CN105726580A - Application of serratia marcescens vaccine in preparing medicine for preventing and/or treating immune system functional defect diseases and diabetes - Google Patents
Application of serratia marcescens vaccine in preparing medicine for preventing and/or treating immune system functional defect diseases and diabetes Download PDFInfo
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Abstract
The invention discloses an application of serratia marcescens and vaccine thereof in preparing a medicine for preventing and/or treating immune system functional defect diseases and/or diabetes. The serratia marcescens is collected in the China General Microbiological Culture Collection Center on January 11, 2016, with collection number CGMCC NO.11987. The serratia marcescens vaccine provided by the invention realizes an obvious effect in preventing and treating immune system functional defect diseases and/or diabetes; and the serratia marcescens vaccine is an inactivated vaccine which is safe without toxic or side effect.
Description
Technical field
The present invention relates to the purposes of serratia marcescens, be specifically related to the application in preparation prevention and/or treatment function of immune system defect class disease and/or diabetes medicament of this Becterin of Serratia marcescens.
Background technology
Function of immune system defect class disease refers to owing to immune organ, tissue, cell and molecule exist birth defect, or the day after tomorrow is secondary to infection, cancer, metabolic disease, malnutrition or the factor such as physics and chemistry, caused immunologic inadequacy syndrome, including PID and secondary immunodeficiency disease.
Treatment for function of immune system defect class disease at present adopts immunosuppressive therapy more, as adopted treatment allergy disease, autoimmune disease, the lymphoproliferative disease etc. such as Adrenal Glucocorticoid, cell toxicant material, ciclosporin, monoclonal antibody；Or adopt immunomodulating, enhancing and immunity replacement therapy treatment immune deficiency disorder, autoimmune disease etc..But the disease of the immunodeficiency class that above-mentioned Therapeutic Method is treated is often relatively single, and its therapeutic effect is also less desirable.
Diabetes (diabetes) are by inherited genetic factors, immunologic function disorder, microorganism infection and toxin thereof, free radical toxin, the various virulence factor of Nervous and Mental Factors etc. acts on body and causes hypoinsulinism, insulin resistant etc. and the sugar that causes, protein, fat, a series of metabolism disorder syndromes such as power and water Xie Zhi, clinically with hyperglycemia for main feature, model case may occur in which polyuria, polydipsia, polyphagia, the performance such as become thin, i.e. " three-many-one-little " symptom, diabetes (blood glucose) are once control bad to cause complication, cause kidney, eye, the exhaustion pathological changes at the positions such as foot, and cannot cure.The orally-taken blood sugar reducing drug main treating diabetes at present to have biguanides, sulfonylurea drugs, Thiazolidinediones, glinides, alpha-glucosidase inhibitor, dipeptidyl peptidase-4 (DPP-4) inhibitor and injection of insulin agent, but the above-mentioned healing potion to diabetes, often price is high, and efficiency also tends to not fully up to expectations.
Serratia marcescens (Serratiamarcescens), also known as Bacterium prodigiosum, is a kind of antibacterial producing scarlet pigment, and its form of diverse is distributed widely in nature, is often occupy flora in water and soil earth.Both at home and abroad to the research of serratia marcescens relatively early, but in prevention and treatment function of immune system defect class disease and diabetes and be not belonging to popular research field.Research is used for the preparation of prodigiosin mostly using serratia marcescens as engineering bacteria；Some reports (patent 2012103989308) are also had to be used for preventing and treating Coptotermes formosanus Shtrari. by serratia marcescens；And utilize serratia marcescens to prepare biological dye (patent 2012101038897)；The less report being related to serratia marcescens is used for prevent and treat function of immune system defect class disease；Have no the report that serratia marcescens is used for preventing and treating diabetes.The medication effect of existing treatment immune deficiency disorder and diabetes is not good enough, and patient generally requires extra medication, brings inconvenience to patient, and adds medical treatment cost.
