CN109825497A - A kind of preparation method and applications of novel tubercle bacillus fusant bacterial strain - Google Patents
A kind of preparation method and applications of novel tubercle bacillus fusant bacterial strain Download PDFInfo
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Abstract
The invention discloses a kind of preparation method and applications of novel tubercle bacillus fusant bacterial strain, optimize to the regeneration condition of BCG Strain Protoplast and H37Ra Strain Protoplast, turn out good, the well-grown regeneration strain of activity.A kind of method of fusant preparing BCG Strain Protoplast and H37Ra Strain Protoplast is provided, it makes two kinds of protoplast fusions using electroporation technology, and its best fusion conditions is explored, while regeneration culture is carried out to fusant, obtain well-grown regeneration strain.The novel tuberculosis vaccine fusant bacterial strain that the present invention utilizes Protoplast fusion technology successfully to construct using BCG and two bacterial strain of H37Ra by parent strain; it is provided simultaneously with BCG vaccine and the excellent biological nature of H37Ra bacterial strain and inhereditary feature; a possibility that having probed into the immune protection effectiveness and safety of this fusant bacterial strain, having become tuberculosis candidate vaccine for the further explore and study fusant bacterial strain, which lays the foundation, provides foundation.
Description
Technical field
The invention belongs to recombinations, protoplast technical field of vaccines, are related to a kind of novel tubercle bacillus fusant bacterial strain
Preparation 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 present
The 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%), children
1000000 (34%).This six countries of India, Indonesia, China, Nigeria, Pakistan and South Africa account for neopathy
The 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 prevention
Occur.
Effective measures lungy are controlled still to be to effective prevention lungy, it is currently the only to be applied to clinical prevention
Vaccine lungy is still BCG vaccine (bacille calmette-guerin, BCG), it be by mycobacterium bovis not
Attenuated 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 made
With 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 culture
Simply, the shortcomings that cheap, safety is relatively preferable, but people gradually have found it in long-term application process and not
Foot: 1.WHO thinks that the inoculation of BCG vaccine tuberculosis serious for children such as tubercular meningitis and Military tuberculosis have preferably
Preventive effect, but have no protective effect at human tuberculosis, but adult is main propagation source lungy, therefore block and be situated between
Seedling inoculation can not reduce transmission of tuberculosis.2. Vaccine effectiveness is simultaneously unstable, and has opposite using contraindication and adverse reaction.It grinds
Studying 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 acquired
Immunodeficiency patient is likely to result in serious complication or disseminated tuberculosis disease after bcg vaccination；4. in long-term biography
Some important antigen genes are lost during generation, immune protection effectiveness reduces, in addition can also be to tuberculosis sufferer after bcg vaccination
The 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 to
Develop novel tuberculosis vaccine.Many emerging tuberculosis vaccines are in the different tested stages, up to the present still without one kind
Vaccine 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 strain
It 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 simultaneously
The 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 had
Research 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 vaccine
Preferable 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 utilization
Lack textual criticism.
