CN109897813A - The construction method of helicobacter pylori cagA gene Inactivating mutations strain - Google Patents
The construction method of helicobacter pylori cagA gene Inactivating mutations strain Download PDFInfo
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- CN109897813A CN109897813A CN201910218753.2A CN201910218753A CN109897813A CN 109897813 A CN109897813 A CN 109897813A CN 201910218753 A CN201910218753 A CN 201910218753A CN 109897813 A CN109897813 A CN 109897813A
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Abstract
This application involves gene engineering technology fields, specifically disclose the construction method of helicobacter pylori cagA gene Inactivating mutations strain, include the following steps: that (1) scraping is grown in 11639 thallus of the H.Pylori addition electrotransformation buffer of Columbia agar, the concentration for being diluted to 11639 thallus of H.Pylori is 1012A/L obtains solution A；(2) it takes 100 μ L solution As to be added in ice-cold 0.2cm BioRed electricity revolving cup, and 50 μ g of pHG1 targeting vector is added；(3) electric revolving cup is placed on the ice cube below liquid taking device；(4) enough SOC culture mediums are added into SOC culture medium storage tank, liquid flowing tube is protruded into electric revolving cup；(5) Colombia's blood plate is fixed with cylinder, and makes to brush and is contacted with the surface of Colombia blood plate, the input starting time is 10~15min on the controller；(6) after completing to Colombia's blood plate brushing, Colombia's blood plate is removed.By applying liquid taking device, it is possible to reduce manual operation bring error simultaneously improves efficiency.
Description
Technical field
The present invention relates to gene engineering technology fields, and in particular to the structure of helicobacter pylori cagA gene Inactivating mutations strain
Construction method.
Background technique
Helicobacter pylori (Helicobacter pylori, H.pylori) is the master of gastritis, gastric ulcer and incidence gastric cancer
Want one of inducement.By cag pathogenicity island in H.pylori genome (cagpathogenicity is-land, PAI) coding
CagA albumen (Cytotoxin-associatedgene A antigen, CagA) be H.pylori most important virulence because
Son.PAI long 40kb, the multiple gene cluster comprising about 31 genes, cagA gene are located at its C-terminal.IV-the type point of PAI coding
It secretes system and the CagA of coding is injected into gastric epithelial cell, can be I by H.pylori points according to the difference of the protein-coding region CagA
Type and II type.I island bacterium PAI type H.pylori encodes CagA albumen familial combined hyperlipidemia excretory system, II island type H.pylori bacterium PAI
Code area is imperfect, does not encode CagA albumen.Research shows that I type H.pylori is external and toxicity in vivo and pathogenic is much higher than II
Type H.py-lori, therefore generally believe that CagA albumen is the most important virulence factor of H.pylori.However, the function of CagA albumen
Can may be more complicated than the imagination, obtain cagA gene inactivation H.pylori bacterial strain be further investigation CagA albumen virulence,
The important premise of pathogenesis.At present by homologous recombination gene method for deactivating, helicobacter pylori is successfully constructed
(H.pylori) cagA gene Inactivating mutations strain comprising following technical step.
Step (1): the culture of 11639 bacterial strain of H.pylori containing 10g/L vancomycin, 5g/L amphotericin B and
It is inoculated with 11639 plants of bacterium of H.pylori in Colombia's blood agar culture-medium of 5g/L metopycide, is placed in micro- aerobic
(10% sheep blood, 5%O2,85%N2,10%CO2) is cultivated in environment.
Step (2): antibiotics sensitivity detection wild type H.pylori11639 bacterial strain is coated on contains 2 × 10 respectively? 2G/L chloramphenicol, erythromycin, streptomysin, kanamycins, on tetracycline taxi driver brother's rival subculture blood plate, is set ampicillin
In 37 DEG C of micro- aerobic cultures, every observation helicobacter pylori growing state for 24 hours.
Step (3): pHG1 cagA gene targeting plasmid construction and Select to use pMD19-T (Takara) plasmid conduct
11639 cagA gene of H.pylori inactivates plasmid backbone, the upstream cagA-up-F/R primer amplification cagA gene internal 500bp
Homology arm, the downstream cagA-down-F/R primer amplification cagA gene internal 500bp homology arm；Use overlap PC R method
Upstream and downstream homology arm is connected, and is transferred to acquisition pHG0 plasmid on pMD19-T carrier, cat-F/R primer is for cloning
Gro-EL-cat chloramphenicol acetyltransferase expression cassette on pHK plasmid, by the bis- enzymes of expression cassette BamHI-Eco RI
PHG0 carrier is connected to after cutting.
