CN1318573C - Mycobacterium smegmatis preparation and use thereof - Google Patents

Abstract

The present invention provides a preparation of smegmatis mycobacteria, which is prepared by that the smegmatis mycobacteria are crushed and split, such as a high-pressure crushing method or an ultrasonic crushing method. The preparation is a safe and effective immune regulating agent. The preparation can enhance the immune function of normal organisms, can promote the recovery of the immune function of the organisms with low immunity and can inhibit over strong immune response of the organisms with immune hyperfunction, namely that the preparation has bidirectional immune regulating effect. The preparation of smegmatis mycobacteria can promote Th1 response and inhibit Th2 response and has potential preventing or/and therapeutic effects on diseases of tuberculosis, hepatitis such as hepatitis B, asthma, etc., and the preparation is a safe and effective vaccine for preventing and treating tuberculosis.

Description

Mycobacterium Smegmatis preparation and application thereof

Technical field

The application relates to biological technical field, field of immunology and microbial product pharmaceutical use field, is specifically related to the immunoloregulation function checking and the purposes in prevention and treatment of diseases such as tuberculosis, asthma, hepatitis B of mycobacterium Smegmatis preparation.

Background technology

Tuberculosis is one of ten big diseases of the harm humans health announced of The World Health Organization (WHO), estimate according to WHO, the people in the whole world existing nearly 1/3 has infected tubercule bacillus, and 2,000 ten thousand active tuberculosis patients are arranged, and annual newly-increased 8,000,000-1,000 ten thousand patients also have 3,000,000 people because of tuberculosis death; If control strategy can not get further improvement, estimation is in the period of 2000-2020, to there be 1,000,000,000 people to infect that tubercule bacillus, 200,000,000 people suffer from tuberculosis, 3,500 ten thousand people die from tuberculosis (WHO report.Basicfacts on TB.World TB Day, 24.Mar, 2002.).China is that 22 tuberculosis height are born one of country in the world, according to the 4th the tuberculosis epidemiology investigation data in the whole nation in 2000, the whole nation 500,000,000 people that have an appointment have infected tubercule bacillus, and wherein 10% people throughout one's life tuberculosis can take place, and this part people is called as the tuberculosis high risk population because of the sickness rate height.

Chemotherapy is most important tuberculosis measure of control always, but along with being widely current of resistance tubercule bacillus, and adds that antituberculosis drugs thing toxic side effect is big, chemotherapy cycles is long etc. and cause that patient dependence is poor, therapeutic process is poor with result of treatment repeatedly.And, in the tubercle bacillus affection person in the whole world nearly 1/3, quite a few latent infection person being arranged, tuberculosis rate is far above the general population.How preventing the latent infection person who has infected tubercule bacillus to develop into the active tuberculosis patient, is the problem that tuberculosis control must solve.Existing tuberculosis prophylaxis goods bacille Calmette-Guerin vaccine only is applicable to the crowd who does not infect as yet, and to infection population is invalid, chemoprophylaxis is difficult to be widely accepted because of above-mentioned drawback again.Therefore, in the face of serious day by day tuberculosis epidemic situation, particularly huge tuberculosis high risk population (tubercule bacillus latent infection person), rely on chemotherapy to be difficult to thoroughly capture this chronic disease merely, people must explore new prophylactico-therapeutic measures.

International anti-consumptive disease meeting and WHO once went out at eighties of last century index futures at the end of the nineties, should classify new generation vaccine as the important direction of preventing and treating lungy in the future.The mankind want final controlling tuberculosis, not only to spare no effort to diagnose and the treatment tuberculosis patient, will more develop generation lungy of effective vaccine prevention and propagation (Ann M.Ginsberg.What ' s new in tuberculosis vaccines? Bulletin of the World Health Orgnization.2002,80 (6)).Some patents and document disclose deactivation cow mycobacterium (M.Vaccae) vaccine to treatment situation lungy.Studying more is Britain Stanford professor, and he prepares cow mycobacterium preparation with radiation deactivation or high-temperature inactivation mode, and employing subcutaneous injection mode.Studies show that said preparation has immunostimulation preferably, but this kind cow mycobacterium preparation is the whole cell preparation, thalli granule greatly, easily forms cenobium, cause the injection back tangible part even whole body side reaction to occur, having limited its continuous several times clinically uses, and single uses this type of preparation obviously not meet the repeatedly ultimate principle of repetitive stimulation of cellullar immunologic response, thereby has limited the immune effect of cow mycobacterium.Clinical study for many years also confirms, the cow mycobacterium preparation of Stanford research is at present to limited (the Mayo RE of protection effect lungy, Stanford JL.Double-blind placebo-controlled trial of Mycobacterium vaccae immunotherapy fortuberculosis in KwaZulu, South Africa, 1991-97.Trans R Soc Trop Med Hyg.2000Sep-Oct; 94 (5): 563-8.Stanford J, Stanford C, Grange J.Immunotherapy withMycobacterium vaccae in the treatment of tuberculosis.Front Biosci.2004 May1; 9:1701-19.)

Analogous products also have the Mycobacterium phlei preparation, but its injection side effect is big, can not carry out multiple injection and has influenced action effect.Further developing safely and effectively, tuberculosis prophylaxis is must needing of people's controlling tuberculosis with the treatment preparation.

Bronchial asthma (asthma) is one of modal chronic respiratory tract disease in the global range, be a kind of by multiple inflammatory cell mastocyte particularly, the chronic inflammatory airway disease that eosinophilic granulocyte and T lymphocyte participate in, this inflammation can cause repeatedly symptoms such as the panting of outbreak, shortness of breath, uncomfortable in chest and cough in the susceptible person, normal with extensive and changeable airflow obstruction, inflammation causes that also air flue increases multiple stimulating factor reactivity.People recognize the important driving effect that the T lymphocyte is play gradually in the asthma inflammatory process in recent years.According to excretory cytokine difference, the T lymphocyte can be divided into Th0, Th1 and three kinds of subclass of Th2.In a short time, the Th0 cell is subjected to influence of various factors such as cytokine, antigenic characteristic, hormone after the antigenic stimulation, and to Th1 or Th2 differentiation, IL-12 impels Th0 to the differentiation of Th1 direction, and the latter secretes IL-2, INF-γ, TNF-β; IL-4 and IL-10 impel Th0 to the differentiation of Th2 direction, and the latter secretes IL-4, IL-5, IL-10, IL-13.Th1 subclass and Th2 subclass suppress mutually, so that IL-4 suppresses the reaction of Th1 type, the reaction of INF-γ inhibition Th2 type.Produce Th2 type cytokines such as IL-4 and IL-5 when having confirmed at present the lymphocyte activator of asthmatic patient lung, and the latter produces for IgE and the eosinophilic granulocyte adding is necessary.This shows that asthmatic patient exists tangible Th2 advantage to reply.Clinical treatment asthma is mainly slowed down inflammation and is replied gas circuit and block by using reflunomide and bronchodilatation medicine, but the life-time service reflunomide may cause unacceptable side reaction.In recent years studies show that it is the core that respiratory system immune disorder class disease conditions such as tuberculosis, asthma and allergic rhinitis reverse that the reaction of Th1 type is raised.

Chronic viral hepatitis B is because not obtaining timely, effective treatment behind the organism infection hepatitis B virus (HBV) causes, this disease is in China's sickness rate height, big, the effective radical cure measure of shortage of harm.Think now the chronic viral hepatitis B patient immunne response particularly cellullar immunologic response reduce, this prosoplasia with the interior Th1/Th2 of body is relevant.The HBV that body continues infects and causes the Th1/Th2 prosoplasia, makes Th1 reply and reduces the companion or reply reinforcement without Th2, and this unusual differentiation is unfavorable for body removing HBV, has promoted the lasting existence of HBV on the contrary.Cytokine plays a significant role in this process.At first, HBV infects precursor cell or the sophisticated DCs of DCs (dendritic cell), and breed therein, damaged function [the 1.Wang FS of DCs, Xing LH, Liu MX, Zhu CL, et al.Dysfunction of peripheral blood dendritic cellsfrom patients wiTh chronic hepatitis B virus infection.World JGastroenterol.2001; 7 (4): 537-41.2.Lohr HF, Pingel S, Bocher WO, etal.Reduced virus specific T helper cell induction by autologous dendriticcells in patients wiTh chronic hepatitis B-restoration by exogenousinterleukin-12.Clin Exp Immunol.2002; 130 (1): 107-14.3.Beckebaum S, Cicinnati VR, Zhang X, et al.Hepatitis B virus-induced defect ofmonocyte-derived dendritic cells leads to impaired T helper type 1response in vitro:mechanisms for viral immune escape.Immunology.2003; 109 (4): 487-95.], the ability of its secretion IL-12 is reduced, limited of the differentiation of Th0 cell to the Th1 cell.Virus-specific t h1 cell relies on directly antiviral or indirect immunoregulation effect of excretory various kinds of cell factor performance, both can directly suppress duplicating of virogene as its excretory IFN-γ, also can promote DC secretion IL-12, the latter further promotes the differentiation of Th1 cell, be to exist mutual positive regeeration [4.Szabo SJ between IFN-γ and the IL-12, Dighe AS, Gubler U, et al.Regulation ofThe interleukin[IL]-12R beta 2 subunit expression in developing T helper1[Th1] and Th2 cells.J Exp Med.1997; 185 (5): 817-24.].The cytokine of the activation of DC cell and Th1 emiocytosis is prerequisite [the 5.Kasahara S that body produces virus-specific CTL, Ando K, Saito K, Sekikawa K, rt al.Lack of tumor necrosisfactor alpha induces impaired proliferation of hepatitis B virus-specificcytotoxic T lymphocytes.J Virol.2003; 77 (4): 2469-76.], the latter is an important effect cell of removing HBV.Research to the Hepatitis B virus vaccine nonresponder also shows, the unresponsiveness of vaccine mainly lacks reactive relevant [6.Chedid MG with Th1, Deulofeut H, Yunis DE, Defect inTh1-like cells of nonresponders to hepatitis B vaccine.Hum Immunol.1997; 58 (1): 42-51.7.Vingerhoets J, VanhamG, Kestens L, et al.DeficientT-cell responses in non-responders to hepatitis B vaccination:absenceof TH1 cytokine production.Immunol Lett.1994; 39 (2): 163-8.].The effect of Th2 cell is opposite, mainly secretes IL-4, IL-5, IL-6, IL-10, participates in B cell proliferation, antibody generation and allergy disease, and simultaneously, IL-4 and IL-10 all suppress the Th1 differentiation, start the Th2 differentiation.There are some researches show chronic viral hepatitis B patient peripheral blood CD4 ⁺In the T cell based on Th0, in liver also based on Th0, secrete low-level IFN-γ, the level of IL-4 is then higher, point out this type of crowd to reply [the 8.Bertoletti A that preponderates with the Th2 type, D ' Elios MM, Boni C, et al.Different cytokine profilesof intraphepatic T cells in chronic hepatitis B and hepatitis C virusinfections.Gastroenterology.1997; 112:193-9.9.Chisari FV, Ferrari C.Hepatitis B virus immunopaThogenesis.Annu Rev Immunol.1995; 13:29-60.], and virus-specific t h2 cell has the persistent infection that is beneficial to HBV.Therefore, searching can activating Th 1 cell, suppress the immunomodulator of Th2 cell, and the control of chronic viral hepatitis B is had very big application prospect.

