CN106591208A - Vector strain of recombinant single-chain antibody expressing DNase I, AIF or integrating toxins, and application of strain - Google Patents
Vector strain of recombinant single-chain antibody expressing DNase I, AIF or integrating toxins, and application of strain Download PDFInfo
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Abstract
The invention provides a vector strain of a recombinant single-chain antibody expressing DNase I, AIF or integrating toxins, and an application of the strain. The technical scheme depends on a single-chain antibody for specifically identifying a TEM1 protein; and an effect of specifically killing tumors is achieved by carrying humanized toxins DNase I and Aif in combination with tumor internal specific aggregation and intracellular invasion characteristics of attenuated salmonella typhimurium VNP20009. Specifically, recombinant plasmids Abvec-Igk-DNase I, Abvec-Igk-T18-Dnase I, Abvec-Igk-Aif and Abvec-Igk-T18-Aif are constructed firstly; the recombinant plasmids are amplified and introduced into a salmonella typhimurium VNP20009 strain in an electric shock transformation way; based on the intracellular invasion characteristic of the attenuated salmonella typhimurium, the protein expression is realized by taking HEK293-T cells as a host; and finally, the attenuated salmonella typhimurium inside and outside cells is killed by a serum culture medium containing penicillin and streptomycin, the recombinant plasmids are released to eukaryocytes, and supernate containing a target protein is obtained after cell disruption.
Description
Technical field
The present invention relates to technical field of molecular biology, enters one and is related to the research of gene recombinaton and transfection method, while relating to
And the expression and its functional verification of destination protein, and in particular to a kind of expression DNase I, AIF is integrated with the restructuring of the toxin
The carrier bacterial strain of single-chain antibody and the application of the bacterial strain.
Background technology
Deoxyribonuclease I (Deoxyribonuclease I, DNase I) is a kind of specific DNA hydrolytic enzyme, by
The monomeric glycoprotein of 260 aminoacid compositions, including two disulfide bond (Cys101-Cys104, Cys173-Cys209) and two
Potential N- connections glycosylation site (Asn18 and Asn106).In Ca2+And Mg2+Deng the pH neutral that bivalent metal ion is present
Under the conditions of can hydrolyze the oligonucleotide that double-stranded DNA generates 5' phosphate terminals and 3' hydroxyl terminal.DNase I as one kind except disappearing
Change enzyme, participate in the degraded of food source DNA；With going deep into for research, it is found that endogenouss DNase I participates in DNA in apoptosis process
Degraded generates DNAladder, while exogenous DNase I can be by portable with its cell surface --- non-cationic dependency
The phosphoric acid of mannose -6/insulin like growth factor (CI-MPR), raises and the closely related Fas of apoptosis as transcription factor
Gene expression；Also, during necrocytosiss, plasma fibrinolysis system activation, collaboration DNase I are into non-viable non-apoptotic cell and attached in core
Nearly aggregation, effectively hydrolyzing non-viable non-apoptotic cell chromatin dna.
AIF (apoptosis inducing factor, AIF), is that a class is present in mitochondrion
Conservative flavoprotein between adventitia, can induce Caspase independence cell apoptosis.The AIF genes of people are located at Xq25-
The length of 26, the mRNA that its transcription is produced is 2.4kb, and encoding the protein for producing has the alternative splicing body of three different lengths,
Their aminoacid number is respectively 613,609 and 326.In addition to inducible apoptosis, AIF has the activity of oxidoreductase concurrently.
After cell is subject to the stimulation of specific apoptosis induction signal, the permeability duct on mitochondrial membrane
(mitochondrial permeability transition pores, MPTP) is opened, it is allowed to which AIF is discharged into from mitochondrion
Endochylema, and in nucleus, and another mitochondrial protein endonuclease G (Endo G) together, cause nuclear dna
Coagulation simultaneously fragments into the fragment of 50kb sizes
In mediating apoptosis process, AIF plays the role of two aspects：One can be as it is apoptotic it is initial because
Son, two can be as the apoptotic direct effect factor.Under normal conditions, AIF is present in mitochondrion, Ke Yiqing
Except intracellular free radical is so as to preventing apoptosis；After cell is subject to apoptotic stimulus.AIF first from mitochondrion out, so
After be transferred in Cytoplasm, be finally transferred to be played in nucleus and promote apoptotic effect.
Endosialin (TEM1/CD248) is I type transmembrane protein, by 80.9KDa's for being glycosylated modification
Protein core district's groups are into the glycoprotein of modification after ripening is about in 175KDa.TEM1 is the recent human tumor label for finding
One of, its infiltration of cell growth angiogenesis and transfer to tumor plays an important role.