Step (4): the production of 11639 competent cell of H.pylori is grown on Colombia's fine jade with electrotransformation condition scraping
11639 thallus of H.Pylori of rouge is resuspended in electrotransformation buffer (10% glycerol, 9% sucrose, remaining is water), is diluted to
Concentration is 1012A/L.It takes 100 μ L to be added in ice-cold 0.2cm Bio Red electricity revolving cup, 50 μ g of pHG1 targeting vector, ice is added
Upper placement 10min.Electricity is adjusted using Bio Red Gene pluser and turns parameter to 2.5kV, 25 μ F, 200 Ω, and electric shock is primary, adds
Enter 100 μ L of SOC culture medium, be applied on Colombia's blood plate of not resistance immediately, is transferred to and contains after micro- aerobic culture 48h
2×10- 272h is cultivated on Colombia's blood plate of g/L chloramphenicol.
In above-mentioned steps (4), needs for electric revolving cup to be put on ice for 10 minutes, after SOC culture medium then is added, need
It is coated on Colombia's blood plate rapidly；Since the time placed on ice is longer, and 10 minutes are also not easy to
Handle other things, when waiting is easy situation that is absent-minded, therefore that standing time time-out on ice often occur.In addition, turning by electricity
Change buffer to be coated on Colombia's blood plate and go forward, need to be rapidly added SOC culture medium, and by electrotransformation buffer and SOC
Culture medium is uniformly mixed；Since duration is shorter, it is easy to appear maloperation phenomenon, to influence experimental result, therefore in this process
It is middle to reduce artificial operating procedure to the greatest extent.
Summary of the invention
The purpose of the present invention is to provide the construction methods of helicobacter pylori cagA gene Inactivating mutations strain, to complete
After the freezing of 11639 thallus of H.Pylori, it can complete to mechanization the electrotransformation to thallus, electricity is added in SOC culture medium
Conversion buffer is simultaneously squeegeed on Colombia's blood plate, to reduce operating error.
It is characterized by comprising following steps for the construction method of helicobacter pylori cagA gene Inactivating mutations strain:
Step (1): 11639 thallus of H.Pylori that scraping is grown on Columbia agar is added in electrotransformation buffer,
The concentration for being diluted to 11639 thallus of H.Pylori is 1012A/L obtains solution A；
Step (2): taking 100 μ L solution As to be added in ice-cold 0.2cm Bio Red electricity revolving cup, and pHG1 target practice is added and carries
50 μ g of body；
Step (3): electric revolving cup is placed on the ice cube below liquid taking device；
Liquid taking device includes driving motor, Qu Ye mechanism, SOC culture medium storage tank, brushing and control driving motor starting
And the controller that Bio Red Gene pluser powers on；Qu Ye mechanism includes axially along cylinder arranged in a vertical direction, cylinder
It is equipped with rotation axis in vivo, the upper end of rotation axis passes through the output axis connection of pneumatic clutch and driving motor；It is set in the middle part of cylinder
The first magnetic piston and the second magnetic piston being sequentially arranged from top to bottom are equipped between partition, the bottom and partition of cylinder,
The magnetic pole of the opposite end of first magnetic piston and the second magnetic piston is opposite；It is air cavity between partition and the first magnetic piston, the
It is imbibition chamber between two magnetic pistons and cylinder body bottom, rotation axis rotation will drive the first magnetic piston to move back and forth；First magnetic
Property piston constantly move back and forth can be formed compressed gas control pneumatic clutch separation, the second magnetic piston reciprocatingly slide can will
SOC culture medium discharge in SOC culture medium storage tank；It is living that the top of partition is equipped with the third for being only capable of sliding along cylinder axial reciprocating
Plug constitutes between third piston and partition and takes sap cavity, and rotation axis is connect with third piston thread, and rotation axis passes through clockwork spring and cylinder
Body connection；Cylinder be equipped with the liquid flowing tube that takes sap cavity be connected to, brush and be fixedly connected with third piston, third reciprocating motion of the pistons
Liquid can be sucked by liquid flowing tube and drains into liquid is brushed on；Second magnetic piston is equipped with the side that one is plane, side
Face is equipped with spherical groove and lead-over groove, and the depth of lead-over groove is greater than the depth of spherical groove, and the lower end of lead-over groove extends to the
The lower surface of two magnetic pistons；Spherical groove is connected to lead-over groove by guide groove；Starting block is slidably connected on cylinder, and is set
There is the reset spring connecting with starting block, starting block is equipped with telescopic gag lever post, and gag lever post can be with spherical groove, lead-over groove
It is formed and is cooperated with guide groove.
