CN106591365A - Attenuated salmonella typhimurium mediated eukaryocyte plasmid transfection method - Google Patents
Attenuated salmonella typhimurium mediated eukaryocyte plasmid transfection method Download PDFInfo
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- CN106591365A CN106591365A CN201611112192.0A CN201611112192A CN106591365A CN 106591365 A CN106591365 A CN 106591365A CN 201611112192 A CN201611112192 A CN 201611112192A CN 106591365 A CN106591365 A CN 106591365A
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- salmonella typhimurium
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- C—CHEMISTRY; METALLURGY
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Abstract
The invention provides an attenuated salmonella typhimurium mediated eukaryocyte plasmid transfection method. According to the transfection method, based on an intracellular invasion characteristic of the attenuated salmonella typhimurium, recombinant plasmids are carried to enter eukaryocytes to realize specific protein expression. Specifically, recombinant eukaryotic expression plasmids are introduced into an attenuated salmonella typhimurium VNP20009 strain in an electric shock transformation way; then recombinant bacteria and the eukaryocytes are cultured in a co-incubation manner; and finally, the attenuated salmonella typhimurium inside and outside cells is killed by a serum culture medium containing penicillin and streptomycin, and the recombinant plasmids are released to the eukaryocytes to realize the specific protein expression. Compared with conventional calcium sulfate transfection, liposome transfection and electro-transformation, the transfection method disclosed by the invention takes viable bacteria as a bearing host of specific plasmids, so that a larger number of target vectors can be obtained by only conventional culture; and in addition, the plasmid transfection operating steps are simplified, the cost is lowered, and the transfection efficiency and the cytotoxicity are comprehensively better than those in the conventional transfection method.
Description
Technical field
The present invention relates to gene recombination technology field, and in particular to a kind of eucaryon of attenuated salmonella typhimurium mediation is thin
Cytoplasmic granules transfection method.
Background technology
Salmonella typhimurium is a kind of aggressive intracellular bacterium, and Salmonella typhimurium is entered behind Host Digestion road, first
Stick and invade the lymph follicle that there is one group of set in intestinal epithelial cell, small intestinal mucosa, be referred to as peyer's patcheses (Peyer '
S Patches, PPs).M cells (Microfold cell) are then the Microfold cells in PPs surfaces sparse distribution, by intestinal
Chrotoplast is tightly surrounded.The higher permeability of cell makes it to transport soluble large molecule, but also can transport
Particulate matter and complete microorganism.Its major function is can to pass through gut epithelium barrier by the antigenic substance of mucous membrane surface
It is transported to intracellular, and angtigen presentation is caused the primary immune response of body to lymphocyte.It is attenuated by some methods
Salmonella typhimurium still have good aggressivity, therefore it equally can Jing cell traffics enter body.
The gene recombination method that exogenous gene imports carrier cell is mainly included into lipofection, disease in prior art
Malicious infection protocol, electric robin etc..Wherein, there is certain injury to cell in the method for being transfected by electricity irritation, although cost is extremely low, but
It is also to greatly reduce transfection efficiency；Virus transfection method is by the exploitation based on human immune deficiency I types virus
Slow virus carrier is planted, it is respectively provided with infection ability to somatoblast and Unseparated Cell, effectively DNA can be incorporated into into host
In genome, efficiency highest is mainly used in the cell of some hardly possible transfections such as primary cell, stem cell, undifferentiated cell, or uses
In the structure of stable cell lines, but technical costss highest in all transfection methods.Lipofection is by cation
After lipid encapsulation DNA with cell membrane fusion transporte to cells in, can high-efficiency transfection, although dropped compared to virus transfection cost
It is low, but the problems such as still suffer from relatively costly, not easy to operate on the whole.
The content of the invention
It is contemplated that for the technological deficiency of prior art, there is provided a kind of eucaryon of attenuated salmonella typhimurium mediation
Bioblast transfection method, with the relatively costly technical problem of the eukaryotic cell plasmid transfection method for solving prior art.
The invention solves the problems that another technical problem be that optional eukaryotic cell plasmid transfection method is more in prior art
Limitation.
The invention solves the problems that another technical problem be prior art eukaryotic cell plasmid transfection method it is less efficient.
