A kind of preparation method and applications of novel tubercle bacillus fusant bacterial strainTechnical field
The invention belongs to recombinations, protoplast technical field of vaccines, are related to a kind of novel tubercle bacillus fusant bacterial strainPreparation method and applications.
Background technique
Tuberculosis is a kind of world caused by being infected by knot branch core bacillus (Mycobacterium Tuberculosis)Sexually transmitted disease and single pathogenic infection lead to the highest infectious diseases of the death rate.Tuberculosis is still global sense at presentThe number one killer of infectious diseases.The new data that " the 2017 global tuberculosis report " of World Health Organization's publication is announced is shown:10,400,000 new cases are estimated to be in world wides in 2017, wherein male 5,900,000 (56%), women 3,500,000 (34%), children1000000 (34%).This six countries of India, Indonesia, China, Nigeria, Pakistan and South Africa account for neopathyThe 60% of example sum.Tuberculosis epidemic situation fails to obtain control, and it is lungy that reason of searching to the bottom is a lack of effective vaccine preventionOccur.
Effective measures lungy are controlled still to be to effective prevention lungy, it is currently the only to be applied to clinical preventionVaccine lungy is still BCG vaccine (bacille calmette-guerin, BCG), it be by mycobacterium bovis notAttenuated live vaccine obtained from 230 generations is passed in stealpass, and since nineteen twenty-one comes out, the whole world, which has more than 4,000,000,000 person-times of inoculations, to be madeWith BCG vaccine, there are about 100,000,000 newborn's uses to inoculate against every year, becomes most widely used vaccine in the world.BCG vaccine cultureSimply, the shortcomings that cheap, safety is relatively preferable, but people gradually have found it in long-term application process and notFoot: 1.WHO thinks that the inoculation of BCG vaccine tuberculosis serious for children such as tubercular meningitis and Military tuberculosis have preferablyPreventive effect, but have no protective effect at human tuberculosis, but adult is main propagation source lungy, therefore block and be situated betweenSeedling inoculation can not reduce transmission of tuberculosis.2. Vaccine effectiveness is simultaneously unstable, and has opposite using contraindication and adverse reaction.It grindsStudying carefully confirms that BCG vaccine differs greatly to preventive effect lungy in different geographical different crowd, and protective rate differs for 0~80%,Therefore whether related BCG vaccine, which effectively disputes on, all exists always.3. the children low for congenital immunity and the day after tomorrow are acquiredImmunodeficiency patient is likely to result in serious complication or disseminated tuberculosis disease after bcg vaccination；4. in long-term biographySome important antigen genes are lost during generation, immune protection effectiveness reduces, in addition can also be to tuberculosis sufferer after bcg vaccinationThe diagnostic result of person generates certain influence.
Traditional BCG vaccine is no longer satisfied the mankind and prevents demand lungy, and various countries researcher is being continually striving toDevelop novel tuberculosis vaccine.Many emerging tuberculosis vaccines are in the different tested stages, up to the present still without one kindVaccine successfully can substitute BCG vaccine and be widely used in clinical prevention.
M. tuberculosis mycobacteria international standard avirulent strain H37Ra bacterial strain is by M. tuberculosis mycobacteria velogen strainIt obtains after more secondary culture attenuations of H37Rv, is had the advantage that in terms of becoming tuberculosis vaccine
First, antigen is complete compared with BCG.
The reproduction speed of second, H37Ra in human macrophages is much lower, but exempts from again with H37Rv bacterial strain simultaneouslyThe abundant activating immune cell of epidemic focus performance, and growth can be colonized in host for a long time, meet the basic demand of live bacterial vaccines.
Third, mankind's tuberculosis are mostly caused by M. tuberculosis mycobacteria, and minority is caused by Mycobacterium tuberculosis var.bovis, are hadResearch finds that the effect of the immune animal resistance mycobacterium bovis of BCG vaccine is better than and resists M. tuberculosis mycobacteria.H37Ra bacterial strain is as tuberculosis vaccine candidate strain also by extensive concern.Although H37Ra bacterial strain has in terms of becoming vaccinePreferable potential, but have many research shows that its virulence is strong compared with BCG vaccine, it is still owed separately as tuberculosis vaccine safety in utilizationLack textual criticism.