Step (4): enough SOC culture mediums being added into SOC culture medium storage tank, and liquid flowing tube is protruded into electric revolving cup；
Step (5): Colombia's blood plate is fixed with cylinder, and is made to brush and be connect with the surface of Colombia blood plate
Touching, the input starting time is 10~15min on the controller；
Step (6): after completing to Colombia's blood plate brushing, Colombia's blood plate is removed.
The beneficial effect of this programme is:
In the present solution, being equipped with timed ports on controller, the input starting time, goes forward side by side on the timed ports of controller
Row confirmation, then controller starts timing, used for conventional timer component；For example, being by monolithic and its programming
The achievable function.
After driving motor brings into operation, driving motor will drive rotation axis rotation, so that rotation axis drives the first magnetism
Reciprocating motion of the pistons.In first cycle of operation that the first magnetic piston moves back and forth, the first magnetic piston will drive the second magnetic
Property piston move back and forth together, at this time this it is achievable into electric revolving cup be added SOC culture medium；And when the first magnetic piston enters the
After two cycles of operation, since gag lever post and spherical groove cooperate, the second magnetic piston is out of service.Simultaneously because rotation axis with
The connection of third piston thread, rotation axis drive third piston upwards；When driving motor drives rotation axis rotation, clockwork spring will store
Can, and after pneumatic clutch is in disengaged position, clockwork spring will drive rotation axis reversion, so that rotation axis drives under third piston
It moves, this process can be by electrotransformation buffer and SOC media transfer to brushing, and is conducive to brush to move down to be applied to brother's human relations
Than sub- blood plate.
By the difference of the frequency run using the first magnetic piston and third piston reciprocating in this programme, to be formed
SOC culture medium is injected into electrotransformation cup, electrotransformation buffer is transferred to the sequencing of brushing, to be conducive to electrotransformation
Buffer and SOC culture medium be uniformly mixed, and mechanization completes experimental procedure.
Preferred embodiment one: advanced optimizing as to base case, and the electricity of the Bio Red Gene pluser turns ginseng
Number is adjusted to 2.5kV, 25 μ F, 200 Ω；Thallus can be made to reach preferably state, in subsequent incubation, embody compared with
Good growth conditions.
Preferred embodiment two: as advanced optimizing to preferred embodiment one, the side wall of the cylinder is equipped with control driving
The shutdown switch that motor is closed, reset spring be with the pressure spring that offsets of starting block, shutdown switch and reset spring respectively with starting
The both ends of block are opposite；In the step (6), before removing Colombia's blood plate, it should pull and open to reset spring side
Motion block makes to start block and shutdown switch separation.
In preferred embodiment two, by the way that shutdown switch is arranged, the stopping of driving motor can be realized automatically.In addition, taking
Before lower Colombia blood plate, starting block should be pulled to guarantee device so that device is in reset state to reset spring side
Next time operates normally.
Preferred embodiment three: as advanced optimizing to preferred embodiment two, the liquid flowing tube uses hose, consequently facilitating taking
Liquid pipe is bent to protrude into electric revolving cup.
Preferred embodiment four: first electric revolving cup is put into and is detested in step (3) as advanced optimizing to preferred embodiment three
In oxygen tank, then anaerobic jar is placed on ice.Since H.Pylori bacterium is anaerobic bacteria, and the time that electric revolving cup is placed on ice
It is longer, therefore electricity is turned to be placed in anaerobic jar, then be put on ice, it can avoid damage H.Pylori thallus.
Preferred embodiment five: as advanced optimizing to preferred embodiment four, the volume accounting of each gas in the anaerobic jar
For 5%O2, 85%N2, 10%CO2.5%O2, 85%N2, 10%CO2Gas content be the optimal growth ring of H.Pylori bacterium
Border.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of liquid taking device in the embodiment of the present invention；
Fig. 2 is the left view of part where starting block in Fig. 1；
Fig. 3 is the cross-sectional view of liquid taking device A-A in Fig. 1.