To realize above technical purpose, the present invention is employed the following technical solutions：
A kind of eukaryotic cell plasmid transfection method of attenuated salmonella typhimurium mediation, comprises the following steps：
1) competent cell of attenuated salmonella typhimurium VNP20009 strains is prepared；
2) plasmid to be transfected being taken, by electric robin steps for importing 1) in the competent cell, screening positive clone is obtained
Recombinant bacteria；
3) HEK293-T cells and step 2 are taken) obtained by recombinant bacteria, with the cell concentration of the two as 1:20～1:200
Ratio mixes in Tissue Culture Plate, cultivates 4～16h, and then the culture medium in Tissue Culture Plate is replaced by containing for Mus
The cell culture medium of the antibiotic of salmonella typhi, cultivates 40～56h.
On the basis of above technical scheme, transfection efficiency can also be further investigated, the pattern of implementing can be according to this
The conventional method in field carries out actual selection, including but not limited to eGFP method, light microscopic directly observe, albumen mark
Sign the conventional transfection efficiency evaluation methodology such as identification, Flow Cytometry.
Preferably, step 3) change culture medium before incubation time be 10h.
Preferably, step 3) in HEK293-T cells addition be 8000～12000/hole.
Preferably, step 3) after the mixing, total amount of liquid is 450～550 μ L in every hole of Tissue Culture Plate.
Preferably, step 3) antibiotic for Salmonella typhimurium is penicillin and streptomycin.
Preferably, step 2) described in electric robin comprise the following steps：Take plasmid to be transfected and step 1) impression
State cell mixes in electric revolving cup, then turns 4.7ms with the condition electricity of voltage 1.8kv, the Ω of resistance 200, electric capacity 25uF
Preferably, the specification of the electric revolving cup is 0.1cm.
Preferably, step 2) screening positive clone be by amicillin resistance screen.
Preferably, step 1) specifically include following operation：
A frozen attenuated salmonella typhimurium VNP20009 strains streak inoculation) is taken in LB solid medium cultures to shape
Into single bacterium colony；
B) picking single bacterium colony is inoculated in 3～7ml LB fluid mediums, 35～39 DEG C of 10～14h of shaken cultivation；
C) take step B) culture obtained by bacterium solution, by 1:80～1:120 volume ratio is inoculated in LB fluid mediums, is shaken
Swing culture and be not less than 0.4 to bacterium solution OD value；
D) after 15～25min of ice bath, centrifuging and taking precipitation is washed with the sterile deionized water of 1/8～1/12 volume pre-cooling, and
Afterwards centrifuging and taking precipitation is with 1/80～1/120 volume pre-cooling, 8～12% (mL/mL) concentration glycerol washing thalline, then be centrifuged it is heavy
Shallow lake is resuspended in 1/80～1/120 volume pre-cooling, 8～12% (mL/mL) glycerol.
It is further preferred on this basis：Prepared competent cell is retained standby under the conditions of -80 DEG C；Step C)
The consumption of middle LB fluid mediums is 100mL；Step D) in sterile deionized water washing number of times be 2 times.
Preferably, the condition of the centrifugation is：10min is centrifuged with the rotating speed of 3000rpm under the conditions of 4 DEG C.
Preferably, the plasmid to be transfected, is with pTriex as expression vector, is integrated with TEM1 antibody expressing genes portion
The recombiant plasmid of burst section；On this basis it is further preferred that the integration is to expand TEM1 total lengths using round pcr, it
Afterwards using enzymes double zyme cutting TEM1 fragments and pTriex expression vectors, TEM1 fragments are inserted into into pTriex using T4 ligases
The multiple clone site of expression vector
In above technical scheme, carrier for expression of eukaryon is not limited to pTriex carriers, all to be made with mammal
Eukaryon expression plasmid for host cell is used equally to the present invention.
Preferably, being also integrated with EGFP gene on the recombiant plasmid；Step 3) after the completion of continue executing with following steps
4)：Using fluorescent protein expression level in fluorescence microscope cell, its deleterious cellular effects and transfection efficiency are determined.
The invention provides a kind of eukaryotic cell plasmid transfection method of attenuated salmonella typhimurium mediation, the technical side
Case using Salmonella typhimurium VNP20009 strains as medium, first by recombiant plasmid by electroporated whole with bacterial genomes
Close, obtain recombinant bacteria, then recycle the antibacterial to realize transfection to the infectivity of virus, finally kill cell using antibiotic
Inside and outside Salmonella typhimurium, the eucaryon plasmid for discharging realizes the expression of specific protein.