Protoplast fusion (Protoplast fusion) technology is that grow up the sixties in last century one is novelMicrobial Breeding technology.Using either physically or chemically induce two parents (only plasmalemma be coated with protoplast) intoRow fusion, recombinates through the exchange between genome, and screening obtains the stable fusant for integrating parents' merit to reachTo the hybridization purpose of gene level, is filtered out after recombinating its inhereditary material and obtain the stabilization for possessing parents' merit and meltZygote, to promote new species to generate and optimize its evolution.The large labor intensity of traditional strain improvement technology is overcome, more wheels are neededThe disadvantages of mutagenesis and screening, the breeding time is long, provides new method for improvement cell trait, especially has to directive breedingImportant meaning.
Summary of the invention
The preparation method and applications for being designed to provide a kind of novel tubercle bacillus fusant bacterial strain of invention, for currentPrevent the urgency that tuberculosis develops novel vaccine, considers that BCG and H37Ra bacterial strain is used separately as vaccine and all lacked in the presence of certainIt falls into but respectively has advantage, and two bacterial strains have certain complementarity in inhereditary feature and biological characteristic, so the present invention is with BCGThe novel tuberculosis vaccine fusant bacterial strain for utilizing Protoplast fusion technology successfully to construct by parent strain with two bacterial strain of H37Ra(B/R bacterial strain) is provided simultaneously with BCG vaccine and the excellent biological nature of H37Ra bacterial strain and inhereditary feature, and has further probed into thisThe immune protection effectiveness and safety of fusant bacterial strain become tuberculosis candidate vaccine for the further explore and study fusant bacterial strainA possibility that lay the foundation foundation be provided.
The purpose of the present invention one provides a kind of method for preparing BCG Strain Protoplast and H37Ra Strain Protoplast, andIts optimum preparating condition is explored, while the regeneration condition of BCG Strain Protoplast and H37Ra Strain Protoplast is carried out excellentChange, turns out good, the well-grown regeneration strain of activity.Prepare the optimal conditions of BCG protoplast: cell age is 18 days, digestsWhen concentration is 12mg/ml, enzymolysis time is 5h；Prepare the optimum condition of H37Ra Strain Protoplast: cell age is 21 days, enzymatic hydrolysisConcentration is 12mg/ml, enzymolysis time 6h；
The purpose of the present invention two is to provide a kind of fusion for preparing BCG Strain Protoplast and H37Ra Strain ProtoplastThe method of son, makes two kinds of protoplast fusions using electroporation technology, and explore its best fusion conditions, while to fusionSon carries out regeneration culture, obtains well-grown regeneration strain.The optimal fusion conditions of fusant bacterial strain (B/R bacterial strain): fusionVoltage E=2.2Kv/cm, shock by electricity time t=0.8ms.
The purpose of the present invention three is to provide the method for a kind of observation and identification fusant bacterial strain (B/R bacterial strain), utilizes fluorescenceLabelling method, and analytical calculation is carried out using laser confocal microscope image analysis software Zeiss LSM Image ExaminerFusion efficiencies.
Its technical solution is as follows:
A kind of preparation method of novel tubercle bacillus fusant bacterial strain, comprising the following steps:
Step 1, the culture of tubercle bacillus and the preparation of tubercle bacillus bacteria suspension
BCG bacterial strain and H37Ra plants are inoculated in respectively in Roche solid medium, and observation in every 7 days is primary, and bacterium colony is faint yellowBacterium colony is grown in particle or cauliflower-like.In Biohazard Safety Equipment, about 2~3 weeks Roche cultured on solid medium states of culture are takenGood BCG bacterium colony is placed in autoclaved bacteria grinder, and the physiological saline containing 0.05%Tween-80 is extremely respectively plus on a small quantityIn bacterial strain dismembyator, after grinding uniformly, carrys out detection bacterium concentration using bacterial turbidity instrument, pass through the physiology of 0.05%Tween-80Salt water come adjust bacterial concentration be 1 × 107/ml.It marks, is saved in -20 DEG C of refrigerators, is spare.
