CN1483812A - M. smegmatics vaccine - Google Patents
M. smegmatics vaccine Download PDFInfo
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- CN1483812A CN1483812A CNA021306877A CN02130687A CN1483812A CN 1483812 A CN1483812 A CN 1483812A CN A021306877 A CNA021306877 A CN A021306877A CN 02130687 A CN02130687 A CN 02130687A CN 1483812 A CN1483812 A CN 1483812A
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Abstract
The present invention discloses a bacterial vaccine of Mycobacterium smegmatis, it is a lytic product of mycobacterium smegmatis, and centralizes the action characteristics of good adjuvant action of bacterial cell wall, antigen specificity of somatic protein and induction-producing cell factor, and richly contains bacterial DNA CpG fragment with strong immune activity. Said bacterial vaccine is obtained by collecting Mycobacterium smegmatis grown on improved Luo's culture medium and adopting high-pressure steam method to make lytic preparation. Said bacterial vaccine can be used for raising and restoring immunological function for curing and preventing tubercle bacillus infection.
Description
Technical field
The present invention relates to a kind of biological products, be meant M. smegmatics (Mycobacterium Smegmatis) CGMCC NO.0795 vaccine especially, belong to technical field of microbe application with immunoregulation effect.
Background technology
Tuberculosis is one of serious infectious diseases of harm humans health, and a large amount of appearance of many resistances tubercule bacillus and sharply increase with the concurrent tuberculosis number that makes of acquired immune deficiency syndrome (AIDS) form the very big one-tenth side of body to human beings'health.Strong reactor in infecting the tubercule bacillus crowd (during the tuberculin experiment, scleroma diameter 〉=15mm or bubble, necrosis are arranged, and remove and send out the patient) incidence probability is high, is called as the tuberculosis infection high risk population.China has nearly half population to infect tubercule bacillus at present, and wherein the high risk population reaches 5,000 ten thousand more than.A large amount of tuberculosis high risk population is a huge hidden danger, and their sickness rate own is high, is again the potential contagium, how to control these crowd's potential endogenous recurrences and exogenous infection again, is one of emphasis of global tuberculosis control.
At present world to control mode lungy mainly is: infection population bcg vaccination is not prevented and clinical patient is adopted long-term chemotherapy.Yet tuberculosis prophylaxis is continued to use bacille Calmette-Guerin vaccine for many years, and its applicable object is the healthy population that does not infect as yet, to high risk population's unprotect effect of having infected; The high risk population that the long-term rule chemotherapy is difficult to again do not fallen ill as yet accepts.Therefore, for the high risk population seeks suitable protective devices, to control lungy with significant.For this reason, we propose new tuberculosis prophylaxis notion: modern prevention not only refers to prevent the infection of pathogenic agent to the host, more should comprise preventing infection crowd's morbidity.
At the end of last century, domestic scholars is successfully developed the M.vaccae vaccine on the basis of professor's Stanford research, and it is obtaining significant curative effect aspect assisting therapy lungy, and this has pointed out the possibility of the tuberculosis high risk population being taked immunoprophylaxis; Yet the M.vaccae vaccine has been subjected to strict patent protection abroad, is unfavorable for further research and application.At present similar products like also has the Mycobacterium phlei preparation, but it is big because of the injection side effect, can not carry out multiple injection and has influenced action effect; M. smegmatics also is a kind of non-virulent, the mycobacterium that grows soon, and body is had stronger immunoregulation effect, does not still have the report of this vaccine both at home and abroad.
Summary of the invention
Main purpose of the present invention is to provide a kind of liquid of M. smegmatics vaccine or the preparation method and its usage of freeze-dried preparation.
The objective of the invention is to be achieved through the following technical solutions:
A kind of M. smegmatics vaccine, this vaccine comprises the split product of M. smegmatics (MycobacteriumSmegmatis).
Cell in the described vaccine has been used the high pressure steam process cracking.
Described vaccine comprises cell walls, the tropina of M. smegmatics and is rich in DNA of bacteria to have the segmental mixture of stronger immunocompetent CpG.
