CN1201464A - Helicobacter pylori protein - Google Patents
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- CN1201464A CN1201464A CN96198105A CN96198105A CN1201464A CN 1201464 A CN1201464 A CN 1201464A CN 96198105 A CN96198105 A CN 96198105A CN 96198105 A CN96198105 A CN 96198105A CN 1201464 A CN1201464 A CN 1201464A
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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Abstract
The present invention provides a novel antigenic protein obtainable from the coccoid form of H. pylori. The use of the antigenic protein in methods of detecting H. pylori, as well as in the preparation of vaccines, is also disclosed. A method of culturing the coccoid form of H. pylori is also provided.
Description
The present invention relates to a kind of neoantigen that derives from the helicobacter pylori of coccoid form, it is generally used in the medicine, and in the vaccine preparation and in the diagnosis of the detection of coccoid form and helicobacter pylori infection, and curee's disease prognosis is measured.
Helicobacter pylori is a kind of gram-negative bacteria, usually relates to (Marshall etc., Australian medical journal, 142:439-444 (1985) in chronic active gastritis and ulcer of digestive system disease; Buck, G.E., clinical microbiology magazine, 3:1-12 (1990)).In vitro culture, there are two kinds of tangible morphology form in helicobacter pylori, educable spiral sample form and coccoid form (Marshall etc., microbiology communication, the 25:83-88 (1984) that can not cultivate; Kung, J.S.L. and HO, B., by F.Megraud and H.Lamouliatte, Bordeaux, France (1988) editor discusses (summary P9) about gastroduodenal pathology and Campylobacter pylori).When spiral helicine bacterial exposure in air the time, is surpassed about 2 hours and then can not survive.Under the condition of discomfort, the spirrillum differentiation becomes coccoid (Vijayakumari and Ho, gastroenterology Belgium journal, 56:101 (1993)).
Up to the present, only have that one piece of coccoid is external successfully to be converted into spiral helicine report, this experiment has proved unrepeatable (Mai etc., edit by F.Megraud and H.Lamouliatte, Elsevier scientific publication merchant (1989), gastroduodenal pathology and Campylobacterpylori, the 28-33 page or leaf.Though coccoid external formation can or be removed nutrition and induce (Nilius etc. by microbiotic, Zbl.Bakt.280:259-272 (1993), but the research of coccoid form is owing to lack about the effect of helicobacter pylori in the information of this form and the life cycle and shortage and be used to obtain any method of synchronized culture of this form and interrupted.Up to now, the form that helicobacter pylori of coccoid form is considered as effectively " death ", can not survive, its function in the life cycle of bacterium is unclear.
The result of this paper discussion shows that the coccoid form can exist with a kind of viable form really, and works really in the life cycle of helicobacter pylori, has shown in this diagnosis that acts on helicobacter pylori infection, prognosis and the treatment.
At present, identified the helicobacter pylori spiral form various antigens (referring to, for example, WO93/22682), and be used for, for example, detect in the diagnostic kit of helicobacter pylori, for example the HELISAL that puts on market by CORTECS company limited
TMMethod of inspection.Yet result described herein shows: for guaranteeing accurately and the diagnosis of (promptly only coccoid infection) fully, coccoid form that can bacterial detection is necessary.Now, do not identify the specific antigens of coccoid form.
Like this, first aspect, the molecular weight that the invention provides the natural PAGE mensuration of usefulness that can be obtained by the helicobacter pylori of coccoid form is the antigen protein of 60kDa.Specifically, this protein can be characterized by further, and it has following N terminal amino acid sequence:
D-T-H-K-S-E-I-A-H-R-F-N-D-L-G。
The uniqueness of supposing this proteinic antigenicity and its coccoid form exists, and it is useful as a kind of means of the existence of the helicobacter pylori that detects the coccoid form.
Therefore, second aspect the invention provides the purposes of antigen of the present invention on the antibody that detects anti-helicobacter pylori (especially detecting the coccoid form).In addition, neoantigen of the present invention can be used in combination with other antigen (particularly those can be obtained by the spiral form of helicobacter pylori), so that the more method of sensitive detection helicobacter pylori to be provided.
In addition, coccoid antigen of the present invention can be used for generation and can be used for detecting the antigen antibody of (comprising the antigen that exists as a complete coccoid part).Therefore, for example, antibody can be labeled, and as detecting coccoid a kind of means utilization in tissue sample and the analogue.The method of utilizing antigen to produce antibody is known to the technician, and it is also like this that mark is used for the means of this antibody-like of these class methods.Like this, the third aspect the invention provides the purposes of coccoid antigen of the present invention in antibody (for example polyclonal antibody) preparation.On the other hand, the invention provides the purposes of such antibody (comprising the antibody that produces at coccoid antigen of the present invention) on the helicobacter pylori that detects the coccoid form.
Antigen of the present invention also finds purposes as the part of antigen composition, and said composition can comprise the antigen of the helicobacter pylori of anti-spiral and two kinds of forms of coccoid.Therefore, the 6th aspect, the invention provides the composition that comprises antigen protein of the present invention, this protein can also can not planted other Heliobacter pylori antigen with one or more and be combined, these one or more plant other antigen and can obtain by the helicobacter pylori of spiral sample or coccoid form.
The 7th aspect the invention provides the method for the helicobacter pylori that detects the coccoid form, and for example by detecting antibody, this comprises the step that antigen of the present invention or antigen composition of the present invention contact with sample.Usually, sample is a biological sample, for example, and blood sample, urine samples or saliva sample.Antigen or antigen composition are directly contacted with biological sample.Can at first handle biological sample so that make it preferably, for example, adjust pH etc. by filtering.The example of the method that is fit to is those described in UK Patent Application 9422991.1.