Summary of the invention
For above-mentioned prior art, it is an object of the invention to provide Becterin of Serratia marcescens application in preparation prevention and/or treatment function of immune system defect class disease and/or diabetes medicament.
The present invention is based on the early-stage Study of inventor, to be derived from the serratia marcescens (preserving number: CGMCC1.0589) of China General Microbiological culture presevation administrative center preservation as starting strain, a strain new strains (preserving number is CGMCCNO.11987) is obtained through ultraviolet mutagenesis, research finds that it has the anti-tumor activity of wide spectrum, and the basis using this bacterial strain as research, carry out a series of technology patulous research.Inventor is when utilizing above-mentioned bacterial strains to carry out clinical antineoplastic research, it is found surprisingly that, this bacterial strain (preserving number is CGMCCNO.11987) not only has anti-tumor activity, and immunodeficiency class disease and diabetes also have prevention and/or therapeutic effect preferably.
The present invention adopts following technical proposals:
According to the first aspect of the invention, it is provided that serratia marcescens application in preparation prevention and/or treatment function of immune system defect class disease and/or diabetes medicament.
Serratieae (Serratiasp.) SM-1 of the present invention, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 01 11st, 2016 and (is called for short CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCCNO.11987.
The morphological feature of this bacterial strain is: gram negative bacilli, is sized to 1.5～0.6um, and flagellum, dynamic all over the body, without pod membrane and spore.Ordinary broth agar culture medium (pH7.2-7.4) well-grown, bacterium colony milky, free from extraneous odour, rounded, smooth, protuberance, neat in edge.
Physio-biochemical characteristics are: glucose fermentation, sucrose, mannitol, inositol, sorbitol, amygdaloside, produce acid not aerogenesis；Azymic rhamnose, arabinose, 6-(.alpha.-D-galactosido)-D-glucose., can produce the outer deoxyribonuclease of born of the same parents.
Cultivate for 4～28 DEG C and produce red pigments, but 37 DEG C are cultivated not chromogenesis, and cultivation temperature is reduced to 28 DEG C of cultivations from 37 DEG C and can produce red pigments.
According to the second aspect of the invention, it is provided that the application in preparation prevention and/or treatment function of immune system defect class disease and/or diabetes medicament of a kind of Becterin of Serratia marcescens.
The active component of described Becterin of Serratia marcescens is pure culture biology of above-mentioned serratia marcescens.
The preparation method of described Becterin of Serratia marcescens is, pure culture biology of serratia marcescens obtained by suitable separation method.
Described suitable separation method is: 4000-6000rpm is centrifuged 30-60min, abandons supernatant, precipitation is carried out inactivation treatment, then is centrifuged, and abandons supernatant, washing, and centrifugation is prepared and be get final product.
Described function of immune system defect class disease includes lupus erythematosus, psoriasis, dry syndrome, ichthyosis, wart class sexually transmitted disease (STD) and asthma.Described diabetes class disease includes type Ⅰ diabetes mellitus and type Ⅱdiabetes mellitus.
Serratia marcescens biology pure culture preparation method be: by single colony inoculation of serratia marcescens slant strains on LB fluid medium, shaker fermentation cultivate；The condition of fermentation culture is: LB fluid medium pH6.0-8.0, and inoculum concentration is 8-15%, ventilation 40-90%, cultivation temperature 32-39 DEG C, incubation time 16-26h, rotating speed 120-200rpm.
In above-mentioned application, it is possible to by Becterin of Serratia marcescens with medically acceptable carrier in the form of compositions, put on the patient that need to treat, make consumption can be changed according to the age of patient, the state of an illness etc..
Described carrier refers to the carrier that pharmaceutical field is conventional, such as diluent, dispersant, stabilizer etc..
Above-mentioned composition can prepare into various dosage form in a conventional way, it is preferred that for: injection formulation or freeze-dried powder.
In above-mentioned injection formulation, the weight content of Becterin of Serratia marcescens is 0.1-99.9%, it is preferred that content is 0.5-90%.