Described mixture is aqueous vaccine or freeze-dried preparation.
Above-mentioned M. smegmatics vaccine prepares from M. smegmatics.
The preparation method of described M. smegmatics vaccine comprises the steps: at least
Step 1: yeast culture: with dissolving under the liquid sub preservation bacterial classification room temperature, be inoculated in modified Russell medium, cultivate after 3-7 days transferred species for 37 ℃ and cultivated 3-5 days for 37 ℃ in modified Russell medium;
Step 2: microorganism collection: when treating thalli growth to logarithmic phase, with physiological saline wash the thalline on Russell medium surface and with above-mentioned thalline with physiological saline washing once or once, centrifugal collection thalline.Centrifugal condition be 6000r/min, 30min, 4 ℃.
Step 3: vaccine preparation: with physiological saline or phosphate buffered saline buffer above-mentioned thalline is made into the bacterium liquid of desired concn, through 121 ℃, 15min high pressure, cracking thalline, the even matter of preparation cell.
Step 4: finished product preparation: the even matter of the above-mentioned cell of packing under the aseptic condition, promptly make the M. smegmatics aqueous vaccine, or the freeze-dried preparation of obtained by freeze drying M. smegmatics vaccine.
The compound method of described modified Russell medium is (2000ml): behind asparagine 4.5g, potassium primary phosphate 3.0g, magnesium citrate 0.65g, sal epsom 0.3g, yam starch 37.6g, glycerol 15.0ml, distilled water 750ml mixing, boiling water bath is to clear and bright, 10 pounds of sterilizations in 20 minutes are matrix; New fresh hen egg cleaned drain, soaked 30 minutes in 75% alcohol, take out, put into sterilisable chamber, dry; Egg liquid 1250ml squeezed into container stirs evenly after sterile gauze is filtered in the big triangular flask that contains matrix, mixing also adds 2% peacock green liquor 20ml mixing, pours in the wide-necked bottle, manages in the packing, about 7ml/ pipe; At 87 ℃-88 ℃, dry under 40 minutes conditions at last.It is standby to store 4 ℃ of refrigerators behind 37 ℃ of aseptic mensuration 24h.
The purposes of above-mentioned M. smegmatics vaccine in a kind of biological products of preparation is: these biological products can be used for preventing tuberculosis high risk population's endogenous recurrence and exogenous infection again; Or the tubercle bacillus affection crowd's who can be used for promoting immunologic function to be in low state immunologic function is recovered; Or can be used for suppressing immunologic function be in hyperfunction state the tubercle bacillus affection crowd cross strong immune response.
These biological products can be used for improving the Th1 para-immunity of normal population and reply.
In sum, the present invention has following advantage:
Vaccine provided by the invention has been concentrated the antigen-specific of the effect of bacteria cell wall good adjuvant, tropina and has been induced and produced the effect of cytokines characteristics, and is rich in DNA of bacteria and has stronger immunocompetent CpG fragment; Solve the problem that external similar articles thalli granule is big, aqueous vaccine easily forms cenobium, avoided or reduced the side effect that may occur after the present conventional vaccine inoculation, improved the security of goods; But multiple injection, having solved external similar articles intradermal routes can only a shot or the long not good enough shortcoming of effect of injecting at interval more than three months.
The M. smegmatics vaccine that the present invention relates to not only can prevent normal population by tubercle bacillus affection, more can control the recurrence of tuberculosis high risk population potential endogenous economical, effectively and exogenously infect again, this to the spreading of controlling tuberculosis, to improve human life quality significant.In addition, this vaccine also has the potential therapeutic value to the tetter of immune-mediated.
Description of drawings
The negative control group of Fig. 1 (physiological saline group) cavy organ disease outward appearance;
Fig. 2 is a M. smegmatics vaccine prevention group cavy organ disease outward appearance of the present invention;
The positive control group of Fig. 3 (M.Vaccae vaccine group) cavy organ disease outward appearance.