TEM1 outer portions are by five globular domains (the c-type Lectin domain of N-terminal, sushi spline structure domain, and three tables
Skin growth factor (EGF) sample repeat) and a mucoprotein sample district's groups into, wherein core protein a large amount of salivas acidifying with thrombosis regulation
Protein similar.For adult tissue, the expression and distribution of TEM1 is such：Uterus>Glomerule>Fallopian tube blood vessel>The heart and lung>Its
Hetero-organization；In addition, TEM1 expressions are relatively little in endothelial progenitor cells.However, TEM1 has high expression in following tumor,
Such as sarcoma, the cerebral tumor, breast carcinoma, skin carcinoma, colon cancer, and experiment shows in ovarian cancer, the infiltration of TEM1 expression and tumor,
Prognosis is closely related.Therefore, TEM1 is not expressed or low expression in normal structure, and in the spy of the high expression of neoplasm vascularity
Point, predictive of TEM1 as the target molecules of tumor diagnosis and therapy potentiality.
The content of the invention
It is contemplated that for the technological deficiency of prior art, there is provided one kind expression DNase I, AIF is integrated with the poison
Element recombinant single chain antibody carrier bacterial strain and the application of the bacterial strain, to solve prior art in people source toxin DNase I, AIF
The relatively low technical problem of preparation efficiency.
The invention solves the problems that another technical problem be people source toxin DNase I, AIF of natural structure to tumor cell
Attack and lack targeting.
The invention solves the problems that another technical problem be obtain be integrated with DNase I or AIF or its both with it is single-stranded
It is numerous to the expression operation of such plasmid in prior art in the case of the recombiant plasmid of the complex expressing gene of antibody
Trivial, protein expression level is relatively low.
To realize above technical purpose, the present invention is employed the following technical solutions：
Express DNase I, AIF or be integrated with the carrier bacterial strain of the recombinant single chain antibody of the toxin, it is characterised in that the bacterium
Strain is prepared by following methods：
1) T18-DNase I expressing gene, sequence of the sequence as shown in SEQ ID No.1 is taken as shown in SEQ ID No.2
T18-AIF expressing genes, sequence such as SEQ ID No.4 institutes as shown in SEQ ID No.3 of DNase I expressing genes, sequence
The AIF expressing genes for showing, are connected respectively by enzyme action mode with Abvec-Igk plasmids, respectively obtain recombiant plasmid Abvec-
Igk-T18-DNase I、Abvec-Igk-DNase I、Abvec-Igk-T18-Aif、Abvec-Igk-Aif；
2) take step 1 respectively) gained recombiant plasmid Abvec-Igk-T18-DNase I, Abvec-Igk-DNase I,
Abvec-Igk-T18-Aif, Abvec-Igk-Aif, carry out plasmid amplification in host cell；
3) respectively collection step 2) recombiant plasmid Abvec-Igk-T18-DNase I, Abvec-Igk-DNase after amplification
I, Abvec-Igk-T18-Aif, Abvec-Igk-Aif, import competent Salmonella typhimurium by electric robin respectively
In bacterium VNP20009 strains, that is, obtain the carrier bacterium of described expression DNase I, AIF or the recombinant single chain antibody for being integrated with the toxin
Strain.
As preferred：
The T18-DNase I expressing genes are with pET302-T18-DNAse I as template, with sequence such as SEQ ID
DNA fragmentation shown in No.6, SEQ ID No.7 is primer, and Jing PCR amplifications are obtained；
The DNase I expressing genes be with pET302-T18-DNAse I as template, with sequence such as SEQ ID No.6,
DNA fragmentation shown in SEQ ID No.9 is primer, and Jing PCR amplifications are obtained；
The T18-AIF expressing genes be with pCDNA3.3C-T18-Aif as template, with sequence such as SEQ ID No.6,
DNA fragmentation shown in SEQ ID No.7 is primer, and Jing PCR amplifications are obtained；
The AIF expressing genes are with pCDNA3.3C-T18-Aif as template, with sequence such as SEQ ID No.6, SEQ ID
DNA fragmentation shown in No.8 is primer, and Jing PCR amplifications are obtained.
Preferably, step 2) host cell is E.coli Top10.
Preferably, step 1) the enzyme action mode is Jing Age I and Bsiw I double digestions, to digestion products after purification,
It is connected in Abvec-Igk plasmids using T4 ligases.
Preferably, step 3) described in electric robin comprise the following steps：Recombiant plasmid is taken respectively with described in impression
The Salmonella typhimurium VNP20009 strains of state mix in electric revolving cup, then with voltage 1.8kv, the Ω of resistance 200, electric capacity 25uF
Condition electricity turn 4.7ms.
