CN1297657C - Pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain - Google Patents
Pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain Download PDFInfo
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- CN1297657C CN1297657C CNB200510059850XA CN200510059850A CN1297657C CN 1297657 C CN1297657 C CN 1297657C CN B200510059850X A CNB200510059850X A CN B200510059850XA CN 200510059850 A CN200510059850 A CN 200510059850A CN 1297657 C CN1297657 C CN 1297657C
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- pseudomonas aeruginosa
- pilus
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- mannose
- mannose sensitive
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Abstract
The present invention relates to a pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain which is characterized in that the strain has mannose sensitive hemagglutination pili. The present invention also relates to a method for culturing, processing, applying and using the pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain, and medical care products prepared by utilizing the pseudomonas aeruginosa mannose sensitive haemagglutination 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 pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain.The present invention also relates to the cultivation, processing and use of this pseudomonas aeruginosa strain.The invention still further relates to the medical product that utilizes this pseudomonas aeruginosa strain preparation.
Background technology
Pseudomonas aeruginosa is a common name clinically, and its formal name used at school is Pseudomonas aeruginosa (Pseudomonasaeruginosa).On taxonomy, Pseudomonas aeruginosa belongs to Rhodopseudomonas (PseudomonasMigula 1894), and referring to the R.E. Buchanan, N.E. Ji Bensi waits volume, the outstanding Bacteria Identification handbook of uncle, the 8th edition, Science Press (1984), the 274th page to the 279th page.According to the common sense of this area, term " Pseudomonas aeruginosa " is with the address of a kind of species under different occasions with " Pseudomonas aeruginosa ".In this application, the more term " Pseudomonas aeruginosa " that used also uses term " Pseudomonas aeruginosa " in the time of necessary, and the difference on this term uses does not constitute the difference of indication microorganism.With regard to essential meaning, the term among the application " Pseudomonas aeruginosa " can replace with " Pseudomonas aeruginosa ".
About 1.5～3.0um is long for the Pseudomonas aeruginosa size, and 0.5～0.8um is wide, and profile is shaft-like.Gram-negative.Have mobility, great majority are single-ended flagellum.Can from soil and water, be separated to, from clinical sample, separate usually, for example separate in wound, burn and the urinary tract infection.The suitableeest culture temperature is about 37 ℃ of degree.Important feature comprises: produce pyocyanin, the oxidase test positive can liquefy gelatin, faint reduces fat, and 41 ℃ of growths or not decomposition glucose for 4 ℃, the arginine dihydrolase test is positive, or the like referring to the R.E. Buchanan, N.E. Ji Bensi waits volume, the outstanding Bacteria Identification handbook of uncle, the 8th edition, Science Press (1984), the 278th page.Owing to can produce pyocyanin, thus green fester formed during infected wound, so be called as Pseudomonas aeruginosa or Pseudomonas aeruginosa.
To Mammals, especially human, Pseudomonas aeruginosa is a kind of conditioned pathogen.Prolonged application hormone, immunosuppressor, chemotherapy of tumors, radiotherapy etc. cause patient immune function's low (unbalance), and the patient of perform the operation back or some treatment operation back (tracheotomy, retention catheterization pipe etc.) easily suffers from the infection of this bacterium.In addition, the environment resistibility is stronger to external world for Pseudomonas aeruginosa, can long-term survival at the humidity place, and insensitive to ultraviolet ray, damp and hot 55 ℃ just were killed in 1 hour.Normal human skin, in especially moist position such as oxter, perineal position and the duct, respiratory tract and enteron aisle all have this bacterium to exist.All once be separated to this bacterium in the hospital on plurality of devices and the apparatus, and propagated by all means and also be one of route of transmission with contacting of patient for patient, patient.Pseudomonas aeruginosa also can cause and the irrelevant infection of hospital environment, in recent years to this existing more understanding, it has become the The main pathogenic fungi that osteomyelitis due to foot puncture infection, endocarditis, the Drug abuse, ocular infection, infection of newborn external otitis, swimming pool etc. cause tetter etc.
Pseudomonas aeruginosa can produce the multiple material relevant with virulence, for example intracellular toxin, exotoxin A, elastoser, collagenase, pancreas peptase etc.The pseudomonas aeruginosa strain type is more, can be divided into 12 serotypes according to O-antigen, can be divided into 7 classes according to the Fisher immunologic pattern.In adopting antibiotic prevention such as penicillins, born of the same parents' class or methods of treatment, Resistant strain constantly appears.Also explored the immune method that strengthens modulate host, such as, Chinese patent 86108380, December 10 1986 applying date, applicant Genetic System Corp., wherein disclosing can be in conjunction with production and the application of the monoclonal antibody of the lipopolysaccharide molecule of Pseudomonas aeruginosa; Chinese patent application 87104581, on August 25 1987 applying date, applicant Genetic System Corp. wherein discloses the monoclonal antibody that can discern the Pseudomonas aeruginosa flagellum.In Chinese patent application 94190491.1, June 2 1994 applying date, applicant First sugar refining Corporation, wherein disclose by repeating purge process and cultivated the attenuated pseudomonas aeruginosa strain and prepare the immune vaccine that contains cell wall protein, the purpose of this technical scheme is to overcome and the relevant problem of pseudomonas aeruginosa strains self toxicity, for example when extracting cell wall protein as antigen with the malicious common Pseudomonas aeruginosa of leniently convicting somebody, have heterologous nucleic acids and toxicity polymer substance, lipopolysaccharides (LPS) etc. is sneaked into wherein, thereby has limited the purposes of cell wall protein immune vaccine.This technical scheme can overcome the danger that self intrinsic toxicity of pseudomonas aeruginosa strains is produced by cultivating attenuated pseudomonas aeruginosa strains.