Beneficial effects of the present invention:
(1) function of immune system defect class disease and diabetes are respectively provided with obvious preventive and therapeutic action by Becterin of Serratia marcescens provided by the invention；And the vaccine that this Becterin of Serratia marcescens is inactivation, safe without toxic side effect.
(2) Becterin of Serratia marcescens provided by the invention proves through clinical application research for many years, its determined curative effect；And the research of existing serratia marcescens rests on laboratory stage more, the prospect of its clinical practice remains in many uncertainties, and therefore, the Becterin of Serratia marcescens of the present invention is more for the value of clinical practice.
Accompanying drawing explanation
Fig. 1: cladogram schematic diagram.
Detailed description of the invention
Instrument involved in following embodiment, reagent, material etc., unless otherwise noted, be in prior art existing conventional instrument, reagent, material etc., can pass through regular being either commercially available.Experimental technique involved in following embodiment, detection method etc., unless otherwise noted, it is in prior art existing normal experiment method, detection method etc..
Embodiment 1: the screening of bacterial strain
1. starting strain screening
Strain is derived from the serratia marcescens of China General Microbiological culture presevation administrative center preservation, preserving number: CGMCC1.0589, under aseptic condition, be respectively coated on LB slant medium after taking 10 times of gradient dilutions of former strain 37 DEG C cultivate 24h.Select the flat board that colony count is about 20, picking is relatively big, distinct, be creamy white single bacterium colony of growing fine starting strain as ultraviolet mutagenesis, take its thalline in the triangular flask of normal saline and bead, make aseptic bacteria suspension 500mL, 30 DEG C, 250rpm water-bath 4h, prepare into 106The mutagenic bacteria suspension of CFU/mL, standby.
2. the determination of strain mutagenesis and fatality rate
Uviol lamp preheating 25min, takes mutagenic bacteria suspension 10mL respectively and is placed in multiple flat board (D=9cm), uniformly places 25cm place under uviol lamp and irradiates the different time (0,10,15,20,25,30,35,40,45,50,55,60s), the bacteria suspension then taking out 1mL respectively from each flat board is settled to 10mL with normal saline, and stepwise dilution is to 10-1、10-2、10-3、10-4、10-5、10-6Times, the diluent 100uL taking each gradient in the dark coats on Solid media for plates, takes out after 37 DEG C of cultivation 24h, and the bacterium colony that picking grows fine, test tube slant solid medium preserves, and carries out the screening of superior strain.Meanwhile, do blank with non-irradiated bacteria suspension, calculate ultraviolet mutagenesis fatality rate.
The clump count of the clump count of fatality rate=ultraviolet mutagenesis bacteria suspension/non-mutagenic bacteria suspension
3. the screening of superior strain
3.1. flat board primary dcreening operation
The mutagenic bacteria suspension normal saline dilution irradiating 25-30s is chosen to 10 according to ultraviolet mutagenesis-1、10-2、10-3、10-4、10-5、10-6Times, it is respectively coated in Solid media for plates, cultivates 24h for 37 DEG C.Choosing single bacterium colony 35 strain with bacterium colony size and growth conditions for observation index, test tube slant goes down to posterity after 3 times, and totally 6 strain of stable growth is respectively designated as SM1-6.
3.2. shaking flask is sieved again
1. strain: SM1, SM2, SM3, SM4, SM5, SM6.
2. method: by above-mentioned bacterial strains with starting strain SM0 respectively by the inoculum concentration of 10%, be placed in the triangular flask (25mL/100mL) equipped with LB fluid medium, cultivate 21h, shaking speed 180rpm for 37 DEG C.
3.3. the mensuration of cell concentration
Adopt turbidimetry for Determination cell concentration, take 1mL fermentation liquid sterile distilled water and dilute 10 times, do equivalent dilution with nonvaccinated culture medium and compare, measure optical density (OD) value of cell suspending liquid at 650nm wavelength place.
3.4. culture presevation
It is stored in-80 DEG C of refrigerators through sieving gained serratia marcescens high yield strain excellent test tube slant again.