The preparation of step 2, BCG Strain Protoplast
Logarithmic growth phase BCG bacterium solution 10mL respectively, adjustment bacterial concentration are 3.5 × 107A/mL, 3500r/min centrifugation5min abandons supernatant and collects thallus, washed twice with PBS, and the pretreating agent of 1ml37 DEG C of preheating is added to final concentration of0.01mol/L, 37 DEG C of water-baths keep the temperature 20min, and 3500r/min is centrifuged 5min, then after being washed twice with PBS, are suspended in thallusIn protoplast stabilizing solution, the lysozyme of preheating, 37 DEG C of water enzyme digestions are added.In water-bath mechanism, take at regular intervalsActivity and shape that FDA fluorescent staining carries out observation protoplast under fluorescence microscope is added in the BCG bacterium solution of a small amount of enzymatic treatmentAt situation.When the formation rate of BCG Strain Protoplast reaches 90% or more, centrifugation is dezymotized, and adds protoplast stabilizing solutionSuspension protoplast.
The preparation of step 3, H37Ra Strain Protoplast
The bacterium solution 10mL of logarithmic growth phase H37Ra bacterial strain respectively, adjustment bacterial concentration are 3.5 × 107A/mL,3500r/min is centrifuged 5min, abandons supernatant and collects thallus, is washed twice with PBS, the pretreating agent that addition 1ml is preheated to final concentration0.01mol/L, 37 DEG C of water-baths keep the temperature 20min, and 3500r/min is centrifuged 5min, then after being washed twice with PBS, are suspended in thallusIn protoplast stabilizing solution, the lysozyme of preheating, 37 DEG C of water enzyme digestions are added.In water-bath mechanism, take at regular intervalsThe observation of rhodamine B fluorescent staining is added in the H37Ra bacterium solution of a small amount of enzymatic treatment.When the formation rate of H37Ra Strain Protoplast reachesWhen to 90% or more, centrifugation is dezymotized, and protoplast stabilizing solution suspension protoplast is added.
Further, in step 2, the cell age of thallus is 18 days, enzymatic hydrolysis concentration is 12mg/ml, enzymolysis time 5h.
Further, in step 3, the cell age of thallus is 21 days, and enzymatic hydrolysis concentration is 12mg/ml, enzymolysis time 6h.
A kind of identification method of novel tubercle bacillus fusant bacterial strain, comprising the following steps:
The preparation and identification of step 1, fusant bacterial strain
The BCG and the two isometric 1:1 of parent's Strain Protoplast of H37Ra that prepare active are uniformly mixed, are placed inIt shocks by electricity in cup, electric shock fusion is carried out in pulse voltage E=2.2KV/c, burst length t=8ms, it is micro- using laser co-focusingSem observation and identification fusant.Since the BCG protoplast for using FDA fluorescent staining to mark shows green fluorescence, rhodamine BThe H37Ra protoplast of fluorescent staining label is displayed in red fluorescence, and the fusant formed after electro' asion is simultaneous with two kindsFluorescence.Therefore, after electro' asion, the fusant of formation is micro- by laser co-focusing for BCG protoplast and H37Ra protoplastSem observation shows yellow fluorescence；
Step 2, BCG and H37Ra Strain Protoplast electro' asion efficiency test
To optimize electro' asion condition, respectively with 1.9,2.0,2.1,2.2,2.3,2.4V/cm totally six groups of fusion voltages progressTest.It can be obtained by laser confocal microscope image analysis software Zeiss LSM Image Examiner analysis, in voltageFusion efficiencies are higher compared with other each groups when V=2.2V/cm.When overtension or too strong pulse strength, original will causeRaw plastid is damaged, to cause the decline of fusion rate, is unfavorable for the regeneration of fusant.If but electric field strength is too small or pulse strengthNot enough, then the protoplast of parents' bacterial strain cannot come into full contact with, and be not easy to form fusant.
Further, in step 2, electro' asion voltage V=2.2V/cm.
Application of the novel tubercle bacillus fusant bacterial strain of the present invention during novel protoplast vaccine preliminary screening.
Beneficial effects of the present invention:
The present invention is successfully constructed using BCG and two bacterial strain of H37Ra by parent strain using Protoplast fusion technology novelTuberculosis vaccine fusant bacterial strain (B/R bacterial strain), is provided simultaneously with BCG vaccine and the excellent biological nature of H37Ra bacterial strain and inhereditary feature,And the immune protection effectiveness and safety of this fusant bacterial strain are further probed into, has been the further explore and study fusant bacterial strainIt lays the foundation as a possibility that tuberculosis candidate vaccine and foundation is provided.
