CN107253987A - With the mycoplasma genitalium MgPa receptor proteins interacted and its separation method and purposes - Google Patents
With the mycoplasma genitalium MgPa receptor proteins interacted and its separation method and purposes Download PDFInfo
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- CN107253987A CN107253987A CN201710432872.9A CN201710432872A CN107253987A CN 107253987 A CN107253987 A CN 107253987A CN 201710432872 A CN201710432872 A CN 201710432872A CN 107253987 A CN107253987 A CN 107253987A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
Abstract
Present invention screening has obtained receptor protein --- the histone H2B positioned at human urethra's epithelial cell membrane surface.The Protein Separation is made up of from the epicyte protein of human urine tract epithelial cell, its nucleotide sequence as shown in SEQ ID NO.1 126 amino acid.The present invention screens acceptors of the MgPa on human urethra's epithelial cell membrane using improved VOPBA methods, to further elucidate, Mg is possible to be sticked and mechanism of causing a disease, stop that Mg sticks or invaded host cell for further design acceptor model molecule and antagonistic molecule, previous experiments basis is provided for Mg preventing and curing infections.Therefore the receptor protein histone H2B can be used in preparing in the targeted drug for treating or preventing Mycoplasma genitalium infection disease.
Description
Technical field
The present invention relates to a kind of receptor protein --- histone H2B (Histone interacted with mycoplasma genitalium MgPa
H2B), the separation method and purposes of the albumen are additionally provided, belongs to bioengineering and medical diagnosis on disease and Prevention Technique field.
Background technology
Mycoplasma genitalium (Mycoplasma genitalium, Mg) is first from a non-gonococcal urethritis patients urine
A kind of urogenital infections mycoplasma isolated in road secretion.Clinical research shows that women infection Mg can cause uterine neck
Scorching, pelvic infecton and infertile etc.；And male's infection can then cause acute and chronic NUG and infertility；In addition, Mg infection is also
Chance infection with human immunodeficiency virus (HIV) is closely related.
The combination of corresponding acceptor is the first step of pathogenic infection host in pathogen and host cell membrane, and can it be felt
Contaminate host cell most important.Research shows that Mg must first attach to urogenital tract or respiratory mucosa epithelial cell ability
Infection host is simultaneously colonized in infection site or invaded intracellular.
Mycoplasma does not have cell membrane, but there is on Mg film more memebrane protein.Mg passes through the memebrane protein on cell membrane
Its field planting is mediated to host cell surface and Infective.Mycoplasma genitalium adhesion protein (Mycoplasma genitalium
Protein of adhesion, MgPa) it is a kind of most memebrane protein of Mg contents, and be a kind of topmost adhesion protein,
Mediation Mg sticks or even plays vital effect during invading host epithelial cells.The acellular of spontaneity sticks
The Mg mutant strains of activity then lack MgPa.Iverson-Cabral SL etc. research also indicates that the persistent infection process in Mg
In, the antibody that body is produced illustrates that MgPa C-terminal and its immunogenicity is close mainly for the C-terminal variable region guarded in MgPa
It is related.The research such as Dehon PM shows that can partly suppress Mg for the antibody of MgPa C-terminals sticks to Hep-2 cells, explanation
MgPa is a kind of important adhesion protein, and mainly attaches to host epithelial cells by its C-terminal.Since MgPa's and Mg is viscous
Attached, Infective is relevant, therefore the receptor protein interacted with MgPa is there may be on host cell.However, being so far
What only, interacted on Mg by acceptor molecule on MgPa and host cell membrane, so as to cause it to stick or intrude into place
Your Majesty's chrotoplast, there is not yet relevant report.
The acceptor of host cell surface is to determine mycoplasma genitalium to the whether susceptible key factor of host cell, and is to determine
Determine one of principal element of pathological characters of intrusion feature, circulation way and host of mycoplasma genitalium.Therefore, to genital branch
The research of substance acceptor help to illustrate from molecular level the pathogenesis of mycoplasma genitalium and its with host cell phase interaction
Relation, the receptor protein reached using labor statement stops mycoplasma genitalium and the combination of its acceptor, is preventing and treating mycoplasma genitalium
The important channel of infection.