In above technical scheme, Salmonella typhimurium is a kind of aggressive intracellular bacterium, and VNP20009 strains are used as current
Using more strain, good aggressivity can be still kept after attenuation, therefore the approach of its invasion body is similar to wild strain,
Body is invaded yet by mucosal epithelial cells approach and non-epithelial cell (dendritic cell) approach two ways.
The present invention's has the technical effect that following several respects：
1, plasmid used in the present invention is present in attenuated salmonella typhimurium VNP20009 living, it is only necessary to subtracting
Malicious Salmonella typhimurium VNP20009 is simply cultivated, you can be used for the purpose bacterium for transfecting in a large number.With traditional phosphorus
Sour calcium, electricity turn the carrier preparation procedure loaded down with trivial details with liposome and compare, simple, convenient, low cost.
2, traditional calcium phosphate, electricity turn and lipofection has very big toxic action to cell, and it is big to sacrifice
Amount host cell is cost.The Salmonella typhimurium that the present invention is used be mammal intracellular bacterial parasite, and host cell category
In symbiosiss, there is no toxic action substantially to host cell, and during antibacterial and cell are incubated altogether, antibacterial can equably by
Mammal intake is intracellular, therefore possesses the advantages of transfection efficiency is homogeneous, murder by poisoning is little.
3, the attenuated salmonella typhimurium VNP20009 containing purpose plasmid used by the present invention can be with liquid and lyophilizing
Powder form is preserved, and if desired for direct activation transfection experiment is can be used for, convenient, fast.
4, the transfection efficiency used by the present invention can reach 80-95%, the 10-20%, lipofection with calcium phosphate method
30-50% compare with electric robin 30-45, have a clear superiority.Transfection method used by the present invention to cytoactive almost without
Any impact, and the motility rate that the motility rate of calcium phosphate method is 4-20%, lipofection is 1- for 50-80% and electricity robin motility rate
20%.
Description of the drawings
Fig. 1 be in the embodiment of the present invention 1 TEM1-GFP albumen in HEK293T cell inner expression fluorescence results figures.
Specific embodiment
The specific embodiment of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
Will not be described in detail to belonging to known structure or function in following examples.In addition to being defined, institute in following examples
Technology and scientific terminology have the identical meanings being commonly understood by with those skilled in the art of the invention.
Test reagent consumptive material used in following examples, if no special instructions, is routine biochemistry reagent；The experiment
Method, if no special instructions, is conventional method；Quantitative test in following examples, is respectively provided with three repetitions and tests, as a result
Average；% in following examples, if no special instructions, is weight/mass percentage composition.
Embodiment 1
1st, the structure of recombiant plasmid pTriex-TEM1-EGFP
TEM1 total lengths are expanded using round pcr, afterwards using enzymes double zyme cutting TEM1 fragments and pTriex expression vectors,
TEM1 fragments are inserted into the multiple clone site of pTriex expression vectors using T4 ligases.TEM1 fragments are located at carrier pTriex
Before middle EGFP gene, the carrier for building is named as pTriex-TEM1-EGFP.
2nd, prepared by attenuated salmonella typhimurium VNP20009 competence
By the attenuated salmonella typhimurium VNP20009 streak inoculations of -80 DEG C of preservations in nonresistant LB flat boards, 37 DEG C
Overnight incubation；Picking single bacterium colony in 5ml LB, 37 DEG C of shaken cultivation 12h；By 1：100 ratios are inoculated in 100ml LB shakes
Culture is swung to antibacterial OD=0.4 or so；After ice bath 20min, 4 DEG C of 3000rpm are centrifuged 10min；Bacterial sediment is pre- with 1/10 volume
Cold sterile deionized water is washed twice, and 4 DEG C of 3000rpm are centrifuged 10min；Bacterial sediment is again with 1/100 volume pre-cooling
10% glycerol washing thalline, 4 DEG C of 3000rpm are centrifuged 10min；Bacterial sediment is resuspended in into 10% glycerol of 1/100 volume pre-cooling
In, make -80 retentions after VNP20009 competence subpackages standby.