4. interpretation of result
4.1. induction mutation of bacterium result
The different time is irradiated at serratia marcescens bacteria suspension 25cm place under uviol lamp, obtains the relation of mutation time and fatality rate, in Table 1.
Table 1 ultraviolet mutagenesis and fatal ends
| Irradiation time | 0 10 20 25 30 40 50 60 |
| Fatality rate (%) | 0 42.1 58.7 71.2 80.3 89.6 97.5 99.4 |
According to the literature, generally there is direct mutation in dosage on the low side in ultraviolet mutagenesis, occurs negative sudden change in higher dosage.Forefathers study and tend to low dosage process cell more, and general fatality rate controls between 70-80%.The irradiation time that this experiment adopts is 0-60s, and result shows: serratia marcescens fatality rate within the scope of ultra-vioket radiation 25-30s is 70-80%.
4.2. superior strain stability the selection result
The good six strain bacterium of SM1, SM2, SM3, SM4, SM5, SM6 stability through Uv-induced screening, carry out shaking flask to sieve again with starting strain SM0 simultaneously, wherein bacterial strain SM1 shake flask fermentation obtains Fungal biodiversity (bacterial population) is 10.65 hundred million/mL, higher than other bacterial strains；And preservation in-80 DEG C of refrigerator, regularly go down to posterity (25d goes down to posterity once).
Table 2 six strain serratia marcescens shaking flask sieves Fungal biodiversity comparative result again
| Bacterial strain | SM0 SM1 SM2 SM3 SM4 SM5 SM6 |
| Fungal biodiversity (108/mL) | 5.13 10.65 7.81 8.36 6.69 9.64 6.16 |
4.3. mitotic stability
Bacterial strain SM-1 was passed for 60 generations continuously, still conforms to serratia marcescens characteristic, illustrate that bacterial strain SM-1 has mitotic stability.
4.4.16SrDNA check order
The 16SrDNA of bacterial strain SM-1 is checked order (authorized company is Hua Da gene), and sequencing result is such as shown in SEQIDNO.1.
TATTATGTTCCTTGTCGTAAGCGCCCTCCCGAGGTTAAGCTAACTACTTCTTTTAGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACATACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATATGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGATTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAATCGACATCGTTTACAGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTCGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACGAGACTCTAGCTTGCCAGTTTCAAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCTGACTTAACAAACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGAGTAACGTCAATTGATGAACGTATTAAGTTCACCACCTTCCTCCTCGCTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCATTACCCCACCTACTAGCTAATCCCATCTGGGCACATCTGATGGCAAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCGACGTTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCGCTCGTCACCCAGGGAGCAAGCTCCCCTGTGCTACCGCTCGACGAGCACGGAAGCGTACTAAGCCAA。(SEQIDNO.1)
Above-mentioned 16SrDNA sequence is done tetraploid rice, draws cladogram as shown in Figure 1.16SrDNA sequence and the 16SrDNA sequence of other bacterium in Genbank of SM-1 bacterial strain is compared with blast.Through comparison, this bacterial strain is 99.79% with Serratiasp.CH-B17 homology in Genbank；With Serratiasp.EBL4 likelihood 99.78%, SerratiamarcescensstrainTCCC11387 likelihood 99.77%, SerratiamarcescensstrainAU1209 likelihood 99.76%, SerratiamarcescensstrainSER1 likelihood 99.437%, these bacterial strains belong to serratia marcescens.
16SrDNA is the conserved sequence of antibacterial, is referred to as " living fossil " sequence.By sequencing, the prompting of sequence alignment result: mutagenesis screening bacterial strain SM-1 is serratia marcesens.This bacterial strain has been carried out preservation by applicant, this strain classification called after Serratieae Serratiasp., is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 01 11st, 2016, and deposit number is CGMCCNO.11987.
