CN101880640A - Klebsiella pneumoniae (Mannose-sensitive hemagglutination) pilus strain - Google Patents
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Abstract
The invention relates to a klebsiella pneumoniae mannose-sensitive hemagglutination pilus strain which is characterized by comprising mannose-sensitive hemagglutination pilus. The invention also relates to the cultivation, treatment, application and using method of the klebsiella pneumoniae mannose-sensitive hemagglutination pilus strain. The invention also relates to a medical product prepared by the klebsiella pneumoniae mannose-sensitive hemagglutination pilus strain.
Description
Technical field
The present invention relates to a kind of microorganism strains of novelty.Particularly, the present invention relates to a kind of Klebsiella pneumonia mannose sensitive haemagglutination pilus strain.The present invention also relates to the cultivation, processing and use of this Klebsiella pneumonia bacterial strain.The invention still further relates to the medical product that utilizes this Klebsiella pneumonia bacterial strain preparation.
Background technology
Klebsiella (Klebsiella) is most important conditioned pathogen except that intestinal bacteria at present, and is very similar to the Hafnia alvei biochemical reaction to Serratia, enterobacter, and some amino acid decarboxylase enzyme test helps to differentiate.
Klebsiella can divide five kinds, promptly Klebsiella pneumonia (K.pneumoniae), acid-producing Klebsiella bacterium (R.oxytoca), separate ornithine klebsiella (K.ornithinolytica), plant living klebsiella (K.planticola) and autochthonal klebsiella (K.terrigena).Wherein Klebsiella pneumonia can divide 3 subspecies again: pneumonia subspecies (subsp.pneumoniae), ozena subspecies (subsp.ozaenae) and nose scleroma subspecies (subsp.rhinoscleromatis).
In the clinical Klebsiella that is separated to, Klebsiella pneumonia accounts for more than 80%, is the most important and common pathogenic bacteria in this genus.
Klebsiella pneumonia can produce pod membrane for the tubbiness bacillus of long 1～2 μ m, wide 0.5～0.8 μ m, and no gemma is unpowered, common end-to-end paired growth.Pod membrane is not chloride polysaccharide material, and is thicker, makes gramstaining and promptly can be observed, but more easily observe with india ink method.The Klebsiella pneumonia optimum growth temperature is 37 ℃, can be 12～43 ℃ of scope growth, and 55 ℃ of 30min can be inactivated.On differential medium, have the look bacterium colony now because of ferment lactose; Then can be the glutinous peptone sample bacterium colony of canescence because of producing a large amount of pod membrane materials on solid medium, bacterium colony easily merges mutually, with easily drawwing wire that transfering loop is chosen; And liquid is obviously sticky cultivate a couple of days in meat soup after.
Klebsiella has O antigen O and kantigen K, presses the composition of kantigen K, and Klebsiella pneumonia can be divided into 80 types, and the pneumonia subspecies belong to pod membrane 3 and 12 types mostly; The ozena subspecies have types such as 4,5,6,15, and are the most common with 4 types; Nose scleroma subspecies then mostly are 3 types.
The klebsiella virulence is stronger, and the Klebsiella pneumonia of minute quantity (100 bacteriums) is injected in mouse peritoneal and can causes dead mouse.
Klebsiella adaptability is strong, can very fast adaptation host environment and survive, various microbiotic are easily produced resistance, and be to produce one of the highest bacterium of extended spectrum rate, often simultaneously to multiple antibiotics resistance.This kind resistance can mediate for karyomit(e), also can propagate by R-plasmid, causes in some ward of hospital and infects localized epidemics.
Pneumonia due to the klebsiella can be divided into primary pneumonia and secondary pneumonia.Both boundary lines sometimes quite difficulty distinguish, it is generally acknowledged that on the basis of original pneumonia infection disease the infection that this bacterium is taken place is a Secondary cases under certain condition of causing a disease.
The virulence of klebsiella may be relevant with its pod membrane, and pod membrane can suppress chemotactic, the phagolysis of scavenger cell, but definite pathogenesis is not illustrated as yet fully.