The content of the invention
The present invention extracts the memebrane protein of human urine tract epithelial cell (SV-HUC-1cell), application enhancements using sonioation method
Virus be coated with western blotting technique (VOPBA), i.e., using prokaryotic expression and purified recombinant protein rMgPa directly with transfer
Epicyte protein component on to pvdf membrane is incubated, and screening has obtained the acceptor egg positioned at human urethra's epithelial cell membrane surface
In vain --- histone H2B.The Protein Separation is from the epicyte protein of human urine tract epithelial cell, its nucleotide sequence such as SEQ ID
Shown in NO.1, it is made up of 126 amino acid.
Present invention also offers above-mentioned receptor protein histone H2B method for separating and preparing, comprise the following steps：
(1), the memebrane protein MgPa to mycoplasma genitalium is expressed, purified, and recombinant protein rMgPa is made；
(2) memebrane protein of human urine tract epithelial cell, is extracted using sonioation method；
(3), it is coated with using improved virus in western blotting technique screening step (2) in obtained memebrane protein and restructuring egg
The memebrane protein of white rMgPa specific bonds, screening obtains the albumen of a treaty 14KDa；
(4) the about 14KDa albumen obtained in step (3), is subjected to mass spectral analysis identification, above-mentioned receptor protein group is produced
Albumen H2B.
In step (2), the human urine tract epithelial cell for taking culture form good, Trypsin Induced 5min adds culture medium
Cell precipitation is collected after terminating digestion, centrifugation 6min；Then add PBS and gently blow and beat cell precipitation, wash three times；Add cell
Lysate ice bath 20min, addition protease inhibitors PMSF, ultrasonication, 10W, 5s/ times, interval 15s, 30 times altogether, then
2500rpm/min centrifuges 10min, takes supernatant to centrifuge 30min, and precipitation is resuspended with PBS, and -80 DEG C of preservations obtain human urethra's epithelium
The Membrane protein extraction thing of cell.
Step (3) are concretely comprised the following steps：
a、SDS-PAGE
The human urethra's epithelial cell memebrane protein that will be extracted in step (2), determines and sample-loading buffer (5 ×) is added after concentration,
5min, 1000g centrifugation 1min are boiled after mixing, takes 10 μ L loadings to carry out SDS-PAGE separation, separation gel is 12%, concentration glue is
5%, deposition condition is：Concentrate glue 80V, separation gel 120V；Control group is set simultaneously；
B, transferring film and closing
(i) balance of the processing of pvdf membrane and gel：A pvdf membrane more bigger than gel fragment is cut, methanol is used
Soak and rinse 10s with deionized water again after 10-15s, 10min is balanced through transferring film buffer solution after, after electrophoresis terminates, by separation gel
In purpose fragment be positioned in transferring film buffer solution and balance 10min；
(ii) in half-dried transferring film instrument, the filter paper balanced, pvdf membrane, gel, filter paper are folded successively from top to bottom
Plus, with glass bar by removal of bubbles, transferring film is carried out in constant-pressure conditions：Condition is 15V, 30min；
(iii) pvdf membrane immersion confining liquid in, 4 DEG C overnight after with TBST rinse 5 times, each 5min；
C, Western blotting
(i) rMgPa albumen is incubated：RMgPa albumen is added, in 4 DEG C of overnight incubations, TBST is rinsed 5 times, each 5min；
(ii) it is incubated primary antibody：Add dilution 1:1000 rabbit-anti rMgPa polyclonal antibody, 37 DEG C of incubation 2h, TBST drifts
Wash 5 times, each 5min；
(iii) it is incubated secondary antibody：Add dilution 1:The goat anti-rabbit igg of 5000 HRP marks, 37 DEG C of incubation 1h, TBST rinsings 5
It is secondary, each 5min；
(iv) develop.