In recent years, the pseudomonas sickness rate increases to some extent.Studies show that pseudomonas Pseudomonas kind is up to more than 140 kinds, major part maybe can cause Plant diseases for saprophytic microorganism, has 25 kinds can infect the mankind approximately.Because false single bag bacterium belongs to aerobic on taxonomy or the amphimicrobian non-fermentative gram-negative bacilli, based on structural general character, also there is the activated protein extract that adopts multiple pseudomonas to be used for prevention and treatment chronic cardiopulmonary disease pulmonary infection and various pseudomonas infection, referring to Chinese patent ZL95119866.1, the December 7 nineteen ninety-five applying date, the surplus state of patentee China, June 26 2002 Granted publication day, wherein described and comprised particulate protein, endotoxin protein, mucus protein and anatoxic pseudomonas active protein need to make the intracellular toxin detoxification in the enforcement, and make extracellular toxin become toxoid.At InfectImmun1980; This is inferior to have reported Japanese scholar on the 40:670, once the dead thalline of finding Pseudomonas aeruginosa has immunoregulation effect, its dead thalline is expelled in the animal body and can excites humoral immunization and cellular immunity, induce and generate Interferon, rabbit and have the mitogen effect, can also induced animal the growth of cancer cells of opposing inoculation, but because this thalline toxicity is too big, immunizing potency is low, so can't practical application.
Therefore, need to seek and provide to have wide spectrum, high immunogenicity, hypotoxic material, be used to prevent and/or treat and the infectation of bacteria diseases associated.But the Pseudomonas aeruginosa of putting down in writing in the prior art is difficult to satisfy the demand.
As mentioned above, Pseudomonas aeruginosa is gram-negative bacterium (G
-).The cell walls internal layer is the peptidoglycan layer, and skin is asymmetrical bilayer lipid membrane.Contain lipopolysaccharides (LPS is also referred to as intracellular toxin endotoxin) in the skin, protein and phosphatide.The side effects limit that toxic ingredient brought such as lipopolysaccharides to the utilization of Pseudomonas aeruginosa.
As mentioned above, Pseudomonas aeruginosa has flagellum.Flagellum mainly is made of a kind of albumen that is called flagellin (flagellin).
Some does not have pili the Pseudomonas aeruginosa that the nature separation obtains, and some has the feathering sex fimbria.
Pili 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.
Other has report, and pili is relevant with twitch sports type (TWiching motility), (sees also
1. Med.M:robiol., 29:321～330,1998.＜micrococcus gonococcus 〉
2. Gene, 192:109～113,1997,＜Pseudomonas aeruginosa〉think all IV type ordinary pilus (belonging to non-mannose sensitive haemagglutination ordinary pilus) that belong to of two bacterium at present more.)
At Sharon N, Eshdat Y, Silverblatt F J, Ofek I, Bacterial adherenceto cell 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-SpecificFimbrial Adhesions of Noninvasive Enterotoxigenic Escherichiacoli Strains, Microbiological Reviews, June 1982, Vol.46, No.2, the character of colibacillary multiple pili has been discussed P.129～161 mannose-sensitive hemagglutination (Mannose-sensitive hemagglutination also has been discussed, mannose sensitive haemagglutination) and seminose resistance hemagglutination (MRHA) and non-hemagglutination (Non-HA) at SpeertDP, Lon BA, Effekbar F, Opsonin-independent phagocytosis ofPseudomonas aeruginosa, Antibiot Chemother.985; 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).Encode from pilin matter, various ways is arranged.Be subjected to the caryoplasm Gene Handling such as I type ordinary pilus protein coding, the sex fimbria protein coding is controlled by plasmid.
In the Pseudomonas aeruginosa that is separated to, if having feathering sex fimbria or end hair sex fimbria, then except sex fimbria was subjected to plasmid control, the gene of other pili also can be positioned on the karyomit(e).Some ordinary pilus does not have mannose-sensitive hemagglutination (mannose sensitive haemagglutination) character.1980 to 1981, have the functional gene that forms mannose sensitive haemagglutination pilus on the karyomit(e) of Israel scholar Gitboa-Garber professor (In adhesion andmicroorganism pathogenicy ciba foundation symposium 80.PitmanMedical p119～141) discovery Pseudomonas aeruginosa, but can only in the Pseudomonas aeruginosa body, synthesize mannose sensitive haemagglutination protein, can not with the mannose sensitive haemagglutination protein secreting outside thalline, can not form mannose sensitive haemagglutination pilus at cell surface.
Professor Gitboa-Garber extracts the mannose sensitive haemagglutination proteantigen after using the broken bacterium thalline of supersonic method, but yields poorly, and is difficult for keeping purity.At Sharon N, Eshdat Y, Silverblatt FJ, Ofek I, Bacterial adherence to cell surface sugars, ciba found Symp.1981; In 80:119～141 Gitboa-Garber professor experimental result there is narration.
Based on the above, desire to make Pseudomonas aeruginosa, perhaps its thalline extract, perhaps its metabolite becomes product useful, safety, such as immunomodulator, must overcome its toxicity.Simultaneously,, should impel its common immunogen of expressing various bacteria, form mannose sensitive haemagglutination pilus such as surface at bacterial cell in order to increase its wide spectrum effect performance.Utilize immunogenic reservation or further strengthened in order to reach preferable effect, to be further noted that.
The present invention has obtained a kind of pseudomonas aeruginosa strain of novelty by strain improvement and transformation, has satisfied above-mentioned needs.
Summary of the invention
First aspect, the invention provides a kind of pseudomonas aeruginosa strain, the called after pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain is also referred to as Pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain (Pseudomonas aeruginosa mannose-sensitive hemagglutination pilusstrain).On taxonomy, this bacterial strain belongs to a kind of novel strain of the Pseudomonas aeruginosa of Rhodopseudomonas (Pseudomonas).This bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on February 5th, 1993, No. 13 Microbe Inst., Chinese Academy of Sciences in north, Zhong Guan-cun, Haidian District, city, BeiJing, China, address.CGMCC is the international depositary institution of biological material specimens that budapest treaty is admitted.The numbering of this bacterial strain is CGMCC No.0190, and the classification name is Pseudomonas aeruginosa (Pseudomonas aeruginosa).
Pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain is characterised in that:
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.