Embodiment
The present invention is described in detail below in conjunction with the drawings and specific embodiments:
M. smegmatics (Mycobacterium Smegmatis) has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 10th, 2002, be called for short CGMCC, and deposit number is CGMCC NO.0795.
The M. smegmatics vaccine is the split product of M. smegmatics, and its preparation method is:
Step 1: yeast culture: with dissolving under the liquid sub preservation bacterial classification room temperature, be inoculated in modified Russell medium, cultivate after 3-7 days transferred species for 37 ℃ and cultivated 3-5 days for 37 ℃ in modified Russell medium;
Step 2: microorganism collection: when treating thalli growth to logarithmic phase, with physiological saline wash the thalline on Russell medium surface and with above-mentioned thalline with physiological saline washing once or once, centrifugal collection thalline.Centrifugal condition be 6000r/min, 30min, 4 ℃.
Step 3: vaccine preparation: with physiological saline or phosphate buffered saline buffer above-mentioned thalline is made into the bacterium liquid of desired concn, through 121 ℃, 15 minutes high pressure, cracking thalline, the even matter of preparation cell.
Step 4: finished product preparation: the even matter of the above-mentioned cell of packing under the aseptic condition, promptly make the M. smegmatics aqueous vaccine, or through lyophilize, make the freeze-dried preparation of M. smegmatics vaccine.
Wherein, the modified Russell medium compound method is (2000ml):
At first, behind asparagine 4.5g, potassium primary phosphate 3.0g, magnesium citrate 0.65g, sal epsom 0.3g, yam starch 37.6g, glycerol 15.0ml, distilled water 750ml mixing, boiling water bath is to clear and bright, and 10 pounds of sterilizations in 20 minutes are matrix.
Secondly, new fresh hen egg (about four jin) cleaned drain, soaked 30 minutes in 75% alcohol, take out, put into sterilisable chamber, dry;
Then, 1250ml squeezes into porcelain cup with egg liquid, stir evenly after sterile gauze is filtered in the big triangular flask that contains matrix, and mixing, and add 2% peacock green liquor 20ml, and mixing is poured in the wide-necked bottle, manages in the packing, and about 7ml/ pipe is dried at 87 ℃-88 ℃ at last; It is standby to store 4 ℃ of refrigerators behind 37 ℃ of aseptic mensuration 24h.
The M. smegmatics vaccine is applied to the experimental result of animal:
The M. smegmatics vaccine prevents the effectiveness experiment of high-risk tuberculosis: with tuberculosis virulent strain inguinal region subcutaneous injection healthy guinea pig, set up the high-risk model of tubercle bacillus affection.Infected back 3 days, and carried out immunoprophylaxis for every cavy intramuscular injection 0.1mg M. smegmatics vaccine, weekly, continuous 4 weeks.Simultaneously make negative control group, make positive controls (M. smegmatics vaccine and M.vaccae vaccine application dose are a person-portion/only) with the M.vaccae vaccine with physiological saline.The result observes: infect the 7th week of back, dissect and respectively organize cavy, observe each internal organs tuberculosis degree such as liver, spleen, lung, lymphoglandula, mark with double-blind method according to " the pathology standard of index behind the pathology experimental technique tubercle bacillus affection "; The spleen of getting each cavy carries out viable bacteria to be separated; Get liver, spleen, the lung of all animals simultaneously and do pathologic finding.Each internal organs appearance results such as Fig. 1, Fig. 2, shown in Figure 3, its pathological score and viable bacteria separating resulting are as follows:
Table 1. cavy organ disease index (X ± SD)
Negative control group M. smegmatics vaccine group M.vaccae group
Pathology index 64.6 ± 21.0 30.0 ± 15.3**, 34.4 ± 22.1**
*: when treatment group and control group compare, p＜0.01
The logarithmic value of table 2. GPS dirty work bacterium separation number (X ± SD)
Negative control group M. smegmatics vaccine group M.vaccae group
The spleen bacterium is counted logarithm 4.69 ± 0.67 3.59 ± 1.66 3.89 ± 1.78
The pathological section result is: the lung of control animals, liver, spleen are popularity millet appearance tubercle or caseous necrosis more.M.vaccae group pathology is lighter, and removing has individual example to be tuberculosis sample pathology, mostly is the class epithelium sample tubercle after the treatment or presents normal tissue slice; M. smegmatics vaccine prevention group pathology is the lightest, presents non-specific hyperplasia, treatment back class epithelium sample tubercle or normal tissue slice.