The pathological characteristic of this genus infectation of bacteria is that the bacterial growth breeding is fast, can form single-shot or multiple abscess at each internal organs, contains the gram negative bacillus that has pod membrane in a large number in the transudate.
The primary lobar pneumonia that Klebsiella pneumonia causes is with gerontal patient, alcoholism and to suffer from serious former disease patient such as diabetes, cancerous swelling, hemopathy be many.Pathological change is different with person due to the streptococcus pneumoniae, and normal downright bad, the liquefaction of alveolus wall forms single or multiple vomicas.Alveolar includes the sticky phlegm of a large amount of courage and uprightness.The vomica surface has the scleroproein exudate to cover more, the early stage pleural adhesion that promptly easily takes place.The incidence of pyothorax is about 25%, is high than pneumococcal pneumonia.Focus dissipates not exclusively normal, causes fibroplasia, the suppurative focus of remnants or bronchiectasis.Bacillus canalis capsulatus infects and accounts for 10% of nosocomial infection, common person such as urinary tract infections, respiratory tract and wound infection etc.Multiple suppuration focus and thoracic cavity, pericardial cavity empyema etc. all can appear in liver, kidney, brain etc. in the septicemia case.Pathology still can cause pseudomembranous enterocolitis the colon person, and idol can cause perforation and diffuse peritonitis.Hard nose knot subspecies and the pathogenic of ozena subspecies are not illustrated as yet fully, and the former can cause chronic granuloma scleroma disease, and the latter may be relevant with the ozena due to nasal mucosa and the atrophy of nasal turbinates, is a kind of opportunistic infection.
The Klebsiella pneumonia of putting down in writing in the prior art is pathogenic bacterium, does not have medical applications and is worth; At present still Klebsiella pneumonia is not used to prepare the report of vaccine.
Fundamental research about bacterial pilli is more.Bacterial pilli is the extremely very thin albumen microfilament in thalline surface, must use electron microscopic observation.Characteristics are: thin, short, straight, hard, many, it is generally acknowledged that the motion of pili and bacterium is irrelevant, and can be divided into ordinary pilus and sex fimbria two classes according to form, 26S Proteasome Structure and Function.The former is with bacterial adhesion and to infect the host relevant, and the latter be a hollow tube, and is relevant with the transmission genetic material.At Sharon N, Eshdat Y, Silverblatt F J, Ofek I, Bacterial adherence tocell surface sugars, ciba Found Symp.1981; In 80:119～141, illustrated to exist with host cell in the pili of intestinal bacteria and Salmonellas adherent material has taken place, and should adhesion can be subjected to sugar, for example inhibition of D-seminose; And the adhesion of glycoprotein mediation is widely general.At Wim Gaastra and Frist K.de Graaf, Host-Specific Fimbrial Adhesions ofNoninvasive Enterotoxigenic Escherichia coli Strains, MicrobiologicalReviews, June 1982, Vol.46, No.2, the character of colibacillary multiple pili has been discussed P.129～161 mannose-sensitive hemagglutination (Mannose-sensitivehemagglutination, mannose sensitive haemagglutination) and seminose resistance hemagglutination (MRHA) and non-hemagglutination (Non-HA) also have been discussed.At Speert DP, Lon BA, Eftekbar F, Opsonin-independent phagocytosis of Pseudomonas aeruginosa, AntibiotChemother.985; In 36:88～94, pointed out the pili part mediation the phagolysis of people's neutrophilic granulocyte to pseudomonas.About the comprehensive argumentation of pili, referring to, Mou Xiya, the new development of bacterial pilli and the research of relevant adhesin, JOURNAL OF MICROBIOLOGY, the 4th volume, the 3rd phase, in November, 1984, the 14th page to the 17th page.The pili that the prompting of above-mentioned public publication has mannose-sensitive hemagglutination (mannose sensitive haemagglutination) character may be present on the multiple genus bacterium, is I type ordinary pilus (being mannose sensitive haemagglutination pilus) such as the modal pili of enterobacteriaceae (Enterobacteriaceae).