Second aspect the invention provides the plasmid that influences bacterium nuclear dna encoding and express mannose sensitive haemagglutination pilus.Contain in the pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain and assist the bacterial nucleus dna encoding to express the proteinic plasmid of mannose sensitive haemagglutination pilus.The plasmid that adopts conventional way to extract from this bacterial strain assists bacterium nuclear dna encoding to express, and is also included within the present invention.Further, adopt this plasmid to help this genus of conversion or the resulting bacterial strain of other bacteriums also to be within the present invention.By the thalline engaging process, the plasmid in the pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain is shifted and nuclear staining body DNA integration, and the microorganism that comprises this plasmid of gained like this is also within the present invention.So adopt the gene of the separated coding mannose sensitive haemagglutination pilus that ordinary skill in the art means obtain, and this gene that exists with existence form in the plasmid or the existence form in the karyomit(e) also within the scope of the invention.
The 3rd aspect the invention provides the dead thalline of pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain, the thalline prepared product, and extract separates the material from thalline.The cell of pseudomonas aeruginosa 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 pseudomonas aeruginosa 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 pseudomonas aeruginosa 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 pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain of the present invention.Pseudomonas aeruginosa 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.The equal tool positive effect of immunoregulatory activity and antitumor action.
The accompanying drawing summary
The relevant preservation information of pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain of the present invention is as follows:
Preservation date: on February 5th, 1993
Depositary institution's title: China Committee for Culture Collection of Microorganisms common micro-organisms center
Depositary institution is called for short: CGMCC
Deposit number: 0190
Fig. 1 is from the isolating Pseudomonas aeruginosa electron micrograph of nature, shows the morphological specificity (feathering, non-mannose sensitive haemagglutination pilus) of the Pseudomonas aeruginosa described in the prior art.
Fig. 2 is the electron micrograph of pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain of the present invention, shows the morphological specificity (peritricha, mannose sensitive haemagglutination pilus) of pseudomonas aeruginosa strain of the present invention.
Fig. 3 is the oxydase comparing result photo of Pseudomonas aeruginosa of the present invention and other bacterium, and wherein upper part shows that the experiment of contrast bacterial oxidation enzyme is negative; Lower part is represented the Pseudomonas aeruginosa oxidase test positive of the present invention.
Fig. 4 is the pigment comparing result photo of Pseudomonas aeruginosa of the present invention.The flat pannel display contrast bacterium on the left side can not form the verdigris color marennin; The flat pannel display Pseudomonas aeruginosa of the present invention on the right has the feature that forms the verdigris pigment.
Fig. 5 shows that pseudomonas aeruginosa strain of the present invention forms N in the glucose nitrate culture-medium
2, performance is positive, and the contrast bacterium is negative findings; Also show in semi-solid sporting trial and present the positive.In Fig. 5, from left to right, No. 1 test tube is represented normal substratum contrast, and No. 2 test tubes represent that Pseudomonas aeruginosa of the present invention has formed bubble, and No. 3 and No. 4 test tubes represent to contrast bacterium; No. 5 test tube is represented the upgrowth situation of Pseudomonas aeruginosa of the present invention in semisolid medium.
Fig. 6 is the mannose sensitive haemagglutination slide aggegation direct viewing of pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain of the present invention, presents the positive photo of mannose sensitive haemagglutination.
Fig. 7 after the mannose sensitive haemagglutination of pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain of the present invention is suppressed by seminose, presents mannose sensitive haemagglutination negative findings photo.
Invent auspicious stating
1. definition and explanation
In this application, following term and explanation are specified.
" bacterium " is the unicellular prokaryotes of a class. Genophore is comprised of a branch of double-stranded DNA filament, without any the boundary of film it and cytoplasm is separated. Lack organelle. Ribosome is 70S. That talk about among the application is " eubacteria (eubacteria) ". Eubacteria can be divided into two types of Gram-positive and Gram-negatives.
" Gram's stain " is by using crystal violet solution and iodine liquid, and be different according to eubacterial cell wall structure, the colouring method that bacterium is distinguished.
" Pseudomonas aeruginosa genus " " listing in the pseudomonadaceae under the aerobic bacillus of Gram-negative and the coccus part in uncle's outstanding Bacteria Identification handbook the 8th edition. Cell is single, straight or curved bacillus. Single flagellum or many flagellums, Gram-negative. Organising can heterotrophic bacteria, respiratory metabolism, never fermentation. Catalase is positive. Type sepecies: pseudomonas aeruginosa (Psudomonas aeruginosa (Schroeter) Migula 1900,884).
" other gramnegative bacteriums of listing in uncle's outstanding Bacteria Identification handbook the 8th edition, it is also relevant with the present invention to be included in the 8th part Gram-negative facultative Bacteroides nodosus, Session 9 Gram-negative anaerobic bacteria, the tenth part Gram-negative coccus and coccobacillus.
" the 279th page of the outstanding Bacteria Identification handbook of uncle the 8th edition, this species different name is Pseudomonas aeruginosa for being described in of pseudomonas aeruginosa (Psudomonas aeruginosa (Schroeter) Migula 1900,884). New model strain: ATCC 10145; NCIB8259; NCTC 10332Opin.36, Jud. Comm.
" flagellum " is the locomotive 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 sex fimbria.
" ordinary pilus " adheres to relevant with bacterial body. Ordinary pilus is of a great variety, and the relatively difference according to its effect in bacterial adhesion can be divided into non-specific adhesion pili and selectivity and adhere to pili.
" non-specific adhesion pili " refers to that those can make the pili of bacterial adhesion on the multiple matrix, on multiple eukaryotic, the red blood cell of many animals for example, leucocyte, epithelial cell, and zooblast, plant cell and fungal cell in other tests. Namely these pili do not have strict selectively to the host, adhere to and have popularity, for example colibacillary 1 type pili (type 1fimbriae). When using the red blood cell in different animals source, non-specific adhesion shows the hemagglutination activity of parallel pattern; And erythroagglutination (hemagglutinaion) is to mannose-sensitive (mannose-sensitive). Therefore, mannose-sensitive erythroagglutination (mannose-sensitive hemagglutination, mannose sensitive haemagglutination) is a feature of non-specific adhesion pili.
" specificity adhesion pili " is meant that those impel the pili of bacterial adhesion on the particular substrate.Just these pili have strict selectivity to the host, and adhesion can only take place between bacterium and special host, such as coli common pili K88.Erythroagglutination does not show mannose-sensitive, i.e. the blood clotting that is not suppressed by seminose.
" mannose sensitive haemagglutination pilus " full name is " a mannose-sensitive erythroagglutination pili ", is meant to promote the non-narrow spectrum adhesion pili of bacterial adhesion on the multiple matrix, can be suppressed by seminose.In this application, mannose sensitive haemagglutination pilus is a generalized concept.Existing research prompting on bacterium, such as Pseudomonas aeruginosa, intestinal bacteria, Salmonellas etc., adheres in the process of host cell, and the sugar mediation adheres to ubiquity.Some is a mannose-sensitive, and some is a L-Fucose sensitivity, D-semi-lactosi sensitivity.At this, " mannose sensitive haemagglutination pilus " comprises mannose-sensitive cell agglutination effect pili, that various other carbohydrates are regulated, with bacterium and host's the relevant pili of non-specificity adhesion.
Existing " Pseudomonas aeruginosa " bacterial strain, comprise that the bacterial strain that is collected in each DSMZ is (such as ATCC10145, deposit number in the Chinese patent application 94190491.1 is KCCM10029, KCCM 10030, KCCM 10031, KCCM 10032, KCCM 10033, the KCCM10034 bacterial strain to KCCM 10035), laboratory study usefulness (such as PAK1244, PAK and PAU
1Bacterial strain).These bacterial strain major parts have strong toxicity, do not have whole body pili, and the pili type is non-mannose sensitive haemagglutination type.
Pseudomonas aeruginosa strains 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 pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain, and is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 5th, 1993, and numbering is CGMCC No.0190.
2. describe in detail
Pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain full name provided by the invention is pseudomonas aeruginosa (Pseudomonas aeruginosa) mannose sensitive haemagglutination pilus strain, it is a kind of Pseudomonas aeruginosa with whole body pili mannose sensitive haemagglutination (Mannose Sensitive Hemagglutination) pili, and this pilus strain has the wide spectrum immunogenicity of striding Pseudomonas.
As described in the background section, after the dead thalline of finding Pseudomonas aeruginosa has immunoregulation effect,, can't actually utilize owing to himself toxic relation.Existing research concentrates on to be managed to utilize on the extract or isolate of certain composition of bacterium or several compositions, for example the protein in the cell walls.Though someone has also carried out seed selection to bacterial classification, purpose is the bacterial cell wall-held protein that reduces bacterial strain toxicity, obtains to have higher-security.Therefore, prior art does not provide the feasible method that the material in the comprehensive utilization Pseudomonas aeruginosa thalline carries 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 making up a strain and have hypotoxicity, the immunogenic pseudomonas aeruginosa 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.
Relevant article and patent application following (the contents intact ground in these public publications is introduced in the present patent application) that the present inventor delivers this problem:
1. Mou Xiya, the foundation of pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain, microorganism journal, 1986,26 (2): 176;
2. Mou Xiya, the foundation of pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain and Preliminary Applications research, medical research communication, 1987,33:83-87;
3. Mou Xiya, Guo Yanqun, Fu Hongwen, the applied research of pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain, Dalian Medical College's journal, 1990,12 (2): 63-65;
4. Chinese patent application 93100114.5, February 6 1993 applying date, applicant's Mou Xiya, denomination of invention pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain and foundation thereof, publication number CN1090602A, open day on August 10th, 1994;
5. Mou Xiya etc., the anti-tumor activity immunoregulation effect of Pseudomonas aeruginosa preparation, Shanghai immunity magazine, 1994,14:218.
The present inventor and creates technology certainly and makes " Pseudomonas aeruginosa preparation " immunomodulator after successfully setting up pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain.
Since " Pseudomonas aeruginosa preparation " launch products, many medical workers and the present inventor further compare Journal of Sex Research work, the article of delivering have following these:
1. Sun Wen is flat, Fu Hongwen, and Liu Ni, etc., the research that its immune indexes changes after 50 routine cancer patientss' use " pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain " immunomodulators, Wu Mengchao chief editor, Chinese clinical tumor yearbook, Urumchi: the Xinjiang People's Press, 1997,330-331;
2. Sun Wen is flat, Fu Hongwen, and Liu Ni, Wu Yu, Mou Xiya, Jin Yan, higher primary school is flat, and the Pseudomonas aeruginosa preparation is to the clinical observation of three kinds of cancer patientss' immunity curative effects, Chinese microbiology and Journal of Immunology, the fourth phase the 20th in 2000 volume;
3. Zhao Yiliang, Yang Wei; Pseudomonas aeruginosa preparation, carboplatin are to treating the clinical observation of postoperative gastric cancer cancer ascites, Chinese traditional Chinese and western medicine magazine, the 4th the 13rd phase of volume of July in 2003.
Making up pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain is that deleterious Pseudomonas aeruginosa is transformed into a kind of key that the bacterial strain of medical value is arranged.The product of this bacterial strain shows beneficial effect on prevention and treatment of diseases.The Pseudomonas aeruginosa preparation is that pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain is after deactivation and the suspension aqueous injection of making has two-way immunomodulatory and wide spectrum anti-infectious function.To tumor-bearing mice, can improve scavenger cell and NK cytoactive, keep t helper cell and T and suppress cell ratio in normal level; Also can improve mouse Pseudomonas aeruginosa, Bacillus proteus, intestinal bacteria and pneumobacillus etc. are infected lethal survival rate; Can adjust the nonequilibrium state of human body fluid immunity and cellular immunization; Adjust the synergy of interleukin II, Interferon, rabbit and antibody.Indication: to all kinds of tumours, as leukemia, malignant lymphoma, liver cancer, cancer of the stomach, bladder cancer etc.; The infection that various pathogenic agent microorganisms cause is as urinary system repeated infection, tubercle bacillus affection, chronic hepatitis B, the infection of intractable oral Candida albicans etc.; Various autoimmune disorders all have prevention and therapeutic action as bronchial asthma, psoriatic, allergic rhinitis, nephrotic syndrome etc.
At the beginning of setting up pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain, the present inventor notices that the dead thalline of Pseudomonas aeruginosa has immunoregulation effect but toxicity is very big and be difficult to practicality simultaneously; The present inventor thinks that by the research of oneself bacterial pilli is important paathogenic factor (delivering immunology meeting for the first time collection of thesis in the whole nation " cause of disease effect of bacterial pilli " in August, 1964 in 1964) already; Existing pseudomonas aeruginosa strain (no matter be cultivate bacterial strain or from the isolating pseudomonas aeruginosa strain of nature) does not all have the Zhousheng pili, and pili all is non-mannose sensitive haemagglutination pilus; Mannose sensitive haemagglutination pilus in intestinal bacteria and Salmonellas has ubiquity, promptly on the various bacteria Pseudomonas mannose sensitive haemagglutination pilus is arranged all.There is mannose sensitive haemagglutination protein in the tenuigenin of known existing pseudomonas aeruginosa strain, but is based on the constructional feature of Pseudomonas aeruginosa itself, can not form mannose sensitive haemagglutination pilus at cell surface.The present inventor proposes to set up hypotoxic, as to have mannose sensitive haemagglutination pilus pseudomonas aeruginosa strain, and wherein the existence of mannose sensitive haemagglutination pilus makes this bacterial strain become a kind of the have strong antigen of practical value, the immunologic stimulant of broad spectrum.
The foundation of pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain of the present invention mainly comprises two aspects: 1. the virulence that reduces Pseudomonas aeruginosa; 2. express mannose sensitive haemagglutination pilus at the bacterial body surface-stable.
The method of the virulence of reduction pseudomonas aeruginosa mannose sensitive haemagglutination can have multiple, and the inventor has adopted continuous passage to reduce toxicity.
To separate from natural strong toxicity Pseudomonas aeruginosa strain as original bacterium sample strain (LD
50=10
7Viable bacteria), adopt and to add soup in the product of ordinary culture medium (as the substratum that the sodium-chlor that adopts by 0.3% meat extract, beef infusion broth, 1% peptone and 0.5% constitutes, pH=7.4～7.6) behind autoclaving again and cultivate and go down to posterity.
This soup can be the antibiotic medicine in the Western medicine, routine gentamicin, how withered rhzomorph etc.; Also can be the clearing heat and detoxicating medicine in the Chinese medicine, as Acalypha australis, Philippine Violet Herb, the root bark of tree peony, Herba Bidentis Bipinnatae, bitter dish and or the Radix Astragali etc.Above-mentioned soup is added to makes it concentration in the ordinary culture medium and be lower than Mlc.The bacteriocidal concentration of these medicines has nothing in common with each other, and can obtain these data with reference to the related tool book of pharmaceutical field.Get its bacteriocidal concentration 1/10th as Mlc, make it to develop immunity to drugs to lower virulence and toxicity.
Repeatedly repeat the above-mentioned operation of going down to posterity, easing down to through its virulence after 200 generations can be for the usefulness of preparation vaccine.Though the toxicity of the more original bacterium sample of its toxicity strain has descended nearly a hundred times, but still keeps original complete immunogenicity, still can form enough specific antibodies and cellular immunization after promptly injecting the non-lethal quantity of small white mouse.
Expressing mannose sensitive haemagglutination pilus at the bacterial body surface-stable is key of the present invention.
At first be to obtain the bacterial cell chromatin dna.For example, can adopt repeatedly the collaborative thalline that destroys of freezing and SDS synthetic method, deproteinize extracts DNA then.
Gained bacterial cell chromatin dna is carried out enzyme cut, obtain the DNA small segment.The chromosomal DNA of getting above-mentioned attenuated strain adopts Pstl, EcoRl and the compound restriction endonuclease of Hpall to cut 1～2 hour at 65 ℃ of enzymes, makes it become small segment.
The DNA small segment engages again at random.Get the liquid that contains the DNA small segment and add 0.3～0.5% ligase enzyme,, make various tiny dna fragmentations be bonded into long-chain DNA (two strands) again at random 0～12 ℃ of effect 1～12 day.Engage situation with submicroscopy.If do not connect, change ligase enzyme and repeat.
Be prepared into single stranded DNA.Add restriction endonuclease to above-mentioned the joint in the liquid that long-chain DNA is arranged again, for example the compound restriction endonuclease of PstI, EcoRI and HPaII was cut about 5 minutes at 65 ℃ of enzymes, made long-chain DNA become strand.
DNA with strand transforms the attenuation thalline.The above-mentioned DNA liquid of separating two strands is joined the CaCl that contains conventional concentration
2In the attenuated strain, in the ordinary culture medium that adds 2% agar (by the mixture of the sodium-chlor of 0.3% meat extract, beef infusion broth, 1% peptone and 0.5%), clone in 37～41 ℃ of common cultivations repeatedly, promptly may obtain pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain.
The relevant method of setting up pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain, particularly has a detailed description in the disclosed Chinese patent application 93100114.5 article that the present inventor's Mou Xiya is delivered.All the elements of Chinese patent application 93100114.5 are introduced herein, as a reference.
After the success of pseudomonas aeruginosa mannose sensitive haemagglutination strain construction, maintain property is stable always.So far go down to posterity 40 times, still keep original characteristics.Also aspect anti-infective, antineoplaston and the immune disorder good result is being arranged all by the vaccine of pseudomonas aeruginosa mannose sensitive haemagglutination bacterial strain preparation.
The principal character of pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain is as follows:
1. form cultural characters
(1) Gram-negative reaction, thalline is the microbend bacillus.Flagella staining shows that thalline one end has single or several flagellums.Hiss capsule stain feminine gender.
(2) the semi-solid power (+) of cultivating
(3) β zone of hemolysis (+) on the blood agar flat board
(4) produce water-soluble pyocyanin (+) on the KingShi agar plate
2. biochemical reaction
(1) glucose oxidase decomposition run (+)
(2) Chinese holly edge hydrochlorate substratum (Xi Menshi) growth test (+)
(3) urease forms test (christensen) substratum (+)
(4) nitrate glucose forms N
2(+)
(5) oxydase forms test (+)
(6) 42 ℃ of incubation growth tests of brain heart infusion meat soup (+)
(7) hydrolysis acetamidase test (+)
3. serum agglutination test
The Pseudomonas aeruginosa blood grouping serum that uses the Chengdu institute of Biological Products to produce is a serotype IV type Pseudomonas aeruginosa with this identification of strains.
Wherein the corresponding test of (+) expression is positive.
4. pili form and character
(1) Fig. 2 is the electron micrograph of bacterial strain of the present invention, can see that the thalline of this bacterial strain has typical Zhousheng sex fimbria.
(2) the part pili is a mannose sensitive haemagglutination pilus.Mannose sensitive haemagglutination and hemagglutination-inhibition test are strong positive, referring to Fig. 6 and Fig. 7.Wherein relevant method of carrying out the mannose sensitive haemagglutination test, referring to Dalian Medical College's journal, 1985,7 (1): 1-3.
Embodiment
Embodiment 1 seed selection pseudomonas aeruginosa mannose sensitive haemagglutination bacterial strain
Get natural non-all pilus strains, the strong toxicity Pseudomonas aeruginosa of non-mannose sensitive haemagglutination strain (LD
50=10
7Individual viable bacteria), the substratum (pH=7.5) that constitutes at the sodium-chlor by 0.3% meat extract, beef infusion broth, 1% peptone and 0.5% adds soup again behind autoclaving, and this soup is that the mixed solution of 2～8% Radixs Astragali and 1% Herba Bidentis Bipinnatae is cultivated.Virulence through the Pseudomonas aeruginosa of going down to posterity for 462 times adopts kind 14～18 gram small white mouses in Kunming to record LD
50=9 * 10
8Individual viable bacteria.Place the distilled water cooling to make it glaciation the Pseudomonas aeruginosa of above-mentioned attenuation, meanwhile also need in distilled water, add the SDS medicine, thalline is destroyed through repeated multiple times afterwards again with its thawing.Add chloroform-primary isoamyl alcohol then in ruined thalline, then protein is flocks and separates out.Centrifugation is removed protein and is left and taken supernatant liquor, uses the diluted hydrochloric acid dissolution particulate matter again.The supernatant liquor that will contain DNA repeatedly washs with aqueous ethanolic solution removes the chromosomal DNA that salt can obtain the attenuated strain of purifying.Have or not the DNA gene with electron microscope (ten thousand times of X100) inspection.Chromosomal DNA to above-mentioned attenuated strain adopts Pstl, EcoRl and the compound restriction endonuclease of HPaII to cut 1～2 hour, make it become small segment, get the liquid that contains the DNA small segment and add 0.3%～0.5% ligase enzyme, 0～12 ℃ of effect 1～12 day, make various tiny dna fragmentations be bonded into long-chain DNA (two strands) again at random.Engage situation with submicroscopy.In above-mentioned recombine has the liquid of long-chain DNA, add Pstl, EooRI and the compound restriction endonuclease of HPaII, cut about 5 minutes at 65 ℃ of enzymes.Use membrane filtration then, filtrate being added to contained in the attenuated strain of calcium chloride of 5% concentration, in substratum, clone the mannose sensitive haemagglutination bacterial strain that promptly may obtain Pseudomonas aeruginosa repeatedly in 37～41 ℃ of co-cultivation by 0.3% meat extract, beef infusion broth, 1% peptone, 0.5% sodium-chlor and 2% agar.
Embodiment 2 pseudomonas aeruginosa mannose sensitive haemagglutination pilus strains
The inventor is resulting bacterial strain, the called after pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain.It meets the identification mark of pseudomonas on the taxonomy, has the desired mannose sensitive haemagglutination pilus of the present invention simultaneously.Mannose sensitive haemagglutination pilus is the distinctive feature that bacterial strain of the present invention is different from existing pseudomonas aeruginosa strain, gives the strong immunity property analysed and the wide spectrum immunogenicity of bacterial strain of the present invention.This pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain is deposited in Chinese microbial preservation management committee common micro-organisms center on February 5th, 1993, and preserving number is CGMCC No.0190.
According to clinical microbiology (higher medical universities and colleges cooperation teaching material), Dalian Medical College printing house, 1987.7 first impressions, the 210-346 page or leaf is identified Pseudomonas aeruginosa of the present invention.Find that Pseudomonas aeruginosa of the present invention has whole mycology features of Pseudomonas aeruginosa, sees Table 1.
Table 1. Pseudomonas aeruginosa of the present invention has whole mycology features of Pseudomonas aeruginosa
| The mycology feature | Pseudomonas aeruginosa strain of the present invention | Non-pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain (is example with intestinal bacteria) |
| Form | Gram negative bacillus end flagellum | The all flagellums of gram negative bacillus |
| Power | The semi-solid top of positive (see figure 5) presents muddiness | Positive semi-solid top presents muddiness |
| Oxidase test | Positive (see figure 3) | Negative |
| The dull and stereotyped king A of pigment substratum | Verdigris pigment (see figure 4) | Non-pigment |
| Glucose oxidase | Positive nonfermented | Negative fermentation (producing sour aerogenesis) |
| The maltose oxidation | Not oxidation nonfermented | Fermentation (producing sour aerogenesis) |
| The wood sugar oxidation | Positive nonfermented | Negative fermentation (producing sour aerogenesis) |
| On Chinese holly wood-rimmed acid substratum | Growth | Do not grow |
| Methionin decarboxylation acid test | Negative | ?- |
| The arginine dihydrolase test | Positive | ?- |
| Ornithine decarboxylase | Negative | ?- |
| In 42 ℃ of brain heart infusion agars | Growth | Do not grow |
| Serological test (Chengdu institute of Biological Products blood grouping serum) | IV type Pseudomonas aeruginosa | Do not react |
Pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain of the present invention has the mycology feature different with the non-mannose sensitive haemagglutination pilus strain of Pseudomonas aeruginosa again, sees Table 2.
The new mycology feature that table 2 Pseudomonas aeruginosa of the present invention has
| The mycology feature | Pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain of the present invention | The non-mannose sensitive haemagglutination pilus strain of Pseudomonas aeruginosa | The relevant document of judgment standard |
| Form | Many very thin and upright and outspoken pili (referring to Fig. 2) are arranged around the thalline | No mannose sensitive haemagglutination pilus | Electron microscope observation |
| The mannose sensitive haemagglutination test | Positive (see figure 6) | Negative | Dalian Medical College's journal 1985,7 (1): 1-3 |
| Flat-plate bacterial colony adheres to the red corpuscle test | Positive | Negative | The opticmicroscope low power is observed (10 * 10) |
| Saccharomycetic direct agglutination test | The MS blood clotting positive | MS blood clotting feminine gender | Opticmicroscope (1000X) |
The cultural characters of this bacterial strain is same as common Pseudomonas aeruginosa, can preserve, go down to posterity and cultivate with reference to the introduction in relevant textbook, the reference book.
The hereditary property of embodiment 3 pseudomonas aeruginosa mannose sensitive haemagglutination pilus strains
Present embodiment is the experiment situation that obtains the mutated site research of whole body mannose sensitive haemagglutination pilus about pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain.
Experiment (1):
Result: extract plasmid DNA from this bacterium peritricha mannose sensitive haemagglutination pilus strain, transmit and modify feathering non-mannose sensitive haemagglutination Pseudomonas aeruginosa pilus strain (the high persister of penbritin), observe non-mannose sensitive haemagglutination pilus strain and whether on the basis of bacterial nucleus DNA reorganization, be subjected to the coordination of plasmid DNA and adjustment may obtain to express peritricha mannose sensitive haemagglutination pilus strain character again.
Method: (a) plasmid DNA is extracted: the peritricha mannose sensitive haemagglutination is in 37 ℃ of cultivations, centrifugal of L-meat soup, put in the sucrose Tritonx-100 damping fluid, collect bacterium, the a plurality of centrifuge tubes of packing, add N,O-Diacetylmuramidase respectively, 10～15 ℃ with 10～20 minutes, and 100 ℃ of water-baths were put ice bath in 30 minutes about 5 minutes again.High speed centrifugation (1.5 ten thousand rpm/ branch) adds propyl alcohol or ethanol and puts 20 ℃ of nights, and high speed centrifugation is got the DNA precipitation and is dissolved in the TE damping fluid again, and RNAase goes RNA.0.7～1.2% agarose electrophoresis to isolate the plasmid DNA of 0.4～10Kb.
(b) plasmid DNA is transmitted test design:
37 ℃ of shaking culture of non-mannose sensitive haemagglutination bacterium LB base 2 hours, ice bath is 10 minutes again, and 4000 rev/mins of centrifugal supernatants that go are got bacterial sediment, suspend with 0.85%NaCl, 4000 leave the heart and add 5%CaCl again
2The suspension thalline was put ice bath after 20 minutes, 4000 left the heart again, abandoned supernatant.At last with sedimentary bacterial suspension in a small amount of 5%CaCl
2In the solution, become and accept to transmit bacterium.
In serial test tube, add the plasmid DNA of 0.4～10Kb respectively., add above-mentioned transmission bacterium simultaneously, promptly non-mannose sensitive haemagglutination bacterium.42 ℃ of water-baths 3 minutes were then put ice bath 60 minutes immediately.Every pipe adds 37 ℃ of vibrations of LB nutrient solution and cultivates a few hours, to bacterium liquid muddiness, electron microscopic observation.
Experiment (2):
The result: by live body peritricha mannose sensitive haemagglutination pilus strain and the mutual post-coitum of the two trafficability characteristic pili of the non-mannose sensitive haemagglutination pilus strain of feathering, the variation of the two research.
Method: the two is inoculated in respectively in the LB substratum with live body peritricha mannose sensitive haemagglutination pilus strain and the non-mannose sensitive haemagglutination pilus strain of feathering, 37 ℃ of shaking culture 2 hours, the ice bath effect is 10 minutes again, 4000 rev/mins of centrifugations, remove supernatant, get the bacterial precipitation thing respectively and suspend 4000 rev/mins of centrifugations again with 0.85%NaCl solution, remove supernatant, add 5%CaCl
2, liquid suspension thalline is put the ice bath effect after 20 minutes again, again 4000 rev/mins centrifugal, remove supernatant.Thalline with centrifugation is suspended in a small amount of 5%CaCl at last
2In the solution.42 ℃ of water-baths 3 minutes were then put ice bath 60 minutes immediately.Add LB nutrient solution (containing the high strength ammonia penicillin G) at last respectively, a few hours are cultivated in 37 ℃ of vibrations, begin muddiness until bacterium liquid.Carry out electron microscopic observation immediately.
Experiment (3):
Result: detect the non-mannose sensitive haemagglutination pilus strain chromosome length of peritricha mannose sensitive haemagglutination pilus strain and feathering difference respectively, and the gene studies of coding peritricha mannose sensitive haemagglutination pilus strain, find that its karyomit(e) all is linear, length is all near 910700bp.
Method: the chloroform phenol hybrid system of introducing in bright 1989 according to woods ten thousand at first proposes DNA of bacteria, uses glass rod to contact gently and can propose thread sedimentary DNA, DNA is dissolved in an amount of 0.1SSC liquid again, observes promptly to show under the Electronic Speculum to be linear state.According to the 1. bacterium thalline sum mean value and the 2. parameter of plasmid 0.4～10Kb and karyomit(e) ratio, inventor's predesigne goes out to be 910700bp.
These tests show that the karyomit(e) of pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain or nuclear are distinguished the big or small basic identical of interior dna molecular and other Pseudomonas aeruginosas.
The vaccine of embodiment 4 preparation pseudomonas aeruginosa mannose sensitive haemagglutination pilus strains
With the pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain inoculation LB semisolid medium of preserving, put 37 ℃ of overnight incubation, the preparation activated spawn.
In the LB culture medium slant, the inoculation activated spawn is cultivated 18～20h at 37 ℃.Draw the moistening whole media surface of formaldehyde (1% formaldehyde PBS) solution 2～3ml with valinche, with the whole sucking-offs of bacterium liquid, move in the empty saline bottle after washing repeatedly, draw 3～4ml formaldehyde PBS again and carry out the flushing second time, be transparence until substratum, obtain the bacterium liquid of deactivation.Collected thalline with 6000xg in centrifugal 10 minutes at 4 ℃.PBS with sterilization dilutes the bacterium liquid that contains 10,000,000,000 dead thalline into about every ml, uses as vaccine.
The composition and the compound method of LB substratum are as follows:
Prepare every liter of substratum, should in the 950ml deionized water, add: microbial culture tryptone 10g, microbial culture yeast extract 5g and NaCl 10g.Shake container to solute and dissolve fully, regulate pH value to 7.0, add deionized water to cumulative volume 1L with 5mol/L NaOH (about 0.2ml).Then, add Bacto-agar according to 7g/L or 15g/L, at 151bf/in
2(1.034 * 10
5Pa) steam sterilizing 20 minutes under the high pressure, preparation semisolid medium and culture medium slant.
The application characteristic of embodiment 5 pseudomonas aeruginosa mannose sensitive haemagglutination pilus strains
Pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain can be made vaccine or immunomodulator.Any toxic reaction is seen at 15,000,000,000 dead thalline of injected in mice end, and the human body usage quantity is 1,800,000,000 dead thalline, only is 1/10 of small white mouse, and clinical treatment is fool proof.Also disclose said preparation nutritive value, excite regulation and control immune effect, the anti-infection ability of striding Pseudomonas, antitumor action, to effect of some inflammation and immune disorder class illness or the like.
The non-mannose sensitive haemagglutination pilus of the existing nature Pseudomonas aeruginosa of pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain provided by the invention has the expression of mannose sensitive haemagglutination pilus on the bacterial body surface again, also has sex fimbria.
Adopt yeast culture, the prepared dead thalline of deactivation thalline can be used as the activeconstituents of preparation vaccine.In this vaccine, have on the non-mannose sensitive haemagglutination pilus of inherent pili part (ligands) can with the glycolipid receptor acting of host cell.A plurality of Pseudomonas such as helicobacter pylori also are to adhere to the glycolipid acceptor, so can block the infection of most of false unit cell bar genus, Helicobacterium, Aeromonas, eisseria, legionella etc. with the vaccine of pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain preparation provided by the invention.Pili part on the mannose sensitive haemagglutination pilus can combine with the glycoprotein receptor of host cell, can block infection and the infection of most of Escherichia, proteus, Klebsiella, Aerobacter, Citrobacter, the inferior Bacillaceae of Hough Buddhist nun, bacillus cloacae genus, salmonella spp, shigella flexneri, Eltor vibrio cholerae etc.Sex fimbria can adhere on the surface receptor of many genus bacteriums, blocks the ability that it produces resistance and virulence.The pili multiple ligand on vaccine surface can directly stick on pathogenic micro-organism and the cancer cells surface, both can block it and adhere to infection and invasion and attack body, can strengthen its immunogenicity in human body again.The pilin multiple ligand of vaccine under the synergy of the flagellum of bacterium thalline, outer membrane protein, somatic cells wall glycolipid antigen, sticks on scavenger cell, T and the bone-marrow-derived lymphocyte surface, activates the high energy mannose receptor, directly activates them.Make it when performance kills and wounds invasion pathogenic micro-organism and cancer cells, and bear the antigen presenting cell function quickly and efficiently.Make the NK cell, the T cell, B cell and humoral immunization protection mobilize fast comprehensively.
Human body is accepted " vaccine " 2～4 weeks, detects, and finds NK, CD3, CD4, cd8 cell number, activity, CD4/CD8 ratio, protectiveness factor fast steering states in a healthy and balanced way such as endogenous interferon-gamma, interleukin II, IgG, IgM, SIgA.
Clinical application shows, stride Pseudomonas anti-infective aspect, opportunistic infection flora such as ectoparasite flora of pair cell such as Escherichia (intestinal bacteria), proteus, Rhodopseudomonas (Pseudomonas aeruginosa), klebsiella spp (pneumobacillus), Citrobacter and L type thereof infect curative ratio and reach more than 90%.The infection of pair cell endoparasitism flora such as tubercule bacillus also has significant curative effect.
Lung cancer, malignant lymphoma, leukemia, bladder cancer etc. all there is significant curative effect.To bladder cancer, adopt the vaccine infusion therapy, the recurrence of 91.9% postoperative end.
Aspect treatment immune disorder illness: asthma, psoriatic, urticaria, lupus erythematosus, bleb-zoster, similar rheumatism all obtain good efficacy.Asthma curative ratio 57～75%, efficient more than 90%.The analgesic effect of rheumatoid arthritis is particularly outstanding.
Claims (4)
1. pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain, preserving number is CGMCC0190.
2. pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain as claimed in claim 1 is characterized in that having pili all over the body.
3. pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain as claimed in claim 1 is characterized in that having non-mannose sensitive haemagglutination pilus.
4. pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain as claimed in claim 1 is characterized in that having sex fimbria.
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| CN101914463B (en) * | 2010-02-09 | 2012-09-05 | 北京万特尔生物制药有限公司 | Proteus vulgaris (mannose-sensitive hemagglutination) piliated strain |
| WO2013004020A1 (en) * | 2011-07-07 | 2013-01-10 | 深圳市宝舜泰生物医药股份有限公司 | Pseudomonas aeruginosa (pa-msha) strain igy and preparation method and use thereof |
| CN103690941B (en) * | 2013-12-30 | 2016-03-23 | 北京万特尔生物制药有限公司 | A kind of Pseudomonas aeruginosa MSHA pilus strain tumor vaccine and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1090602A (en) * | 1993-02-06 | 1994-08-10 | 牟希亚 | Pseudomonos aeruginosa MSHA fimbria strain and foundation thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1090602A (en) * | 1993-02-06 | 1994-08-10 | 牟希亚 | Pseudomonos aeruginosa MSHA fimbria strain and foundation thereof |
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