Comprehensive The above results, as can be seen: with control group relatively, the M. smegmatics vaccine is the same with the M.vaccae vaccine, can significantly alleviate the cavy internal organs lesion degree, reduce the pathology index, suppress or kill the intravital tubercule bacillus of cavy.
Experiment conclusion is: the M. smegmatics vaccine has prophylactic effect to " high-risk " cavy that infects tubercule bacillus.
The pharmaceutical research of M. smegmatics vaccine:
The immunoregulation effect and the mechanism of action thereof of M. smegmatics vaccine have been inquired into respectively with normal, immunologic hypofunction and the hyperfunction animal model of immunologic function.Found that: the M. smegmatics vaccine can improve the immunologic function of intact animal, especially improving the Th1 para-immunity replys, comprise the expression of killer factor (NO) in Th1 type cytokines such as strengthening T lymphproliferation response, delayed hypersensitivity, raising IL-12 and IL-2 and the scavenger cell, this is significant to the disease that infects in the cells such as prevention tuberculosis; Simultaneously, in the animal model of immunologic hypofunction, the M. smegmatics vaccine can promote the recovery of immunologic function, and in the hyperfunction animal model of immunologic function, the M. smegmatics vaccine can suppress strong immunne response.Can find out that thus the M. smegmatics vaccine has two-way immunoloregulation function, this prevention to the tubercle bacillus affection crowd that presents different immunological statuss because of infection period is different is significant.
The toxicologic study of M. smegmatics vaccine:
By the requirement of new drug preclinical study, carried out the M. smegmatics vaccine to the acute toxicity test of mouse, long term toxicity test and systemic anaphylaxis experiment of cavy and the thermal source experiment of rabbit of Beagle dog.Get 3 kinds of dosage (be respectively human dosage 25000 times, 50000 times, 250000 times), through the abdominal cavity, 2 kinds of approach injections of muscle mouse, the result does not all cause the abnormal symptom and the death of experiment mice.Get 3 kinds of dosage (be equivalent to intend 62.5 times, 625 times of clinical people's consumption and 3125 times time), give continuous 14 weeks of intramuscular injection of Beagle dog, before experiment, in, latter stage and decubation, by the detection of indexs such as general behavior, routine blood test, blood biochemistry, routine urinalysis, electrocardiogram(ECG and pathology, do not find that all toxic reaction appears in the Beagle dog.Simultaneously, thermal source experiment and systemic anaphylaxis experiment have proved that respectively the M. smegmatics vaccine does not have heat source response, do not cause the parasexuality reaction of whole body speed.
In a word, M. smegmatics vaccine experimental result shows: this vaccine can improve the cellular immune function of normal mouse, promotes the recovery of immunologic hypofunction immune function of mice; Suppress the superpower immunne response of the hyperfunction cavy of immunologic function; In the animal model of tubercle bacillus affection, this vaccine can significantly suppress the breeding, near and alleviate the lesion degree of internal organs of tubercule bacillus in the infected animals body; In the animal model of porcine blood serum sensitization, this vaccine can significantly suppress the anaphylactoid generation of animal pattern; Simultaneously, this vaccine has good security.
It should be noted last that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement technical scheme of the present invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
Claims (9)
1, a kind of M. smegmatics vaccine is characterized in that: this vaccine comprises the split product of M. smegmatics (Mycobacterium Smegmatis) CGMCC NO.0795.
2, M. smegmatics vaccine according to claim 1, it is characterized in that: the cell in the described vaccine has been used the high pressure steam process cracking.