3rd, recombiant plasmid pTriEx-TEM1-EGFP electricity turns attenuated salmonella typhimurium VNP20009
Recombiant plasmid pTriex-TEM1-EGFP electricity containing green fluorescence gene is rotated into into attenuated Salmonella typhinaurium Salmonella
In bacterium VNP20009, using 0.1cm electricity revolving cups, condition setting is：The Ω 25uF of 1.8kv 200, electricity turns 4.7ms；It is blue or green by ammonia benzyl
Chloramphenicol resistance filters out positive colony, obtains recombination attenuated salmonella typhimurium VNP20009, is named as pTriEx-TEM1-
EGFP-VNP20009。
4th, attenuated salmonella typhimurium VNP20009 intracellulars invasion and attack HEK293T and green fluorescent protein detection
By HEK293T cells according to 10000/ hole, 24 orifice plates are inoculated in (using the culture without penicillin and streptomycin
Base), by recombination attenuated salmonella typhimurium pTriEx-TEM1-EGFP-VNP20009 according to HEK293-T cells：Restructuring is thin
Bacterium=1:20～1:200 ratio is inoculated in 24 orifice plates, the μ L of cumulative volume 500, after culture 4-16h, is replaced by containing penicillin
48h is mixed with the culture medium of streptomycin, directly using whether having fluorescent protein expression in fluorescence microscope cell, really
Fixed its deleterious cellular effects and transfection efficiency, as shown in Figure 1.
Embodiment 2
A kind of eukaryotic cell plasmid transfection method of attenuated salmonella typhimurium mediation, comprises the following steps：
1) competent cell of attenuated salmonella typhimurium VNP20009 strains is prepared；
2) plasmid to be transfected being taken, by electric robin steps for importing 1) in the competent cell, screening positive clone is obtained
Recombinant bacteria；
3) HEK293-T cells and step 2 are taken) obtained by recombinant bacteria, with the cell concentration of the two as 1:20 ratio is thin
Mix in born of the same parents' culture plate, cultivate 4h, then the culture medium in Tissue Culture Plate is replaced by containing for Salmonella typhimurium
Antibiotic cell culture medium, cultivate 40h.
On the basis of above technical scheme, following condition is met：
Step 3) in HEK293-T cells addition be 8000/hole.
Step 3) after the mixing, total amount of liquid is 450 μ L in every hole of Tissue Culture Plate.
Step 3) antibiotic for Salmonella typhimurium is penicillin and streptomycin.
Step 2) described in electric robin comprise the following steps：Taking plasmid to be transfected and step 1) competent cell is in electricity
Mix in revolving cup, then 4.7ms is turned with the condition electricity of voltage 1.8kv, the Ω of resistance 200, electric capacity 25uF
The specification of the electric revolving cup is 0.1cm.
Step 1) specifically include following operation：
A frozen attenuated salmonella typhimurium VNP20009 strains streak inoculation) is taken in LB solid medium cultures to shape
Into single bacterium colony；
B) picking single bacterium colony is inoculated in 3ml LB fluid mediums, 35 DEG C of shaken cultivation 10h；
C) take step B) culture obtained by bacterium solution, by 1:80 volume ratio is inoculated in LB fluid mediums, shaken cultivation
It is 0.4 to bacterium solution OD value；
D) after ice bath 15min, centrifuging and taking precipitation is washed with the sterile deionized water of 1/8 volume pre-cooling, and then centrifuging and taking is sunk
Shallow lake is resuspended in 1/80 volume pre-cooling with 1/80 volume pre-cooling, 8% (mL/mL) concentration glycerol washing thalline, then centrifugation
, in the glycerol of 8% (mL/mL).
The condition of the centrifugation is：10min is centrifuged with the rotating speed of 3000rpm under the conditions of 4 DEG C.
The plasmid to be transfected, is with pTriex as expression vector, is integrated with TEM1 antibody expressing genes Partial Fragments
Recombiant plasmid.
EGFP gene is also integrated with the recombiant plasmid；Step 3) after the completion of continue executing with following steps 4)：Using glimmering
Fluorescent protein expression level in light microscope observation of cell, determines its deleterious cellular effects and transfection efficiency.
Embodiment 3
A kind of eukaryotic cell plasmid transfection method of attenuated salmonella typhimurium mediation, comprises the following steps：
1) competent cell of attenuated salmonella typhimurium VNP20009 strains is prepared；
2) plasmid to be transfected being taken, by electric robin steps for importing 1) in the competent cell, screening positive clone is obtained
Recombinant bacteria；
3) HEK293-T cells and step 2 are taken) obtained by recombinant bacteria, with the cell concentration of the two as 1:200 ratio exists
Mix in Tissue Culture Plate, cultivate 16h, then the culture medium in Tissue Culture Plate is replaced by containing for Salmonella typhimurium
The cell culture medium of the antibiotic of bacterium, cultivates 56h.
