inoculated in 37 DEG C of fluid nutrient medium
Shaken cultivation is stayed overnight, kit extraction Plasmid DNA, restriction enzymes double zyme cutting identification positive plasmid progress sequencing (see
Fig. 2).Sequencing shows successfully building Rv2837c prokaryotic expression carrier, is named as pET-CnpB, complete genome sequence is such as
SEQ ID NO：Shown in 1.
The purifying of Rv2837c activated protein and identification：
The correct recombinant plasmid of sequencing is taken, Transformed E .coli BL21 (DE3) is coated on the LB of kalamycin resistance
Plate, 37 DEG C of cultures to bacterium colony generate.Single bacterium colony is transferred the LB liquid medium into 5mL containing kanamycins on picking plate
In, 37 DEG C of shaken cultivations are stayed overnight, with 1:Overnight culture is forwarded to fresh antibiotic LB liquid medium by 100 ratio
In, continue 37 DEG C of shaken cultivations to OD600=0.6, IPTG is added to final concentration to 1mmol/L, continues 37 DEG C of culture 4h.It collects
Culture, 6 000rpm are centrifuged 5min, collect thallus, and 12%SDS-PAGE gel electrophoresis analysis shows band of expression about 36kDa
(see Fig. 3 wherein M：Marker；1：Induction bacterium；2：Non- induction bacterium).After PAGE gel electrophoresis, half-dried electrotransfer electrophoresis is used
Albumen is gone on pvdf membrane, 5%BSA37 DEG C of closing 1h, PBST are washed three times, are added 1:1 000 anti-His tag antibodies, 37
DEG C be incubated for 1h, PBST washing after, be added HRP mark 37 DEG C of incubations 45min of goat anti-mouse igg secondary antibody, addition chemiluminescent substrate
Liquid colour developing, BIO-RAD chemiluminescence fluoroscopic imaging systems analysis, it is seen that have specific band (see Fig. 4 wherein M at about 36kDa：
Marker；1：DH5α；2：Non- induction bacterium), show in Escherichia coli successful expression Rv2837c albumen.
For obtain great amount of soluble destination protein, by overnight culture 1:100 transfer into the fresh LB of 500mL, 37 DEG C of trainings
It supports to OD600=0.6, culture temperature is down to room temperature by water-bath, and the IPTG of final concentration of 0.05mmoL/L, 16 DEG C of cultures are added
Overnight；Bacterial precipitation is collected by centrifugation, -80 DEG C freeze 2 hours or more；Bacterial precipitation is taken out, weight is weighed, is added according to the wet bacterium of 1g
The ratio of 20ml lysate A (50mM Tris-HCl, 150mM sodium chloride, 10mM imidazoles, 10% glycerol, protease inhibitors),
Bacterial precipitation is resuspended, is incubated for 30min on ice；By bacterial suspension be placed in ice bath with 40% cleavage rate ultrasound 10min (5 seconds crack,
10 seconds intervals)；By ultrasonic lysate in 4 DEG C of 12 000rpm high speed centrifugation, supernatant is left and taken；Take 2mL Ni ion affinity chromatography column
Filler is balanced with the lysate of 5 times of volumes, is placed in appropriate containers, and supernatant is added, and 4 DEG C of slow oscillations combine 2 hours；It will mix
Close object be transferred to chromatographic column, respectively with the washing lotion B of 20 times of column volumes (50mM Tris-HCl, 150mM sodium chloride, 20mM imidazoles),
Washing lotion C (50mM Tris-HCl, 150mM sodium chloride, 50mM imidazoles) and washing lotion D (50mM Tris-HCl, 150mM sodium chloride,
75mM imidazoles) cleaning Ni column；It is eventually adding eluent E ((50mM Tris-HCl, 150mM sodium chloride, 300mM imidazoles), segmentation
Efflux after collecting elution；It is sampled from Fractional Collections liquid, 12%SDS-PAGE electrophoresis detection purifying protein；Collection contains mesh
Albumen all collection liquids, be transferred to bag filter and be put into 500mL PBS, 4 DEG C of stirrings are dialysed 30min, are transferred to containing 10% glycerol
Dialyse 30min again in the 500mL PBS of pre-cooling, -80 DEG C of preservations after packing.It is purified using Ni ion affinity chromatography and obtains purity
>95% soluble recombinant protein (see Fig. 5).Purifying protein BCA standard measure, yield are 17.4mg/L culture.