Eight aspect the invention provides the method that is used for diagnosing helicobacter pylori infection, comprises the step that antigen protein of the present invention is contacted with the biological sample that derives from the curee.As described herein, antigen can provide with the form of antigen composition.Usually, diagnostic method of the present invention also will comprise other detection of antigens step of the helicobacter pylori that can derive from spirrillum or coccoid form.A kind of more sensitive method of diagnosing helicobacter pylori infection in this way, is provided.Suitably be that diagnostic method of the present invention carries out on blood sample, urine samples or saliva sample.
Suitably be that diagnostic method of the present invention will utilize testing apparatus or test kit (HELISAL for example
TMUse in the test) carry out.Therefore, on the other hand, the invention provides the test kit that is used for diagnosing helicobacter pylori infection that comprises antigen protein of the present invention.Usually, test kit of the present invention will comprise that also one or more plant the antigen that other can derive from the helicobacter pylori of spirrillum or coccoid form.
Identifying that unique antigen of the present invention also makes provides the anti-helicobacter pylori vaccine to become possibility, and this vaccine is activated on the bacterium of anti-spiral and two kinds of forms of coccoid.Therefore, the tenth aspect, the present invention comprises the of the present invention antigenic vaccine of planting adjuvant and/or carrier with one or more for the infectious prevention of helicobacter pylori or treatment provide.In this embodiment preferred on the one hand, vaccine comprises that one or more derive from the antigen of the helicobacter pylori of spiral form.Be that these additional antigens will comprise at least a antigen to spiral sample form uniqueness aptly.
In an alternative embodiment, vaccine can comprise the helicobacter pylori self (kill or " live ") of coccoid form, because cell can dissolve induction of immunity reaction then in gi tract.In addition, can on expressing the basis of neoantigen, they utilize in the form of the helicobacter pylori that exists between real coccoid form and the real spiral sample form.In other words, the helicobacter pylori of coccoid form or intermediate form is used to send as carrier and passs neoantigen to obtain immune response.
In addition, determine: neoantigen of the present invention can be used for detecting the IgM antibody that children produce in the reaction to helicobacter pylori infection.Therefore, the tenth on the one hand, the invention provides the method that detects the IgM antibody of the helicobacter pylori of anti-coccoid form among the children, and this method comprises the step that antigen of the present invention is contacted with the biological sample that obtains from children.Be that biological sample is blood sample, urine samples or saliva sample aptly.
As described herein, now designed the method for the helicobacter pylori that is used to cultivate the coccoid form, so it is to survive.Like this, last aspect, the invention provides the method for the helicobacter pylori of cultivating the coccoid form, this method includes increases the step of carbonic acid gas in the substratum of the helicobacter pylori that comprises spiral sample form regularly, so that the transformation to the coccoid form takes place, wherein, the coccoid form of acquisition is to survive.Aptly, added twice CO in one day at least
2, and make nine weeks of cultivation to guarantee transformation.
Describe the present invention with reference to the following example, these embodiment should not be construed as limiting the invention.Embodiment relates to accompanying drawing, wherein:
Fig. 1: show to be grown in typical growth curve with the helicobacter pylori in pH and the simultaneously-measured chemostat of urase;
Fig. 2: be presented at (a) spirrillum that phase microscope, amplification * 1000 time see and (b) coccoid wet preparation.The density of spirrillum cell is uniformly, and coccoid has two types: (A) have dense cytoplasmic densification and (B) have those of loose kytoplasm as " blood shadow " cell;
Fig. 3: show coccoid transmission electron microscope photo, amplify * 80,000;
Fig. 4: show coccoid transmission electron microscope photo, amplify * 80,000 with flagellum;
Fig. 5: show silver-colored painted SDS-PAGE protein profile.Swimming lane: 1. high molecular mark (Sigma): 2. lower molecular weight mark (Sigma): the 3. spiral helicine V of the coccoid NCTC 11637:5. of spiral helicine NCTC 11637:4.
2: 6. coccoid V
2
Fig. 6: show (a) spirrillum and (b) the painted smear of periodic acid schiff of coccoid modification, amplify * 1000;
Fig. 7: show the gramstaining of coccoid modification, wherein, (a) handle, (b) do not carry out this processing with the ptyalin αDian Fenmei;
Fig. 8: the DNA that shows helicobacter pylori.The spiral helicine V of the coccoid NCTC 11637:4. of the spiral helicine NCTC 11637:3. of λ DNA:2. that the logical 1.HindIII of swimming cuts
2: 5. coccoid V
2
Fig. 9: show the Albert coloration result of modifying, amplify * 1000;
Figure 10: show the detection of the urease activity on the PAGE.Swimming lane: 1. high molecular mark (Pharmacia): the 2. spiral helicine V of the coccoid NCTC 11637:4. of spiral helicine NCTC:11637:3.
2: 5. coccoid V
2
Figure 11: show the painted natural PAGE protein profile of silver.Swimming lane: 1. high molecular mark (Pharmacia): the 2. spiral helicine V of the coccoid NCTC 11637:4. of spiral helicine NCTC 11637:3.
2: 5. coccoid V
2
Figure 12: show under the non-sex change condition, with the antigenic Western immunoblotting of coccoid.Swimming lane: 1. molecular weight marker (Pharmacia): the anti-spirrillum serum of anti-spirrillum serum: B. before the A. immunity: the anti-coccoid serum of anti-coccoid serum: D. before the C. immunity;
Figure 13: show coccoid indirect fluorescent antibody test, amplify * 1000, wherein can see, as the spirillum sample, coccoid fluorescence under the UV-light, the surface antigen that shows them is similar.
(a) preparation of bacterial isolates and helicobacter pylori differentiated form.
Utilization is from the helicobacter pylorus bacteria strain V of the isolating locality of patient of non-ucler dyspepsia
2The initial cultivation at chocolate blood agar (CBA) of this bacterial strain gone up to check purity.
Then, plate culture is used as the inoculum that 250ml comprises flat round vase of Schott of 30ml BHIH (with 10% horse serum and the additional brain heart perfusion of 0.4% yeast extract), and cultivated 72 hours at 37 ℃.This becomes the inoculum or the batch culture of chemostat conversely.