On the basis of above technical scheme, following condition is met：
Step 3) in HEK293-T cells addition be 12000/hole.
Step 3) after the mixing, total amount of liquid is 550 μ L in every hole of Tissue Culture Plate.
Step 2) described in electric robin comprise the following steps：Taking plasmid to be transfected and step 1) competent cell is in electricity
Mix in revolving cup, then 4.7ms is turned with the condition electricity of voltage 1.8kv, the Ω of resistance 200, electric capacity 25uF
Step 1) specifically include following operation：
A frozen attenuated salmonella typhimurium VNP20009 strains streak inoculation) is taken in LB solid medium cultures to shape
Into single bacterium colony；
B) picking single bacterium colony is inoculated in 7ml LB fluid mediums, 39 DEG C of shaken cultivation 14h；
C) take step B) culture obtained by bacterium solution, by 1:120 volume ratio is inoculated in LB fluid mediums, shaken cultivation
It is 0.6 to bacterium solution OD value；
D) after ice bath 25min, centrifuging and taking precipitation is washed with the sterile deionized water of 1/12 volume pre-cooling, and then centrifuging and taking is sunk
Form sediment with 1/120 volume pre-cooling, 12% (mL/mL) concentration glycerol washing thalline, then centrifugation to be resuspended in 1/120 volume pre-
In the glycerol of cold, 12% (mL/mL).
The plasmid to be transfected, is with pTriex as expression vector, is integrated with TEM1 antibody expressing genes Partial Fragments
Recombiant plasmid.
Embodiment 4
A kind of eukaryotic cell plasmid transfection method of attenuated salmonella typhimurium mediation, comprises the following steps：
1) competent cell of attenuated salmonella typhimurium VNP20009 strains is prepared；
2) plasmid to be transfected being taken, by electric robin steps for importing 1) in the competent cell, screening positive clone is obtained
Recombinant bacteria；
3) HEK293-T cells and step 2 are taken) obtained by recombinant bacteria, with the cell concentration of the two as 1:100 ratio exists
Mix in Tissue Culture Plate, cultivate 10h, then the culture medium in Tissue Culture Plate is replaced by containing for Salmonella typhimurium
The cell culture medium of the antibiotic of bacterium, cultivates 48h.
On the basis of above technical scheme, following condition is met：
Step 3) antibiotic for Salmonella typhimurium is penicillin and streptomycin.
Step 1) specifically include following operation：
A frozen attenuated salmonella typhimurium VNP20009 strains streak inoculation) is taken in LB solid medium cultures to shape
Into single bacterium colony；
B) picking single bacterium colony is inoculated in 5ml LB fluid mediums, 37 DEG C of shaken cultivation 12h；
C) take step B) culture obtained by bacterium solution, by 1:100 volume ratio is inoculated in LB fluid mediums, shaken cultivation
It is 0.5 to bacterium solution OD value；
D) after ice bath 20min, centrifuging and taking precipitation is washed with the sterile deionized water of 1/10 volume pre-cooling, and then centrifuging and taking is sunk
Form sediment with 1/100 volume pre-cooling, 10% (mL/mL) concentration glycerol washing thalline, then centrifugation to be resuspended in 1/100 volume pre-
In the glycerol of cold, 10% (mL/mL).
Embodiment 5
A kind of eukaryotic cell plasmid transfection method of attenuated salmonella typhimurium mediation, comprises the following steps：
1) competent cell of attenuated salmonella typhimurium VNP20009 strains is prepared；
2) plasmid to be transfected being taken, by electric robin steps for importing 1) in the competent cell, screening positive clone is obtained
Recombinant bacteria；
3) HEK293-T cells and step 2 are taken) obtained by recombinant bacteria, with the cell concentration of the two as 1:100 ratio exists
Mix in Tissue Culture Plate, cultivate 10h, then the culture medium in Tissue Culture Plate is replaced by containing for Salmonella typhimurium
The cell culture medium of the antibiotic of bacterium, cultivates 48h.
Embodiments of the invention have been described in detail above, but the content is only presently preferred embodiments of the present invention,
Not to limit the present invention.All any modification, equivalent and improvement made in the application range of the present invention etc., all should
It is included within protection scope of the present invention.