Detailed description of the invention
The preparation condition of Fig. 1: BCG protoplast；
A: the forming amount of different cell age BCG Strain Protoplasts；B: the formation of different lysozyme concentration BCG protoplastsAmount；C: the forming amount of different enzymolysis time BCG protoplasts；
The preparation condition of Fig. 2: H37Ra protoplast；
A: the forming amount of different cell age H37Ra Strain Protoplasts；B: the shape of different lysozyme concentration H37Ra protoplastsCheng Liang；C: the forming amount of different enzymolysis time H37Ra protoplasts；
Fig. 3: BCG protoplast FDA dyes (200 ×)；
A:BCG protoplast dark field；B:BCG protoplast bright-field；The superposition of the protoplast light and shade visual field C:BCG；
Fig. 4: H37Ra Strain Protoplast rhodamine B dyes (200 ×)；
A:H37Ra Strain Protoplast dark field；B:H37Ra Strain Protoplast bright-field；C:H37Ra bacterial strain plasmThe body light and shade visual field is folded；
The fusant (200 ×) of Fig. 5: BCG protoplast and H37Ra Strain Protoplast；
A:BCG protoplast dark field；B:H37Ra protoplast dark field；C:BCG and H37Ra Strain Protoplast is brightThe visual field；The superposition of the D:BCG and H37Ra Strain Protoplast fusant bacterial strain light and shade visual field；
Fig. 6: influence of the fusion voltage to protoplast fusion efficiency.
Specific embodiment
Technical solution of the present invention is described in more detail with reference to the accompanying drawings and detailed description.
The preparation of 1 mycobacterium tuberculosis strain protoplast of embodiment
1. materials and methods:
1.1 main agents materials:
1.1.1 main material: H37Ra plants of mycobacterium tuberculosis international standard velogen strain (H37Ra plants of abbreviation) and BCG vaccineBacterial strain (abbreviation BCG bacterial strain) is provided by Chinese drug biological products assay institute, this laboratory saves；
Beta -mercaptoethanol is purchased from Shanghai Sheng Gong biotechnology Co., Ltd；Tris alkali (Tris, base) and lysozymePurchased from Amresco company；FDA is purchased from Sigma company；Rhodamine B is purchased from Solarbio company；Sodium glutamate is purchased from Shang HaiyuanLarge Bioisystech Co., Ltd；The cup that shocks by electricity is purchased from Eppendorf company, Germany.
1.1.2 key instrument equipment: by Shihezi of Xinjiang's University Medical College " the Xinjiang place and national high fall ill " Ministry of EducationKey lab provides.
Generic centrifuge is purchased from Changsha Xiang Yi centrifuge Instrument Ltd.；Centrifuge tube, culture dish, pipette are purchased from CangzhouFu Kang medical supplies company；10 μ l, 20 μ l, 100 μ l, 200 μ l, 1ml pipettor are purchased from Eppendof company, Germany；791 type magneticPower blender is purchased from Shanghai Nanhui telecommunication instrument factory；Inverted microscope XD-101-2B and laser confocal microscope are purchased from JapanOlympus company；Electroporation is purchased from Eppendorf company, Germany.
1.1.3 experimental animal: 6 weeks or so adults, health C57BL/6 mouse, half male and half female, weight is about 18 ± 2g,It is provided by Shihezi Univ's animal center.It raises in medical college, Shihezi Univ " the high-incidence Zoonosis infectiousness disease in west areaDisease prevention and treatment " collaborative innovation central laboratory (BSL-3).
1.2 experimental method
1.2.1 the culture of tubercle bacillus and the preparation of tubercle bacillus bacteria suspension
BCG bacterial strain and H37Ra plants are inoculated in respectively in Roche solid medium, and observation in every 7 days is primary, and bacterium colony is faint yellowBacterium colony is grown in particle or cauliflower-like.In Biohazard Safety Equipment, about 2~3 weeks Roche cultured on solid medium states of culture are takenGood BCG bacterium colony is placed in autoclaved bacteria grinder, and the physiological saline containing 0.05%Tween-80 is extremely respectively plus on a small quantityIn bacterial strain dismembyator, after grinding uniformly, carrys out detection bacterium concentration using bacterial turbidity instrument, pass through the physiology of 0.05%Tween-80Salt water come adjust bacterial concentration be 1 × 107/ml.It marks, is saved in -20 DEG C of refrigerators, is spare.