Protoplast fusion (Protoplast fusion) technology is that grow up the sixties in last century one is novel
Microbial Breeding technology.Using either physically or chemically induce two parents (only plasmalemma be coated with protoplast) into
Row fusion, recombinates through the exchange between genome, and screening obtains the stable fusant for integrating parents' merit to reach
To the hybridization purpose of gene level, is filtered out after recombinating its inhereditary material and obtain the stabilization for possessing parents' merit and melt
Zygote, to promote new species to generate and optimize its evolution.The large labor intensity of traditional strain improvement technology is overcome, more wheels are needed
The disadvantages of mutagenesis and screening, the breeding time is long, provides new method for improvement cell trait, especially has to directive breeding
Important 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 current
Prevent 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 certain
It 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 BCG
The 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 this
The immune protection effectiveness and safety of fusant bacterial strain become tuberculosis candidate vaccine for the further explore and study fusant bacterial strain
A 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, and
Its optimum preparating condition is explored, while the regeneration condition of BCG Strain Protoplast and H37Ra Strain Protoplast is carried out excellent
Change, turns out good, the well-grown regeneration strain of activity.Prepare the optimal conditions of BCG protoplast: cell age is 18 days, digests
When concentration is 12mg/ml, enzymolysis time is 5h；Prepare the optimum condition of H37Ra Strain Protoplast: cell age is 21 days, enzymatic hydrolysis
Concentration 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 Protoplast
The method of son, makes two kinds of protoplast fusions using electroporation technology, and explore its best fusion conditions, while to fusion
Son carries out regeneration culture, obtains well-grown regeneration strain.The optimal fusion conditions of fusant bacterial strain (B/R bacterial strain): fusion
Voltage 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 fluorescence
Labelling method, and analytical calculation is carried out using laser confocal microscope image analysis software Zeiss LSM Image Examiner
Fusion 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 yellow
Bacterium 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 taken
Good 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 quantity
In bacterial strain dismembyator, after grinding uniformly, carrys out detection bacterium concentration using bacterial turbidity instrument, pass through the physiology of 0.05%Tween-80
Salt 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 centrifugation
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 of
0.01mol/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 thallus
In protoplast stabilizing solution, the lysozyme of preheating, 37 DEG C of water enzyme digestions are added.In water-bath mechanism, take at regular intervals
Activity 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 treatment
At situation.When the formation rate of BCG Strain Protoplast reaches 90% or more, centrifugation is dezymotized, and adds protoplast stabilizing solution
Suspension 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 concentration
0.01mol/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 thallus
In protoplast stabilizing solution, the lysozyme of preheating, 37 DEG C of water enzyme digestions are added.In water-bath mechanism, take at regular intervals
The 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 reaches
When 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 in
It 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-focusing
Sem observation and identification fusant.Since the BCG protoplast for using FDA fluorescent staining to mark shows green fluorescence, rhodamine B
The H37Ra protoplast of fluorescent staining label is displayed in red fluorescence, and the fusant formed after electro' asion is simultaneous with two kinds
Fluorescence.Therefore, after electro' asion, the fusant of formation is micro- by laser co-focusing for BCG protoplast and H37Ra protoplast
Sem 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 progress
Test.It can be obtained by laser confocal microscope image analysis software Zeiss LSM Image Examiner analysis, in voltage
Fusion efficiencies are higher compared with other each groups when V=2.2V/cm.When overtension or too strong pulse strength, original will cause
Raw 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 strength
Not 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 novel
Tuberculosis 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 strain
It 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 protoplasts
Amount；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 protoplasts
Cheng 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 plasm
The 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 bright
The 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 vaccine
Bacterial 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 lysozyme
Purchased from Amresco company；FDA is purchased from Sigma company；Rhodamine B is purchased from Solarbio company；Sodium glutamate is purchased from Shang Haiyuan
Large 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 Education
Key lab provides.
Generic centrifuge is purchased from Changsha Xiang Yi centrifuge Instrument Ltd.；Centrifuge tube, culture dish, pipette are purchased from Cangzhou
Fu 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 magnetic
Power blender is purchased from Shanghai Nanhui telecommunication instrument factory；Inverted microscope XD-101-2B and laser confocal microscope are purchased from Japan
Olympus 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 area
Disease 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 yellow
Bacterium 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 taken
Good 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 quantity
In bacterial strain dismembyator, after grinding uniformly, carrys out detection bacterium concentration using bacterial turbidity instrument, pass through the physiology of 0.05%Tween-80
Salt 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 from
Heart 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 of
0.01mol/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 thallus
In protoplast stabilizing solution, the lysozyme of preheating, 37 DEG C of water enzyme digestions are added.In water-bath mechanism, take at regular intervals
Activity 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 treatment
At situation.When the formation rate of BCG Strain Protoplast reaches 90% or more, centrifugation is dezymotized, and adds protoplast stabilizing solution
Suspension 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 5h
BCG 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 concentration
0.01mol/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 thallus
In protoplast stabilizing solution, the lysozyme of preheating, 37 DEG C of water enzyme digestions are added.In water-bath mechanism, take at regular intervals
The 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 reaches
When 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 6h
H37Ra 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, room
About 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 added
Under 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, set
In 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-focusing
Micro mirror observation and identification fusant.Since the BCG protoplast for using FDA fluorescent staining to mark shows green fluorescence, rhodamine B
The H37Ra protoplast of fluorescent staining label is displayed in red fluorescence, and the fusant formed after electro' asion is simultaneous with two kinds
Fluorescence.Therefore, after electro' asion, the fusant of formation is micro- by laser co-focusing for BCG protoplast and H37Ra protoplast
Sem 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 progress
Test.It can show that Fig. 6 can be seen by laser confocal microscope image analysis software Zeiss LSM Image Examiner analysis
In 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 or
Pulse 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' asion
Most 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 into
Row.