Summary of the invention

The contriver finds that by document and experimental analysis to many mycobacteriums M. smegmatics has the distinct advantages as good immunomodulator.M. smegmatics (Mycobacterium Smegmatis) is a kind of fast growth, non-virulent mycobacterium, have stronger immunogenicity, present research for M. smegmatics concentrates on and utilizes the research that the M. smegmatics growth is fast, non-virulent is applied to recombinant protein as the carrier of foreign gene.Yet a kind of biological products want real service in human health, except having tangible validity, also must have reliable security.Cow mycobacterium preparation though have immunostimulation preferably, because of process technology limit, makes security limited as described in the background art, and then has limited continuous several times clinically and used, influenced its action effect.Can itself has good immunogenicity M. smegmatics, but be successfully applied to clinically with the mycobacterium Smegmatis preparation of this bacterium preparation, and security is an important factor.

As everyone knows, the bacterial cell wall polysaccharide has good immunostimulation, tropina and has preferably that antigenicity, bacterial nucleic acid have excellent immunoloregulation function because of being rich in the CpG segment, comprehensively the advantage of mycobacterium cell wall polysaccharide, tropina and nucleic acid three big compositions might be prepared good immunomodulator.Yet, the whole cell preparation that contains these three big compositions have a fatal weakness be exactly thalli granule big, cause the toxic side effect that is difficult to accept, mycobacterium particularly, because of lipid content height, cell wall structures complexity, conventional bacteria breaking mode is difficult to make the abundant cracking of thalline.Therefore, want fully to use the immunoregulation effect of mycobacterium, it is clinical that it really is applied to, and the selection of technology is most important.The contriver is by research for many years, comparison, self-examination and further experiment to experimental result are found, the broken thalline of elder generation in cracking process, as pressure breaking or ultrasonication technology, better the cracking mycobacterium makes the abundant cracking of its cell walls, discharges tropina and nucleic acid, and make split product reach a kind of homogeneous, solvable state, and adopt the freeze-drying assistant agent freeze-dried products of low levels forming simultaneously, make goods keep satisfactory stability.Mycobacterium Smegmatis preparation through above prepared is compared with other thalline goods (as the whole cell preparation of radiation deactivation preparation or the part cracking thalline preparation of high pressure steam cracking preparation), when keeping the good immunity of mycobacterium, because of making the abundant cracking of thalline, and finally obtain homogeneous, soluble preparation, have better security.

Mycobacterium Smegmatis preparation provided by the invention; preferred freeze-dried preparation; it is a kind of safe and effective immunomodulator; can strengthen normal body immunologic function, promote the recovery of immunocompromised body's immunity and suppress the strong excessively immunne response of immune hyperfunction body promptly have two-way immunoregulation effect.Mycobacterium Smegmatis preparation can promote Th1 to reply, suppress Th2 to reply that diseases such as tuberculosis, hepatitis and asthma are had the potential prevention or/and result of treatment.

The invention provides mycobacterium Smegmatis preparation is used for immunoregulatory medicine in preparation application; wherein said immunomodulatory for the immunologic function that improves normal body, promote the recovery of immunocompromised body's immunity or suppress the strong excessively immunne response of immune hyperfunction body; promote delayed type hypersensitivity, promote the lymphocytic propagation of T; concrete, described immunomodulatory shows as and promotes the Th1 para-immunity to reply or suppress the Th2 para-immunity and reply.The present invention further provides mycobacterium Smegmatis preparation and be used for the treatment of application in the medicine of tuberculosis, hepatitis such as hepatitis B or asthma in preparation.The present invention also provides mycobacterium Smegmatis preparation to be used to strengthen antigen presenting cell to antigenicly engulfing with processing power, promote scavenger cell secrete GM-CSF or IL-1 cytokine, improving the expression amount of IL-12, IL-2, IFN-γ in preparation, or reduce IL-4 expression amount medicine and be used to promote body to kill and wound and the removing ability to entering intracellular microorganism, as the application in the medicine of the generation that promotes NO.

Mycobacterium Smegmatis preparation provided by the invention is prepared from through cracking by M. smegmatics, and at first broken thalline can adopt high pressure crush method and sonioation method in cracking process, preferred high pressure crush method.The present invention is based on the cracking preparation that above-mentioned breaking method is applied to mycobacterium Smegmatis preparation, its concrete steps and parameter are selected not as limitation of the present invention, and those skilled in the art can carry out conventional the selection and not influence the effect of preparation.Preferred implementation of the present invention; the high pressure crush method is centrifugal collection M. smegmatics; with frozen-dried protective liquid dilution after high-pressure homogeneous crusher machine thalline, then through 110 ℃-135 ℃ steam treatment 10-40 minute, preferred 121 ℃ of steam treatment made mycobacterium Smegmatis preparation in 20 minutes.Frozen-dried protective liquid is a phosphate buffered saline buffer; those skilled in the art know multiple frozen-dried protective liquid; be not limited to the concrete reagent that the present invention uses; the phosphate buffered saline buffer that the present invention preferably uses has following proportioning: add 3.75 weight part monosodium glutamates in 1500 parts by weight of deionized water; 3.75 weight part sucrose; 10.8 weight part sodium-chlor; 6.525 the weight part anhydrous potassium dihydrogenphosphate is made into potassium liquid; in 1500 parts by weight of deionized water, add 3.75 weight part monosodium glutamates; 3.75 weight part sucrose; 10.8 weight part sodium-chlor; 27.45 the weight part disodium hydrogen phosphate,anhydrous is made into sodium liquid; getting 1500 weight part potassium liquid and 1100 weight part sodium liquid mixes; the pH that makes mixed solution is 6.0-8.0; preferred 7.0-7.4; most preferably 7.2; mixed solution by the G3 funnel filter back 110 ℃-135 ℃ steam treatment 10-40 minute, preferred 115 ℃ of steam sterilizings were phosphate buffered saline buffer in 30 minutes.Being prepared into wet bacteria concentration with the frozen-dried protective liquid dilution is the bacteria suspension of 1-1000mg/ml; the preferred concentration of wet bacterium is according to the concrete instrument that uses; to reach good crushing effect is good; the bacteria suspension of preferred 10mg/ml; the broken thalline condition of the high pressure draft shearing technique homogenate of high pressure homogenizer is 400-2000bar, preferred 1200bar.Sonioation method is centrifugal collection M. smegmatics; with frozen-dried protective liquid dilution back ultrasonication thalline; then through 110 ℃-135 ℃ steam treatment 10-40 minute, preferred 121 ℃ of steam treatment 20 minutes or 60Co irradiation made mycobacterium Smegmatis preparation in 15 minutes.Frozen-dried protective liquid is a phosphate buffered saline buffer; it is the bacteria suspension of 1-1000mg/ml that the frozen-dried protective liquid dilution is prepared into wet bacteria concentration; preferred concentration is according to the concrete instrument that uses; to reach good crushing effect is good; the bacteria suspension of preferred 100mg/ml; the broken thalline condition of Ultrasonic Cell Disruptor 200-2000W, broken 5 seconds intermittently 5 seconds for once, broken 30-200 time altogether, preferably the 1000W fragmentation is 180 times.

The invention provides the pharmaceutical composition or the vaccine that are used to prevent or treat diseases such as tuberculosis, hepatitis, asthma, described pharmaceutical composition or vaccine comprise the mycobacterium Smegmatis preparation that obtains through aforesaid method.

The application's embodiment selects for use bacterial strain number for the M. smegmatics of CGMCC NO.0795 is that example describes, but not as restriction of the present invention, any other M. smegmatics can be obtained same effect.