First molecule that acceptor runs into when being Mycoplasma genitalium infection host cell, discloses the species and structure of acceptor,
Help to further elucidate the mechanism of causing a disease of mycoplasma genitalium.The present invention screens MgPa in human urine using improved VOPBA methods
Acceptor on tract epithelial cell film, to further elucidate, Mg is possible to be sticked and mechanism of causing a disease, is further design acceptor simulation
Molecule and antagonistic molecule stop that Mg sticks or invaded host cell, and previous experiments basis is provided for Mg preventing and curing infections.Therefore it is described
Receptor protein histone H2B can be used in preparing in the targeted drug for treating or preventing Mycoplasma genitalium infection disease.
Brief description of the drawings
Fig. 1 is SDS-PAGE to human urethra's epithelial cell membrane analysis of protein figure；
Fig. 2 is the rMgPa associated proteins figures that improved VOPBA technology screenings are arrived；
Fig. 3 is the specific binding figure that indirect ELISA identifies rMgPa and H2B；
Fig. 4 is the combination figure that Western blotting identify H2B in rMgPa and epicyte protein；
Fig. 5 is positioning figure of the H2B albumen on human urine tract epithelial cell；
Fig. 6 is the indirect immunofluorescence Adherence inhibition lab diagram after rMgPa is handled by H2B；
Fig. 7 is the indirect immunofluorescence Adherence inhibition lab diagram after Mg is handled by H2B.
Embodiment
At present, analytical technique of mass spectrum plays a significant role in the research of protein-protein group.It can be utilized
Protein molecule charge-mass ratio (M/Z) difference and different Separation of Proteins is come, so as to analyze and identify agnoprotein matter.This
Mass spectral analysis is carried out to about 14kDa albumen in patent of invention, as a result shows that 14kDa receptor proteins and histone H2B have stronger
Correlation.
Obtaining receptor protein and receptor protein antibody contributes in the characteristic and function of research receptor protein, the present invention to enter one
Step utilizes H2B albumen and H2B antibody, through ELISA, Far-western blotting technique studies rMgPa and receptor protein
Interaction.As a result show that rMgPa can interact and specific bond with H2B；Specific protein in about 14kDa target stripe
Also it can be combined with rMgPa, illustrate to contain histone H2B in about 14KDa albumen.The present invention utilizes indirect immunofluorescence assay detection
Positioning of the histone H2B in human urethra's epithelial cells.As a result show, a part of fluorescence signal is on film, and a part is thin
In kytoplasm, illustrate that histone H2B is distributed on human urethra's epithelial cell membrane, because the acceptor of pathogen is concentrated mainly on host
Cell membrane surface, thus immunofluorescent test result from side reflect histone H2B albumen be probably can be mutual with rMgPa
The acceptor of effect.
Also further checking histone H2B is rMgPa acceptor in the present invention, i.e. histone H2B can mediate rMgPa and Mg
Human urine tract epithelial cell is attached to, and proves that histone H2B can suppress rMgPa and Mg to human urethra's epithelium with Adherence inhibition experiment
Sticking for cell, illustrates that histone H2B can be used to treat infectious diseases caused by Mg.
Specific embodiment described in above content is as follows：
1st, Prepare restructuring albumen rMgPa
The condition groped according to inventor's early stage, is expressed recombinant protein rMgPa, is purified, be concentrated by ultrafiltration, identify and
Endotoxin is gone to handle.It is about 2000 μ g/mL through BCA kit measurement protein concentrations.