1.1 zooperies: 204 C57BL/6 mouse are randomly divided into four groups: physiological saline group (36), BCG group (56
Only), 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 inoculation
The 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 connecing
After 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 won
Complete lungs, spleen；Organs and tissues are made to be homogenized and be inoculated on modified Russell medium, in 37 DEG C of bacterium constant incubators
Clump 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 outside
All 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 out
After weigh and record) production pathological section row HE dyeing, observation each group mouse lung, liver, spleen each time point pathology
Situation 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 respectively
BCG, 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 infection
The apoptosis rate of the group of cells of hour, 12 hours, 24 hours.B: it extracts and collects normal C57BL/6 mouse peritoneal primary macrophage
In vitro culture infects cultivated Turnover of Mouse Peritoneal Macrophages with the bacteria suspension of BCG, B/R, H37Ra bacterial strain respectively, and is infecting
Macrophage is cracked with 1%TritonX-100 lysate within the 0th, 48,96 hour afterwards, 1000 times of liquid diluting that cracking is obtained
After 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 H37Ra
Bacterial 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 with
H37Ra 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 strain
Weaken.
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 ripe
Know those skilled in the art within the technical scope of the present disclosure, the letter for the technical solution that can be become apparent to
Altered or equivalence replacement are fallen within the protection scope of the present invention.
Claims (6)
1. a kind of preparation method of novel tubercle bacillus fusant bacterial strain, which comprises 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 yellow bacterium colony
It is grown in particle or cauliflower-like；In Biohazard Safety Equipment, take about 2~3 weeks Roche cultured on solid medium of culture in good condition
BCG bacterium colony, be placed in autoclaved bacteria grinder, respectively plus on a small quantity the physiological saline containing 0.05%Tween-80 to bacterial strain
In dismembyator, after grinding uniformly, carrys out detection bacterium concentration using bacterial turbidity instrument, pass through the physiological saline of 0.05%Tween-80
It is 1 × 10 to adjust bacterial concentration7/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 × 107/mL, 3500r/min centrifugation
5min abandons supernatant and collects thallus, washed twice with PBS, 1ml37 DEG C of pretreating agent preheated of addition to final concentration of 0.01mol/
L, 37 DEG C of water-baths keep the temperature 20min, and 3500r/min is centrifuged 5min, then after being washed twice with PBS, thallus are made to be suspended in protoplast
In stabilizing solution, the lysozyme of preheating, 37 DEG C of water enzyme digestions are added；In water-bath mechanism, taken at a small amount of enzyme at regular intervals
The activity and formational situation that FDA fluorescent staining carries out observation protoplast under fluorescence microscope is added in the BCG bacterium solution of reason；When
When the formation rate of BCG Strain Protoplast reaches 90% or more, centrifugation is dezymotized, and adds protoplast stabilizing solution suspension plasm
Body；
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 × 107/mL, 3500r/min
It is centrifuged 5min, supernatant is abandoned and collects thallus, washed twice with PBS, the pretreating agent that addition 1ml is preheated to final concentration 0.01mol/L,
37 DEG C of water-baths keep the temperature 20min, and 3500r/min is centrifuged 5min, then after being washed twice with PBS, so that thallus is suspended in protoplast steady
Determine in liquid, the lysozyme of preheating, 37 DEG C of water enzyme digestions are added；In water-bath mechanism, a small amount of enzymatic treatment is taken at regular intervals
H37Ra bacterium solution be added rhodamine B fluorescent staining observation；When the formation rate of H37Ra Strain Protoplast reaches 90% or more
When, centrifugation is dezymotized, and protoplast stabilizing solution suspension protoplast is added.
2. the preparation method of novel tubercle bacillus fusant bacterial strain according to claim 1, which is characterized in that in step 2, bacterium
The cell age of body is 18 days, enzymatic hydrolysis concentration is 12mg/ml, enzymolysis time 5h.
3. the preparation method of novel tubercle bacillus fusant bacterial strain according to claim 1, which is characterized in that in step 3, bacterium
The cell age of body be 21 days, enzymatic hydrolysis concentration be 12mg/ml, enzymolysis time 6h.
4. the identification method of novel tubercle bacillus fusant bacterial strain described in a kind of claim 1, which is characterized in that including following step
It is rapid:
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, electric shock is placed in
In cup, electric shock fusion is carried out in pulse voltage E=2.2KV/c, burst length t=8ms, is seen using laser confocal microscope
Examine and identify fusant；Since the BCG protoplast for using FDA fluorescent staining to mark shows green fluorescence, rhodamine B fluorescence dye
The H37Ra protoplast of color marker is displayed in red fluorescence, and the fusant formed after electro' asion is simultaneous with two kinds of fluorescence；Cause
This, after electro' asion, the fusant of formation passes through confocal laser scanning microscope for BCG protoplast and H37Ra protoplast
Show 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, tested by totally six groups of fusion voltages by 2.4V/cm；
It can be obtained by laser confocal microscope image analysis software Zeiss LSM Image Examiner analysis, in voltage V=
Fusion efficiencies are higher compared with other each groups when 2.2V/cm；When overtension or too strong pulse strength, plasm will cause
Body is damaged, to cause the decline of fusion rate, is unfavorable for the regeneration of fusant；But if electric field strength is too small or pulse strength not
Enough, then the protoplast of parents' bacterial strain cannot come into full contact with, and be not easy to form fusant.
5. the identification method of novel tubercle bacillus fusant bacterial strain according to claim 4, which is characterized in that in step 2, electricity melts
Close voltage V=2.2V/cm.
6. novel tubercle bacillus fusant bacterial strain answering during novel protoplast vaccine preliminary screening described in claim 1
With.
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5504005A (en) * | 1987-03-02 | 1996-04-02 | Albert Einstein College Of Medicine Of Yeshiva University | Recombinant mycobacterial vaccine |
| CN1304451A (en) * | 1998-04-07 | 2001-07-18 | 科里克萨公司 | Fusion proteins of i(mycobacterium tuberuculosis) antigens and their uses |
| CN1688335A (en) * | 2002-02-06 | 2005-10-26 | 洛曼动物健康两合公司 | Continuous cell line for the production of vaccines |
| CN101495509A (en) * | 2005-07-08 | 2009-07-29 | 比奥根艾迪克Ma公司 | Sp35 antibodies and uses thereof |
| CN102776275A (en) * | 2012-05-03 | 2012-11-14 | 石河子大学 | Visual detection primer set for tubercle bacillus and kit of primer set |
| CN103045583A (en) * | 2012-12-18 | 2013-04-17 | 广东大华农动物保健品股份有限公司 | Method for constructing swine escherichia coli and haemophilus parasuis attenuated fusion strain |