As describing among Ho and the Vijayakumari, set up the 1.5L fermentor tank (microorganism, 76:59-66 (1993)) that comprises 540ml BHIH.Substratum with the helicobacter pylori culture of 2 * 30ml, 3 ages in days with 1: 10 (inoculum: ratio inoculation culture).Therefore, supply with twice of every day carbonic acid gas, each five seconds, and the feedback control maintenance dissolved oxygen of the impeller speed by about 35-40rpm.With timed interval extraction sample, and the activity of inspection urase, pH, viability, and microscopy metamorphosis.
Culture keeps reaching 3 months under such condition, during this period, continues to monitor every day cell.When observing homogeneous phase/synchronized culture thing, with 10,000g was by centrifugal 40 minutes harvested cells, and washing once more.Then, utilize the glycine method (Ho, B., and Jiang, B., the European magazine of gastroenterology and hepatopathy, 7:121-124 (1995)) of modifying that precipitation is used to prepare coccoid antigen.
Also can use the 1L Schott round-bottomed bottle or side arm and the Erlenmeyer flask that the driving fit rubber stopper is housed of 1L band that comprise 270ml BHIH.Drill diameter is that the hole of 7mm is to hold the disposable filtering unit that comprises 0.22 μ m filter paper (for example Gelman) with 50mm diameter.The BHIH of each 270ml inoculates with the 3 age in days helicobacter pylori cultures of 30ml.Filter paper by 0.22 μ m is supplied with carbonic acid gas, twice of every day.
Culture at the incubation in the incubator (New Brunswick) that shakes that keeps reaching 37 ℃ of nine weeks with 90rpm, then, by with 10, centrifugal 40 minutes of 000g, harvested cell.As mentioned above, the washed cell precipitation prepares antigen once more.
The culture of cultivating 48 hours on chocolate blood agar is used to provide " synchronously " spirillum sample.
(ii) serum sample.
The serum of 50 gastroduodenal disease patients and 50 blood donors is provided by the D Kuperan doctor close friend of the national university hospital of the A/Prof K M Fock of Toa Payoh hospital and Singapore respectively.
(iii) ELISA and Western immunoblotting.
According to Khia and Ho, the method for Biomed.Letts.50:71-78 (1994) is carried out indirect ELISA and Western immunoblotting.
The result
Fig. 1 shows the specific activity of viability, pH and the urase of typical culture.After can seeing for 9 weeks from this figure, culture has become coccoid culture.This microscopy of being gone up the spiral of growth by CBA does not go out to confirm.Spirillum sample form is divided into the continuous supply of the time-dependent of coccoid form in carbonic acid gas.
Be clear that also the coccoid of two kinds of forms is arranged.A kind of have thick kytoplasm, and another kind of presentation just like " blood shadow ".A kind of form in back is considered to inviable.(Nilius etc., down together) compare with other report, and chemostat is cultivated and shown that great majority are thick coccoids.Also gather in the crops these coccoids, and be suspended among the BHIH that replenishes with 20% glycerine.Then, under-80 ℃, preserve the coccoids of suspension up to needs.
ELISA.
By ELISA, the helicobacter pylori IgG antibody of 37 (74%) is seropositive (table 1) to the antigen of coccoid and spirillum sample among 50 patients.In 50 blood donors, find that 16 (32%) and 14 (28%) is serum reactivity (Table I) to coccoid and the antigenic IgG antibody of spirillum sample respectively.Utilize spirillum sample antigen standard as a comparison, IgG ELISA is respectively 98 and 94% to antigenic sensitivity of coccoid and specificity.
Table 1. with ELISA detect to coccoid and
The serum IgG antibody of spirillum sample Heliobacter pylori antigen
| Patient (n=50) | Blood donor (n=50) | |||
| Spirillum sample helicobacter pylori | Positive | Negative | Positive | Negative |
| Positive | ????36 | ????1 | ???14 | ????2 |
| Negative | ????1 | ????12 | ????- | ????34 |
Sensitivity: 98%
Specificity: 94%
These results show: when using coccoid specific antigens and spiral antigen, reached the verification and measurement ratio greater than 4% in the sample that uses.Therefore, be clear that better diagnosis and treatment will be caused by the utilization of coccoid specific antigens.
The Western engram analysis
By the Western engram analysis, serum IgG demonstrates the similar main protein band of identification in coccoid and spirillum sample antigen.Conservative protein matter band in detected two kinds of antigens is 128,116,110,95,91,66,60,54,50 and 33kDa.
Bacterial isolates
Two bacterial strains of helicobacter pylori have been utilized, local helicobacter pylorus bacteria strain V
2(mention in the foregoing description 1, separate) and reference culture NCTC 11637 from non-ucler dyspepsia disease patient.
The culture preparation
Prepare coccoid and spirillum sample culture as the method described in the embodiment 1.For coccoid, (Ho and Vijayakumari, down together) as described makes the batch culture growth.One little duplicate samples is sterilely shifted out at a certain time interval to be evaluated at cultivating one's ability on the chocolate blood agar, and the survival number utilizes Miles and Misra technology (Miles and Misra, hygiology magazine, 38:732-738 (1938) counting.Coccoid per-cent utilizes Neubauer bacterial cell counting chamber to assess under phase microscope by the number of the relative spirillum sample of coccoid in calculating three parts.Utilize the specific activity of the phenol spectrophotometry urase of Hamilton-Miller and Gergan (together following), monitor the pH of substratum simultaneously.
The opticmicroscope of culture and electron microscope microscopy
Utilize phase microscope to observe the motility and the morphology of culture.The Gram's stain dyeing of the smear utilization improvement of air dried culture was wherein carried out 10-30 minute rather than common 1 minute with the pinkish red negative staining of the phenol of dilution.
In the periodic acid schiff dyeing of polysaccharide, the method of Hotchkiss (biochemical information, 16:131-141 (1948)) is used with respect in the pinkish red sulphite normal 15-45 minute and improve with 45 minutes the longer 90 minutes dyeing times of negative staining that malachite green prolongs.
The painted Laybourn of the Albert of volutin granules's agent improves (Cruickshank, medical microbiology: the laboratory diagnosis of infection and control guide, pp656-657 (1968)) to coccoid with 45 minutes longer dyeing time rather than be generally used for further improvement in 3-5 minute of spirillum sample.
, dry on copper mesh the cell fixation that is used for transmission electron microscope in 4% glutaraldehyde, the phospho-wolframic acid with 0.5% (pH6.8) negative staining.Utilize Philips JOEL-JEM-1200EX transmission electron microscope observation grid.
Biochemical measurement
Utilize commercial band API ZYM test kit 2520 to determine the existence and the biotype (Kung etc., medical microbiology magazine, 29:203-206 (1989)) of 19 different enzymatic reactions.The urase specific activity utilizes the phenol spectrophotometer method of Hamilton-Miller and Gergan quantitatively to determine (the urology of investigation, 15:327-328 (1979)), and Protein content is by improved Lowry test determination (Schacterle and Pollack, analytical biochemistry, 51:654-655 (1973)).ATP utilizes bioluminescence assay test kit quantitative assay (biological track, Finland), and polysaccharide content is by measuring (carbohydrate analysis: a kind of feasible method, pp1-2 as the L-hcy thiolactone acidity test method of being described by Chaplin and Kennedy.Edit the Oxford University by M.F.Chaplin and J.F.Kennedy: IRL press (1986)).
The extraction of DNA and microdetermination
Extract the DNA (infecting and immunity 57:623-629 (1988)) of two kinds of forms and electrophoresis on 1% sepharose according to the method for Clayton etc.Total dna content of each cell is measured (analytical biochemistry, 83:252-257 (1977)) according to Kapuscinski and Skoczylas method.
Protein profile and Western immunoblotting assay
The protein profile is illustrated (nature, 227:680-685 (1970)) according to the method for Laemmli by polyacrylamide gel electrophoresis (PAGE).In the natural PAGE of non-sex change, all protein of the whole cell preparation of 30 μ g is electrophoresis on 6% isolating gel and 5% gel that piles up.In the PAGE of sodium lauryl sulphate (SDS) sex change, the protein of same amount is electrophoresis on 10% isolating gel and 5% gel that piles up.The reference protein that is used in operation under the deposition condition is separately determined relative molecular weight.Method (molecular cloning: laboratory manual, the 2nd edition, cold spring port, New York:, show two types gel cold spring harbor laboratory (1989)) according to Sambrock etc. by silver dyeing.In addition, natural and protein belt SDSPAGE utilize the improved method of Towbin etc., in the transfer dyeing (Proc. Natl. Acad. Sci.USA, 76:4350-4354 (1979)) that powers on of Immobilon P (Millipore) film.
Anti-spirillum sample that produces in rabbit or coccoid antibody are used as probe to identify two kinds of specificity and immunogenic proteins in the form.
Haemagglutination and haemagglutination restraining effect are measured
The titer plate of carrying out Morgan etc. with the 2%v/v red blood cell (people or rabbit) of 20 μ l is measured the trickle improvement of (clinical microbiology magazine 29:395-397 (1991)), wherein red blood cell is joined in the 25 μ l bacterial culturess, in each microtitre hole, comprise 10
7-10
12Cell/ml.Before reading the haemagglutination pattern, every kind of mixture is incubated overnight under 4 ℃ in quadruplicate.Under 37 ℃ with 1mg.ml
-1(pronase e, Sigma) pre-treatment bacterium 60 minutes or 60 ℃ of down heating 10 minutes carries out the haemagglutination restraining effect and measures proteolytic enzyme.Similarly, before haemagglutination is measured, red blood cell with 4.0 μ g ml
-1Neuraminidase (Sigma) or 1mg ml
-1Proteolytic enzyme was 37 ℃ of following pre-treatment 60 minutes.
Indirect fluorescent antibody test
Coccoid and spirillum sample smear before with the positive IgG antibody treatment of the anti-helicobacter pylori of human serum slide glass (HTC Wellcome) go up do quick-fried.After 30 minutes the incubation, smear at every turn with phosphate buffered saline (PBS) (PBS-pH7.6) washing, was washed three times ten minutes totally.Then, 1: 40 diluent of the FITC (Wellcome) that puts together with goat Anti-Human class IgG was handled smear 30 minutes.Reach 15 minutes four times with PBS washing smear at every turn,, utilize the Reichert-Jung fluorescent microscope under UV-light, to observe with the sealing of buffering glycerine.
Result and discussion
Coccoid " synchronously " culture
In the chemostat environment, successfully prepared coccoid " synchronously " culture.Fig. 1 has shown the typical growth curve of helicobacter pylori in the chemostat.Be similar to the growth of describing by Ho and Vijayakumari (down together) in a growth in two weeks.Show later stationary phase: viable count reduces to 10 gradually in two weeks of following
5CFU/ml.Thereafter, the decline phase of decline,, five all internal linear were lasting afterwards.At the whole cultivation period in about nine weeks, coccoid percentages show and spirillum sample are inversely proportional to.
The pH of substratum drops to 6.58 at first three days from neutrality, remains on 6.53+/-0.13 stationary phase around then.Then, rise to 6.98 maximum value in the 7th week, after this, in two weeks of following, pH is stabilized in 6.84+/-0.02.Catrenich and the Makin (Gastroenterology of Scandinavia, 6 (supplementary issue 181): 58-64 (1991)) reported the similar inverse relation between pH and the viable count, and to propose to lose viability and change coccoid into be because by the alkaline pH of the endogenous generation of deaminase active.Yet, in our research, as (commenting microorganism academic year by Marshall etc. (down with) and Dick, 44:249-269 (1990)) reports, because but the pH of substratum remains in the tolerance range (pH6.5-7.5) of helicobacter pylorus bacteria growing, it most possibly is because the restraining effect of nutrient depletion or metabolite but not the variation of pH that the spirillum sample changes coccoid into.
The mean value of urase specific activity (USA) by the 4.90+ of exponential phase/-4.42U mg
-1Protein increase to the 11.58+ of stationary phase/-10.03U mg
-1Protein.In the meantime, can be observed the peak of three kinds of patterns, be respectively the the 18th, the 156th and the 333rd hour climax.Then, USA is with linear minimizing of the decline phase that descends.When incubation period finishes, USA be 0.18+/-0.03U mg
-1Protein.This shows: urease activity is valuable to active regeneration spirillum sample and reduces with coccoid formation.Marshall etc. (gastroenterology, 99:697-702 (1990)) report: the premiere feature of external helicobacter pylori urease activity is that the protection bacterium makes it to avoid acid.Like this, stationary phase USA increase may be since metabolism spirillum sample to the adaptation reaction of the sour pH that increases.Companion's row ground, when pH rose, USA reduced in decrement phase.
Microscopy
Under phase microscope, the spirillum sample occurs with the bending of homogeneous contrast in the kytoplasm density or S shape rod that (Fig. 2 a).On the other hand, coccoid is a cyclic, and forms by two types: one type shows the fine and close thick kytoplasm that has, and another kind of type has loose kytoplasm, and the outward appearance (Fig. 2 b) of " blood shadow " cell is arranged.Utilize sucrose density gradient centrifugation, the coccoid that separates two types is impossible.Under transmission electron microscope, our observation is similar to the more early stage report by (1984 times together) such as Marshall, wherein, the spirillum sample is shown as cell size with 0.3-0.5 μ * 1.0-3.0 μ and the curved rod with the 1-6 flagellum clump that ends up with ball tip.Relatively, coccoid is a cyclic, and diameter is in the 200-300nm scope, and complete cytolemma (Fig. 3) is arranged.
When under phase microscope, observing, be different from spirillum sample with the distinctive motility of advancing by leaps and bounds, coccoid is immotile.On the other hand, transmission electron microscope has shown exist (Fig. 4) of flagellum in some coccoids.This may mean or flagellum is an in fact amphitrichous of the resistates (Marshall etc., 1984 times with) of spirillum sample or coccoid, but because they are in inactive state or lack energy drives it but non-activity.Owing to cloned flagellin gene, (bacteriology magazine such as Suerbaum, 175:3278-3288 (1993)) sudden change of observing main flagellin gene has caused immotile, helicobacter pylori with flagellum, and the sudden change of accessory flagellin gene produces the reactivity bacterium with flagellum usually.In this research, sex change SDS PAGE protein profile shows: the less important flagellin protein of 58kDa is present in spirillum sample and the coccoid with equality strength, but main 52kDa flagellin protein is with respect to the spirillum sample, and intensity reduces (Fig. 5) in coccoid.
Because less important flagellin is positioned at flagellum hook (flagellum adheres to required structure) nearside (Kostrzynska etc., bacteriology magazine, 173:937-946 (1991)), result of study can be explained flagellum complete on the coccoid, but lacks mobility.
By improved gramstaining, the spirillum sample shows as Gram-negative with negative staining after 10 minutes, even and coccoid still keeps faint Gram-negative in negative staining after 30 minutes.In the dyeing of improved periodic acid schiff, the spirillum sample is dyed green, and (Fig. 6 a), but coccoid is dyed shiny red, and this has shown exist (Fig. 6 b) of on its cell walls polysaccharide.This reaction is used in the fuzzy halo of the about 50-60nm radius around the coccoid and keeps (as observed under at transmission electron microscope) (Fig. 3).Contribution may be exactly the reason of coccoid dyeing difference in improved gramstaining greater than this polysaccharide layer of 50% coccoid cellular component.Consistent therewith is: handle coccoid with the ptyalin αDian Fenmei before the dyeing and improved Gram-reaction (Fig. 7) widely with the polysaccharide content of dyspepsiacoccus sample.In addition, coccoid polysaccharide content is Duoed 10 times (table 2) than the spirillum sample.Someone points out: the existence of glycocalyx can help bacterium to survive under the hostile environment condition.Moisture absorption layer can be used as the pilot-gas exchange and stops the cell damping fluid of the excessive absorption or the forfeiture (this can cause lysis and death) of liquid (Wilkinson, bacteriology summary, 22:46-73 (1958)).
Table 2: spirillum sample and coccoid integral part
| Integral part (μ g/ cell) | ?????????????????NCTC?11637 | ??????????????????V 2 | ||
| The spirillum sample | Coccoid | The spirillum sample | Coccoid | |
| Protein | ????7.84×10 -9 | ????3.92×10 -9 | ????7.86×10 -9 | ????4.62×10 -9 |
| ????DNA | ????6.21×10 -10 | ????3.12×10 -10 | ????7.25×10 -10 | ????3.79×10 -9 |
| Polysaccharide | ????6.38×10 -11 | ????5.01×10 -10 | ????2.95×10 -11 | ????2.15×10 -10 |
| ????ATP * | ????1.02×10 -16 | ????1.27×10 -18 | ????6.19×10 -16 | ????8.40×10 -18 |
* be unit with μ mol/ cell
Like this, coccoid might be survived at human external by the protection that the thick polysaccharide layer from atmospheric layer oxygen tension and adverse environment provides.Provide the similar observations (use and environmental microbiology, 52:531-538 (1985)) of campylobacter jejuni by Rollins and Colwell, wherein, when coccoid conversion is carried out, can see the increase of culture suspension viscosity from the spirillum sample.They think: the generation of extracellular mucopolysaccharide as a kind of adaptive variation to guarantee the extensive existence of campylobacter jejuni.
In the helicobacter pylori culture, do not observe the increase on this viscosity, but granulous spirillum sample culture tends to become floss.Under phase microscope, " synchronously " coccoid occurs with big clump.The polysaccharide layer can work and make the coccoid generation floss that flocks together.
Dna content
Can extract complete chromosomal DNA from coccoid, this shows: these forms may be (Fig. 8) that can survive.Total dna content of spirillum sample approximately is 5.22 * 10
-7The ng/ cell, and coccoid total dna content approximately is 3.13 * 10
-7Ng/ cell (table 2).Compare with the spirillum sample, the DNA that is reduced in coccoid can be by observed coccoid colony's explanation (Fig. 2 b) with loose and seepage kytoplasm under the phase microscope.On the other hand,, half of DNA total amount only arranged in the coccoid, also can show the dormancy or the surviving policy of this differentiated form with the comparison of spirillum sample.
The dna content that Novitsky and Morita (use and environmental microbiology, 33:635-641 (1977)) are reported in the vibrios ANT 300 that hungry existence plunges into the commercial sea living reduces by 48%.They think: this may be the strategy of energy conservation that causes the degraded of external or the DNA that duplicates of part.
Biochemical character
The spirillum sample is oxydase and catalase male, but coccoid does not show any visible reaction (table 3) in these are tested qualitatively.In API ZYM test, spirillum sample and coccoid have all shown the similar enzyme profile (medical microbiology magazine, 29:203-206 (1989)) that belongs to by the biotype II of descriptions such as Kung.What is interesting is, in coccoid and spirillum sample, observe the acidity and the alkaline phosphatase activities (table 3) of equivalent.The existence of these enzymes in the coccoid of dormancy can be worked to the generation of the energy of the transhipment of inorganic phosphate and ATP form.Coccoid comprises ATP, but lacks 100 times (table 2) than the spirillum sample.This means: coccoid be a kind of can survive but the form of dormancy.Similarly, the vibrios ANT 300 that given birth to by the sea of 99% reduction on endogenous is breathed shows (Novitsky and Morita use and environmental microbiology 32:617-622 (1976)) as the part of the strategics for survival under the long-term nutrition hunger.
Table 3: spirillum sample and coccoid enzyme profile
| Enzyme | The spirillum sample | Coccoid |
| Oxydase | ??????????+ | ?????????- |
| Catalase | ??????????+ | ?????????- |
| Urase | + 3.61U/mg protein | W 0.18U/mg protein |
| Acid phosphatase | ???????+(≥40nM) | ??????+(≥40nM) |
| Alkaline phosphatase | ???????+(≥40nM) | ??????+(≥40nM) |
| Napthol ASB1 phosphohydrolase | ???????+(20nM) | ??????+(20nM) |
| The leucine Arylamidase | ???????+(≥40nM) | ??????+(5nM) |
+=the positive
-=feminine gender
W=is weak positive
In addition, nearest work is illustrated: formation (Bode etc., general JOURNAL OF MICROBIOLOGY, the 139:3029-3033 (1993) of intracytoplasmic polyphosphate under unfavourable condition in the spirillum sample of helicobacter pylori; Caselli etc., Gut, 34:1507-1509 (1993)).When the ATP undersupply, the energy that these structure representatives are stored and the storage vault of phosphorus, and can regard alternative energy source as.Volutin granules's agent (polyphosphate) is observed (Fig. 9) by Albert dyeing in coccoid, and can constitute surviving policy.In addition, work in the metabolism that exists in these polyphosphates of phosphohydrolase.
Have 0.18+/-0.03U mg
-1The coccoid average urase specific activity ratio of protein active have 3.61+/-0.52U mg
-1Few 20 times of the average urase specific activity of the spirillum sample of protein active.In the coccoid low urease activity may or because the urase enzymic activity more than the coccoid of the enzyme of residual effect or dormancy does not need as active regeneration spirillum sample in the coccoid.Even this can explain that also coccoid quick urease test still is negative after 48 hours, the urease activity band can not be gone up sensitive dyeing by PAGE with the method for Shaik and detect (Shaik etc., analytical biochemistry, 103:140-143 (1980)) (showing) as Figure 10.Yet seemingly (as showing on the sex change SDS PAGE) of conservation (Fig. 5) in coccoid to be respectively the urase subunit A of 29kD and 66kD and B.What is interesting is and notice:, in spirillum sample and coccoid, still observe the band strength that these two kinds of urase subunits equate although urease activity hangs down 20 times in the coccoid.
In the coccoid only the dominance of urase A and B subunit exist and can explain in the helicobacter pylori urease activity (Labigne etc., bacteriology magazine, 173:1920-1931 (1991)) that reduces when urease activity also crucial urase subunit C and D lacked.
The protein profile
Also observe the molecular weight that shows this form dormancy, non-activity state in the coccoid as (Fig. 5) that shows among the SDS-PAGE and be＜14〉200kD the minimizing of range protein band on quantity and intensity.This situation only is that half (table 2) this fact of the total protein content found in the spirillum sample is further strengthened by the total protein content of each cell in the coccoid.It is necessary that Reeve etc. (bacteriology magazine, 160:1041-1046 (1984)) elaboration protein degradation is survived to the hunger of intestinal bacteria and Salmonella as the part of energy conservation.On the other hand, the existence of the conservation of possible urase subunit A and B and flagellin matter may mean that urase and flagellum change the spirillum sample into to coccoid and in gastric environment the later existence of spirillum sample be necessary.
In natural PAGE, observe exist the tangible band (Figure 11) with 60kDa of three kinds of new proteins of 955kDa, 871kDa and 661kDa with trace.The appearance of new protein band will appear to show that coccoid has the certain significance, and having more than is the denatured form of representing helicobacter pylori.In addition, the Western immunoblotting shows that 60kDa protein is specific and immunogenic (Figure 12) to coccoid.
Haemagglutination and haemagglutination restraining effect are measured
But most of bacterial strain aggegations of known helicobacter pylori comprise people and rabbit various red blood cells (Morgan etc., 1991, down with; Taylor etc., medical microbiology magazine, 37:299-303 (1992)).In this research, spirillum sample and coccoid are with greater than 10
8The bacterial concentration of individual cell is aggegation people red blood cell fully equally.For the rabbit red blood cell, the spirillum sample is with minimum 10
8Individual cell agglutination, and coccoid needs 10
9Individual cell is to produce the visible haemagglutination.Haemagglutination is suppressed by the heating and the protease treatment of red blood cell.This shows: the haemagglutination in the coccoid (being similar to by (communication of FEMS microbiology, 56:109-1l2 (1988)) such as Huang observed in the spirillum sample) is a protein, and acceptor is not a protein, is sialic acid.Therefore, as by Wadstrom etc. also observed (gastroenterology and hepatology Europe magazine, 5 (supplementary issue 2): S12-S15 (1993)), the haemagglutination character of spirillum sample remains in the coccoid.
Indirect fluorescent antibody test
Coccoid demonstrates fluorescence (Figure 13) under UV-light, this shows that its surface protein is complete and is similar to the spirillum sample.The specificity that this character can be used for the testing environment helicobacter pylori exists and existence, can help to illustrate simultaneously pipeline.Use similar character can study the Salmonella enteritides in environment survival but the form (Roszak etc. that can not cultivate, Canadian Journal of Microbiology, the existence of 30:334-338 (1983) and vibrio cholerae and intestinal bacteria (Xu etc., microbial ecology 8:313-323 (1982)).
It seems that from the result coccoid form can exist this and former think opposite with the form of survival.It has the existence of complete DNA, atpase activity, new and existence conservative protein matter, thick glycocalyx to protect it under the unfavourable condition in environment.
The spirillum sample to the character of the strict demand of nutrition and physiological requirements stoped their existence in environment (Marshall etc., 1984, down with; Dick, 1990, down together).Microaerophilic spirillum sample is to the oxygen sensitivity, and has the people to report if ingress of air surpasses 2 hours, just becomes nonviable (Soltesz etc., clinical microbiology magazine, 30:1453-1456 (1992)).Like this, coccoid modal state as an alternative can be the link of (also may in the transfer mode of helicobacter pylori) in the cell cycle.The similarly survivable but differentiated form that can not cultivate in other microorganism (as yellow myxococcus (White etc., the bacteriology magazine, 95:2186-2197 (1968)), vibrios ANT300 (Novitsky and the Morita of Hai Sheng, 1977, down with) with become brilliant Arthrobacter (Boylen and Ensign, bacteriology magazine, 103:569-577 (1970)) observes in cell cycle, and demonstrate under unfavourable condition in their surviving policy and have complete effect.
Many reports quoted by non-cultural method (as
3H thymidine picked-up research) detect (Shahamat etc. in water, Klin.Wochenschr., 67:62-62 (1989)), by (the Mapstone etc. of polymerase chain reaction detection in ight soil, Lancet, 341:447 (1993)) and detect (Westblom etc. in the water source of Peru, Acta Enterolgica Belgica, 56 (supplementary issues): 47 (1993)) helicobacter pylori.By
3The C urea breath test is to the universal research of helicobacter pylori also relevant with the consumption of river (Klein etc., Lancet, 337:1503-1508 (1991)).These Indirect Detecting Method are not got rid of the coccoid possibility that detects in environment, because this studies show that the existence of DNA in coccoid.And, from the ight soil that the people has just excreted, isolate helicobacter pylori and do not get rid of such possibility: promptly because the spirillum sample survival in normal atmosphere of its little aerobic character and oxygen toxicity is no more than 2 hours (Soltesz etc., 1992, (Krieg and Hoffman down together), microorganism academic year comments, 40:107-130 (1989)).These early stage reports only can be used for establishing the following fact: the helicobacter pylori that has more than one single forms.
Claims (29)
1. antigen protein, this antigen protein have the molecular weight of 60kDa when measuring by natural PAGE, this protein can be available from the helicobacter pylori (H.pylori) of coccoid form.
2. as the desired antigen protein of claim 1, this protein has following-terminal amino acid sequence: D-T-H-K-S-E-I-A-H-R-F-N-D-L-G.
3. as the purposes of the antigen protein defined in claim 1 or claim 2, this purposes is the antibody that is used to detect anti-helicobacter pylori.
4. as the desired purposes of claim 3, wherein said detection of antibodies is used to detect the existence of the helicobacter pylori of coccoid form.
5. as the desired purposes of claim 3, the Heliobacter pylori antigen protein of wherein said coccoid form can be used in combination available from other antigen of the helicobacter pylori of spirillum sample or coccoid form with one or more.
6. as the purposes of the antigen protein defined in claim 1 or the claim 2, this purposes is to be used for Antibody Preparation.
7. as desired purposes in the claim 6, wherein said antibody is polyclonal antibody.
8. as the purposes of the antibody of definition in claim 6 or the claim 7, this purposes is used to detect the helicobacter pylori of coccoid form.
9. test kit that is used for as the method defined in the claim 8, this test kit comprise as the antibody defined in claim 7 or the claim 8.
10. composition, said composition comprise as antigen protein defined in claim 1 or the claim 2 and dispensable one or more other Heliobacter pylori antigen.
11. as the desired composition of claim 10, wherein said dispensable one or more other Heliobacter pylori antigen can available from the helicobacter pylori of spirillum sample or coccoid form or they both.
12. a method that is used to detect helicobacter pylori, this method comprise the step that the antigen protein defined in claim 1 or claim 2 is contacted with sample.
13. as the desired method of claim 12, this method is used to detect the helicobacter pylori of coccoid form.