Description of drawings

The IL-12 that the peritoneal macrophage vitro culture of Fig. 1 mycobacterium Smegmatis preparation immune mouse produces

0: negative control group 1:0.1mg mycobacterium Smegmatis preparation group

2:0.25mg mycobacterium Smegmatis preparation group 3:0.5mg mycobacterium Smegmatis preparation group

^*Refer to that shame dirt group compares p＜0.05 with control group ^*Refer to that shame dirt group compares p＜0.01 with control group

Fig. 2. the IL-2 that the splenic T lymphocyte vitro culture of mycobacterium Smegmatis preparation immune mouse produces

0: negative control group 1:0.1mg mycobacterium Smegmatis preparation group

2:0.25mg mycobacterium Smegmatis preparation group 3:0.5mg mycobacterium Smegmatis preparation group

^*Refer to that shame dirt group compares p＜0.01 with control group

Fig. 3. the IFN-γ that the splenic T lymphocyte vitro culture of mycobacterium Smegmatis preparation immune mouse produces

(N.S refers to the physiological saline control group, and M.S refers to the mycobacterium Smegmatis preparation immune group)

*: when referring to that the splenic T lymphocyte is external and stimulating with PPD, shame dirt group and control group be p＜0.05 relatively

The IL-4 that the splenic T lymphocyte vitro culture of Fig. 4 mycobacterium Smegmatis preparation immune mouse produces

(N.S refers to the physiological saline control group, and M.S refers to the mycobacterium Smegmatis preparation immune group)

☆: when referring to that the splenic T lymphocyte is external and stimulating with ConA, shame dirt group and control group be p=0.065 relatively

Fig. 5. the GM-CSF that the peritoneal macrophage vitro culture of mycobacterium Smegmatis preparation immune mouse produces

0: negative control group 1:0.1mg mycobacterium Smegmatis preparation immune group

2:0.5mg mycobacterium Smegmatis preparation immune group 3:1.0mg mycobacterium Smegmatis preparation immune group

*: refer to that shame dirt group compares p＜0.01 with control group

Fig. 6. the IL-1 that the peritoneal macrophage vitro culture of mycobacterium Smegmatis preparation immune mouse produces

0: negative control group 1:0.1mg mycobacterium Smegmatis preparation immune group

2:0.5mg mycobacterium Smegmatis preparation immune group 3:1.0mg mycobacterium Smegmatis preparation immune group

*; Refer to that shame dirt group compares p＜0.05 with control group

Fig. 7. the NO that the peritoneal macrophage vitro culture of mycobacterium Smegmatis preparation immune mouse produces

0: negative control group 1:0.1mg mycobacterium Smegmatis preparation immune group

2:0.5mg mycobacterium Smegmatis preparation immune group 3:1.0mg mycobacterium Smegmatis preparation immune group

*: refer to that shame dirt group compares p＜0.01 with control group

Fig. 8. mycobacterium Smegmatis preparation is to the influence of the T lymphproliferation response of immunologic hypofunction mouse

0: negative control group 1:0.1mg mycobacterium Smegmatis preparation immune group

2:0.5mg mycobacterium Smegmatis preparation immune group 3:1.0mg mycobacterium Smegmatis preparation immune group

4: normal control group * *: refer to that each group and negative control group compare p＜0.05

Embodiment

Embodiment 1: the preparation of mycobacterium Smegmatis preparation

Can adopt different physics cleavage methods to prepare mycobacterium Smegmatis preparation, most preferably the high pressure crush method.The bacterial strain of selecting for use is M. smegmatics (Mycobacterium smegmatis), it is that example describes that present embodiment is selected the M. smegmatics of bacterial strain CGMCC NO.0795 for use, and CGMCC NO.0795 bacterial strain is putting down in writing in first to file CN1483812A of having announced.Other M. smegmatics bacterial strain is suitable equally.

1, the high pressure crush method prepares mycobacterium Smegmatis preparation

Get the M. smegmatics of fresh culture on the modified Russell medium; collect thalline with the physiological saline wash-out; again for several times through the physiological saline centrifuge washing; accurately weigh; being prepared into wet bacteria concentration with frozen-dried protective liquid (phosphate buffered saline buffer) dilution is the bacteria suspension of 10mg/ml; through the high pressure homogenizer (Homogenizer that Denmark APV company produces; Model-2000) the broken thalline of high pressure draft shearing technique homogenate; condition is 1200bar; broken 4 times; the low temperature low-speed centrifugal is removed the solids precipitation thing; 2-8 ℃ of low temperature low-speed centrifugal condition; 1000-6000 rev/min centrifugal 10-30 minute; present embodiment adopts 4 ℃; 3000 rev/mins centrifugal 10 minutes, supernatant liquid was mycobacterium Smegmatis preparation in 20 minutes through 121 ℃ of steam treatment.For keeping better stability, preferably above-mentioned mycobacterium Smegmatis preparation vacuum freezedrying is prepared into the M. smegmatics freeze-dried preparation.

This method is except using the high pressure draft shearing technique of high pressure homogenizer, other similar high pressure crushing technology all can, as adopting French Pressure cell lysing cell (400-12000Ib/in ²) back low temperature low-speed centrifugal.

2, sonioation method prepares mycobacterium Smegmatis preparation

Get the M. smegmatics of fresh culture on the modified Russell medium; collect thalline with the physiological saline wash-out; again for several times through the physiological saline centrifuge washing; accurately weigh; being prepared into wet bacteria concentration with frozen-dried protective liquid (phosphate buffered saline buffer) dilution is the bacteria suspension of 100mg/ml; through the broken thalline of Ultrasonic Cell Disruptor (the JY98-3 ultrasonic cell disruptor that Ningbo Xin Zhike device institute produces); select condition 1000W for use; broken 5 seconds intermittently 5 seconds for once; broken 180 times altogether; the low temperature low-speed centrifugal is removed the solids precipitation thing; 2-8 ℃ of low temperature low-speed centrifugal condition; 1000-6000 rev/min centrifugal 10-30 minute; present embodiment adopts 4 ℃; 3000 rev/mins centrifugal 10 minutes, supernatant liquid through 121 ℃ of steam treatment 20 minutes (or ⁶⁰ Co irradiation 15 minutes) is mycobacterium Smegmatis preparation.For keeping better stability, preferably above-mentioned mycobacterium Smegmatis preparation vacuum freezedrying is prepared into the M. smegmatics freeze-dried preparation.

The present invention preferably uses phosphate buffered saline buffer as frozen-dried protective liquid, and its composition and proportioning are as follows:

1) potassium liquid:

Monosodium glutamate 3.75g

Sucrose 3.75g

Sodium-chlor 10.8g

Anhydrous potassium dihydrogenphosphate 6.525g

Deionized water 1500ml

2) sodium liquid:

Monosodium glutamate 3.75g

Sucrose 3.75g

Sodium-chlor 10.8g

Disodium hydrogen phosphate,anhydrous 27.45g

Deionized water 1500ml

3) frozen-dried protective liquid:

Get 1500ml potassium liquid and 1100ml sodium liquid and mix, the pH that makes mixed solution is 7.2, and mixed solution is frozen-dried protective liquid through 115 ℃ of steam sterilizing 30min after filtering by the G3 funnel again.

The mycobacterium Smegmatis preparation of above-mentioned high pressure crush method or sonioation method preparation is used for the prevention of tuberculosis, hepatitis B, asthma or the preferred dose of treatment is 0.01-10mg mycobacterium Smegmatis preparation/agent, preferred 0.05-5mg/ agent, most preferably 0.1-1mg/ agent.0.1-1mg, the 2mg that mentions among described 0.01-10mg, 0.05-5mg or 0.1-1mg mycobacterium Smegmatis preparation and the following embodiment, 5mg, 10mg, 50mg mycobacterium Smegmatis preparation are meant the preparation that the split product of M. smegmatics after the aforesaid method cracking by respective wet bacterium weight obtains.

The high pressure steam cracking process of mentioning in first to file CN1483812A mainly utilizes high temperature to kill M. smegmatics, high temperature has certain splitting action to bacterium simultaneously, but because the cell wall cracking is insufficient, most bacterium thalline outward appearances after the cracking are not destroyed, but can a small amount of albumen of limited release and nucleic acid.High pressure fragmentation of the present invention utilizes the high pressure draft shearing technique, the pressure generation acute variation that bacterium is born in moment, make the thorough disintegration of bacterial cell wall, fully discharge tropina and nucleic acid, thalline or the inadequate cell walls of cracking that minute quantity is not broken are removed through the low temperature low-speed centrifugal again, finally obtain homogeneous, solvend.

By finding out the difference of two kinds of methods more intuitively to the M. smegmatics lytic effect to the detection of serial index in the preparation process.

Two kinds of preparation methods' of table 1 mycobacterium Smegmatis preparation comparison

Sequence number	Project	The high pressure steam cracking process	The high pressure crush method
				1 2 3	The protein content of bacterium liquid after the OD405 fragmentation of the broken back of light microscopy (acid-fast stain) the bacterium liquid of broken back bacterium liquid	More acid-fast stain male mycobacterium 0.254 60.9 μ g/ml is arranged	Before centrifugal is a slice homogeneous, seldom sees not broken thalline; Centrifugal back is equal pledge 0.530 679 g/ml entirely

Illustrate: the original bacterial concentration described in the table 1 is 10mg/ml.