| US20150073026A1 (en) * | 2010-03-12 | 2015-03-12 | Council Of Scientific & Industrial Research | 1, 2, 4-triazole derivatives and their anti-microbial activity |
| CN105087421A (en) * | 2015-04-23 | 2015-11-25 | 甘肃长业生态生物科技集团有限公司 | Fusant mixture, preparation method of fusant mixture, and method for producing organic fertilizer |
| CN106591167A (en) * | 2015-10-20 | 2017-04-26 | 天津农学院 | Method for preparing and regenerating sphingomonas sp protoplast |
| CN110079490A (en) * | 2019-03-29 | 2019-08-02 | 石河子大学 | A kind of building and application thereof of BCG vaccine PhoPR gene overexpression bacterial strain |
| CN110305889A (en) * | 2019-07-02 | 2019-10-08 | 石河子大学 | A kind of building and its application of tubercle bacillus Pup deletion mutant bacterial strain |
-
2019
- 2019-01-25 CN CN201910071380.0A patent/CN109825497A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5504005A (en) * | 1987-03-02 | 1996-04-02 | Albert Einstein College Of Medicine Of Yeshiva University | Recombinant mycobacterial vaccine |
| CN1304451A (en) * | 1998-04-07 | 2001-07-18 | 科里克萨公司 | Fusion proteins of i(mycobacterium tuberuculosis) antigens and their uses |
| CN1688335A (en) * | 2002-02-06 | 2005-10-26 | 洛曼动物健康两合公司 | Continuous cell line for the production of vaccines |
| CN101495509A (en) * | 2005-07-08 | 2009-07-29 | 比奥根艾迪克Ma公司 | Sp35 antibodies and uses thereof |
| US20150073026A1 (en) * | 2010-03-12 | 2015-03-12 | Council Of Scientific & Industrial Research | 1, 2, 4-triazole derivatives and their anti-microbial activity |
| CN102776275A (en) * | 2012-05-03 | 2012-11-14 | 石河子大学 | Visual detection primer set for tubercle bacillus and kit of primer set |
| CN103045583A (en) * | 2012-12-18 | 2013-04-17 | 广东大华农动物保健品股份有限公司 | Method for constructing swine escherichia coli and haemophilus parasuis attenuated fusion strain |
| CN105087421A (en) * | 2015-04-23 | 2015-11-25 | 甘肃长业生态生物科技集团有限公司 | Fusant mixture, preparation method of fusant mixture, and method for producing organic fertilizer |
| CN106591167A (en) * | 2015-10-20 | 2017-04-26 | 天津农学院 | Method for preparing and regenerating sphingomonas sp protoplast |
| CN110079490A (en) * | 2019-03-29 | 2019-08-02 | 石河子大学 | A kind of building and application thereof of BCG vaccine PhoPR gene overexpression bacterial strain |
| CN110305889A (en) * | 2019-07-02 | 2019-10-08 | 石河子大学 | A kind of building and its application of tubercle bacillus Pup deletion mutant bacterial strain |
Non-Patent Citations (9)
| Title |
|---|
| HARSHAD THACORE等: "THE FORMATION OF SPHEROPLASTS OF MYCOBACTERIUM TUBERCULOSIS IN TISSUE CULTURE CELLS", 《AM REV RESPIR DIS》 * |
| LI WEI等: "The Roles of Host Noncoding RNAs in Mycobacterium tuberculosis Infection", 《FRONT IMMUNOL》 * |
| 姚文兵主编: "《生物技术制药概论》", 31 August 2015, 中国医药科技出版社 * |
| 柳小玲等: "以卡介苗和MTB H37Ra的原生质体为亲本制备融合菌株的实验研究", 《中国病理生理杂志》 * |
| 柳小玲等: "新型结核病疫苗融合菌株制备及其免疫学特性的研究", 《中国病原生物学杂志》 * |
| 梁粟等: "新型结核病疫苗菌株在小鼠体内的毒性及安全性研究", 《中国病原生物学杂志》 * |
| 梁粟等: "新型结核病疫苗菌株的免疫保护效力及安全性研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑（电子期刊》 * |
| 樊超: "构建及选育新型结核病疫苗菌株的实验研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑（电子期刊）》 * |
| 樊超等: "卡介苗菌株和结核分枝杆菌H37Ra菌株原生质体的制备研究", 《石河子大学学报(自然科学版)》 * |
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