In order to prevent and/or treat and the infectation of bacteria diseases associated, need to seek and provide to have wide spectrum, high immunogenicity, hypotoxic material.The present invention is by the Klebsiella pneumonia bacterial strain of screening from obtaining clinically, obtained a kind of mannose sensitive haemagglutination pilus strain, and found to have the purposes that stimulates the immunity of organism effect with the split vaccine of this Klebsiella pneumonia mannose sensitive haemagglutination pilus strain preparation.So, the invention provides the Klebsiella pneumonia mannose sensitive haemagglutination pilus strain that has use value, can be used to produce vaccine.
Summary of the invention
Summary of the invention
First aspect, the invention provides a kind of Klebsiella pneumonia bacterial strain, called after Klebsiella pneumonia mannose sensitive haemagglutination pilus strain, Latin is called " Klebsiella pneumoniae (Mannose sensitive hemagglutination) pilus strain ".On taxonomy, this bacterial strain belongs to a kind of novel strain of the Klebsiella pneumonia (Klebsiella pneumoniae) of Klebsiella (Klebsiella).
This bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), No. 3, No. 1 institute in North Star West Road, Chaoyang District, city, BeiJing, China, address, Microbe Inst., Chinese Academy of Sciences on 01 22nd, 2010.CGMCC is the international depositary institution of biological material specimens that budapest treaty is admitted.The numbering of this bacterial strain is CGMCC No.3605, and the classification name is Klebsiella pneumonia (Klebsiella pneumoniae).
Klebsiella pneumonia mannose sensitive haemagglutination pilus strain of the present invention is characterised in that following one or more:
A., many very thin and upright and outspoken pili are arranged around thalline,
B. the mannose sensitive haemagglutination test presents strong positive,
C. flat-plate bacterial colony adheres to red corpuscle test strong positive,
D. saccharomycetic direct agglutination test is the MS blood clotting,
E. has non-mannose sensitive haemagglutination pilus.
Second aspect the invention provides the dead thalline of Klebsiella pneumonia mannose sensitive haemagglutination pilus strain, cellular lysate composition, the thalline prepared product, extract separates the material from thalline, and it can be used as the activeconstituents of vaccine or immunomodulator or other medical articles separately.The cell of Klebsiella pneumonia mannose sensitive haemagglutination pilus strain comprises structures such as cell walls, cytoplasmic membrane, tenuigenin and organoid, nuclear district or nucleoid, and cell walls comprises internal layer and skin again, and there is an end flaggellation in the extracellular, and has whole body pili.The pili kind is various, comprising mannose sensitive haemagglutination pilus, and non-mannose sensitive haemagglutination pilus and sex fimbria.Say that from composition dna molecular is arranged, RNA molecule, protein, lipid molecule or the like.In the culturing process, also can produce meta-bolites.
In one embodiment, based on Klebsiella pneumonia mannose sensitive haemagglutination pilus strain of the present invention,, obtain dead thalline through cultivating and deactivation.Dead thalline can be used as the body immune system stimulator that animal body comprises human body, obtains beneficial effect.Preferably, dead thalline can use behind the purifying from substratum or nutrient solution.Also other reagent or medicament, adjuvant, assistant agent etc. be can add therein, better immunostimulation or regulating effect obtained.
In one embodiment, based on Klebsiella pneumonia mannose sensitive haemagglutination pilus strain of the present invention, steps such as process cultivation, extraction, purifying can obtain a certain or several or arbitrary combination in said structure or the above-mentioned substance, be used as the body stimulator that animal body comprises human body, obtain useful effect.
Purposes about Klebsiella pneumonia mannose sensitive haemagglutination pilus strain of the present invention.Klebsiella pneumonia mannose sensitive haemagglutination pilus strain toxicity of the present invention is low, and immunization is strong, and has the characteristic of wide spectrum effect.Cellular immunization and immunity system with the vaccine or the immunomodulator of this bacterial strain preparation can activate the regulation and control human body comprehensively produce resistant function to various bacteria and fungi.The equal tool positive effect of immunoregulatory activity and antitumor action.