As can be seen from the above table, the smegmatis mycobacterium of same weight only is 1/10 of a high pressure crush method through the protein content that the high pressure steam cracking process discharges, and other composition burst sizes also obviously reduce, and cause the light absorption value of split product on the low side; Broken back bacterium liquid microscopy also can be found out the homogeneous degree difference of the two, the smegmatis mycobacterium preparation of high pressure crush method preparation is more more even than the smegmatis mycobacterium preparation of high pressure steam cracking process preparation, and the preparation behind the low temperature low-speed centrifugal has not contained tangible solids precipitation thing.Therefore, the smegmatis mycobacterium preparation of high pressure crush method preparation obviously has better security, and can be by the principle continuous several times use clinically of cellular immunization, to reach the ideal immune effect.

Embodiment 2: mycobacterium Smegmatis preparation is to the influence of mouse delayed type hypersensitivity and T lymphopoiesis function

The T lymphocyte is important adjusting cell and effector cell of immune response, and delayed type hypersensitivity and T lymphproliferation response are used for estimating body's immunological function as the basic parameter of the inside and outside cellular immune function of body respectively.Behind this example explanation mycobacterium Smegmatis preparation immune mouse to the influence of delayed type hypersensitivity and T lymphproliferation response.

Give experimental mice and control group mice subcutaneous injection 0.25mg mycobacterium Smegmatis preparation and isopyknic physiological saline respectively, every 2 week injections 1 time, inject 2 times altogether.2 weeks after the last immunity, attack the sufficient pad in right side with 10 μ gPPD/20 μ l, compare with BSA simultaneously, with the sufficient pad in same dosage injection mouse left side, measure sufficient mat thickness respectively with attack back 24h before attacking.To attack forward and backward footpad swelling (mm) is the intensity that index is judged the DTH reaction.The results are shown in Table 2.

Table 2 mycobacterium Smegmatis preparation is to the influence (mm) of mouse delayed type hypersensitivity

	Negative control group	Shame dirt preparation experimental group
			Foot relatively about right foot (PPD) left side foot (BSA)	0.175±0.093 0.088±0.074 p＞0.05	0.342±0.058 * 0.056±0.042 p＜0.01

Annotate: ^*Refer to relatively p＜0.05 of shame dirt preparation group and control group

Give experimental mice and control group mice subcutaneous injection 0.25mg mycobacterium Smegmatis preparation and isopyknic physiological saline respectively, injected for 3 weeks once in a week, continuously.A week after the last immunity, aseptic spleen, the preparation mononuclearcell suspension got, making cell concn is 2 * 10 ⁶/ ml, packing 96 orifice plates, every hole 200 μ l add 20 μ lConA or PPD or perfect medium (making blank) then, and ConA and PPD final concentration in the hole is respectively 5 μ g/ml and 15 μ g/ml, and each is handled and repeats the 2-3 hole; 37 ℃, 5%CO ₂Behind the complete moistening cultivation 70h, every hole adds 10 μ lMTT (5mg/ml), after continuing to cultivate 4h, and the centrifugal supernatant that goes.Every hole adds 100 μ l20%SDS-DMF cell pyrolysis liquids, and 37 ℃ are spent the night, and puts microplate reader (Wellscan MK3) and goes up colorimetric, and the mensuration wavelength is 570nm, and reference wavelength is 630nm.The OD value mean value that calculates multiple hole is with reaction T cell proliferation intensity.The results are shown in Table 3.

Table 3 mycobacterium Smegmatis preparation is to the influence of mouse T lymphocyte propagation function

	Negative control group	Shame dirt preparation experimental group
			ConA PPD	0.173±0.030 0.010±0.004	0.355±0.062 ** 0.093±0.024 **

Annotate: ^*Refer to relatively p＜0.01 of shame dirt preparation group and control group

Conclusion: The above results shows that mycobacterium Smegmatis preparation can promote delayed type hypersensitivity and the T lymphopoiesis function of mouse, promptly raises the immunologic function of normal mouse.

Embodiment 3: mycobacterium Smegmatis preparation is to the influence of mouse Th1 and the generation of Th2 type cytokines

The Th cell is important adjusting cell and effector cell of immunity system, and according to excretory cytokine difference, the Th cell is divided into Th1 and Th2 two big classes, the former main secretion of gamma-IFN and IL-2 etc., and the latter mainly secretes IL-4, IL-5 and IL-10 etc.The running balance of Th1/Th2 is subjected to multifactor adjusting, and wherein IL-12 is considered to the initiation factor that Th1 replys, and promotes the Th0 cell to the Th1 cytodifferentiation.Th1/Th2 replys and keeps a running balance, and is most important to modulation on immune status.Have now found that; the multiple disease of respiratory system is unbalance relevant with Th1/Th2's; for tuberculosis; the Th1 advantage is replied the mediate protection immune response, and the Th2 advantage is replied the reaction that then mediates based on tardy parasexuality reaction, is accompanied by pathologic immunologic injury (MosmannTR; Sad S.The expanding universeof T-cell subsets:Th1; Th2 and more.Immunol Today, 1996,17:138-146.)

This example to the influence that Th1, Th2 type cytokines produce, illustrates the regulating effect that mycobacterium Smegmatis preparation is replied body Th1/Th2 by the research mycobacterium Smegmatis preparation.

With the mycobacterium Smegmatis preparation immune mouse of various dose, inject another group mouse in contrast with isopyknic physiological saline simultaneously.In 1 week after the last immunity, aseptic taking-up spleen lymphocyte and peritoneal macrophage are adjusted cell concn and are respectively 2.0 * 10 ⁶/ ml and 2.5 * 10 ⁶/ ml, packing 24 orifice plates, every hole 1ml, and add 100 μ lConA or PPD, 37 ℃, 5%CO ₂Centrifugal behind the complete moistening cultivation 72h, get supernatant ,-30 ℃ of preservations.Use corresponding cytokine content in mouse IL-2, IL-4 and the IFN-γ ELISA kit measurement splenic lymphocyte culture supernatant respectively, with IL-12 content in the mouse IL-12 ELISA kit measurement peritoneal macrophage culture supernatant.Measured value is learned processing by statistics, the results are shown in Figure 1-4.

This routine presentation of results mycobacterium Smegmatis preparation can improve the expression amount of Th1 type cytokines such as IL-12, IL-2 and IFN-γ, reduces the expression amount of Th2 type cytokines such as IL-4.The deducibility mycobacterium Smegmatis preparation can raise the Th1 class and replys, suppresses the Th2 class and reply thus, and this control to immunologically mediated diseases such as tuberculosis, asthma, hepatitis B is significant.

Embodiment 4: mycobacterium Smegmatis preparation is to the influence of mouse macrophage secretion IL-1 and GM-CSF

Scavenger cell is a kind of important antigen presenting cell and immune effector cell, can secrete various kinds of cell factor performance panimmunity function.Except inflammatory reaction is had important regulatory role, also can strengthen the phagocytic function of granulocyte and scavenger cell as GM-CSF, induce maturing dendritic cell and distribution of functionality; IL-1 is a kind of important inflammatory mediator, has vital role in anti-microbial infection, has important immunoregulation effect simultaneously, but the enhancement antigen presenting cells is to antigenic activate the phagocytic capacity and submission ability etc.This example illustrates the regulating effect of said preparation to the antigen presentation system by inquiring into the influence of mycobacterium Smegmatis preparation to the scavenger cell secrete cytokines.Experimental technique the results are shown in Figure 5,6 with embodiment 3.

Above presentation of results mycobacterium Smegmatis preparation can promote cytokines such as mouse macrophage secrete GM-CSF and IL-1, and the deducibility said preparation is expected to strengthen antigen presenting cell and engulfs and processing power antigenic thus.

Embodiment 5: the influence that mycobacterium Smegmatis preparation produces mouse body intracellular nitric oxide

NO is a kind of important signaling molecule and strong effector molecule in vivo, and its level raising helps body to colonizing in killing and wounding and scavenging(action) of bacterium in the cell, virus.Macrophages in vitro is cultivated through former the inducing of suitable stimulation and is produced NO, and the NO of generation is oxidized to NO fast ^- ₂, by detecting NO ^- ₂The generation level that can reflect NO.Behind this example explanation mycobacterium Smegmatis preparation immune mouse to the influence of peritoneal macrophage NO production.

With the mycobacterium Smegmatis preparation immune mouse of various dose, inject another group mouse in contrast with isopyknic physiological saline simultaneously.1 week after the last immunity, aseptic taking-up peritoneal macrophage, adjusting cell concn is 2.5 * 10 ⁶/ ml, packing 24 orifice plates, every hole 1ml, and add 100 μ l LPS, 37 ℃, 5%CO ₂Centrifugal behind the complete moistening cultivation 24h, get supernatant ,-30 ℃ of preservations.Detect the content of NO in the supernatant with the Griess reagent color developing method.The results are shown in Figure 7.

This example shows that mycobacterium Smegmatis preparation can promote the generation of NO, and this will help body and kill and wound and scavenging(action) entering microorganism performances such as intracellular tubercule bacillus.

Embodiment 6: mycobacterium Smegmatis preparation is to the immunoregulation effect of immunologic hypofunction body

This example explanation mycobacterium Smegmatis preparation is to caused by cyclophosphamide immunocompromised effect of immunologic function.40 Balb/c mouse are divided into 5 groups at random, are respectively the basic, normal, high dosage group of mycobacterium Smegmatis preparation, negative control group and normal control group, 8 every group.The normal control group is not given any medicine in entire test; The basic, normal, high dosage treated animal of mycobacterium Smegmatis preparation at first respectively with 0.1mg, 0.5mg and the immunity of 1.0mg mycobacterium Smegmatis preparation once, negative control treated animal injection equal-volume physiological saline, give basic, normal, high dosage group of mycobacterium Smegmatis preparation and negative control group animal subcutaneous injection endoxan 100mg/kg after one week, cause the immunologic hypofunction model, behind the injection endoxan the 1st day and the 3rd day distinguish again booster immunization once, dosage and the same first immunisation of mode.In a week behind the injection endoxan, with embodiment 2 aseptic separation and Culture splenic lymphocyte, every hole adds 20 μ l5mg/ml MTT behind the cultivation 70h, continues to cultivate 4h, detects lymphopoiesis intensity.The results are shown in Figure 8.