Purifying protein is taken, following reaction systems are formed：50mM Tris-HCl (pH 7.5), 1mM MnCl2,10mM
NaCl, 1mM c-di-AMP, 3 μM of Rv2837c, totally 10 μ L.37 DEG C of incubation 1h are added 1 μ L 0.5M EDTA and terminate reaction, add
Enter 40 μ L deionized waters, after mixing, isometric HPLC grade methanol is added.4 DEG C save standby inspection.20 μ L samples are taken, C- is injected into
18 chromatographic columns (250 × 4.6mm, Vydac) carry out reverse phase HPLC chromatography HPLC analysis.Flow visualizing is：buffer A
(100mM KH2PO4, 4mM 4-butyl ammonium hydrogen sulfate, pH5.9) and buffer B (75%buffer A, 25% methanol).Separate journey
Sequence：Time (minute) and mobile phase (percentage of buffer B) are respectively：0.0,0；2.5,0；5.0,30；10.0 60；
14.0 100；21.0 100；22.0 50；23.0 0.Flow velocity is 0.7mL/min.254nm length ultraviolet light detection product generates.
HPLC testing result shows that Rv2837c can degrade c-di-AMP (see Fig. 6).
Rv2837c Protein Detection infection animal and tuberculosis patient serum：
It takes 5 μ g/mL of purifying protein to be coated with 96 hole elisa plates, after PBS washing, 5%BSA-PBS room temperature is added and closes 1h, sense
After contaminating mice serum, tuberculosis patient and consumptive's dilution, it is separately added into each 100 hole μ L/ of hole, 37 DEG C of incubation 1h, with
HRP is added afterwards and marks 37 DEG C of incubation 45min of goat anti-mouse igg secondary antibody, 100 hole μ L/ of substrate (TMB) developing solution is added, after colour developing
50 μ L 2M H are added2SO4Terminate liquid measures OD450Value, and draw corresponding chart.
Testing result is shown, after mouse infection 2w, can be detected out specific antibody (P in serum<0.05) when, with infection
Between extension, serum antibody titer switchs to after chronic infection (12w), Rv2837c specific antibody titres in significantly increasing trend
Highest, and it is higher than Ag85B antibody level (see Fig. 7).It is coated with elisa plate with purifying protein, detects tuberculosis patient and pulmonary tuberculosis
Patient's serum, according to OD450Value draws ROC curve, and area under the curve 0.65 shows with diagnostic (see Fig. 8).
The detection of Rv2837c protein immunization response level：
1, Rv2837c protein immunization humoral immunity measures
In ELISA method detection immune serum specific antibody the result shows that, Rv2837c is immune can be examined after two weeks
Antibody is measured, antibody level gradually rises (P after booster immunization<0.01), antibody level and strong immunogene Ag85B are quite (see figure
9)。
2, Rv2837c protein immunization mouse cell immunoassays
(1) spleen lymphocyte proliferation situation detects
4 weeks after the completion of mouse immune, the sterile separation splenic lymphocytes of immune mouse are taken, with the RPMI containing 10%FCS
Isolated lymphocyte concentration is separately adjusted to angularly 1 × 10 by 1640 complete culture solutions6(100 μ l/ in 96 orifice plates are added in a/mL
Hole), experimental group is separately added into the diluted PPD (25mg/L) and recombinant protein of 100 μ L 1640 complete culture solutions of RPMI
(25mg/L), 100 μ L culture solutions are added in negative control group, while the blank control wells that splenic lymphocytes are not added are arranged, and all groups
It is all provided with 3 multiple holes.5%CO2After 37 DEG C of culture 72h of incubator, the MTS (5mg/mL) in 20 holes μ L/ is added, continues to cultivate 4h, add
Enter 25 hole μ L/ 10%SDS, measures A490 value.As a result it is indicated with proliferation index SI：SI=A490 (experimental group-blank control)/
A490 (negative control-blank control).Testing result shows that recombinant protein is immune to can significantly promote spleen lymphocyte proliferation (P<
0.05, see Figure 10).