2nd, sonioation method extracts human urethra's epithelial cell memebrane protein
(1) the human urine tract epithelial cell about 10 for taking culture form good7/ mL, Trypsin Induced 5min, add culture medium
Cell precipitation is collected after terminating digestion, 1000g, centrifugation 6min；
(2) add PBS and gently blow and beat cell precipitation, wash three times；
(3) addition cell pyrolysis liquid ice bath 20min, addition protease inhibitors PMSF, ultrasonication (10W, 5s/ times,
Have a rest 15s, 30 times altogether) and then 2500rpm/min centrifugation 10min, take the 000g of supernatant 16 to centrifuge 30min, precipitate with 300 μ L's
PBS is resuspended, -80 DEG C of preservations；
Extracted using sonioation method and SDS-PAGE analyses are carried out after human urethra's epithelial cell memebrane protein, as a result such as Fig. 1 institutes
Show in the range of 12~55KDa, all there is obvious band to occur, the memebrane protein being resuspended after ultracentrifugation determines it through BCA methods
Concentration is 600 μ g/mL, illustrates the memebrane protein for successfully extracting human urine tract epithelial cell.
3rd, the epicyte protein that improved VOPBA methods screening interacts with rMgPa
3.1 SDS-PAGE
By human urethra's epithelial cell memebrane protein of extraction, determine and take 20 μ L to add 5 μ L sample-loading buffers (5 ×) after concentration,
5min, 1000g centrifugation 1min are boiled after mixing, takes 10 μ L loadings to carry out SDS-PAGE separation.Separation gel is 12%, and concentration glue is
5%, deposition condition is：Concentrate glue 80V, separation gel 120V；Control group is set simultaneously.
3.2 transferring films and closing
(1) balance of the processing of pvdf membrane and gel：A pvdf membrane more bigger than gel fragment is cut, is put in methanol
Rinsed again with deionized water after immersion 10-15s, pvdf membrane is balanced into 10min with transferring film buffer solution.After electrophoresis terminates, by purpose
Fragment is positioned in transferring film buffer solution and balances 10min.
(2) in half-dried transferring film instrument, the filter paper balanced, pvdf membrane, gel, filter paper are folded successively from top to bottom
Plus, in constant-pressure conditions carry out transferring film after excluding bubble with glass bar：Condition is 15V, 30min.
(3) pvdf membrane immersion confining liquid in, 4 DEG C overnight after with TBST rinse 5 times, each 5min.
3.3 Western blotting
(1) rMgPa albumen is incubated：RMgPa albumen is added, in 4 DEG C of overnight incubations, TBST is rinsed 5 times, each 5min；
(2) it is incubated primary antibody：Add dilution 1:1000 rMgPa polyclonal antibody, 37 DEG C are incubated 2h, and TBST is rinsed 5 times,
Each 5min；
(3) it is incubated secondary antibody：Add dilution 1:The goat anti-rabbit igg of 5000 HRP marks, 37 DEG C of incubation 1h, TBST rinsings 5
It is secondary, each 5min；
(4) develop.
As a result it is as follows：
It is incubated by the memebrane protein of extraction after SDS-PAGE analyses and transferring film with rMgPa, and rabbit-anti rMgPa antibody is one
Anti-, the goat anti-rabbit igg of HRP marks is secondary antibody.As a result show, the swimming lane through being incubated with rMgPa is about to have at 14KDa in molecular weight
One obvious band occurs, and does not have obvious band to occur with the rMgPa swimming lanes being incubated, as shown in Fig. 2 explanation about 14KDa
Albumen may be the target protein interacted with rMgPa.
4th, mass spectral analysis identification target stripe
(1) taking the human urethra epithelial cell membrane protein 10 μ L after measure concentration to add 2.5 μ L, (5 × protein electrophoresis loading is delayed
Fliud flushing), 5min, 1000g centrifugation 1min are boiled after mixing, takes 10 μ L loadings to carry out SDS-PAGE separation.Separation gel is 12%, dense
Contracting glue is 5%, and deposition condition is：Concentrate glue 80V, separation gel 120V.
(2) band muzzles carries out tearing glue open with Thin film glove；
(3) coomassie brilliant blue staining liquid dyeing 3h, destainer decolouring 2h, high-visible to target stripe；
(4) the blade wash clean of glue will be cut, the band scaled off, which is washed with deionized water, to be brushed；
(5) target stripe is put into import EP pipes, sealed EP pipes with sealed membrane, normal temperature transport；
(6) LC-MS identifications are carried out by brightness fine horse biotechnology.