14. as claim 12 or the desired method of claim 13, wherein said antigen protein is as the composition forms defined in claim 10 or the claim 11.
15. as each desired method of claim 12-14, wherein said sample is a biological sample.
16. as the desired method of claim 15, wherein said biological sample is blood sample, urine samples or the saliva sample that obtains from the curee.
17. comprising, a method that is used for diagnosing helicobacter pylori infection, this method make the step that contacts with the biological sample that from the curee, is obtained as the antigen protein defined in claim 1 or the claim 2.
18. as the desired method of claim 17, wherein said antigen protein is as the composition forms defined in claim 10 or the claim 11.
19. as each desired method of claim 12-18, this method comprises that also detection can be available from other antigenic step of the helicobacter pylori of spirillum sample or coccoid form.
20. as the desired method of claim 19, wherein said biological sample is blood sample, urine samples or saliva sample.
21. a test kit that is used for diagnosing helicobacter pylori infection, this test kit comprise as the antigen protein defined in claim 1 or the claim 2.
22. as the desired test kit of claim 21, this test kit also comprises one or more can be available from other antigen of the helicobacter pylori of spirillum sample or coccoid form.
23. a vaccine that is used for the prevention or the treatment of helicobacter pylori infection, this vaccine comprise as antigen protein and one or more adjuvants and/or carrier defined in claim 1 or the claim 2.
24. as the desired vaccine of claim 23, this vaccine also comprises one or more can be available from other antigen of the helicobacter pylori of spirillum sample or coccoid form.
25. as claim 23 or the desired vaccine of claim 24, wherein said antigen protein provides with the helicobacter pylori of coccoid form.
26. as claim 23 or the desired vaccine of claim 24, wherein said antigen protein provides with coccoid of helicobacter pylori and the intermediate form between the spirillum sample form, this intermediate form is carried antigen.
27. the method for the IgM antibody of a helicobacter pylori that detects anti-coccoid form in children, this method comprise the step that the antigen protein defined in claim 1 or claim 2 is contacted with biological sample available from said children.
28. method of cultivating the helicobacter pylori of coccoid form, this method may further comprise the steps: add carbonic acid gas regularly in the substratum of the helicobacter pylori that comprises spirillum sample form, so that take place to the transformation of coccoid form, wherein the coccoid form of said acquisition can be survived.
29. as the desired method of claim 28, wherein said carbonic acid gas supplies with twice every day, nearly 3 months time.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9519865.1A GB9519865D0 (en) | 1995-09-29 | 1995-09-29 | Protein |
| GB9519865.1 | 1995-09-29 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1201464A true CN1201464A (en) | 1998-12-09 |
Family
ID=10781468
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96198105A Pending CN1201464A (en) | 1995-09-29 | 1996-09-27 | Helicobacter pylori protein |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP0859788A1 (en) |
| JP (1) | JP2000500123A (en) |
| KR (1) | KR19990063833A (en) |
| CN (1) | CN1201464A (en) |
| AU (1) | AU7137896A (en) |
| BR (1) | BR9610953A (en) |
| CA (1) | CA2233328A1 (en) |
| GB (1) | GB9519865D0 (en) |
| NO (1) | NO981407L (en) |
| WO (1) | WO1997012910A1 (en) |
| ZA (1) | ZA968185B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6599509B2 (en) | 1997-09-02 | 2003-07-29 | Massachusetts Institute Of Technology | Compositions and methods comprising helicobacter antigens for treatment and prevention of inflammatory bowel disease |
| MY120554A (en) * | 1997-10-29 | 2005-11-30 | Honda Motor Co Ltd | Valve operating system in internal combustion engine |
| IT1299312B1 (en) * | 1998-02-13 | 2000-03-16 | Consortia Lab Srl | DOSAGE IN BIOLOGICAL LIQUIDS OF DIRECT ANTIBODIES AGAINST ONE OR MORE ANTIGENS OF THE HELICOBACTER PYLORI BY IMMUNOLOGICAL METHOD |
| GB9806039D0 (en) * | 1998-03-20 | 1998-05-20 | Cortecs Ltd | Therapy |
| KR101600867B1 (en) * | 2008-06-03 | 2016-03-10 | 삼성전자주식회사 | Refrigerator |
-
1995
- 1995-09-29 GB GBGB9519865.1A patent/GB9519865D0/en active Pending
-
1996
- 1996-09-27 CN CN96198105A patent/CN1201464A/en active Pending
- 1996-09-27 CA CA002233328A patent/CA2233328A1/en not_active Abandoned
- 1996-09-27 KR KR1019980702303A patent/KR19990063833A/en not_active Application Discontinuation
- 1996-09-27 WO PCT/GB1996/002404 patent/WO1997012910A1/en not_active Application Discontinuation
- 1996-09-27 BR BR9610953-0A patent/BR9610953A/en not_active Application Discontinuation
- 1996-09-27 JP JP9514063A patent/JP2000500123A/en active Pending
- 1996-09-27 AU AU71378/96A patent/AU7137896A/en not_active Abandoned
- 1996-09-27 ZA ZA9608185A patent/ZA968185B/en unknown
- 1996-09-27 EP EP96932692A patent/EP0859788A1/en not_active Withdrawn
-
1998
- 1998-03-27 NO NO981407A patent/NO981407L/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| CA2233328A1 (en) | 1997-04-10 |
| ZA968185B (en) | 1998-03-27 |
| NO981407D0 (en) | 1998-03-27 |
| JP2000500123A (en) | 2000-01-11 |
| GB9519865D0 (en) | 1995-11-29 |
| NO981407L (en) | 1998-05-28 |
| AU7137896A (en) | 1997-04-28 |
| KR19990063833A (en) | 1999-07-26 |
| WO1997012910A1 (en) | 1997-04-10 |
| EP0859788A1 (en) | 1998-08-26 |
| BR9610953A (en) | 1999-12-21 |
| MX9802486A (en) | 1998-10-31 |
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