(2) splenic lymphocytes CD4+/CD8+Cells ratio detection：
4 weeks after the completion of mouse immune, the sterile separation splenic lymphocytes of immune mouse are taken, 5mL erythrocyte cracked liquid is added
5min is acted on, RPMI 1640 culture medium of the 5mL without serum is added and terminates reaction, 1 500rpm is centrifuged 10min, and PBS washing is thin
Born of the same parents 3 times (5mL/ pipe).Cell precipitation uses the RPMI 1640 culture medium without serum to be resuspended, and FITC: CD4/RPE: CD8 bis- marks are kept away
Light dyes 30min.It ibid washs cell 3 times, it is to be checked that cell fixer 0.5mL/ pipe is added.Flow cytomery cell sample
Product, the results showed that：Protein immunization group mouse, CD4+/CD8+Cells ratio is in increasing trend, and with especially heavy to Intracellular Infection
The CD8 wanted+Based on cell number increase, the tulase that this removes parasitism intracellular to immunity of organism is advantageous (see Figure 11).
(3) Secreted by Mouse Splenic cytokines measurement is immunized
Mouse spleen lymphocyte suspension concentration is adjusted to 2 × 106/ ml is added separately in 96 orifice plates by 100 holes μ L/,
Every group sets 3 multiple holes, 2 positive controls and 2 negative controls, while setting the blank control wells and reagent controls that cell is not added
Hole；Experimental port is separately added into the diluted PPD (25mg/L) and recombinant protein (25mg/ of 100 μ L 1640 complete culture solutions of RPMI
L), 100 μ L culture solutions are added in negative control group.Set 37 DEG C of 5%CO2It is incubated for 72h, collects cells and supernatant, ELISA method detection
The concentration of cell factor in supernatant, the results showed that, recombinant protein can induce splenic lymphocytes Th1 cytokines IFN-γ and
IL-2 release increases, without improving Th2 cytokines IL-10 secretion level (see Figure 12).
(4) detection of mouse lung secrete cytokines is immunized
4 weeks after the completion of mouse immune, mouse is put to death, takes lung tissue segment, is ground after liquid nitrogen frozen is added, Trizol method
Total serum IgE is extracted, ultraviolet specrophotometer is quantitative.Taking 500ng RNA reverse transcription is cDNA, and real-time quantitative PCR detects cell factor
Transcriptional level, the results show that the immune inducible mouse secretion of gamma-IFN of recombinant protein and IL-10 cell factor increase, wherein
IFN-γ secretion level is significantly higher than Ag85B and (P is immunized<0.05, see Figure 13), show the immune inducible lung's lymph of recombinant protein
Cell cytokine release increases, and it is immune to facilitate body resisting tuberculosis infection.
Claims (1)
1. a kind of mycobacterium tuberculosis Rv2837c albumen of Recombinant protein expression is in preparation diagnosis or prevents system lungy
Application in agent, it is characterised in that：The amino acid sequence of the mycobacterium tuberculosis Rv2837c albumen such as SEQ ID NO：2 institutes
Show.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610011743.8A CN105567660B (en) | 2016-01-08 | 2016-01-08 | A kind of method and its application of Recombinant protein expression mycobacterium tuberculosis Rv2837c activated protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610011743.8A CN105567660B (en) | 2016-01-08 | 2016-01-08 | A kind of method and its application of Recombinant protein expression mycobacterium tuberculosis Rv2837c activated protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105567660A CN105567660A (en) | 2016-05-11 |
| CN105567660B true CN105567660B (en) | 2018-11-20 |
Family
ID=55878290
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610011743.8A Active CN105567660B (en) | 2016-01-08 | 2016-01-08 | A kind of method and its application of Recombinant protein expression mycobacterium tuberculosis Rv2837c activated protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105567660B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107217026B (en) * | 2017-06-14 | 2020-10-09 | 中国人民解放军第四军医大学 | Recombinant mycobacterium smegmatis strain with c-di-AMP catabolic enzyme knocked out and application thereof |
| CN108752438A (en) * | 2018-06-20 | 2018-11-06 | 遵义医学院附属医院 | Mycobacterium tuberculosis 38KD protein Ds NA extractions, construction of recombinant vector expression |