Preferably, step 3) the competent Salmonella typhimurium VNP20009 strains are by with lower section
Prepared by method：
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.
Meanwhile, present invention also offers preparing DNase I, AIF using above-mentioned bacterial strains or being integrated with the restructuring list of the toxin
The method of chain antibody, comprises the following steps：HEK293-T cells and bacterial strain described in claim 1 are taken, the cell concentration with the two is
1:80～1:120 ratio mixes in Tissue Culture Plate, cultivates 4～16h, then changes the culture medium in Tissue Culture Plate
It is the cell culture medium containing the antibiotic for Salmonella typhimurium, cultivates 40～56h, in then smudge cellses collection
Clearly.
Preferably, the addition of HEK293-T cells is 8000～12000/hole.
Preferably, HEK293-T cells mix in Tissue Culture Plate with bacterial strain described in claim 1 after per liquid in hole
Body cumulative volume is 380～420 μ L.
Preferably, the antibiotic for Salmonella typhimurium is penicillin and streptomycin.
Preferably, after the collection supernatant, continuing executing with following steps：Take TEM1 positive cell SJSA-1 and TEM1 cloudy
Sexual cell HOS is inoculated in Tissue Culture Plate with the inoculum concentration of 6000/ hole cell, and during to cell concentration 30% or so 100 μ L are added
The supernatant, cultivate to cell occur broken or dead when, using the spy of protein on cells in supernatant described in MTT colorimetric determinations
Different in nature lethal effect.
In above technical scheme, the Abvec-Igk plasmids are expressly defined to：Numbering is in NCBI GenBank
The plasmid of FJ475056, the gene order of the plasmid is as shown in SEQ ID No.5.Therefore heretofore described Abvec-Igk matter
Not there is grain other direction actions or overview to act on.
In above technical scheme, the T18 is a kind of single-chain antibody of specific recognition TEM1, the title of the antibody
(i.e. T18) belongs to from wound vocabulary.The T18-DNase I expressing genes and T18-Aif expressing genes refer respectively to be integrated with
The expressing gene of the recombinant single chain antibody T18 of DNase I expressing genes and Aif expressing genes.As a kind of from wound vocabulary, T18-
The technical characteristic of DNase I expressing genes and T18-Aif expressing genes is respectively the DNA by sequence as shown in SEQ ID No.1
With the DNA signs as shown in SEQ ID No.3；In the case where the particular sequence of the two is known, the two can be by this
Conventional DNA synthetic methods are obtained in field, naturally it is also possible to obtained using the PCR method disclosed in above optimal technical scheme
.
The invention provides the carrier bacterial strain of a kind of expression DNase I, AIF or the recombinant single chain antibody for being integrated with the toxin
And the application of the bacterial strain.The technical scheme relies on a kind of single-chain antibody of specific recognition TEM1 albumen, with reference to attenuation Mus wound
The intra-tumor specific accumulation of cold Salmonella VNP20009 and the characteristic of intracellular invasion and attack, carrier source toxin DNase I and Aif
Reach the effect of specific killing tumor.From the point of view of specifically, the present invention constructs first Abvec-Igk-DNase I, Abvec-
Igk-T18-DNase I, Abvec-Igk-Aif, Abvec-Igk-T18-Aif recombiant plasmid, Jing after amplification by electroporated
Mode import Salmonella typhimurium VNP20009 strains, using attenuated salmonella typhimurium intracellular attack characteristic, with
HEK293-T cells realize the expression of albumen for host, finally kill cell using the blood serum medium containing penicillin, streptomycin
Inside and outside attenuated salmonella typhimurium, makes recombiant plasmid discharge into eukaryotic cell, obtains containing mesh Jing after cell breakage
Albumen supernatant.
In the present invention, the single-chain antibody T18 of Binding experiment room early stage screening is used as bionavigation, and T18 is to utilize yeast
Screening of phage antibody library is isolated can be in combination with the high-affinity of the mankind and muroid TEM1, specific single-chain antibody, by realizing it
With the amalgamation and expression of humanization toxin DNase I and Aif, the final purpose of apoptosis of tumor cells is realized.
Meanwhile, the present invention using attenuated salmonella typhimurium VNP20009 as therapy vector, integrated application attenuation Mus
Property of the characteristic and specific accumulation of salmonella typhi intracellular invasion and attack in solid tumor, further expands in the present invention and swells
The specificity that tumor is killed.