As shown in the figure, normal control group T lymphopoiesis intensity obviously is better than negative control group, and immunologic hypofunction modelling success is described; Mycobacterium Smegmatis preparation immune group T lymphopoiesis intensity obviously is better than negative control group even is better than the normal control group, illustrates that mycobacterium Smegmatis preparation has the restitution that promotes the immunologic hypofunction body's immunity.

Embodiment 7: mycobacterium Smegmatis preparation is to the immunoregulation effect of the hyperfunction body of immunologic function

This example is an example with I type and the super quick model of IV type, and the immunoregulation effect of mycobacterium Smegmatis preparation to the super quick animal of immunity is described.

The disease index of table 4 porcine blood serum sensitized mice

	Negative control group	Mycobacterium Smegmatis preparation
					0.1mg	0.5mg	1.0mg
		Disease index P value	2.400±0.821	1.053±0.941 p＜0.01				0.684±0.478 p＜0.01	1.105±0.966 p＜0.01

The super quick model of I type: the Balb/c mouse is divided into basic, normal, high dosage group of mycobacterium Smegmatis preparation and negative control group at random, and the dilute serum of every mouse peritoneal injection 0.5ml markization carries out sensitization, the next day once, totally 3 times, set up the super quick model of I type.Next day after the last sensitization, inject 0.1mg, 0.5mg and 1.0mg mycobacterium Smegmatis preparation respectively for the basic, normal, high dosage group of mycobacterium Smegmatis preparation mouse, the next day once, continuous 7 times, give negative control group injected in mice equal-volume physiological saline simultaneously.Next day after the last injection, attack for all mouse end of line veins with the high density porcine blood serum, 1ml/ is only.According to the disease index standard of keeping the score, judge the mouse invasion situation, record morbidity number of animals and disease index (score value is morbidity 1 fen above person) are (with reference to " Chinese biological goods rules " (2000 editions).The results are shown in Table 4.

The super quick model of IV type: in first, the mid-term of tubercle bacillus affection, body's immunological function is in hyperfunction state, and (allergy of IV type DTH) strengthens to show as skin allergic reaction.The cavy subcutaneous injection tubercule bacillus 1100cfu/0.5ml/ that gives PPD skin experiment feminine gender only sets up the delayed type hypersensitivity model.Then animal pattern is divided at random basic, normal, high dosage group of mycobacterium Smegmatis preparation and negative control group, 3d behind tubercle bacillus affection, 10d, 17d, 24d give the basic, normal, high dosage group of M. smegmatics cavy every intramuscular injection 0.1mg, 0.25mg and 0.5mg mycobacterium Smegmatis preparation respectively, give every injection of negative control group cavy 0.5ml physiological saline.6w behind tubercle bacillus affection gives guinea pig back intradermal injection PPD 0.1ml/ only, observes, measures the red and swollen and scleroma of injection site behind the 48h, writes down longitudinal and transverse footpath, with the power of longitudinal and transverse footpath mean value indication DTH.The results are shown in Table 5.

The delayed type hypersensitivity of table 5 tubercle bacillus affection cavy

	Negative control group	Mycobacterium Smegmatis preparation
					0.1mg	0.5mg	1.0mg
		Scleroma diameter (mm) P value	12.67±1.47	9.87±3.36 p＜0.01				7.04±3.09 p＜0.01	7.25±3.70 p＜0.01

Illustrate: 48h scleroma size after the injection of testing in the table, unit is mm.

Conclusion: mycobacterium Smegmatis preparation can suppress the strong excessively immunne response of immune hyperfunction body, to reduce the pathologic damage of body.

Embodiment 2-7 proof mycobacterium Smegmatis preparation has good immunoregulation effect; except improving the immunologic function of normal body; still can promote the recovery of immunocompromised body's immunity and suppress the strong excessively immunne response of immune hyperfunction body; be that mycobacterium Smegmatis preparation has two-way immunoregulation effect, this to tuberculosis etc. along with the control of the different disease of course of disease difference, body's immunity is significant.And, mycobacterium Smegmatis preparation is replied, is reduced Th2 with adjusted Th1 in performance during immunoregulation effect and replys and be main mode, infectious diseases and asthma etc. in the cells such as tuberculosis, hepatitis such as hepatitis B is replied with Th2 increase the class disease unusually and have prevention and result of treatment.

Embodiment 8: mycobacterium Smegmatis preparation is to prevention effect lungy

After this example explanation is attacked cavy with tubercule bacillus, with the protection effect of mycobacterium Smegmatis preparation immunity.

Behind the tubercle bacillus affection cavy, weekly, continuous 4 weeks are given one of following prepared product of cavy intramuscular injection:

1) physiological saline (negative control)

2) 0.25mg mycobacterium Smegmatis preparation

Infect the 7th week of back, kill cavy and dissection, observe each internal organs tuberculosis degree such as liver, spleen, lung, lymphoglandula, and carry out the pathology scoring with double-blind method; Aseptic homogenate spleen, test mycobacterium tuberculosis colony-forming unit; And get liver, spleen, the lung of cavy, do the pathology inspection after fixing.The result is as follows:

Internal organs outward appearance: a large amount of, tangible tuberculose focus is arranged on negative control group Guinea pig lung and the spleen, also can see typical tuberculose focus on the liver; The animal that tuberculose focus obviously reduces, has on mycobacterium Smegmatis preparation group Guinea pig lung and the spleen almost loses tuberculose focus.Its pathology scoring statistical result sees Table 6.

Table 6 mycobacterium Smegmatis preparation is to the protection effect of tubercle bacillus affection animal

	The pathology index (X ± SD)	Spleen tubercule bacillus lotus bacterium amount logarithmic value (X ± SD)
			Negative control group shame dirt preparation group	49.6±16.7 29.6±12.3**	4.940±0.411 4.000±1.390*

Illustrate: *: refer to relatively p＜0.05 of shame dirt preparation group and negative control group

*: refer to relatively p＜0.01 of shame dirt preparation group and negative control group

Pathological examination: the lung of negative control treated animal, liver, spleen are popularity miliary tuberculosis kitchen range or caseous necrosis more, each organ disease of mycobacterium Smegmatis preparation group all is lighter than negative control group, the tuberculosis scope is limited to, degree is lighter, mostly is proliferative tubercle, nonspecific inflammation or almost normal tissue slice.

Spleen tubercule bacillus lotus bacterium amount: make serial dilution after the spleen homogenate, inoculate modified Russell medium, counting tubercule bacillus colony-forming unit calculates its logarithmic value, takes statistics to learn and handles.The results are shown in Table 6.

Embodiment 2-7 has prevention effect from immunoregulatory angle proof mycobacterium Smegmatis preparation to tuberculosis,

Embodiment 8 goes up directly from the tuberculosis animal pattern, and the proof mycobacterium Smegmatis preparation has unusual effect to control lungy, from then on example especially as can be seen said preparation the body that infects tubercule bacillus is had good prevention effect, this prevention to China and even the huge tuberculosis latent infection person in the whole world is significant.

Embodiment 9: the acute toxicity test of mycobacterium Smegmatis preparation

This example by with the disposable large bolus injection mouse of mycobacterium Smegmatis preparation, observe explanation said preparations such as its toxic reaction, death condition and do not have the acute toxicity effect.The results are shown in Table 7.

Table 7 mycobacterium Smegmatis preparation is to the acute toxicity test of mouse

Dosage (mg/ only)	Injecting pathway	Size of animal (only)	Survival number (only)	Death toll (only)	Have or not abnormal symptom
						Contrast 1.0 2.0 10.0	Abdominal cavity, abdominal cavity, abdominal cavity, abdominal cavity	20 20 20 20	20 20 20 20	0 0 0 0	Do not have

Dosage (mg/ only)	Injecting pathway	Size of animal (only)	Survival number (only)	Death toll (only)	Have or not abnormal symptom
						Contrast 1.0 2.0 10.0	Muscle muscle muscle muscle	20 20 20 20	20 20 20 20	0 0 0 0	Do not have

Conclusion: above-mentioned 3 kinds of dosage injection mouse does not all cause abnormal symptom and death; From 10mg/20g/ mouse of maximum injection amount, the minimum lethal dose of deducibility mouse is greater than 500mg/kg.

Embodiment 10: the local irritant effect of mycobacterium Smegmatis preparation

This example in 2 rabbit (1mg/ml/ only), forms contrast ground with the mycobacterium Smegmatis preparation intramuscular injection, gives other 2 rabbit intramuscular injection 1ml physiological saline.Injection back 48h puts to death rabbit, takes out injection site muscle, vertically cuts, and observes the injection site irritant reaction, and gets injection site fritter muscle and do pathologic finding.Result: abnormal change such as the same with the physiological saline control group, mycobacterium Smegmatis preparation group rabbit injection site myofiber do not see degeneration necrosis, it is hemorrhage not see, oedema and inflammatory cell infiltration.Conclusion: mycobacterium Smegmatis preparation does not have bad hormesis to injection site.

Embodiment 11: mycobacterium Smegmatis preparation systemic allergy test

This example explanation mycobacterium Smegmatis preparation is applied to body and whether can causes systemic anaphylaxis.