Mass Spectrometric Identification result
By LC-MS identification, ncbi database is retrieved, while measured albumen is done second mass analysis, is passed through
Protein compares the matching analysis, and histone H2B albumen scores are very high, it may be possible to the receptor protein that can be interacted with rMgPa.
5th, indirect ELISA identifies rMgPa and H2B combination situation
(1) TBS dilutes rMgPa to 100 μ g/mL, takes 150 μ L to be coated with elisa plate, and 4 DEG C of wet box are stayed overnight；Control is set simultaneously
Group；
(2) coating buffer is got rid of, is cleaned with TBST 3 times, is filled it up with and blockade liquid, 4 DEG C are blockaded 2h；
(3) board-washing 3~5 times, H2B albumen (1 is diluted with liquid is blockaded:1000), 100 μ L/ holes, 37 DEG C, 2h；
(4) board-washing 3~5 times, H2B protein antibodies (1 are diluted with liquid is blockaded:1000), 100 μ L/ holes, 37 DEG C, 2h；
(5) board-washing 6 times, add the goat anti-rabbit igg (1 of HRP marks:5000), 100 μ L/ holes, 37 DEG C, 1h；
(6) 37 DEG C, after 1h, TBST is washed 6~8 times；
(7) A is added, B developers, 37 DEG C of lucifuges are incubated 1h；
(8) terminate liquid is added, with survey light absorption value A at ELIASA 450nm.
As a result it is as follows：
RMgPa and histone H2B specific bond is detected using indirect ELISA, is as a result shown：With blank control group phase
Than rMgPa and the light absorption value of histone H2B incubation groups are more than 1.0, as shown in figure 3, positive control rabbit-anti rMgPa antibody incubations
The light absorption value of group is also greater than 1.0, and the absorbance of blank control group is less than 0.5, with statistical significance (P<0.05), these
As a result illustrate that rMgPa can interact with H2B and occur specific bond.
6th, Western blotting identify rMgPa and histone H2B specific bond
6.1 identification rMgPa and histone H2B directly in conjunction with
(1)SDS-PAGE
Take 20 μ L rMgPa to add 5 μ L (5 × protein electrophoresis sample-loading buffer), 5min, 1000g centrifugations are boiled after mixing
1min, adds 10 μ L loadings to carry out SDS-PAGE separation per hole.
(2) transferring film and closing
Transferring film is carried out in constant current conditions：Condition is 0.03A, 30min, and remaining operating procedure is shown in 3.2.
(3) Western blotting
1. histone H2B albumen is incubated：Add H2B albumen (1:1000), it is incubated 6h at 4 DEG C or stays overnight, TBST rinsings 5
It is secondary, each 5min.
2. it is incubated primary antibody：Add dilution 1:1000 histone H2B antibody, 37 DEG C incubation 2h, TBST rinse 5 times, every time
5min。
3. it is incubated secondary antibody：Add the goat anti-rabbit igg (1 of the HRP marks of dilution:5000), 37 DEG C of incubation 1h, TBST rinsings 5
It is secondary, each 5min.
4. develop
6.2 identification rMgPa and epicyte protein combination
(1)SDS-PAGE
Take 20 μ L rMgPa to add 5 μ L (5 × protein electrophoresis sample-loading buffer), 5min, 1000g centrifugations are boiled after mixing
1min, adds 10 μ L loadings to carry out SDS-PAGE separation per hole.
(2) transferring film and closing
Transferring film is carried out in constant current conditions：Condition is 0.03A, 30min, and remaining operating procedure is shown in 3.2.
(3) Western blotting
1. human urethra's epithelial cell memebrane protein is incubated：Memebrane protein is added, 4h is incubated at 4 DEG C or is stayed overnight, TBST is rinsed 5 times,
Each 5min.