Now, clinically for the treatment of cancer, it is proposed that antibody drug conjugates (Antibody Drug
Conjugates, ADC) the novel medicine of a class, the present invention applies this concept, and Binding experiment early stage basis, is ADC
The development of Drug therapy is there is provided certain experimental data basis.
Description of the drawings
Fig. 1 is that people source toxin processes HOS cell micrograph results figures in the specific embodiment of the invention；
Fig. 2 is that people source toxin processes SJSA-1 cell micrograph results figures in the specific embodiment of the invention.
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
With pET302-T18-DNAse I as template, with SEQ ID No.6 and SEQ ID No.7 sequences as primer, PCR expands
Increase the T18-DNase I fragments shown in acquisition SEQ ID No.1；With SEQ ID No.6 and SEQ ID No.9 sequences as primer,
PCR amplifications obtain the DNase I fragments shown in SEQ ID No.2.With pCDNA3.3C-T18-Aif as template, with SEQ ID
No.6 and SEQ ID No.7 sequences are primer, and PCR amplifications obtain the T18-Aif fragments shown in SEQ ID No.3；With SEQ ID
No.6 and SEQ ID No.8 sequences are primer, and PCR amplifications obtain the Aif fragments shown in SEQ ID No.4.Wherein T18 is experiment
Room early stage has screened the single-chain antibody of specific recognition TEM1 for obtaining, is named as T18.
Acquired T18-DNase I, DNase I, T18-Aif, Aif fragment and plasmid Abvec-Igk, Jing Age I and
Bsiw I double digestions, digestion products after purification, are connected in Abvec-Igk (SEQ ID No.5) plasmid using T4 ligases, weight
Group plasmid is named as Abvec-Igk-T18-DNase I, Abvec-Igk-DNase I and Abvec-Igk-T18-Aif, Abvec-
Igk-Aif.Such as Fig. 1.
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.
By acquired recombiant plasmid Abvec-Igk-T18-DNase I, Abvec-Igk-DNase I and Abvec-Igk-
T18-Aif, Abvec-Igk-Aif electricity is rotated in attenuated salmonella typhimurium VNP20009, using 0.1cm electricity revolving cups, bar
Part is set to：The Ω 25uF of 1.8kv 200, electricity turns 4.7ms；Positive colony is filtered out by amicillin resistance, is recombinated
Attenuated salmonella typhimurium VNP20009, is named as Abvec-Igk-T18-DNase I-VNP20009 and Abvec-Igk-
DNase I-VNP20009 and Abvec-Igk-T18-Aif-VNP20009, Abvec-Igk-Aif-VNP20009.
By HEK293T cells according to 10000/ hole, 24 orifice plates are inoculated in (using the culture without penicillin and streptomycin
Base), by four kinds of recombination attenuated salmonella typhimurium VNP20009 according to HEK293-T cells：Recombinant bacteria=1：100 ratio
Example is inoculated in 24 orifice plates, the μ L of cumulative volume 400, after culture 8h, is replaced by the mixing training of the culture medium containing penicillin and streptomycin
Foster 48h, Jing after piping and druming is broken, collects supernatant, as containing T18-DNase I, DNase I, T18-Aif, Aif albumen supernatant
Liquid.
TEM1 positive cell SJSA-1 and TEM1 negative cells HOS is inoculated with into 6000/ hole cell in 24 orifice plates to cell
During amount 30% or so, 100 μ L T18-DNase I of addition, DNase I, T18-Aif, Aif albumen supernatant cultivate to cell
When now crushing or be dead, using the specific killing action of four kinds of protein on cells of MTT colorimetric determinations.
Embodiment 2
Express DNase I, AIF or be integrated with the carrier bacterial strain of the recombinant single chain antibody of the toxin, the bacterial strain is by following
Prepared by method：
1) T18-DNase I expressing gene, sequence of the sequence as shown in SEQ ID No.1 is taken as shown in SEQ ID No.2
T18-AIF expressing genes, sequence such as SEQ ID No.4 institutes as shown in SEQ ID No.3 of DNase I expressing genes, sequence
The AIF expressing genes for showing, are connected respectively by enzyme action mode with Abvec-Igk plasmids, respectively obtain recombiant plasmid Abvec-
Igk-T18-DNase I、Abvec-Igk-DNase I、Abvec-Igk-T18-Aif、Abvec-Igk-Aif；
2) take step 1 respectively) gained recombiant plasmid Abvec-Igk-T18-DNase I, Abvec-Igk-DNase I,
Abvec-Igk-T18-Aif, Abvec-Igk-Aif, carry out plasmid amplification in host cell；
3) respectively collection step 2) recombiant plasmid Abvec-Igk-T18-DNase I, Abvec-Igk-DNase after amplification
I, Abvec-Igk-T18-Aif, Abvec-Igk-Aif, import competent Salmonella typhimurium by electric robin respectively
In bacterium VNP20009 strains, that is, obtain the carrier bacterium of described expression DNase I, AIF or the recombinant single chain antibody for being integrated with the toxin
Strain.