Specific embodiment
It is further described below by specific embodiment:
Appended drawing reference in Figure of description include: cylinder 10, the first magnetic piston 20, air cavity 21, third check valve 22,
Second tracheae 23, the 4th check valve 24, the second magnetic piston 30, imbibition chamber 31, first pipe 32, second one-way valve 33, spherical surface
Groove 34, guide groove 36, the first check valve 37, third piston 40, takes sap cavity 41, the 6th check valve 42, pipette at lead-over groove 35
43, the 5th check valve 44, catheter 45, clockwork spring 50, brush 60, Colombia's blood plate 70, starting block 80, shutdown switch 81,
First pressure spring 82, gag lever post 83, the second pressure spring 84, rotation axis 90.
Embodiment is basic as shown in attached drawing 1, Fig. 2 and Fig. 3:
In the detailed process that the construction method of helicobacter pylori cagA gene Inactivating mutations strain is implemented, one kind need to be applied to
Liquid taking device.Liquid taking device includes driving motor, Qu Ye mechanism, SOC culture medium storage tank, brushes 60 and control driving motor and open
Dynamic controller.Controller be equipped with connection Bio Red Gene pluser interface and input panel, display screen and really
Recognize button, Bio Red Gene pluser is connect with controller, and the starting time is inputted by input panel, and presses really
Recognize key, display screen will start timing；When the number zero on display screen, driving motor and Bio Red Gene pluser will
It is also turned on power supply and starts.
Qu Ye mechanism includes that the central axes of cylinder 10 are equipped with to be rotated with cylinder 10 axially along cylinder 10 arranged in a vertical direction
The rotation axis 90 of connection, the upper end of rotation axis 90 pass through the output axis connection of pneumatic clutch and driving motor, thus pneumatically from
When clutch combines, driving motor can drive rotation axis 90 to rotate together.The middle part of cylinder 10 is set to be integrally formed with cylinder 10
Partition, between the bottom and partition of cylinder 10 be equipped with magnetic piston, magnetic piston include the first magnetic piston 20 and the second magnetic
Property piston 30, the magnetic pole of the opposite end of the first magnetic piston 20 and the second magnetic piston 30 is on the contrary, so that first is magnetic living
Plug 20 and the second magnetic piston 30 attract each other.First magnetic piston 20 and the second magnetic piston 30 are set gradually from top to bottom,
It is air cavity 21 between partition and the first magnetic piston 20, is imbibition chamber 31 below the second magnetic piston 30.Rotation axis 90 is through the
One magnetic piston 20 and the second magnetic piston 30, and the first magnetic piston 20 and rotation axis 90 constitute cylindrical cam structure；And the
Two magnetic pistons 30 are rotatablely connected with rotation axis 90, and the second magnetic piston 30 can also be slided relative to rotation axis 90.Partition
Top is equipped with third piston 40, constitutes between third piston 40 and partition and takes sap cavity 41, and the upper end of rotation axis 90 is living through third
Plug 40, and rotation axis is threadedly coupled with third piston 40.In addition the cross section of the inner cavity of cylinder 10 is square, and first is magnetic living
It fills in the 20, second magnetic piston 30 and third piston 40 to cooperate with the inner cavity of cylinder 10, therefore the first magnetic piston 20, the second magnetic
Property piston 30 and third piston 40 can only sliding axially along cylinder 10, and cannot relative to cylinder 10 rotate；To rotate
When axis 90 rotates, rotation axis 90 will drive third piston 40 to slide upward or downward, and drive the first magnetic piston 20 back and forth sliding
It is dynamic.