Embodiment 2: the preparation of Becterin of Serratia marcescens
The preparation process condition of Becterin of Serratia marcescens is:
(1) slant strains is passed:
Being inoculated in by novel bacterial SM-1 on Kolle flask LB agar culture medium (pH7.2-7.4), inoculation of going down to posterity for continuous 1-2 time, strain goes down to posterity less than 5 generations, cultivates 21h for 37 DEG C.Take out, put-80 DEG C of refrigerators are stored standby；
(2) cultivation and fermentation:
The former slant strains of serratia marcescens, take 3 single bacterium colonies, mixing, it is inoculated on the LB fluid medium being contained in triangular flask, upper shaking table cultivation and fermentation.Condition is: LB fluid medium pH7.2-7.4, the inoculum concentration of 10%, ventilation 75%, temperature 37 DEG C, 21h, rotating speed 180rpm.
(3) collection bacterium inactivation washing
Carrying out the purebred inspections such as serratia marcescens morphological characteristic before collection bacterium, 4000-6000rpm is centrifuged 30min, abandons supernatant, precipitates with 0.5% formalin in 37 DEG C of inactivation 48h that suspend；By the Serratieae of inactivation with the centrifugal 30min of 5000rpm, abandon supernatant；Wash Serratieae (removal residual formaldehyde) 2 times with physiological saline solution, abandon supernatant, precipitation, weigh, prepare and obtain Becterin of Serratia marcescens.
Embodiment 3: the preparation of Becterin of Serratia marcescens injection
The preparation method of serratia marcescens injection is as follows:
(1) slant strains is passed: serratia marcescens (embodiment 1 screens the bacterial strain SM-1 obtained) is inoculated in Kolle flask LB agar culture medium (pH7.2), cultivates 21h for 37 DEG C.Take out, put-80 DEG C of refrigerators are stored standby.
(2) cultivation and fermentation: the former slant strains of serratia marcescens, takes 3 single bacterium colonies, mixing, is inoculated on the LB fluid medium being contained in triangular flask, cultivation and fermentation.Condition is: LB fluid medium pH7.2, the inoculum concentration of 10%, ventilation 75%, temperature 37 DEG C, 21h, rotating speed 180rpm.
(3) collection bacterium disinfecting scrub: carrying out the purebred inspections such as serratia marcescens morphological characteristic before collection bacterium, 4000-6000rpm is centrifuged 30min, abandons supernatant, precipitates with 0.5% formalin in 37 DEG C of inactivation 48h that suspend；By the Serratieae of inactivation with the centrifugal 30min of 5000rpm, abandon supernatant；Washing Serratieae (removal residual formaldehyde) 2 times with physiological saline solution, abandon supernatant, precipitation is weighed.
(4) preparation fill: take l000ml water for injection and suspended by 320mg Becterin of Serratia marcescens, mix homogeneously, sterilization is configured to the injection of 0.32mg/ml, and turbidimetry surveys Serratieae concentration between 1 × 109CFU/ml ± 10%, finished product is made in bottling fill, puts 4 DEG C and saves backup.
In order to verify above-mentioned technological process and working condition, whether science is feasible, between 3 years, inventor adopts these conditions to produce 6 batches of products continuously, and every batch of product has been carried out quality investigation, and including ne ar, the activation of yield, spleen, concentration, toxicity, tumor-inhibiting action and the index such as aseptic.Its stability having been investigated, it is shown that the product produced by new technology, yield is stable, favorable reproducibility simultaneously, and tumor-inhibiting action is strong, and spleen activates strong, and all other indexs all meet quality criteria requirements.
Embodiment 4: acute toxicity testing
(1) animal packet: 40 kunming mices are randomly divided into 4 groups, often group 10, respectively Normal group, acute toxicity heavy dose group, dosage group, acute toxicity small dose group in acute toxicity.
(2) dosage: the dosage that the normal test dose of the Becterin of Serratia marcescens injection of embodiment 3 preparation provides by consigner, namely 2.5 × 108Cfu/ only, 50 times, 30 times, 10 times of acute toxicity testing large, medium and small dosage respectively normal dose, namely 1.25 × 1010、7.5×109、2.5×109Only, disposable celiac drug administration by injection, each treated animal death condition is in Table 3 for cfu/.