Cavy is divided into 3 groups at random, injects one of following material:

1) positive controls: the next day intramuscular injection BSA 0.5mg/0.5ml/ only

2) negative control group: the next day intramuscular injection physiological saline 0.5ml/ only

3) mycobacterium Smegmatis preparation group: the next day intramuscular injection mycobacterium Smegmatis preparation 5mg/0.5ml/ only

1 time weekly, inject altogether 3 times.After sensitization first, attacked (only attack BSA 4mg/ml/ for the positive controls cavy, only attack mycobacterium Smegmatis preparation 10mg/ml/) from the hind leg intravenous injection respectively in the 14th day and the 21st day to mycobacterium Smegmatis preparation group and negative control group cavy.In back 1 hour of attack, the close observation cavy has or not allergic symptoms such as grabbing nose, perpendicular hair, expiratory dyspnea, spasm, shock even death, and by the symptom classification, symptom is serious more, and progression is high more.

Table 8 is respectively organized the cavy reaction result

As shown in table 8, the positive controls cavy is dead after having 8 typical allergic symptom to occur after the attack, all the other 4 appearance continuously, cough repeatedly, with tangible allergic symptoms such as expiratory dyspnea or spasm, after shame dirt preparation group and negative control group cavy are attacked, any abnormal response all do not occur, illustrate that mycobacterium Smegmatis preparation can not cause systemic anaphylaxis.

Embodiment 12: the long term toxicity test of mycobacterium Smegmatis preparation

This example explanation mycobacterium Smegmatis preparation high dosage, for a long time, repeatedly be applied to whether the Beagle dog can accumulate and to effect of body toxigenicity and severity thereof.

With various dose mycobacterium Smegmatis preparation intramuscular injection Beagle dog (1mg/ only, 10mg/ only or 50mg/ only), another treated animal injecting normal saline is made negative control, for 1 time weekly, injected for 14 weeks continuously.Whole experimental session: observe outward appearance sign, behavioral activity, feed and amount of drinking water, the ight soil proterties of each treated animal every day and have or not death etc., per 1 week is measured a body weight, early stage, mid-term, latter stage and decubation in experiment are measured Beagle dog blood cell, serum biochemistry, electrocardiogram(ECG detection and routine urinalysis respectively, and work in 24 hours is killed the part animal and is dissected after last administration, lives extremely and dissection in all the other animals continuation observation 2 week backs.Each internal organs situation of the comprehensive careful observation of when dissected, core again, liver, spleen, lung, kidney, suprarenal gland, Tiroidina, brain, thymus gland, ovary, uterus, testis etc. weigh, calculate organ coefficient, get the major organs of each system such as stomach, intestines (duodenum, jejunum, ileum), pancreas, lymphoglandula, bladder, prostate gland simultaneously, do the pathology histological examination.

Result: see Table 9-17.In the whole experiment, the single index of individual animal has accidental one to cross the property variation, but all in the normal mobility scale of animal, and do not have dose-dependently, and hence one can see that, and this variation is not a drug-induced.Compare with control group, experiment preceding, in, latter stage and decubation, each dosage treated animal outward appearance sign of mycobacterium Smegmatis preparation, behavior are all normal, the every index of blood cell, serum biochemistry, electrocardiogram(ECG and routine urinalysis is all in normal range, administration group and control group compare, no difference of science of statistics; Each internal organs outward appearance of when dissected and pathological examination there is no unusually.

Conclusion: mycobacterium Smegmatis preparation be less than or equal under the used dosage of test, for a long time, repeatedly using can the toxigenicity effect.

The long-term intramuscular injection of table 9 mycobacterium Smegmatis preparation reaches out the influence (X ± SD) in clotting time mutually to Beagle dog peripheral blood

Detection time	Group	Dog number (only)	WBC (10 9/L)	?RBC ?(10 12/L)	HGB (g/L)	?HCT	?PLT (10 9/L)	Go out the clotting time (S)
									Administration preceding ten days	Dosage group high dose group in the control group low dose group	?6 ?6 ?6 ?6	13.03±4.57 14.53±3.98 11.30±1.63 14.23±2.16	?5.07±0.78 ?4.90±0.47 ?5.26±0.66 ?4.77±0.36	?123.3±15.9 ?121.8±11.6 ?124.8±13.0 ?113.3±7.90	?0.38±0.05 ?0.33±0.04 ?0.38±0.06 ?0.31±0.03	?351.0±171 ?276.5±56 ?298±145 ?226±71.1	53.67±6.50 55.50±3.73 58.83±3.06 56.33±9.42
Administration a few days ago	Dosage group high dose group in the control group low dose group	?6 ?6 ?6 ?6	10.55±5.72 14.23±2.77 12.47±2.25 13.92±3.37	?5.42±0.59 ?5.27±0.54 ?5.76±0.71 ?4.97±0.40	?126.8±12.8 ?125.2±12.9 ?135.7±13.6 ?121.5±6.38	?0.39±0.03 ?0.36±0.06 ?0.39±0.06 ?0.35±0.05	?321.0±132 ?292.8±71 ?312±107 ?400±106	59.00±4.05 63.17±8.91 61.67±5.89 58.67±6.11
									Administration mid-term	Dosage group high dose group in the control group low dose group	?6 ?6 ?6 ?6	13.53±3.42 16.60±2.87 14.93±2.59 15.62±1.85	?5.94±0.43 ?5.87±0.48 ?5.77±0.70 ?5.49±0.50	?143.5±8.69 ?138.3±10.5 ?136.2±12.2 ?128.8±14.8	?0.39±0.03 ?0.39±0.04 ?0.39±0.05 ?0.38±0.05	?276.8±63 ?302.8±56 ?295.2±54 ?279.5±17	56.83±3.31 57.83±8.47 55.50±4.97 56.00±5.14
Administration latter stage	Dosage group high dose group in the control group low dose group	?6 ?6 ?6 ?6	12.18±2.76 13.92±2.02 12.90±1.45 15.18±1.19 *	?5.94±0.34 ?6.09±0.63 ?5.95±0.41 ?5.95±0.33	?141.17±5.3 ?143.0±14.6 ?141.8±7.76 ?137.8±12.0	?0.40±0.03 ?0.40±0.04 ?0.40±0.05 ?0.43±0.06	?277.0±55 ?282.8±20 ?254.7±55 ?265.3±20	65.67±5.35 63.17±4.36 63.83±5.34 61.17±4.36
									Decubation	Dosage group high dose group in the control group low dose group	?2 ?2 ?2 ?2	11.95±1.34 12.10±1.56 13.40±2.97 12.45±2.05	?5.93±0.62 ?5.27±0.04 ?5.74±0.46 ?5.39±0.74	?147.5±3.54 ?132.0±5.66 ?138.5±9.19 ?124.5±17.7	?0.37±0.03 ?0.34±0.03 ?0.37±0.02 ?0.36±0.04	?180.0±20 ?207.5±0.7 ?174.0±37 ?213.5±19	63.50±0.71 66.50±4.95 63.00±2.83 62.50±2.12

Annotate: *: compare P≤0.05 with control group; All the other each groups compare P＞0.05 with control group.

The influence that the long-term intramuscular injection of table 10 mycobacterium Smegmatis preparation is classified to the Beagle canine leucocyte (X ± SD)

Detection time	Group	Dog number (only)	?WBC ?(10 9/L)	Lymphocyte (%)	Neutrophilic granulocyte (%)	Eosinophilic granulocyte (%)	Monocyte (%)	Reticulocyte (%)
									Administration preceding ten days	Dosage group high dose group in the control group low dose group	6 6 6 6	?13.03±4.57 ?14.53±3.98 ?11.30±1.63 ?14.23±2.16	?21.33±4.59 ?22.50±2.88 ?28.00±6.03 ?21.83±4.36	?76.83±5.27 ?76.17±3.43 ?70.67±5.47 ?77.0±4.60	?1.00±0.89 ?0.67±0.52 ?1.17±0.41 ?1.00±0.63	?0.67±0.52 ?0.67±0.82 ?0.33±0.52 ?0.33±0.82	1.05±0.19 1.17±0.29 1.15±0.16 1.83±0.23
Administration a few days ago	Dosage group high dose group in the control group low dose group	6 6 6 6	?10.55±5.72 ?14.23±2.77 ?12.47±2.25 ?13.92±3.37	?26.00±5.66 ?26.00±2.28 ?25.83±6.31 ?24.17±4.40	?72.17±6.11 ?72.83±2.64 ?72.83±6.31 ?74.50±4.23	?1.50±0.84 ?1.00±0.63 ?1.00±0.89 ?1.17±0.75	?0.33±0.52 ?0.17±0.41 ?0.33±0.52 ?0.17±0.41	1.33±0.45 1.65±0.22 1.05±0.35 1.25±0.24
									Administration mid-term	Dosage group high dose group in the control group low dose group	6 6 6 6	?13.53±3.42 ?16.60±2.87 ?14.93±2.59 ?15.62±1.85	?36.33±4.93 ?31.33±3.50 ?36.50±7.06 ?36.67±4.63	?62.83±4.54 ?67.67±3.20 ?62.00±6.66 ?62.17±4.71	?0.67±0.82 ?0.83±0.41 ?1.33±0.52 ?1.00±0.63	?0.17±0.41 ?0.17±0.41 ?0.17±0.41 ?0.17±0.41	1.77±0.36 2.27±0.42 1.57±0.50 1.72±0.41
Administration latter stage	Dosage group high dose group in the control group low dose group	6 6 6 6	?12.18±2.76 ?13.92±2.02 ?12.90±1.45 ?15.18±1.19 *	?30.67±6.31 ?30.83±2.32 ?28.67±3.14 ?30.33±4.08	?68.00±5.69 ?68.00±2.61 ?69.67±3.92 ?68.00±4.15	?0.67±0.82 ?0.83±0.41 ?1.33±0.82 ?1.17±0.75	?0.67±0.82 ?0.33±0.52 ?0.33±0.52 ?0.50±0.55	1.67±0.16 1.78±0.37 1.83±0.19 1.55±0.21
									Decubation	Dosage group high dose group in the control group low dose group	2 2 2 2	?11.95±1.34 ?12.10±1.56 ?13.40±2.97 ?12.45±2.05	?36.0±11.30 ?34.00±9.90 ?38.5±0.707 ?31.00±5.66	?62.50±10.6 ?64.50±7.78 ?60.50±0.71 ?67.50±4.95	?1 ?1 ?1 ?1	?0.5 ?0.5 ?0 ?0.5	1.70±0.42 2.00±0.57 1.70±0.28 1.85±0.07

Annotate: *: compare P≤0.05 with control group; All the other each groups compare P＞0.05 with control group.