1.2.2 the preparation of BCG Strain Protoplast
(adjustment bacterial concentration is 3.5 × 10 to logarithmic growth phase BCG bacterium solution 10mL respectively7A/mL), 3500r/min fromHeart 5min abandons supernatant and collects thallus, washed twice with PBS, and the pretreating agent of 1ml37 DEG C of preheating is added to final concentration of0.01mol/L, 37 DEG C of water-baths keep the temperature 20min, and 3500r/min is centrifuged 5min, then after being washed twice with PBS, are suspended in thallusIn protoplast stabilizing solution, the lysozyme of preheating, 37 DEG C of water enzyme digestions are added.In water-bath mechanism, take at regular intervalsActivity and shape that FDA fluorescent staining carries out observation protoplast under fluorescence microscope is added in the BCG bacterium solution of a small amount of enzymatic treatmentAt situation.When the formation rate of BCG Strain Protoplast reaches 90% or more, centrifugation is dezymotized, and adds protoplast stabilizing solutionSuspension protoplast.
As a result: being prepared under the optimum condition when cell age is 18 days, enzymatic hydrolysis concentration is 12mg/ml, enzymolysis time is 5hBCG protoplast.As shown in Figure 1.
1.2.3 the preparation of H37Ra Strain Protoplast
(adjustment bacterial concentration is 3.5 × 10 to the bacterium solution 10mL of logarithmic growth phase H37Ra bacterial strain respectively7A/mL),3500r/min is centrifuged 5min, abandons supernatant and collects thallus, is washed twice with PBS, the pretreating agent that addition 1ml is preheated to final concentration0.01mol/L, 37 DEG C of water-baths keep the temperature 20min, and 3500r/min is centrifuged 5min, then after being washed twice with PBS, are suspended in thallusIn protoplast stabilizing solution, the lysozyme of preheating, 37 DEG C of water enzyme digestions are added.In water-bath mechanism, take at regular intervalsThe observation of rhodamine B fluorescent staining is added in the H37Ra bacterium solution of a small amount of enzymatic treatment.When the formation rate of H37Ra Strain Protoplast reachesWhen to 90% or more, centrifugation is dezymotized, and protoplast stabilizing solution suspension protoplast is added.
As a result: being 21 days in cell age, enzymatic hydrolysis concentration is 12mg/ml, is prepared under the optimum condition that enzymolysis time is 6hH37Ra Strain Protoplast.As shown in Figure 2.
1.2.4 BCG protoplast FDA fluorescent staining marks
The protoplast solution 1ml of the BCG bacterial strain prepared is taken, 25 μ LFDA liquid are added, after mixing gently, are protected from light, roomAbout 5~10min is dyed under the conditions of temperature, is observed under inverted fluorescence microscope using blue light exciting light.
As a result: BCG protoplast shows green fluorescence.As shown in Figure 3.
1.2.5 the fluorescent staining of H37Ra Strain Protoplast rhodamine B marks
The protoplast solution 0.5ml of the H37Ra bacterial strain prepared is taken, 100 μ L rhodamine B liquid, room temperature condition is addedUnder be protected from light dyeing about 5~10min, observed under inverted fluorescence microscope using green light exciting light.
As a result: H37Ra Strain Protoplast shows red fluorescence.As shown in Figure 4.
The identification of embodiment 2BCG and H37Ra Strain Protoplast electro' asion and fusant bacterial strain
1. experimental method:
The preparation and identification of 1.1 fusant bacterial strains
Active BCG and two parent's Strain Protoplast of H37Ra isometric (1:1) will be prepared to be uniformly mixed, setIn electric shock cup, electric shock fusion is carried out in pulse voltage E=2.2KV/c, burst length t=8ms, it is aobvious using laser co-focusingMicro mirror observation and identification fusant.Since the BCG protoplast for using FDA fluorescent staining to mark shows green fluorescence, rhodamine BThe H37Ra protoplast of fluorescent staining label is displayed in red fluorescence, and the fusant formed after electro' asion is simultaneous with two kindsFluorescence.Therefore, after electro' asion, the fusant of formation is micro- by laser co-focusing for BCG protoplast and H37Ra protoplastSem observation shows yellow fluorescence.As shown in Figure 5.