The each treated animal death condition of table 3 acute toxicity testing
As can be seen from the above table, the median lethal dose(LD 50) of said preparation is close to middle dosage, and it is safe that prompting adopts lower than middle dosage 10 multiple dose.
Clinical trial:
Through thousands of example observation on Clinical Application for many years, Becterin of Serratia marcescens prepared by the present invention has definite curative effect and non-evident effect；Immune deficiency disorder there is dual regulation, especially to the therapeutic effect of lupus erythematosus, psoriasis, dry syndrome, ichthyosis, wart class sexually transmitted disease (STD) and asthma up to 90%；Diabetes are also had good therapeutic effect.Studying confirmation by clinical observation, Becterin of Serratia marcescens Clinical practice prepared by the present invention is safe and reliable.
Model case:
1, Zhao so-and-so, female, 56 years old, financial staff (Daliang City of Xian Zhu Liaoning Province), type Ⅱdiabetes mellitus, polydipsia, polyphagia, polyuria companion become thin 16 years；Nighttime sleep quality defect, spirit is not good enough.The Becterin of Serratia marcescens treatment that in mid-November, 2015 begins to use the present invention to prepare, has completed basis course of therapy, and existing just carrying out strengthens course of therapy (having injected 45 pins), and after treatment, every Index for examination is obviously improved, and has disabled antidiabetic drug.Current blood glucose is effectively controlled, can orthobiosis.Nighttime sleep quality is high, and spirit is good good.
2, Zhang, man, 65 years old, university teacher, type Ⅰ diabetes mellitus, polyuria, polyphagia, polydipsia 11 years；Visual deterioration, weak with microvascular complication with neuropathy.The Becterin of Serratia marcescens treatment beginning to use the present invention to prepare at the beginning of 2015 10 months, has completed basis course of therapy, and existing just carrying out strengthens course of therapy (having injected 75 pins), and after treatment, every Index for examination is obviously improved, and has disabled insulin.Current glycemic control still can, can orthobiosis.
3, Zhu, female, 45 years old, suffer from systemic lupus erythematosus (sle), the double; two oxter of the Becterin of Serratia marcescens beginning to use the present invention to prepare in May, 2014 and double inguinal groove lymph node subcutaneous injection, once in a week, each 0.32mg, complete Primary Care, just continuing to strengthen treatment, after medication, patient is without significant discomfort, without special untoward reaction, transference cure after symptomatic treatment, existing patient's condition is good.
4, Song, man, 45 years old, suffer from psoriasis, the double; two oxter of the Becterin of Serratia marcescens that in February, 2015 begins to use the present invention to prepare and double inguinal groove lymph node subcutaneous injection, twice weekly, each 0.32mg, altogether injection 50 times, current scaling of skin, desquamation decortication takes a turn for the better completely, and immunoglobulin inspection is obviously improved.
5, Lee, man, 38 years old, suffers from condyloma acuminatum, uses Becterin of Serratia marcescens intramuscular injection prepared by the CYP-III microwave integrated treatment associating present invention in January, 2015.Each 0.16mg, once in a week, treatment 20 times altogether, current wart body all disappears, without recurrence.
6, Jiang, female, 55 years old, suffer from sjogren syndrome, the double; two oxter of the Becterin of Serratia marcescens that in November, 2014 begins to use the present invention to prepare and double inguinal groove lymph node subcutaneous injection, twice weekly, each 0.32mg, altogether injection 50 times, current conscious xerostomia, dry lip, eye xeromycteria, thirsty, dispute burn feeling is obviously improved, and parotid gland enlargement disappears, and candida albicans infection disappears, skin pruritus, decortication disappears.Lower lip case biopsy shows that lymphocyte stove disappears.