Claims (10)
1. the eukaryotic cell plasmid transfection method that a kind of attenuated salmonella typhimurium is mediated, it is characterised in that including following step
Suddenly：
1) competent cell of attenuated salmonella typhimurium VNP20009 strains is prepared；
2) plasmid to be transfected being taken, by electric robin steps for importing 1) in the competent cell, screening positive clone is recombinated
Antibacterial；
3) HEK293-T cells and step 2 are taken) obtained by recombinant bacteria, with the cell concentration of the two as 1:20～1:200 ratio
Mix in Tissue Culture Plate, cultivate 4～16h, then the culture medium in Tissue Culture Plate is replaced by containing for mouse typhuss
The cell culture medium of the antibiotic of Salmonella, cultivates 40～56h.
2. the eukaryotic cell plasmid transfection method that a kind of attenuated salmonella typhimurium according to claim 1 is mediated, its
Be characterised by step 3) in HEK293-T cells addition be 8000～12000/hole.
3. the eukaryotic cell plasmid transfection method that a kind of attenuated salmonella typhimurium according to claim 1 is mediated, its
It is characterised by step 3) after the mixing, total amount of liquid is 450～550 μ L in every hole of Tissue Culture Plate.
4. the eukaryotic cell plasmid transfection method that a kind of attenuated salmonella typhimurium according to claim 1 is mediated, its
It is characterised by step 3) antibiotic for Salmonella typhimurium is penicillin and streptomycin.
5. the eukaryotic cell plasmid transfection method that a kind of attenuated salmonella typhimurium according to claim 1 is mediated, its
Be characterised by step 2) described in electric robin comprise the following steps：Taking plasmid to be transfected and step 1) competent cell is in electricity
Mix in revolving cup, then 4.7ms is turned with the condition electricity of voltage 1.8kv, the Ω of resistance 200, electric capacity 25uF.
6. the eukaryotic cell plasmid transfection method that a kind of attenuated salmonella typhimurium according to claim 5 is mediated, its
The specification for being characterised by the electric revolving cup is 0.1cm.
7. the eukaryotic cell plasmid transfection method that a kind of attenuated salmonella typhimurium according to claim 1 is mediated, its
It is characterised by step 1) specifically include following operation：
A frozen attenuated salmonella typhimurium VNP20009 strains streak inoculation) is taken in the culture of LB solid mediums to forming list
Bacterium colony；
B) picking single bacterium colony is inoculated in 3～7ml LB fluid mediums, 35～39 DEG C of 10～14h of shaken cultivation；
C) take step B) culture obtained by bacterium solution, by 1:80～1:120 volume ratio is inoculated in LB fluid mediums, vibration training
Support to bacterium solution OD value and be not less than 0.4；
D) after 15～25min of ice bath, centrifuging and taking precipitation washed with the sterile deionized water of 1/8～1/12 volume pre-cooling, then from
The heart takes 1/80～1/120 volume pre-cooling, 8～12% (mL/mL) concentration glycerol washing thalline of precipitation, then centrifugation weight
In being suspended from 1/80～1/120 volume pre-cooling, 8～12% (mL/mL) glycerol.
8. the eukaryotic cell plasmid transfection method that a kind of attenuated salmonella typhimurium according to claim 7 is mediated, its
The condition for being characterised by the centrifugation is：10min is centrifuged with the rotating speed of 3000rpm under the conditions of 4 DEG C.
9. the eukaryotic cell plasmid transfection method that a kind of attenuated salmonella typhimurium according to claim 1 is mediated, its
The plasmid to be transfected is characterised by, is with pTriex as expression vector, is integrated with TEM1 antibody expressing genes Partial Fragments
Recombiant plasmid.
10. the eukaryotic cell plasmid transfection method that a kind of attenuated salmonella typhimurium according to claim 9 is mediated, its
It is characterised by also being integrated with EGFP gene on the recombiant plasmid；Step 3) after the completion of continue executing with following steps 4)：Using glimmering
Fluorescent protein expression level in light microscope observation of cell, determines its deleterious cellular effects and transfection efficiency.
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| CN111172088A (en) * | 2019-12-26 | 2020-05-19 | 南昌大学 | Recombinant attenuated salmonella typhimurium for expressing HER2 single-chain antibody and function verification method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107656056A (en) * | 2017-08-29 | 2018-02-02 | 山东师范大学 | A kind of method increased based on bacterium to the quick microscopy of bacterium |
| CN111172088A (en) * | 2019-12-26 | 2020-05-19 | 南昌大学 | Recombinant attenuated salmonella typhimurium for expressing HER2 single-chain antibody and function verification method thereof |
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