1.2 BCG and H37Ra Strain Protoplast electro' asion efficiency tests
To optimize electro' asion condition, respectively with 1.9,2.0,2.1,2.2,2.3,2.4V/cm totally six groups of fusion voltages progressTest.It can show that Fig. 6 can be seen by laser confocal microscope image analysis software Zeiss LSM Image Examiner analysisIn voltage V=2.2V/cm, fusion efficiencies are higher compared with other each groups out.When overtension or too strong pulse strength,It will cause protoplast breakage, to cause the decline of fusion rate, be unfavorable for the regeneration of fusant.If but electric field strength it is too small orPulse strength is inadequate, then the protoplast of parents' bacterial strain cannot come into full contact with, and is not easy to form fusant.It to sum up analyzes, electro' asionMost suitable voltage V=2.2V/cm.
The immune protection effectiveness of 3 fusant bacterial strain of embodiment (B/R bacterial strain) and the Primary Study of safety.
1. experimental method
The research of immune protection effectiveness and safety to B/R bacterial strain respectively from zoopery and cell experiment two parts intoRow.
1.1 zooperies: 204 C57BL/6 mouse are randomly divided into four groups: physiological saline group (36), BCG group (56Only), B/R group (56), H37Ra group (56), difference intradermal vaccination sterile saline 0.1ml, BCG bacteria suspension 0.1ml,B/R bacterial strain bacteria suspension 0.1ml, H37Ra bacterial strain bacteria suspension 0.1ml (is 1 × 10 containing viable count6It is a).A: each group after observation inoculationThe general survival state of mouse and survival rate, the 3rd, 6,9,12,15,18 week mouse weight numerical value after record inoculation.B: respectively at connecingAfter grabbing BCG group, B/R group, H37Ra group mouse each 4 de- necks execution when the 2nd, 3,6,9,12 week after kind at random, mouse is wonComplete lungs, spleen；Organs and tissues are made to be homogenized and be inoculated on modified Russell medium, in 37 DEG C of bacterium constant incubatorsClump count (CFU) is counted after culture 20 days.C: 3rd, 6,9,12,15,18 week after immune, at random crawl physiological saline group,BCG group, B/R group, H37Ra group mouse each 6 de- necks are weighed after putting to death；Wherein the 3rd, 6,9 week mouse plucks eyeball before putting to death and takes outsideAll blood separated plasmas survey the level of interleukin 2 (IL-2) and interferon (IFN-γ) in mice plasma with ELISA method；Taking lungs, liver and spleen to fix 48 hours in tissue fixative solution in mouse opening thoracic cavity, the abdominal cavity after execution, (spleen takes outAfter weigh and record) production pathological section row HE dyeing, observation each group mouse lung, liver, spleen each time point pathologySituation of change.D: the Spleen coefficient at each group mouse each time point is calculated according to each group mouse weight and spleen weight recorded.
1.2 cell experiments: a: recovering and cultivates macrophage strain RAW264.7, selects cell in good condition, uses respectivelyBCG, B/R, H37Ra bacterial strain bacteria suspension infection cell are detected using the bis- dye methods of Annexin V-FITC/PI respectively the 6th after infectionThe apoptosis rate of the group of cells of hour, 12 hours, 24 hours.B: it extracts and collects normal C57BL/6 mouse peritoneal primary macrophageIn vitro culture infects cultivated Turnover of Mouse Peritoneal Macrophages with the bacteria suspension of BCG, B/R, H37Ra bacterial strain respectively, and is infectingMacrophage is cracked with 1%TritonX-100 lysate within the 0th, 48,96 hour afterwards, 1000 times of liquid diluting that cracking is obtainedAfter take and be inoculated on Russell medium in right amount, culture counted clump count after 20 days in 37 DEG C of bacterium constant incubators.
1.3 results and conclusion:
1.3.1 the general growth and development of mouse and life span are not influenced after B/R strain inoculated C57BL/6 mouse.
1.3.2 B/R bacterial strain is at least colonized 3 months in C57BL/6 Mice Body, and field planting power enhances compared with BCG, compared with H37RaBacterial strain is weak.
1.3.3 B/R bacterial strain inducing cellular immune response level in C57BL/6 Mice Body is significantly increased compared with BCG, compared withH37Ra bacterial strain is weak.
1.3.4 cell experiment the result shows that the virulence and BCG of B/R bacterial strain without significant difference, it is obvious compared with H37Ra bacterial strainWeaken.
The foregoing is only a preferred embodiment of the present invention, the scope of protection of the present invention is not limited to this, it is any ripeKnow those skilled in the art within the technical scope of the present disclosure, the letter for the technical solution that can be become apparent toAltered or equivalence replacement are fallen within the protection scope of the present invention.