7, Dong so-and-so, female, 24 years old, suffer from bronchial asthma, the double; two oxter of the Becterin of Serratia marcescens that in May, 2014 begins to use the present invention to prepare and double inguinal groove lymph node subcutaneous injection, twice weekly, each 0.32mg, after treatment, asthma is not reaccessed, and pulmonary function is obviously improved.
Claims (9)
1. serratia marcescens application in the medicine of preparation prevention and/or treatment function of immune system defect class disease and/or diabetes.
Described serratia marcescens was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 01 11st, 2016, and preserving number is CGMCCNO.11987.
2. Becterin of Serratia marcescens application in the medicine of preparation prevention and/or treatment function of immune system defect class disease and/or diabetes；The active component of described Becterin of Serratia marcescens is pure culture biology of serratia marcescens in claim 1.
3. apply as claimed in claim 2, it is characterised in that the preparation method of described Becterin of Serratia marcescens is, pure culture biology of serratia marcescens obtained by suitable separation method；
Described suitable separation method is: 4000-6000rpm is centrifuged 30-60min, abandons supernatant, precipitation is carried out inactivation treatment, then is centrifuged, and abandons supernatant, washing, and precipitation is prepared and be get final product.
4. apply as claimed in claim 3, it is characterised in that serratia marcescens biology pure culture preparation method be: by single colony inoculation of serratia marcescens slant strains on LB fluid medium, shaker fermentation cultivate；The condition of fermentation culture is: LB fluid medium pH6.0-8.0, and inoculum concentration is 8-15%, ventilation 40-90%, cultivation temperature 32-39 DEG C, incubation time 16-26h, rotating speed 120-200rpm.
5. apply as claimed in claim 3, it is characterised in that the method for described inactivation treatment is: with 0.5% formalin in 37 DEG C of inactivation 48h that suspend.
6. applying as claimed in claim 4, it is characterised in that the condition of fermentation culture is: LB fluid medium pH7.2-7.4, inoculum concentration is 10%, ventilation 75%, cultivation temperature 37 DEG C, incubation time 21h, rotating speed 180rpm.
7. apply as claimed in claim 4, it is characterized in that, the preparation method of described serratia marcescens slant strains is: be inoculated on the LB agar culture medium that pH is 6.0-8.0 by the serratia marcescens strain that preserving number is CGMCCNO.11987, go down to posterity for continuous 1-2 time inoculation, strain goes down to posterity less than 5 generations, cultivates 16-26h for 32-39 DEG C.
8. apply as claimed in claim 1 or 2, it is characterised in that described function of immune system defect class disease includes lupus erythematosus, psoriasis, dry syndrome, ichthyosis, wart class sexually transmitted disease (STD) and asthma.
9. apply as claimed in claim 1 or 2, it is characterised in that described diabetes class disease includes type Ⅰ diabetes mellitus and type Ⅱdiabetes mellitus.
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| CN201610184183.6A Active CN105726580B (en) | 2016-03-28 | 2016-03-28 | Application of serratia marcescens vaccine in preparing medicine for preventing and/or treating immune system function deficiency diseases and diabetes |
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| CN1465353A (en) * | 2002-06-05 | 2004-01-07 | 靳 海 | Becterin of Serratia marcescens for curing |
| CN1772001A (en) * | 2005-11-03 | 2006-05-17 | 中国人民解放军海军医学研究所 | Application of viscid serratia vaccine in preparing medicine for treating bronchial asthma |
| CN102181382A (en) * | 2011-01-27 | 2011-09-14 | 黑龙江大学 | Serratia marcescens |
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| CN1465353A (en) * | 2002-06-05 | 2004-01-07 | 靳 海 | Becterin of Serratia marcescens for curing |
| CN1772001A (en) * | 2005-11-03 | 2006-05-17 | 中国人民解放军海军医学研究所 | Application of viscid serratia vaccine in preparing medicine for treating bronchial asthma |
| CN102181382A (en) * | 2011-01-27 | 2011-09-14 | 黑龙江大学 | Serratia marcescens |
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| CN105726580B (en) | 2020-01-03 |
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