| CN114984870A (en) * | 2022-03-15 | 2022-09-02 | 齐欣 | Recombinant protein microsphere and preparation method and application thereof |
| CN116875520B (en) * | 2023-07-12 | 2024-06-14 | 吉林农业大学 | Lactic acid bacteria expressing ribosome inactivating protein and application thereof in resisting rotavirus |
| CN118416207A (en) * | 2024-07-02 | 2024-08-02 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Paratuberculosis subunit vaccine and preparation method and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2655190T3 (en) * | 2011-12-22 | 2018-02-19 | The University Of Sydney | Prevention and treatment of Mycobacterium infection |
| KR20150058152A (en) * | 2012-07-10 | 2015-05-28 | 트랜스진 에스아이 | Mycobacterial antigen vaccine |
| IN2015DN03962A (en) * | 2012-10-23 | 2015-10-02 | Statens Seruminstitut |
-
2016
- 2016-01-08 CN CN201610011743.8A patent/CN105567660B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN105567660A (en) | 2016-05-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105567660B (en) | A kind of method and its application of Recombinant protein expression mycobacterium tuberculosis Rv2837c activated protein | |
| Rao et al. | Leptospirosis in India and the rest of the world | |
| Twine et al. | BALB/c mice, but not C57BL/6 mice immunized with a ΔclpB mutant of Francisella tularensis subspecies tularensis are protected against respiratory challenge with wild-type bacteria: association of protection with post-vaccination and post-challenge immune responses | |
| CN102836425A (en) | A tuberculosis vaccine comprising antigens expressed during a latent infection phase | |
| Qi et al. | Development of a rapid and visual nucleotide detection method for a Chinese epidemic strain of Orientia tsutsugamushi based on recombinase polymerase amplification assay and lateral flow test | |
| WO2016201825A1 (en) | Mycobacterium tuberculosis antigens and applications thereof | |
| Chen et al. | Novel recombinant RD2-and RD11-encoded Mycobacterium tuberculosis antigens are potential candidates for diagnosis of tuberculosis infections in BCG-vaccinated individuals | |
| CN102180974B (en) | Tubercle bacillus fusion protein and preparation method and application thereof | |
| Long et al. | Detection and evaluation of antibodies against neutrophil-activating protein of Helicobacter pylori in patients with gastric cancer | |
| Zhang et al. | Identification of RD5‐encoded Mycobacterium tuberculosis proteins as B‐cell antigens used for serodiagnosis of tuberculosis | |
| Luo et al. | Identification of a novel immunodominant antigen Rv2645 from RD13 with potential as a cell-mediated immunity-based TB diagnostic agent | |
| CN104292315A (en) | Preparation for Rv2645 protein, and application thereof on aspects of tuberculosis diagnosis and reorganized BCG vaccines | |
| Wang et al. | Identification and evaluation of the novel immunodominant antigen Rv2351c from Mycobacterium tuberculosis | |
| Wang et al. | Immunogenicity and efficacy analyses of EPC002, ECA006, and EPCP009 protein subunit combinations as tuberculosis vaccine candidates | |
| CN103063846B (en) | Application of SHSP60 for preparing diagnostic reagent for monitoring comprehensive hepatic pathology injury degree of schistosomiasis patient | |
| CN106226520B (en) | The application of antigen of mycobacterium tuberculosis albumen Rv0865 and its B cell epitope peptide | |
| Chen et al. | Recombinant 56-kilodalton major outer membrane protein antigen of Orientia tsutsugamushi Shanxi and its antigenicity | |
| Xiao et al. | Immunological evaluation of a novel mycobacterium tuberculosis antigen Rv0674 | |
| CN106405107A (en) | Use of mycobacterium tuberculosis antigen protein Rv2941 and its T cell epitope peptide | |
| Manosroi et al. | Determination and geographic distribution of Orientia tsutsugamushi serotypes in Thailand by nested polymerase chain reaction | |
| CN107530414B (en) | Mycobacterium tuberculosis antigen for distinguishing active tuberculosis infection and latent tuberculosis infection and application thereof | |
| CN115920021A (en) | Tuberculosis subunit vaccine and preparation method thereof | |
| Song et al. | A combined application of molecular docking technology and indirect ELISA for the serodiagnosis of bovine tuberculosis | |
| EP3953709B1 (en) | Diagnostic reagents | |
| CN101805397A (en) | Mycobacterium tuberculosis ESAT-6 recombinant dipolymer, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |