CN104634979A - Method for screening and identifying microbial cell surface antigen - Google Patents
Method for screening and identifying microbial cell surface antigen Download PDFInfo
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- CN104634979A CN104634979A CN201310574194.1A CN201310574194A CN104634979A CN 104634979 A CN104634979 A CN 104634979A CN 201310574194 A CN201310574194 A CN 201310574194A CN 104634979 A CN104634979 A CN 104634979A
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Abstract
The invention discloses a method for screening and identifying a microbial cell surface antigen, and relates to a method for conveniently and fast screening and identifying the microbial cell surface antigen. The method comprises the following steps: immuno-precipitating and separating a cell surface protein antibody by using microbial whole cells, capturing an antigen-antibody complex by using protein A/G agarose, and eluting to obtain the antigen-antibody complex, and analyzing an immuno-precipitated protein by using SDS-PAGE; analyzing the abundance of immunogen, the affinity of the antibody to the antigen and the determination of the immunogen, and determining the protein type of the antigen; and establishing an immunoprecipitation-based proteomic technology for performing high-flux screening on the protein with immunogenicity as candidate target position of multivalent vaccine. The possibility of the antigen as a vaccine protein can be determined according to the abundance of the immunogen and the affinity of the antibody to the antigen. The protein with the immunogenicity on the certain microbial cell surface can be fast determined, and the method is efficient, fast, economic and practical. The method has universality, and can be used for identifying both bacteria, fungus and other microbial immunogens.
Description
Technical field
The present invention relates to a kind of method of easy, quick screening and identification microbial cell surface proteantigen.
Background technology
Already proved, and adopted vaccine inoculation to be the immunologic intervention approach preventing microbial diseases effective, economic.Microbial cell surface albumen is owing to being positioned cell surface, directly contact with host immune system, its stimulate body produce specific antibody and corresponding microbial cell surface protein combination after, infected by microbes is suppressed by neutralization, or the engulfing of mediated immunity cell, can play a role in the pathogen invasion initial period.Therefore microbial cell surface albumen is good vaccine candidate albumen, is also the focus of research.As scholar finds that vibrios cell surface protein OmpA, OmpU etc. have good immunogenicity and immune protective, can be used as the candidate albumen of vaccine.In the screening of microbial cell surface proteantigen, what routine immunization protein science adopted is first separate microorganism cell surface protein, then carries out immunoblotting analysis screening, but extraction cell surface protein, particularly there is certain difficulty in outer membrane protein, and be separated the two dimensional electrophoresis complex operation of outer membrane protein, poor repeatability, can not show low copy albumen, extreme isoelectric point albumen.And existing extraction and analysis microbial cell surface albumen, destroy the natural consequence of cell surface protein, immunoreactive process in body can not be reflected very well.This limits its application to a certain extent.
Summary of the invention
The present invention aim to provide a kind of simply, the method for screening and identification microbial cell surface antigen efficiently, its technology is the microbial cell surface protein antibodies generation immune response utilizing immunoprecipitation to make in microbe whole-cell and microorganism antiserum, ultrasonic process antibody and microbe whole-cell compound, then use albumin A/G agarose capture antigen antibody complex.SDS-PAGE simple separation antigen antibody complex, determines to have immunogenic protein, finally adopts these immunogenes of mass-spectrometric technique analysis and identification.
The concrete steps of the screening and identification method of the present invention's said microbial cell surface antigen are as follows:
1) cell surface protein antibody is isolated with microbe whole-cell immunoprecipitation: microbial cell collects rear and microorganism antiserum antiserum by volume: microorganism=1:500 adds antiserum ice bath and hatches, negative serum in contrast, centrifugal segregation antiserum cell surface antibodies other compositions outer, collect microbial cell and cell surface antibodies compound;
2), by isolated microbial cell and cell surface antibodies compound, after ultrasonic disruption, hatch with albumin A/G agarose and ice bath, capture antigen antibody complex, after washing three times, wash-out antigen antibody complex;
3) the antigen antibody complex SDS-PAGE after wash-out analyzes, observations is contaminated with silver, negative control does not have or seldom, experimental group has or a lot of protein bands, namely all albumen in this band have immunogenicity, re-use super filter tube to antigen antibody complex carry out concentrated after carrying out SDS-PAGE electrophoresis, the positive band that coomassie brilliant blue staining occurs prepares to carry out mass spectrum;
4) immunogenic qualitative: take out to have and immunogenicly all examine dsred protein band, mass spectrum sample disposal route carries out sample preparation routinely, then carries out mass spectrum and analysis of biological information, to determine the kinds of protein of antigen.
Said microorganism is bacterium, fungi.
The output power adopted during ultrasonication, time, number of times and the time interval can adjust by research object, generally can adopt each 3 seconds, 4 seconds, interval, ultrasonic 2min.
Said microorganism antiserum is the specific antisera obtained with microbe whole-cell immune animal.
The strategy that the present invention takes is different from traditional immunoproteomics, first carry out microbial cell surface albumen and antiserum carries out immune response, simple separation has immunogenic microbial cell surface albumen again, finally utilizes mass-spectrometric technique and bioinformatic analysis to determine the kind of these microbial surface protein antigens.Thus establish a kind of microbial cell surface proteantigen triage techniques based on immunoprecipitation newly, there is immunogenic microbial cell surface protein using the candidate's target position as polyvaccine for high flux screening.According to immunogenic abundance and the affinity with antibody, its effect as immunogenic possibility and effect thereof can be determined.What the present invention can determine rapidly certain microbial cell surface has immunogenic all protein, thus reaches efficient, quick, economic, practical preferred immunogenic object.The present invention can have immunogenic vaccine candidate site by high flux screening, for determining that the gene as polyvaccine target position is laid a good foundation, improves the efficiency of preparation polyvaccine target spot.Because all microorganisms are as after antigen-immunized animal, body all can produce the specific antibody for its antigen, and these antibody can be used for immunoprecipitation experiment, screening immunogene, and the present invention is without two dimensional electrophoresis isolated cell surface protein, greatly experimental procedure must be simplified.Therefore the principle of this experiment and method have ubiquity, operation steps simple and fast, can efficient for the qualification of the microbial cell surface such as bacterium, fungi antigen.
The present invention has further developed the application of immunoprecipitate, namely establishes a kind of microbial cell surface antigen selection technology based on immunoprecipitation, thus can determine the microbial cell surface protein that reacts with microorganism antiserum.Because this design is based on the simplest experimental technique, therefore not only can determine the cell surface antigen that certain Institute of Micro-biology has fast, can also learn that it is in the abundance of cell surface and the affinity with antibody.According to these immunogenic cell surface abundance and the affinity with antibody, determine as multivalence target position vaccine possibility and judge its action effect.
Accompanying drawing explanation
Fig. 1 is the experiment flow figure of microbial cell surface proteantigen screening
Fig. 2 is the electrophoresis result of pathogenic microorganism vibrio parahaemolytious VPL4-90 surface protein antigen screening.
Wherein, the silver dye SDS-PAGE collection of illustrative plates of A, vibrio parahaemolytious VPL4-90 surface protein antigen screening, comprises control group and experimental group; The experimental group of B, vibrio parahaemolytious VPL4-90 surface protein antigen screening examines dye SDS-PAGE collection of illustrative plates.
Fig. 3 is the electrophoresis result of pathogenic microorganism vibrio mimicus ATCC33653 surface protein antigen screening.
Wherein, the silver dye SDS-PAGE collection of illustrative plates of A, vibrio mimicus ATCC33653 surface protein antigen screening, comprises control group and experimental group; The experimental group of B, vibrio mimicus ATCC33653 surface protein antigen screening examines dye SDS-PAGE collection of illustrative plates.
Embodiment
Below implement to be described further processing step of the present invention and outstanding effect thereof by reference to the accompanying drawings.
Embodiment 1
1, microbe growth, counting and collection: microorganism adopts vibrio parahaemolytious VPL4-90, for bacterial classification is preserved in this laboratory.VPL4-90 is inoculated in LB nutrient culture media, 18h cultivated by 28 DEG C of shaking tables.After cultivation terminates, get 2mL bacterium liquid and carry out plate count.All the other nutrient solutions, in the centrifugal 10min of 5000rpm, collect thalline, with brine thalline 3 times.Adjusting its concentration with physiological saline is 10
9cFU/mL.
2, the sero-fast preparation of anti-microbial pathogen: the VPL4-90 thalline suspension to preparation adds volume fraction 0.025% formaldehyde, act on 4h at 30 DEG C, make deactivation cause of disease, 5000rpm rotating speed 10min, use brine again three times, finally adjusting its concentration with physiological saline is again 10
9cFU/mL, injection new zealand white rabbit, every 1mL, control group injecting normal saline; Inject week about once, within the 4th time immune the 7th day, put to death White Rabbit and get blood, leave standstill; The centrifugal 10min of 4000rpm, takes out serum, packing ,-20 DEG C of preservations.
3, the immunoprecipitation of microbial cell surface protein antibodies, 10 of 5mL
9cFU/mLVPL4-90PBS suspension adds 1 μ LVPL4-90 thalline antiserum, and 5mLVm PBS suspension adds 1 μ LVm thalline antiserum vibration mixing, and ice bath, 50rpm incubator overnight, adds negative serum in contrast simultaneously separately.Collected by centrifugation thalline, PBS washs three times, and the immunoprecipitation lysate respectively adding 0.5ml is resuspended, ultrasonication (300W, 4s, 3s, 2min), adds in immunoprecipitation post resin being housed and clog with stopper, ice bath, 50rpm8h.Immunoprecipitation post immunoprecipitation cleansing solution washs three times, with condition buffer solution once, then hatch wash-out after 5min with eluent.
4, SDS-PAGE electrophoresis and silver dye are observed: the immunity of collecting is sunk to the bottom compound and negative control carries out SDS-PAGE electrophoresis, and after electrophoresis terminates, with cma staining, scanning, exports photo.See Fig. 1-A, result shows, and except heavy chain of antibody and light chain connect except a band, more clearly albumen one positive protein bar is with 8.
5, sample concentration and examine dye mass spectrum: the sample collecting the immunoprecipitation post of 10 experimental group, with super filter tube after 5000rpm30min is concentrated 10 times, loading carries out SDS-PAGE electrophoresis, and after electrophoresis terminates, scanning, exports photo.See Fig. 1-B, except heavy chain of antibody and light chain connect except a band, more clearly bar is with 8.Cut out and deliver to contaminating 6 consistent bands with silver Tandem Mass Spectrometry Analysis and the qualification that Hua Da gene and Shanghai Bo Yuan carry out matrix assisted laser desorption ionization, qualification result is in table 1.6 protein bands identify 6 albumen, wherein there are 4 Outer membrane protein antigens being vibrio parahaemolytious cell surface, 2 is vibrio parahaemolytious intracellular protein, but show in some researchs, analyze from proteomics, some intracellular proteins also find at microbial cell surface, and its mechanism principle awaits further investigation.
The MALDI-TOF-TOF/MS qualification of table 1 vibrio parahaemolytious cell surface antigen
Embodiment 2 is except bacterium changes vibrio mimicus ATCC33653, the clear interpolation of 1:250 by volume into, and all the other are all identical with embodiment 1.Result is as follows
1, the SDS-PAGE collection of illustrative plates of the cell surface protein of vibrio mimicus immunoprecipitation, in the running gel examining dye, has 6 protein bands consistent with the positive band that silver contaminates, is numbered a ~ h respectively.The silver dye of vibrio mimicus immunoprecipitation complex is observed and is examined dye and mass spectrographicly the results are shown in Figure 2-A and 2-B.
2,2 be the results are shown in Table to the Mass Spectrometric Identification of 6 protein bands.6 protein bands identify 8 albumen, wherein have 4 Outer membrane protein antigens for vibrio mimicus cell surface, and 2 is the inner membrane protein antigen of vibrio mimicus, and 1 is the protein translation correlation factor that possible be positioned at vibrio mimicus surface, and 1 albuminous cell location is unknown.
The MALDI-TOF-TOF/MS qualification of table 2 vibrio mimicus cell surface antigen
Claims (5)
1. the selection systems method of microbial cell surface antigen, is characterized in that its step is as follows:
1) cell surface protein antibody is isolated with microbe whole-cell immunoprecipitation: microbial cell collects rear and microorganism antiserum antiserum by volume: microorganism=1:500 adds antiserum ice bath and hatches, negative serum in contrast, centrifugal segregation antiserum cell surface antibodies other compositions outer, collect microbial cell and cell surface antibodies compound;
2), by isolated microbial cell and cell surface antibodies compound, after ultrasonic disruption, hatch with albumin A/G agarose and broken liquid ice bath, capture antigen antibody complex, after washing three times, wash-out antigen antibody complex;
3) the antigen antibody complex SDS-PAGE after wash-out analyzes, and contaminates observations, determine positive band with silver, re-use super filter tube to antigen antibody complex carry out concentrated after carrying out SDS-PAGE electrophoresis, coomassie brilliant blue staining prepares to carry out mass spectrum;
4) immunogenic qualitative: take out to have and immunogenicly all examine dsred protein band, mass spectrum sample disposal route carries out sample preparation routinely, then carries out mass spectrum and analysis of biological information, to determine the kinds of protein of antigen.
2. the selection systems method of microbial cell surface antigen as claimed in claim 1, is characterized in that said microorganism is bacterium, fungi.
3. the selection systems method of microbial cell surface antigen as claimed in claim 1, is characterized in that adopting microbe whole-cell immunoprecipitation to be separated from antiserum by the antibody of microbial cell surface albumen.
4. the selection systems method of microbial cell surface antigen as claimed in claim 1, is characterized in that each 3s of ultrasonication, interval 4s, ultrasonic 2min.
5. the selection systems method of microbial cell surface antigen as claimed in claim 1, is characterized in that determining immunogenic protein band with the observation of silver dye, carries out mass spectrophotometry with coomassie brilliant blue staining.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003025568A2 (en) * | 2001-09-14 | 2003-03-27 | Reinhard Zeidler | Identification of antigens by xenogenic, allogenic or autologous antibody-mediated precipitation |
| CN1563982A (en) * | 2004-04-14 | 2005-01-12 | 厦门大学 | Bolting and identifying method for microbial cross antigen |
| CN101143216A (en) * | 2007-07-10 | 2008-03-19 | 中山大学 | Escherichia coli TolC antibody targeting effect improving drug-resistant bacteria sensitivity to antibiotic technology |
| CN102251013A (en) * | 2011-02-22 | 2011-11-23 | 北京市肿瘤防治研究所 | Antibody and antigen for recognizing tumor initiator cell and application thereof |
| CN103160587A (en) * | 2013-04-02 | 2013-06-19 | 南开大学 | Genetic typing chip of 10 common pathogenic legionella and detection kit |
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2013
- 2013-11-15 CN CN201310574194.1A patent/CN104634979A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003025568A2 (en) * | 2001-09-14 | 2003-03-27 | Reinhard Zeidler | Identification of antigens by xenogenic, allogenic or autologous antibody-mediated precipitation |
| US20050037437A1 (en) * | 2001-09-14 | 2005-02-17 | Reinhard Zeidler | Identification of antigen by xenogenic, allogenic or autologous antibody-mediated precipitation |
| CN1563982A (en) * | 2004-04-14 | 2005-01-12 | 厦门大学 | Bolting and identifying method for microbial cross antigen |
| CN101143216A (en) * | 2007-07-10 | 2008-03-19 | 中山大学 | Escherichia coli TolC antibody targeting effect improving drug-resistant bacteria sensitivity to antibiotic technology |
| CN102251013A (en) * | 2011-02-22 | 2011-11-23 | 北京市肿瘤防治研究所 | Antibody and antigen for recognizing tumor initiator cell and application thereof |
| CN103160587A (en) * | 2013-04-02 | 2013-06-19 | 南开大学 | Genetic typing chip of 10 common pathogenic legionella and detection kit |
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