2. it is incubated primary antibody：Add dilution 1:1000 histone H2B antibody, 37 DEG C incubation 2h, TBST rinse 5 times, every time
5min。
3. it is incubated secondary antibody：Add the goat anti-rabbit igg (1 of the HRP marks of dilution:5000), 37 DEG C of incubation 1h, TBST rinsings 5
It is secondary, each 5min.
4. develop.
The combination of 6.3 about 14kDa target proteins and histone H2B antibody
(1)SDS-PAGE
Take 20 μ L epicyte protein to add 5 μ L (5 × protein electrophoresis sample-loading buffer), 5min, 1000g are boiled after mixing
1min is centrifuged, adds 10 μ L loadings to carry out SDS-PAGE separation per hole.
(2) transferring film and closing
Transferring film is carried out in constant-pressure conditions：Condition is 15V, 30min, and remaining operating procedure is shown in 3.2.
(4) Western blotting
1. it is incubated primary antibody：Add dilution 1:1000 histone H2B antibody, 37 DEG C incubation 2h, TBST rinse 5 times, every time
5min。
2. it is incubated secondary antibody：Add the goat anti-rabbit igg (1 of the HRP marks of dilution:5000), 37 DEG C of incubation 1h, TBST rinsings 5
It is secondary, each 5min.
3. develop.
Identification and analysis result
RMgPa can interact with the histone H2B in epicyte protein and occur specific bond
RMgPa is carried out after SDS-PAGE and transferring film, detects that can it with epicyte protein with Western blotting
In H2B combine, as a result as shown in figure 4, after being incubated with memebrane protein and rMgPa, addition histone H2B antibody adds two
Anti- colour developing, there is specific band appearance at about 37kDa；And control group does not have band appearance, illustrate that rMgPa can be with cell membrane egg
Histone H2B specific bonds in white.
Detect positioning of the H2B albumen in human urine tract epithelial cell with indirect immunofluorescence assay, as a result as shown in figure 5,
The cell membrane of human urine tract epithelial cell, cytoplasm and nuclear area have red fluorescence, nucleus it can be seen from Fig. 5
For blueness；Illustrate in the cell with all there is H2B albumen on cell membrane.
7th, histone H2B sticks the inhibitory action of human urine tract epithelial cell to rMgPa and Mg
(1) take one bottle of growth conditions good, be paved with the human urine tract epithelial cell of Tissue Culture Flask bottom 80%~90%, carefully
Born of the same parents' cleaning solution is cleaned 3 times, Trypsin Induced, then adds 5mL culture mediums, and piping and druming is uniform；
(2) 500 μ L nutrient solutions are added into the hole (band cell climbing sheet) of 24 orifice plates, then draws the human urethra of 50 μ L suspensions
Epithelial cell is placed in 37 DEG C, 5%CO into each hole2Cultivated in incubator；
(3) rMgPa (30 μ g) and the independent preincubates of histone H2B, 4 DEG C, 2h；
(4) 100 μ L Mg suspension (1 × 107CCU/mL) with the independent preincubates of H2B, 37 DEG C, 30min；
(5) PBS (pH7.4) washs cell 3 times, and cell, 4 DEG C, 30min are then fixed with 4% paraformaldehyde；
(6) discard 4% paraformaldehyde, PBS washs 3 times, adds the closing of F-12K culture mediums, 37 DEG C, 1h；
(7) confining liquid is discarded, PBS is washed 3 times, adds histone H2B antibody into the hole of 24 orifice plates, be placed in 37 DEG C, incubate
Educate 2h；
(8) rMgPa albumen preincubated mixture and 100 μ L Mg suspension preincubated mixtures to 24 orifice plates are separately added into
Hole in, be placed in 37 DEG C, 2h；
(9) the step of adding primary antibody and secondary antibody and observation sample are identical with rMgPa and Mg adhesion assay.
As a result it is as follows：
In order to further verify that histone H2B is receptor protein that can be with rMgPa interactions, by rMgPa and histone
After H2B preincubates, then detect that rMgPa sticks situation to human urine tract epithelial cell.As a result it is as shown in Figure 6：Compared with control group,
The red fluorescence on human urethra's epithelial cell membrane surface is significantly reduced, and be there occurs during declaratives rMgPa preincubates with histone H2B
Specific bond, causes rMgPa to stick reduction to human urine tract epithelial cell.These results illustrate that histone H2B can partly suppress
RMgPa sticks to human urine tract epithelial cell.
Then, by after Mg and histone H2B preincubates, Mg pairs can be suppressed with indirect immunofluorescene assay histone H2B
Sticking for human urine tract epithelial cell, is observed under fluorescence microscope：Compared with control group, human urine tract epithelial cell in test group
Film surface and intracytoplasmic red fluorescence are significantly reduced, and the Mg for illustrating to stick and invading human urine tract epithelial cell is reduced, such as Fig. 7 institutes
Show.These results show that histone H2B albumen can partly suppress mycoplasma genitalium sticking and invade to human urine tract epithelial cell.
<110>：University Of Nanhua
<120>：With the mycoplasma genitalium MgPa receptor proteins interacted and its separation method and purposes
<160>：1
<210>：1
<211>：126
<212>：Prt
<213>：Artificial sequence
<400>：1
Met Pro Glu Pro Ala Lys Ser Ala Pro Ala Pro Lys Lys Gly Ser Lys
1 5 10 15
Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys Lys Arg Lys Arg
20 25 30
Ser Arg Lys Glu Ser Tyr Ser Ile Tyr Val Tyr Lys Val Leu Lys Gln
35 40 45
Val His Pro Asp Thr Gly Ile Ser Ser Lys Ala Met Gly Ile Met Asn
50 55 60
Ser Phe Val Asn Asp Ile Phe Glu Arg Ile Ala Gly Glu Ala Ser Arg
65 70 75 80
Leu Ala His Tyr Asn Lys Arg Ser Thr Ile Thr Ser Arg Glu Ile Gln
85 90 95
Thr Ala Val Arg Leu Leu Leu Pro Gly Glu Leu Ala Lys His Ala Val
100 105 110
Ser Glu Gly Thr Lys Ala Val Thr Lys Tyr Thr Ser Ala Lys
115 120 125
Claims (5)
1. a kind of receptor protein interacted with mycoplasma genitalium MgPa, it is characterised in that：The Protein Separation is from human urethra
The memebrane protein of epithelial cell, is histone H2B, and its nucleotide sequence is made up of as shown in SEQ ID NO.1 126 amino acid.
2. the method for separating and preparing of the receptor protein described in claim 1, it is characterised in that comprise the following steps：
(1), the memebrane protein MgPa to mycoplasma genitalium is expressed, purified, and obtains recombinant protein rMgPa；
(2) memebrane protein of human urine tract epithelial cell, is extracted using sonioation method；
(3), it is coated with using improved virus in the memebrane protein obtained in western blotting technique screening step (2) and recombinant protein
The memebrane protein of rMgPa specific bonds, screening obtains the albumen of a treaty 14KDa；
(4), by the about 14KDa albumen obtained in step (3) carry out mass spectral analysis identification, that is, obtain described in claim 1 by
Body protein histone H2B.
3. the method for separating and preparing of receptor protein according to claim 2, it is characterised in that：In step (2), culture shape is taken
The good human urine tract epithelial cell of state, Trypsin Induced 5min is added after culture medium terminates digestion, centrifugation 6min and is collected cell
Precipitation；Then add PBS and gently blow and beat cell precipitation, wash three times；Cell pyrolysis liquid ice bath 20min is added, protease is added
Inhibitor PMSF, ultrasonication, 10W, 5s/ times, interval 15s, 30 times altogether, then 2500rpm/min centrifugations 10min, takes
Heart 30min is cleared, precipitation is resuspended with PBS, -80 DEG C of preservations obtain the Membrane protein extraction thing of human urine tract epithelial cell.