The vaccine of Klebsiella pneumonia mannose sensitive haemagglutination pilus strain of the present invention is particularly suitable for immune body, is used to prevent the infectation of bacteria of enterobacteriaceae.
Description of drawings
The relevant preservation information of Klebsiella pneumonia mannose sensitive haemagglutination pilus strain of the present invention is as follows:
Preservation date: on January 22nd, 2010;
Depositary institution's title: China Committee for Culture Collection of Microorganisms common micro-organisms center;
Depositary institution is called for short: CGMCC;
Deposit number: CGMCC No.3605.
Fig. 1 is from Klebsiella pneumonia mannose sensitive haemagglutination pilus strain electron micrograph provided by the invention (48000X), shows the morphological specificity (feathering, mannose sensitive haemagglutination pilus) of described Klebsiella pneumonia.
Detailed Description Of The Invention
1. definition and explanation
In this application, following term and explanation are specified.
" bacterium " is the unicellular prokaryotes of a class. The gene carrier is comprised of a branch of double-stranded dna fiber silk, without any the boundary of film it and cytoplasm is separated. Lack organelle. Ribosome is 70S. That talk about among the application is " true bacterium (eubacteria) ". True bacterium can be divided into two types of Gram-positive and Gram-negatives.
" Gram’s staining method " is by using the purple solution of crystallization and iodine liquid, and be different according to the cell wall structure of true bacterium, the colouring method that bacterium is distinguished.
" flagellum " is the motion organ of some bacterium, mainly is made of flagellin.
" pili " is the fibril accessory structure of bacterium surface, and be shorter, more straight than flagellum, and carefully much smaller. Pili is made of protein. Pili is divided into ordinary pilus and property pili.
" ordinary pilus " adheres to relevant with bacterial body. Ordinary pilus is of a great variety, and the relatively difference according to its effect in bacterium adheres to can be divided into non-specific adhesion pili and selectivity and adhere to pili.
" non-specific adhesion pili " refers to that those can make bacterium adhere to pili on the multiple matrix, on multiple eukaryotic, the red blood cell of many animals for example, leucocyte, upper chrotoplast, and zooblast, plant cell and fungi cell in other tests. It is strict selective that to be exactly these pili do not have the host yet, adheres to have popularity, for example colibacillary 1 type pili (type1 fimbriae). When using the red blood cell in different animals source, non-specific adhesion shows the hemagglutination activity of parallel pattern; And red blood cell agglutination (hemagglutination) is to mannose-sensitive (mannose-sensitive). Therefore, mannose-sensitive red blood cell agglutination (mannose-sensitive hemagglutination, mannose sensitive haemagglutination) is a feature of non-specific adhesion pili.
" selectivity adhesion pili " refers to that those impel bacterium to adhere to pili on the particular substrate. It is strict selective that also to be exactly these pili have the host, and adhesion can only occur between bacterium and special host, such as coli common pili K88. The red blood cell agglutination does not show mannose-sensitive, and the blood that is not namely suppressed by mannose coagulates.
" mannose sensitive haemagglutination pilus " full name is " mannose-sensitive red blood cell agglutination pili ", refers to promote that bacterium adheres to the non-specific adhesion pili on the multiple matrix, can be suppressed by mannose. In this application, mannose sensitive haemagglutination pilus is a generalized concept. Existing research prompting on bacterium, such as the false monad of verdigris, Escherichia coli, salmonella etc., adheres in the process of host's cell, and the sugar mediation adheres to generally and exists. Some is mannose-sensitive, and some is that L-fucose is responsive, D-galactolipin sensitivity. At this, " mannose sensitive haemagglutination pilus " comprises mannose-sensitive cell agglutination pili, various other carbohydrates pili that regulate, relevant with bacterium and host's non-specific adhesion.
" Klebsiella " is one and belongs to name, and Friedlanders bacillus wherein is the condition pathogenic bacteria. The present invention provides a kind of Friedlanders bacillus bacterial strain with medical application by seed selection. The Friedlanders bacillus bacterial strain with medical application with same invention thinking screening is contained in the present invention.
2. describe in detail
Friedlanders bacillus bacterial strain of the present invention shows hypotoxicity, has whole body pili, and mannose sensitive haemagglutination pilus is arranged in the pili. The present invention is this bacterial strain called after Friedlanders bacillus mannose sensitive haemagglutination pilus strain, and is deposited in Chinese microorganism strain preservation administration committee common microorganism center on January 22nd, 2010, and deposit number is CGMCC No.3605.
Friedlanders bacillus full name provided by the invention is the Friedlanders bacillus mannose sensitive haemagglutination pilus strain, it is a kind of Friedlanders bacillus with whole body pili mannose sensitive haemagglutination (Mannose SensitiveHemagglutination) pili, and this pilus strain has strides the broad-spectrum immunogenicity that bacterium belongs to.
As described in the background section, in view of Klebsiella pneumonia is pathogenic bacterium, nobody utilizes Klebsiella pneumonia thalline or material wherein to carry out disease prevention and treatment.
The present inventor has noticed pili in bacterium and the active importance of host, notices that mannose sensitive haemagglutination pilus is in bacterium and the active ubiquity of host; Strive finding that a strain has hypotoxicity, the immunogenic Klebsiella pneumonia mannose sensitive haemagglutination pilus strain of wide spectrum, and utilize material, particularly mannose sensitive haemagglutination pilus in the thalline to carry out immunostimulation and conditioning.
The inventor passes through this Klebsiella pneumoniae mannose sensitive haemagglutination pilus strain has been determined in the screening of many Klebsiella pneumoniaes of clinical acquisition and the experiment in mouse.The product of this bacterial strain shows beneficial effect on prevention and treatment of diseases, can stimulating immune system.
After Klebsiella pneumonia mannose sensitive haemagglutination bacterial strain obtained, maintain property was stable always.Also aspect anti-infective, antineoplaston and the immune disorder good result is being arranged all by the vaccine of Klebsiella pneumonia mannose sensitive haemagglutination bacterial strain preparation.
The principal character of Klebsiella pneumonia mannose sensitive haemagglutination pilus strain is as follows:
1. form cultural characters and biochemical investigation: meet the Klebsiella pneumonia test rating.
2. pili form and character
(1) Fig. 1 is the electron micrograph of bacterial strain of the present invention, can see the Zhousheng pili that the thalline of this bacterial strain has.
(2) the part pili is a mannose sensitive haemagglutination pilus.Through mannose sensitive haemagglutination and hemagglutination-inhibition test, be strong positive, referring to Fig. 1.Wherein relevant method of carrying out the mannose sensitive haemagglutination test, referring to Dalian Medical College's journal, 1985,7 (1): 1-3.
Embodiment
The source and the evaluation of embodiment 1. Klebsiella pneumonia mannose sensitive haemagglutination bacterial strains
The inventor passes through isolated 6 Klebsiella pneumoniaes from clinical samples, with reference to (Mou Xiya, the foundation of pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain, the microorganism journal, 26 (2): 176-179,1986) method in is screened and seed selection, obtains a Klebsiella pneumoniae mannose sensitive haemagglutination bacterial strain.
Main identification mark is as follows:
Show coloured bacterium colony at differential medium top fermentation lactose;
On solid medium, be the glutinous peptone sample bacterium colony of canescence;
Cultivate a couple of days in meat soup after, liquid is obviously sticky;
Gram-negative.
This bacterial strain is carried out electron photomicrograph, as shown in Figure 1, shown the Zhousheng pili.Mannose sensitive haemagglutination and hemagglutination-inhibition test are strong positive.Wherein the method for mannose sensitive haemagglutination and hemagglutination-inhibition test is known and skillfully operations and judgement easily of those of ordinary skills, referring to for example Dalian Medical College's journal, 1985,7 (1): 1-3.
Embodiment 2. strain culturing and vaccine preparation
Klebsiella pneumonia mannose sensitive haemagglutination pilus strain of the present invention was cultivated 18 hours in 37 ℃ on the bacterium flat board, collected culture, and with the formaldehyde solution sterilization, repetitive scrubbing is made bacterin, stroke-physiological saline solution is used in dilution, adds 1% phenylcarbinol as sanitas, the preparation vaccine.
The experimentation on animals of embodiment 3. vaccines
Adopt the dull and stereotyped cultivation of bacterium, 1% formaldehyde physiological saline kill bacteria, with stroke-physiological saline solution washing 5 times, spectrophotometer detects bacterial concentration, checks then to have or not viable bacteria, is diluted to 18 to 2,000,000,000/ml, makes vaccine.
The experimental animal that experimentation on animals is adopted is 18～20 gram small white mouses.Subcutaneous injection 3 times, per injection 0.3ml.Shot to shot turnaround is a week.Last injection back the 7th day, the cross agglutination MSHA antibody titer of the small white mouse of detection immunity.
Table 1. small white mouse is through after " vaccine " of the present invention immunity, the antigenic cross agglutination of MSHA of the antibody in its blood and two kinds of bacteriums the detection of tiring
Immunoprotection test immunity successively is totally 43 small white mouses.Through active immunity in 17 days, most of mouse became thin in the immunologic process, successively was total to dead 7.In the mouse of survival, the heaviest person 24 grams, the lighter's 15 grams.Immunity back mouse show immunoprotection.The result is as follows in the active immunity protection test:
Table 2 immunoprotection test-results
| Animal groups | The immunity fate | Attack bacterial strain | The immune group mortality ratio | The control group mortality ratio | The P value |
| ??1 | 17 days | Corresponding bacterium | ??1％ | ??100％ | ?P＜0.01 |
| ??2 | 17 days | Intestinal bacteria | ??37.5％ | ??80％ | ?P＜0.05 |
| ??3 | 17 days | Pneumobacillus | ??25％ | ??100％ | ?P＜0.01 |
| ??4 | 17 days | Pseudomonas aeruginosa | ??12.5％ | ??100％ | ?P＜0.01 |
The result shows: the mortality ratio of immune group is starkly lower than control group, and expression has significant immune protective effect.
As what it will be apparent to those skilled in the art that, can make many modifications and variations of the present invention and do not deviate from its spirit and scope.Specific embodiments described herein only provides in the mode of example, and entire area restriction the present invention of the equivalent that only is awarded together with these claims with claims.
Claims (10)
1. Klebsiella pneumonia mannose sensitive haemagglutination pilus strain, preserving number is CGMCC No.3605.
2. Klebsiella pneumonia pilus strain as claimed in claim 1 is characterized in that having pili all over the body.
3. Klebsiella pneumonia pilus strain as claimed in claim 1 is characterized in that having non-mannose sensitive haemagglutination pilus.
4. as dead thalline, cellular lysate composition or the thalline extract of claim 1-3 Klebsiella pneumonia pilus strain as described in each.
5. use as each described Klebsiella pneumonia pilus strain of claim 1-3 or its dead thalline, cellular lysate composition or the prepared goods of thalline extract.
6. vaccine or immunomodulator, comprising the dead thalline as claim 1-3 Klebsiella pneumonia hair strain as described in each, the dead thalline of described Klebsiella pneumonia hair strain is the activeconstituents of described vaccine or immunomodulator.
7. vaccine or immunomodulator, comprising the cellular lysate composition just like each described Klebsiella pneumonia hair strain of claim 1-3, described cellular lysate composition is the activeconstituents of described vaccine or immunomodulator.
8. vaccine or immunomodulator, comprising the thalline extract just like each described Klebsiella pneumonia of claim 1-3, described thalline extract is the activeconstituents of described vaccine or immunomodulator.
9. vaccine production method, described method comprises:
Cultivation is made vaccine as claim 1-3 Klebsiella pneumonia as described in each with thalline.
10. the purposes that is used for immunomodulatory, anti-infective or anti-tumor drug as each described Klebsiella pneumonia of claim 1-3 or its dead thalline, cellular lysate composition or thalline extract in preparation.
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