SOC culture medium storage tank is connected to imbibition chamber 31 by first pipe 32, and it is unidirectional that first is equipped in first pipe 32
Valve 37, the first check valve 37 make fluid be only capable of flowing from SOC culture medium storage tank to imbibition chamber 31；10 bottom of cylinder is equipped with and suction
The outlet that sap cavity 31 is connected to, outlet is interior to be equipped with second one-way valve 33, and the fluid in imbibition chamber 31 is only capable of unidirectional by second
Valve 33 is discharged.Gas tank is bolted on 10 outer wall of cylinder, gas tank is connected to by the second tracheae 23 with air cavity 21, and second
Third check valve 22 is equipped in tracheae 23, third check valve 22 makes fluid be only capable of flowing from air cavity 21 to gas tank.In addition, air cavity
21 side wall is equipped with the air inlet being connected to air cavity 21, is equipped with the 4th check valve 24 in air inlet, the 4th check valve 24 makes
Air-flow is only capable of entering air cavity 21 by air inlet.Gas tank is connect by controlling pipeline with pneumatic clutch, is controlled and is installed on pipeline
There is 2/2-way Pneumatic reversal valve, gas tank and pneumatic clutch can be made to disconnect or connect by switching 2/2-way Pneumatic reversal valve
It is logical.Overflow valve is installed, and overflow valve is connected to 2/2-way Pneumatic reversal valve on gas tank, when the pressure in gas tank reaches certain
After value, overflow valve overflow simultaneously pushes the spool of 2/2-way Pneumatic reversal valve mobile, so that gas tank and pneumatic clutch connect
Logical, so that pneumatic clutch is in disengaged position, i.e. the output shaft of rotation axis 90 and driving motor is detached from.
The same side wall of second magnetic piston 30 is equipped with spherical groove 34 and lead-over groove 35, and the depth of lead-over groove 35 is big
In the depth of spherical groove 34, the lower end of lead-over groove 35 extends to the lower surface of the second magnetic piston 30.Spherical groove 34 and mistake
Aqueduct 35 is connected to by guide groove 36, and guide groove 36 is parallel to horizontal plane.And the slide over side walls of cylinder 10 is connected with starting block
80, starting block 80 is equipped with the first pressure spring 82 close to 35 one end of lead-over groove；And starting block 80 is equipped with telescopic gag lever post 83, limit
In 83 regracting of the bar starting block 80 of position, the second pressure spring 84 to offset with gag lever post 83 is equipped in starting block 80.83 direction of gag lever post
One end of spherical groove 34 is set as in spherical surface and embeddable spherical groove 34, guide groove 36 and lead-over groove 35.When gag lever post 83 is embedding
When entering spherical groove 34, starting block 80 is pulled to 35 one end of lead-over groove, then gag lever post 83 will slide into lead-over groove 35 and compress the
One pressure spring 82, and the depth of lead-over groove 35 is greater than the depth of spherical groove 34, therefore under the action of the first pressure spring 82, gag lever post
83 will not return in spherical groove 34；At this point, gag lever post 83 is to the second magnetic piston 30 if the second magnetic piston 30 moves upwards
Without position-limiting action；After gag lever post 83 and lead-over groove 35, which are detached from, to be cooperated, starting block 80 will under the action of the first pressure spring 82
It returns.
In addition, the shutdown switch 81 that control driving motor is closed is additionally provided on the side wall of cylinder 10,81 He of shutdown switch
First pressure spring 82 is opposite with the starting both ends of block 80 respectively, then starting when block 80 returns under the action of the first pressure spring 82 will squeeze
Shutdown switch 81 is pressed, so that driving motor is out of service.
Liquid flowing tube is installed, liquid flowing tube is connected to sap cavity 41 is taken on cylinder 10；It brushes 60 and is slidably connected at the outer of cylinder 10
On wall, and brushing 60 can only reciprocatingly slide along the vertical direction.Brush 60, catheters fixed by catheter 45 and third piston 40
The 5th check valve 44 is equipped in 45, the 5th check valve 44 to take the liquid in sap cavity 41 to be only capable of flowing to brushing by catheter 45
60；And liquid flowing tube is hose, and the 6th check valve 42 is equipped in liquid flowing tube, the 6th check valve 42 makes liquid be only capable of passing through liquid flowing tube
Flow direction takes in sap cavity 41.Catheter 45 extends through always to the bottom surface of third piston 40, and the bottom surface of third piston 40 is fixed
There is a rubber tube being connected to catheter 45, rubber tube is that hose is bent and extend to the bottom for taking sap cavity 41.
Rotation axis 90 is connect by clockwork spring 50 with cylinder 10, i.e., one end of clockwork spring 50 and rotation axis 90 are fixed, clockwork spring 50
The other end and cylinder 10 are fixed.When driving motor drives rotation axis 90 to rotate, 50 accumulation of energy of clockwork spring；And pneumatic clutch is in and divides
After state, clockwork spring 50 will drive rotation axis 90 to invert.With brush 60 opposite positions on also set up Colombia's blood plate
Fixed mechanism, 40 slide downward of third piston, which will drive, brushes 60 movements, while third piston 40 will take the liquid in sap cavity 41
Indentation brush 60 in, so as to by liquid applicator on Colombia's blood plate 70.