The long-term intramuscular injection of table 11 mycobacterium Smegmatis preparation is to the influence of each biochemical indicator of Beagle dog serum (X ± SD)

Detection time	Group	Dog number (only)	GPT (U/L)	GOT (U/L)	ALP (U/L)	BUN (mN)	CRE (uM)
								Administration preceding ten days	Dosage group high dose group in the control group low dose group	6 6 6 6	35.67±7.71 36.33±7.66 31.50±7.92 31.00±6.03	34.17±4.54 37.67±6.71 34.00±2.45 36.50±2.66	122.5±27.3 165.5±35.6 151.7±44.7 130.0±11.9	3.90±0.89 4.33±0.88 4.31±0.76 3.98±0.91	74.18±6.46 71.62±9.35 174.0±242 69.18±4.65
Administration a few days ago	Dosage group high dose group in the control group low dose group	6 6 6 6	34.00±7.67 33.00±5.62 30.17±7.47 29.83±4.02	30.83±4.71 31.67±4.08 30.50±2.35 33.67±6.35	116.2±19.4 150.0±34.1 141.0±44.0 124.0±10.6	4.63±1.06 4.95±1.37 4.92±0.62 4.48±1.00	73.55±5.87 72.20±8.16 74.90±6.73 70.63±5.50
								Administration mid-term	Dosage group high dose group in the control group low dose group	6 6 6 6	40.17±6.40 45.00±10.5 35.00±13.5 33.00±4.00 *	35.33±3.67 44.17±7.05 * 37.17±4.12 40.33±5.92	90.50±18.5 131.0±28.3 106.0±26.7 104.7±7.75	4.97±0.89 5.70±2.12 4.30±0.96 4.65±0.58	76.98±5.21 80.13±6.41 77.57±9.10 75.23±5.45
Administration latter stage	Dosage group high dose group in the control group low dose group	6 6 6 6	42.50±7.79 45.3±10.40 40.50±9.67 37.83±8.47	32.33±2.58 32.67±6.92 32.17±2.64 31.83±2.23	86.00±25.5 118.0±23.3* 92.00±17.2 91.50±20.1	6.00±1.50 6.58±1.52 5.50±1.27 5.08±0.71	83.87±5.51 88.07±6.40 88.53±5.14 80.60±4.89
								Decubation	Dosage group high dose group in the control group low dose group	2 2 2 2	43.5±17.70 46.0±24.00 34.00±8.49 36.00±4.24	29.50±2.12 31.00±4.24 34.50±2.12 29.00±1.41	?64.50±20.5 ?93.00±12.7 ?73.50±24.7 ?66.00±2.83	5.35±0.07 6.80±2.69 7.15±1.48 4.90±0.71	80.25±4.45 82.40±16.3 96.00±1.98 80.25±2.47

Annotate: *: compare P≤0.05 with control group; All the other each groups compare P＞0.05 with control group.

The long-term intramuscular injection of table 12 mycobacterium Smegmatis preparation is to the influence of each biochemical indicator of Beagle dog serum (X ± SD)

Detection time	Group	Dog number (only)	CHO (mM)	GLU (mM)	T.P (g/L)	ALB (g/L)	BIL (uM)
								Administration preceding ten days	Dosage group high dose group in the control group low dose group	6 6 6 6	3.672±0.86 4.040±0.48 3.652±0.23 3.998±0.80	4.400±0.48 4.550±0.48 4.450±0.45 3.917±0.54	60.98±8.23 58.15±3.98 56.07±1.16 57.47±4.14	32.72±1.33 32.78±1.40 33.00±1.33 31.62±0.94	1.767±0.12 2.017±0.59 1.700±0.26 1.883±0.35
Administration a few days ago	Dosage group high dose group in the control group low dose group	6 6 6 6	3.647±0.63 3.887±0.37 3.748±0.29 4.043±0.73	5.250±0.40 5.717±0.26 5.300±0.47 5.050±0.57	61.55±8.14 57.62±4.90 56.83±1.04 58.47±3.81	33.08±1.85 33.12±1.57 33.48±0.97 32.18±0.90	1.983±0.17 1.983±0.71 1.900±0.28 2.017±0.28
								Administration mid-term	Dosage group high dose group in the control group low dose group	6 6 6 6	3.152±0.66 3.412±0.17 3.213±0.19 3.460±0.68	3.317±0.38 3.317±0.94 3.517±0.74 3.333±0.31	64.77±5.80 63.53±3.23 59.57±1.44 61.58±5.78	34.43±1.68 34.35±1.96 33.47±1.84 32.43±1.63	2.167±0.50 2.317±0.50 2.200±0.43 2.100±0.30
Administration latter stage	Dosage group high dose group in the control group low dose group	6 6 6 6	3.530±0.69 3.530±0.13 3.282±0.22 3.615±0.49	4.400±0.46 4.667±0.31 4.467±0.21 4.400±0.39	66.30±7.06 65.45±4.12 61.45±0.65 63.42±5.67	33.77±1.51 34.58±1.49 34.22±1.50 32.38±2.00	2.400±0.66 2.683±0.53 2.650±0.85 2.183±0.62
								Decubation	Dosage group high dose group in the control group low dose group	2 2 2 2	3.61±0.057 3.695±0.38 3.710±0.13 3.845±0.13	4.400±0.00 4.750±0.07 4.550±0.21 4.400±0.42	61.95±0.92 58.35±2.19 60.90±3.96 57.40±2.83	35.20±0.85 33.70±1.41 35.05±0.21 31.95±0.21	2.200±0.14 1.950±0.35 2.050±0.07 1.450±0.21

Annotate: each dosage group of mycobacterium Smegmatis preparation and control group be P＞0.05 relatively

The long-term intramuscular injection of table 13 mycobacterium Smegmatis preparation is to the Electrocardiographic influence of Beagle dog (X ± SD)

Detection time	Group	Dog number (only)	Heart rate (inferior/minute)	P-R interval (S)	QRS interval (S)	Q-T interval (S)
							Administration first two weeks	Dosage group high dose group in the control group low dose group	6 6 6 6	94.20±14.3 90.83±4.92 92.50±13.3 90.00±6.32	0.159±0.016 0.162±0.015 0.153±0.014 0.174±0.028	0.079±0.004 0.082±0.006 0.082±0.006 0.079±0.003	0.197±0.015 0.190±0.013 0.198±0.016 0.193±0.012
Administration the last week	Dosage group high dose group in the control group low dose group	6 6 6 6	180.0±47.7 195.0±16.4 164.2±26.0 170.8±10.2	0.067±0.008 0.057±0.008 0.058±0.008 0.055±0.005*	0.058±0.008 0.041±0.004* 0.052±0.008 0.048±0.010	0.167±0.020 0.162±0.019 0.171±0.016 0.177±0.016
							Administration mid-term	Dosage group high dose group in the control group low dose group	6 6 6 6	156.7±30.1 155.8±24.6 133.3±16.3 133.3±22.5	0.083±0.015 0.093±0.010 0.083±0.008 0.097±0.012	0.045±0.008 0.043±0.008 0.048±0.010 0.053±0.008	0.187±0.024 0.187±0.033 0.193±0.027 0.190±0.013
Administration latter stage	Dosage group high dose group in the control group low dose group	6 6 6 6	163.3±43.7 171.7±42.6 151.7±16.0 145.8±18.0	0.083±0.005 0.083±0.008 0.080±0.011 0.082±0.013	0.043±0.008 0.040±0.000 0.040±0.000 0.038±0.004	0.110±0.013 0.107±0.033 0.107±0.021 0.118±0.026
							Decubation	Dosage group high dose group in the control group low dose group	2 2 2 2	180.0±0.00 150.0±42.4 120.0±14.1 135.0±21.2	0.095±0.007 0.110±0.014 0.090±0.014 0.100±0.028	0.040±0.000 0.030±0.000 0.030±0.000 0.040±0.000	0.110±0.014 0.105±0.007 0.120±0.000 0.115±0.021

Annotate: * and control group be P＜0.05 relatively; All the other each groups compare P＞0.05 with control group.