4. the method for separating and preparing of receptor protein according to claim 2, it is characterised in that：The specific steps of step (3)
For：
a、SDS-PAGE
The human urethra's epithelial cell memebrane protein that will be extracted in step (2), determines and protein electrophoresis sample-loading buffer (5 is added after concentration
×), 5min, 1000g centrifugation 1min are boiled after mixing, takes 10 μ L loadings to carry out SDS-PAGE separation, separation gel is 12%, concentration
Glue is 5%, and deposition condition is：Concentrate glue 80V, separation gel 120V；Control group is set simultaneously.
B, transferring film and closing
(i) balance of the processing of pvdf membrane and gel：A pvdf membrane more bigger than gel fragment is cut, it is soaked with methanol
10s is rinsed with deionized water again after 10-15s, 10min is balanced through transferring film buffer solution after, after electrophoresis terminates, by separation gel
Purpose fragment is positioned in transferring film buffer solution and balances 10min；
(ii) in half-dried transferring film instrument, the filter paper balanced, pvdf membrane, gel, filter paper are sequentially overlapped from top to bottom, used
Removal of bubbles is carried out transferring film by glass bar in constant-pressure conditions：Condition is 15V, 30min；
(iii) in pvdf membrane immersion confining liquid, 4 DEG C overnight, and TBST is rinsed 5 times, each 5min；
C, Western blotting
(i) rMgPa albumen is incubated：RMgPa albumen is added, in 4 DEG C of overnight incubations, TBST is rinsed 5 times, each 5min；
(ii) it is incubated primary antibody：Add dilution 1:1000 rabbit-anti rMgPa polyclonal antibody, 37 DEG C of incubation 2h, TBST rinsings 5
It is secondary, each 5min；
(iii) it is incubated secondary antibody：Add dilution 1:The goat anti-rabbit igg of 5000 HRP marks, 37 DEG C are incubated 1h, and TBST is rinsed 5 times,
Each 5min；
(iv) develop.
5. the purposes of the receptor protein described in claim 1, it is characterised in that：Using histone H2B or its model molecule with
Antagonistic molecule prepares the purposes in the targeted drug for treating or preventing Mycoplasma genitalium infection disease.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004067752A1 (en) * | 2003-01-31 | 2004-08-12 | Dnavec Research Inc. | Paramyxovirus vector encoding ribozyme and utilization thereof |
| CN101759781A (en) * | 2008-12-25 | 2010-06-30 | 上海市第六人民医院 | Protein adhered to surface layers of bacteria and application thereof |
| CN101782585A (en) * | 2010-02-10 | 2010-07-21 | 广州医学院 | Lung cancer tissue protein blotting membrane and preparation method thereof |
| WO2015117057A2 (en) * | 2014-01-31 | 2015-08-06 | The Scripps Research Institute | Protein m related immunoglobulin-binding polypeptides |
-
2017
- 2017-06-09 CN CN201710432872.9A patent/CN107253987B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004067752A1 (en) * | 2003-01-31 | 2004-08-12 | Dnavec Research Inc. | Paramyxovirus vector encoding ribozyme and utilization thereof |
| CN101759781A (en) * | 2008-12-25 | 2010-06-30 | 上海市第六人民医院 | Protein adhered to surface layers of bacteria and application thereof |
| CN101782585A (en) * | 2010-02-10 | 2010-07-21 | 广州医学院 | Lung cancer tissue protein blotting membrane and preparation method thereof |
| WO2015117057A2 (en) * | 2014-01-31 | 2015-08-06 | The Scripps Research Institute | Protein m related immunoglobulin-binding polypeptides |
Non-Patent Citations (1)
| Title |
|---|
| TACHIWANA,H.等: "Chain D, Histone H2b Type 1-j，PDB: 3A6N_D", 《GENBANK数据库》 * |
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