3, M. smegmatics vaccine according to claim 1 is characterized in that: described vaccine comprises cell walls, the tropina of M. smegmatics and is rich in DNA of bacteria to have the segmental mixture of stronger immunocompetent CpG.
4, M. smegmatics vaccine according to claim 3 is characterized in that: described mixture is aqueous vaccine or freeze-dried preparation.
5, the preparation method of the described M. smegmatics vaccine of above-mentioned arbitrary claim, the M. smegmatics vaccine prepares from M. smegmatics.
6, the preparation method of M. smegmatics vaccine according to claim 5 is characterized in that: which comprises at least following steps:
Step 1: yeast culture: with dissolving under the liquid sub preservation bacterial classification room temperature, be inoculated in modified Russell medium, cultivate after 3-7 days transferred species for 37 ℃ and cultivated 3-5 days for 37 ℃ in modified Russell medium;
Step 2: microorganism collection: when treating thalli growth to logarithmic phase, with physiological saline wash the thalline on Russell medium surface and with above-mentioned thalline with physiological saline washing once or once, centrifugal collection thalline.Centrifugal condition be 6000r/min, 30min, 4 ℃.
Step 3: vaccine preparation: with physiological saline or phosphate buffered saline buffer above-mentioned thalline is made into the bacterium liquid of desired concn, through 121 ℃, 15min high pressure, cracking thalline, the even matter of preparation cell.
Step 4: finished product preparation: the even matter of the above-mentioned cell of packing under the aseptic condition, promptly make the M. smegmatics aqueous vaccine, or the freeze-dried preparation of obtained by freeze drying M. smegmatics vaccine.
7, the preparation method of M. smegmatics vaccine according to claim 6, it is characterized in that: the compound method of described modified Russell medium is (2000ml): behind asparagine 4.5g, potassium primary phosphate 3.0g, magnesium citrate 0.65g, sal epsom 0.3g, yam starch 37.6g, glycerol 15.0ml, distilled water 750ml mixing, boiling water bath is to clear and bright, 10 pounds of sterilizations in 20 minutes are matrix; New fresh hen egg cleaned drain, soaked 30 minutes in 75% alcohol, take out, put into sterilisable chamber, dry; Egg liquid 1250ml squeezed into container stirs evenly after sterile gauze is filtered in the big triangular flask that contains matrix, mixing also adds 2% peacock green liquor 20ml mixing, pours in the wide-necked bottle, manages in the packing, about 7ml/ pipe; At 87 ℃-88 ℃, dry under 40 minutes conditions at last.It is standby to store 4 ℃ of refrigerators behind 37 ℃ of aseptic mensuration 24h.
8, the purposes of the described M. smegmatics vaccine of above-mentioned arbitrary claim in a kind of biological products of preparation, these biological products can be used for preventing tuberculosis high risk population's endogenous recurrence and exogenous infection again; Or the tubercle bacillus affection crowd's who can be used for promoting immunologic function to be in low state immunologic function is recovered; Or can be used for suppressing immunologic function be in hyperfunction state the tubercle bacillus affection crowd cross strong immune response.
9, M. smegmatics vaccine according to claim 8 is characterized in that in the purposes of preparation in a kind of biological products: these biological products can be used for improving the Th1 para-immunity of normal population and reply.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02130687 CN1234845C (en) | 2002-09-18 | 2002-09-18 | M. smegmatics vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02130687 CN1234845C (en) | 2002-09-18 | 2002-09-18 | M. smegmatics vaccine |
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| Publication Number | Publication Date |
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| CN1483812A true CN1483812A (en) | 2004-03-24 |
| CN1234845C CN1234845C (en) | 2006-01-04 |
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| CN 02130687 Expired - Fee Related CN1234845C (en) | 2002-09-18 | 2002-09-18 | M. smegmatics vaccine |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1318573C (en) * | 2005-06-13 | 2007-05-30 | 中国药品生物制品检定所 | Mycobacterium smegmatis preparation and use thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1318573C (en) * | 2005-06-13 | 2007-05-30 | 中国药品生物制品检定所 | Mycobacterium smegmatis preparation and use thereof |
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