Specific implementation process includes the following steps:
Step (1): 11639 thallus of H.Pylori that scraping is grown on Columbia agar is added in electrotransformation buffer,
The concentration for being diluted to 11639 thallus of H.Pylori is 1012A/L obtains solution A；
Step (2): taking 100 μ L solution As to be added in ice-cold 0.2cm Bio Red electricity revolving cup, and pHG1 target practice is added and carries
50 μ g of body；
Step (3): it is 5%O that electric revolving cup, which is placed in environment,2, 85%N2, 10%CO2Anaerobism pipe in, and be placed on and take liquid
On ice cube below device, it will be connected to inside the outlet of imbibition chamber 31 and electric revolving cup；
Step (4): enough SOC culture mediums being added into SOC culture medium storage tank, and liquid flowing tube is protruded into electric revolving cup；
Step (5): Colombia's blood plate 70 is located on Colombia's blood plate fixed mechanism so that brush 60 with
The surface of Colombia's blood plate 70 contacts, and the input starting time is 10min on the controller, and presses acknowledgement key；
Step (6): completing after brushing 60 to Colombia's blood plate 70, pulls starting block 80 to reset spring side, makes
It obtains device and is in reset state, and remove Colombia's blood plate 70.
The detailed process of liquid taking device operation is, after driving motor brings into operation, driving motor will drive 90 turns of rotation axis
Dynamic, then rotation axis 90 drives the first magnetic piston 20 to move back and forth, the first magnetic piston 20 by 21 space of reciprocating compression air cavity, from
And the high pressure gas in gas tank.First cycle of operation that first magnetic piston 20 moves back and forth, the absorption of the first magnetic piston 20
Firmly the second magnetic piston 30 drives one with moving back and forth, and imbibition chamber 31 is cultivated the SOC in SOC culture medium storage tank is sucked at this time
Base is simultaneously injected into electric revolving cup.After first magnetic piston 20 enters second cycle of operation, gag lever post 83 and spherical groove 34
Cooperation limits the second magnetic piston 30, then the first magnetic piston 20 and the separation of the second magnetic piston 30, the second magnetic piston 30
It is out of service.
Rotation axis 90 is threadedly coupled with third piston 40, and during rotation axis 90 rotates, rotation axis 90 will drive the
Three pistons 40 move upwards, so that imbibition chamber 31 will suck the liquid in electric revolving cup by pipette 43；Since imbibition chamber 31 is inhaled
Portion gas can be carried while entering liquid, so that liquid will be in spurting, to facilitate electrotransformation when entering imbibition chamber 31
The uniform mixing of buffer and SOC culture medium.
After the pressure in gas tank reaches certain value, overflow valve overflow simultaneously opens 2/2-way Pneumatic reversal valve, then gas tank
Interior gas, which quickly enters, makes the separated state of pneumatic clutch in pneumatic clutch, while rotation axis 90 will be in clockwork spring 50
It is rotated backward under effect, the pressure in gas tank also reduces rapidly.Third piston 40 will be driven to move down when rotation axis 90 inverts, and the
The bottom surface of three pistons 40 is equipped with the rubber tube being connected to catheter 45, and rubber tube extends to the bottom of imbibition chamber 31, thus once
Third piston 40 moves down, and can be pressed into the mixed liquor of electrotransformation buffer and SOC culture medium in figure brush, thus brushing under 60
During shifting, Colombia's blood plate 70 can be coated on.
What has been described above is only an embodiment of the present invention, and the common sense such as well known specific structure and characteristic are not made herein in scheme
Excessive description.It, without departing from the structure of the invention, can be with it should be pointed out that for those skilled in the art
Several modifications and improvements are made, these also should be considered as protection scope of the present invention, these all will not influence what the present invention was implemented
Effect and patent practicability.The scope of protection required by this application should be based on the content of the claims, in specification
The records such as specific embodiment can be used for explaining the content of claim.