The long-term intramuscular injection of table 14 mycobacterium Smegmatis preparation is to the influence of Beagle dog routine urinalysis (X ± SD)

Detection time	Group	Dog number (only)	PH value (X ± SD)	Urinary nitrogen (NIT)	Urine protein (PRO)	Glucose in urine (GLU)	Urine ketone (KET)	Urobilinogen (URO)	Urine bilirubin (BIL)	ERY (BLD)
											Administration first two weeks	Dosage group high dose group in the control group low dose group	6 6 6 6	7.83±0.75 7.50±1.52 6.67±1.51 6.83±1.33	- - ± ±	- - ± -	- - - -	- - - -	- - - -	- - - -	- ± ± ±
Administration the last week	Dosage group high dose group in the control group low dose group	6 6 6 6	7.17±1.17 7.83±0.98 7.33±1.21 8.33±0.82	- ± - -	- - - -	- ± - -	- - - -	- - - -	- - - -	± ± - -
											Administration mid-term	Dosage group high dose group in the control group low dose group	6 6 6 6	?5.83±0.75 ?5.83±1.17 ?5.83±0.98 ?7.17±1.47	± ± ± ±	- - - -	- - - -	- - - -	- - - -	- - - -	- ± - ±
Administration latter stage	Dosage group high dose group in the control group low dose group	6 6 6 6	?7.67±1.51 ?6.33±1.03 ?6.33±0.52 ?6.67±1.37	- - + ±	± - -	- - -	- - -	- - -	- - -	++ ++ ++ ±
											Decubation	Dosage group high dose group in the control group low dose group	2 2 2 2	?6.50±0.71 ?7.00±0.00 ?8.00±1.41 ?5.50±0.71	± ± - ±	- - - -	- - - -	- - - -	- - - -	- - - -	- - - ±

Annotate: each dosage group of mycobacterium Smegmatis preparation and control group be P＞0.05 relatively

The long-term intramuscular injection of table 15 mycobacterium Smegmatis preparation is to the influence of each main organs coefficient of Beagle dog (X ± SD) (administration latter stage; 4/group)

Annotate: each dosage group of mycobacterium Smegmatis preparation and control group be P＞0.05 relatively

The long-term intramuscular injection of table 16 mycobacterium Smegmatis preparation is to the influence of each main organs coefficient of Beagle dog (X ± SD) (decubation; 2/group)

Annotate: each dosage group of mycobacterium Smegmatis preparation and control group be P＞0.05 relatively

The long-term intramuscular injection of table 17 mycobacterium Smegmatis preparation is to each main organs histological examination result of Beagle dog

Animal	The heart	Liver	Lung	Spleen	Kidney and suprarenal gland	Thymus gland	Tiroidina	Stomach	Brain	Reproductive system
											High dosage-3 administration phase high dosage-2 administration phase high dosage-1 administration phase administration phase high dosage-4	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --
Administration interim dosage-1 administration interim dosage-2 administration interim dosage-3 administration interim dosage-4	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --
											-1 administration phase of contrast administration phase-2 administration phases of contrast-3 administration phases of contrast contrast-4	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --	-- -- -- --
Decubation high dosage-1 decubation high dosage-2	-- --	-- --	-- --	-- --	-- --	-- --	-- --	-- --	-- --	-- --
											Dosage in decubation-1 dosage-2 in decubation	-- --	-- --	-- --	-- --	-- --	-- --	-- --	-- --	-- --	-- --
Decubation contrast-1 decubation contrast-2	-- --	-- --	-- --	-- --	-- --	-- --	-- --	-- --	-- --	-- --

Annotate: each dosage group of mycobacterium Smegmatis preparation and each internal organs outward appearance of control animals and pathologic finding there is no unusually

Embodiment 9-12 has made the safety evaluation of system from acute toxicity, local excitation, systemic anaphylaxis and long term toxicity equal angles to mycobacterium Smegmatis preparation, and the presentation of results said preparation does not have above-mentioned toxicity.

Comprehensive 12 embodiment; the result shows that mycobacterium Smegmatis preparation is a kind of safe and effective immunomodulator; has good immunoregulation effect; remove the immunologic function that can strengthen normal body; also can promote the recovery of immunocompromised body's immunity and suppress the strong excessively immunne response of immune hyperfunction body; promptly have two-way immunoregulation effect, to tuberculosis etc. along with the control of course of disease difference, body's immunity inhomogeneity disease is significant.Mycobacterium Smegmatis preparation is replied with Th2 infectious diseases and asthma etc. in the cells such as tuberculosis, hepatitis B and is increased the class disease unusually and have potential to prevent or/and result of treatment to promote Th1 to reply, reduce regulative mode that Th2 replys.Specific tubercule bacillus protection effect test illustrates that more intuitively mycobacterium Smegmatis preparation can provide good protection effect to the body that infects tubercule bacillus, shows that mycobacterium Smegmatis preparation is a kind of tuberculosis prophylaxis safely and effectively and therapeutic vaccine.

Claims (31)

1. mycobacterium Smegmatis preparation, described preparation is prepared from through cracking by M. smegmatics, it is characterized in that: at first adopt the broken thalline of high pressure crush method or sonioation method in the cracking process.

2. preparation as claimed in claim 1, described preparation are freeze-dried preparation.

3. preparation as claimed in claim 1, described high pressure crush method are centrifugal collection M. smegmatics, with after the frozen-dried protective liquid dilution, through high-pressure homogeneous crusher machine thalline, then through 110 ℃-135 ℃ steam treatment 10-40 minute.

4. preparation as claimed in claim 3, described steam treatment condition are 121 ℃ of steam treatment 20 minutes.

5. preparation as claimed in claim 1, described sonioation method are centrifugal collection M. smegmatics, with after the frozen-dried protective liquid dilution, the ultrasonication thalline, then through 110 ℃-135 ℃ steam treatment 10-40 minute or ⁶⁰Co irradiation 15 minutes.

6. preparation as claimed in claim 5, described steam treatment condition be 121 ℃ of steam treatment 20 minutes or ⁶⁰Co irradiation 15 minutes.

7. as claim 3 or 5 described preparations, described frozen-dried protective liquid is a phosphate buffered saline buffer.

8. preparation as claimed in claim 7, described phosphate buffered saline buffer has following weight proportion: add 3.75 weight part monosodium glutamates in 1500 parts by weight of deionized water, 3.75 weight part sucrose, 10.8 weight part sodium-chlor, 6.525 the weight part anhydrous potassium dihydrogenphosphate is made into potassium liquid, in 1500 parts by weight of deionized water, add 3.75 weight part monosodium glutamates, 3.75 weight part sucrose, 10.8 weight part sodium-chlor, 27.45 the weight part disodium hydrogen phosphate,anhydrous is made into sodium liquid, getting 1500 weight part potassium liquid and 1100 weight part sodium liquid mixes, the pH that makes mixed solution is 6.0-8.0, after mixed solution filters by the G3 funnel, 110 ℃-135 ℃ steam treatment 10-40 minute.

9. preparation as claimed in claim 8, the pH of described phosphate buffered saline buffer are 7.0-7.4.

10. preparation as claimed in claim 9, the pH of described phosphate buffered saline buffer are 7.2.

11. preparation as claimed in claim 8, described steam treatment condition are 115 ℃ of steam sterilizings 30 minutes.

12. a pharmaceutical composition comprises each described mycobacterium Smegmatis preparation of claim 1-11.

13. pharmaceutical composition as claimed in claim 12, described pharmaceutical composition are used for prevention or treatment tuberculosis.

14. a vaccine comprises each described mycobacterium Smegmatis preparation of claim 1-11.

15. vaccine as claimed in claim 14, described vaccine are used for prevention or treatment tuberculosis.

16. the application that each described mycobacterium Smegmatis preparation of claim 1-11 is used for preparing immunoregulatory medicine.

17. application as claimed in claim 16, wherein said immunomodulatory is for improving the immunologic function of normal body.

18. application as claimed in claim 17, wherein said immunomodulatory kills and wounds and the removing ability entering intracellular microorganism antigenic engulfing with processing power or promotion body for promoting delayed type hypersensitivity, the lymphocytic propagation of promotion T, reinforcement antigen presenting cell.

19. the generation that application as claimed in claim 17, described immunomodulatory are used to promote scavenger cell secrete GM-CSF or IL-1 cytokine or promote NO.

20. application as claimed in claim 16, wherein said immunomodulatory are to promote the recovery of immunocompromised body's immunity or suppress the strong excessively immunne response of immune hyperfunction body.

21. application as claimed in claim 16, wherein said medicine are used to promote the Th1 para-immunity to reply or suppress the Th2 para-immunity and reply.

22. the expression amount that application as claimed in claim 21, described medicine are used to improve the expression amount of IL-12, IL-2, IFN-γ or reduce IL-4.

23. application as claimed in claim 16, the effective dose of described mycobacterium Smegmatis preparation are the 0.01-10mg/ agent.

24. application as claimed in claim 23, the effective dose of described mycobacterium Smegmatis preparation are the 0.05-5mg/ agent.

25. application as claimed in claim 24, the effective dose of described mycobacterium Smegmatis preparation are the 0.1-1mg/ agent.

26. the application that each described mycobacterium Smegmatis preparation of claim 1-11 is used for preparing prevention or treats the medicine of tuberculosis, hepatitis or asthma.

27. application as claimed in claim 26, wherein said medicine are used for prevention or treatment tuberculosis.

28. application as claimed in claim 26, wherein said medicine are used for prevention or treatment hepatitis B or asthma.

29. application as claimed in claim 26, the effective dose of described mycobacterium Smegmatis preparation are the 0.01-10mg/ agent.

30. application as claimed in claim 29, the effective dose of described mycobacterium Smegmatis preparation are the 0.05-5mg/ agent.

31. application as claimed in claim 30, the effective dose of described mycobacterium Smegmatis preparation are the 0.1-1mg/ agent.

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