Claims (6)
1. the construction method of helicobacter pylori cagA gene Inactivating mutations strain, it is characterised in that include the following steps:
Step (1): the H.Pylori11639 thallus that scraping is grown on Columbia agar is added in electrotransformation buffer, dilution
Concentration to H.Pylori11639 thallus is 1012A/L obtains solution A；
Step (2): it takes 100 μ L solution As to be added in ice-cold 0.2cm BioRed electricity revolving cup, and 50 μ of pHG1 targeting vector is added
g；
Step (3): electric revolving cup is placed on the ice cube below liquid taking device；
Liquid taking device include driving motor, Qu Ye mechanism, SOC culture medium storage tank, brushing and control driving motor starting and
The controller that BioRed Gene pluser powers on；Qu Ye mechanism includes axially along cylinder arranged in a vertical direction, cylinder
Interior to be equipped with rotation axis, the upper end of rotation axis passes through the output axis connection of pneumatic clutch and driving motor；The middle part of cylinder is set to
Partition is equipped with the first magnetic piston and the second magnetic piston being sequentially arranged from top to bottom between the bottom and partition of cylinder, the
The magnetic pole of the opposite end of one magnetic piston and the second magnetic piston is opposite；It is air cavity between partition and the first magnetic piston, second
It is imbibition chamber between magnetic piston and cylinder body bottom, rotation axis rotation will drive the first magnetic piston to move back and forth；First is magnetic
Piston, which constantly moves back and forth, can form compressed gas control pneumatic clutch separation, and the second magnetic piston reciprocatingly slides can be by SOC
SOC culture medium discharge in culture medium storage tank；The top of partition is equipped with the third piston for being only capable of sliding along cylinder axial reciprocating, the
It is constituted between three pistons and partition and takes sap cavity, rotation axis is connect with third piston thread, and rotation axis is connected by clockwork spring and cylinder
It connects；Cylinder be equipped with the liquid flowing tube that takes sap cavity be connected to, brush and be fixedly connected with third piston, third reciprocating motion of the pistons can lead to
It crosses liquid flowing tube sucking liquid and drains into liquid and be brushed on；Second magnetic piston is equipped with the side that one is plane, on side
Equipped with spherical groove and lead-over groove, the depth of lead-over groove is greater than the depth of spherical groove, and the lower end of lead-over groove extends to the second magnetic
The lower surface of property piston；Spherical groove is connected to lead-over groove by guide groove；Slidably connect starting block on cylinder, and be equipped with
Start the reset spring of block connection, starting block is equipped with telescopic gag lever post, gag lever post can with spherical groove, lead-over groove and lead
It is formed and is cooperated to slot；
Step (4): enough SOC culture mediums being added into SOC culture medium storage tank, and liquid flowing tube is protruded into electric revolving cup；
Step (5): Colombia's blood plate is fixed with cylinder, and is made to brush and be contacted with the surface of Colombia blood plate,
The input starting time is 10~15min on controller；
Step (6): after completing to Colombia's blood plate brushing, Colombia's blood plate is removed.
2. the construction method of helicobacter pylori cagA gene Inactivating mutations strain according to claim 1, it is characterised in that:
It is to 2.5kV, 25 μ F, 200 Ω that the electricity of the BioRed Gene pluser, which turns parameter regulation,.
3. the construction method of helicobacter pylori cagA gene Inactivating mutations strain according to claim 2, it is characterised in that:
The side wall of the cylinder is equipped with the shutdown switch that control driving motor is closed, and reset spring is the pressure spring to offset with starting block,
Shutdown switch and reset spring are opposite with the starting both ends of block respectively；In the step (6), Colombia's blood plate is being removed
Before, starting block should be pulled to reset spring side, make to start block and shutdown switch separation.
4. the construction method of helicobacter pylori cagA gene Inactivating mutations strain according to claim 3, it is characterised in that:
The liquid flowing tube uses hose.
5. the construction method of helicobacter pylori cagA gene Inactivating mutations strain according to claim 4, it is characterised in that:
In step (3), first electric revolving cup is put into anaerobic jar, then anaerobic jar is placed on ice.
6. the construction method of helicobacter pylori cagA gene Inactivating mutations strain according to claim 5, it is characterised in that:
The volume accounting of each gas is 5%O in the anaerobic jar2, 85%N2, 10%CO2。
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP1194576A1 (en) * | 1999-06-25 | 2002-04-10 | CHIRON S.p.A. | Delivery system |
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