On the basis of above technical scheme, following condition is met：
The T18-DNase I expressing genes are with pET302-T18-DNAse I as template, with sequence such as SEQ ID
DNA fragmentation shown in No.6, SEQ ID No.7 is primer, and Jing PCR amplifications are obtained；
The DNase I expressing genes be with pET302-T18-DNAse I as template, with sequence such as SEQ ID No.6,
DNA fragmentation shown in SEQ ID No.9 is primer, and Jing PCR amplifications are obtained；
The T18-AIF expressing genes be with pCDNA3.3C-T18-Aif as template, with sequence such as SEQ ID No.6,
DNA fragmentation shown in SEQ ID No.7 is primer, and Jing PCR amplifications are obtained；
The AIF expressing genes are with pCDNA3.3C-T18-Aif as template, with sequence such as SEQ ID No.6, SEQ ID
DNA fragmentation shown in No.8 is primer, and Jing PCR amplifications are obtained.
Step 2) host cell is E.coli Top10.
Step 1) the enzyme action mode is Jing Age I and Bsiw I double digestions, connected using T4 to digestion products after purification
Enzyme is connected in Abvec-Igk plasmids.
Step 3) described in electric robin comprise the following steps：Recombiant plasmid is taken respectively with the competent mouse typhuss
The strain of Salmonella VNP20009 mixes in electric revolving cup, is then turned with the condition electricity of voltage 1.8kv, the Ω of resistance 200, electric capacity 25uF
4.7ms。
Step 3) the competent Salmonella typhimurium VNP20009 strains, it is prepared by the following method：
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).
A kind of method that application above-mentioned bacterial strains prepare DNase I, AIF or are integrated with the recombinant single chain antibody of the toxin, bag
Include following steps：HEK293-T cells and bacterial strain described in claim 1 are taken, with the cell concentration of the two as 1:80 ratio is in cell
Mix in culture plate, cultivate 4h, then the culture medium in Tissue Culture Plate is replaced by containing for Salmonella typhimurium
The cell culture medium of antibiotic, cultivates 40h, and then smudge cellses collect supernatant.
On the basis of above technical scheme, following condition is met：
The addition of HEK293-T cells is 8000/hole；HEK293-T cells are with bacterial strain described in claim 1 in cell
Total liquid volume is 380 μ L in every hole after mixing in culture plate.
The antibiotic for Salmonella typhimurium is penicillin and streptomycin.
After the collection supernatant, following steps are continued executing with：Take TEM1 positive cell SJSA-1 and TEM1 negative cells HOS
It is inoculated in Tissue Culture Plate with the inoculum concentration of 6000/ hole cell, during to cell concentration 30% or so supernatant described in 100 μ L is added,
Cultivate to cell occur broken or dead when, using the specific killing of protein on cells in supernatant described in MTT colorimetric determinations
Effect.
Embodiment 3
Express DNase I, AIF or be integrated with the carrier bacterial strain of the recombinant single chain antibody of the toxin, the bacterial strain is by following
Prepared by method：
1) T18-DNase I expressing gene, sequence of the sequence as shown in SEQ ID No.1 is taken as shown in SEQ ID No.2
T18-AIF expressing genes, sequence such as SEQ ID No.4 institutes as shown in SEQ ID No.3 of DNase I expressing genes, sequence
The AIF expressing genes for showing, are connected respectively by enzyme action mode with Abvec-Igk plasmids, respectively obtain recombiant plasmid Abvec-
Igk-T18-DNase I、Abvec-Igk-DNase I、Abvec-Igk-T18-Aif、Abvec-Igk-Aif；
2) take step 1 respectively) gained recombiant plasmid Abvec-Igk-T18-DNase I, Abvec-Igk-DNase I,
Abvec-Igk-T18-Aif, Abvec-Igk-Aif, carry out plasmid amplification in host cell；
3) respectively collection step 2) recombiant plasmid Abvec-Igk-T18-DNase I, Abvec-Igk-DNase after amplification
I, Abvec-Igk-T18-Aif, Abvec-Igk-Aif, import competent Salmonella typhimurium by electric robin respectively
In bacterium VNP20009 strains, that is, obtain the carrier bacterium of described expression DNase I, AIF or the recombinant single chain antibody for being integrated with the toxin
Strain.
On the basis of above technical scheme, following condition is met：
Step 2) host cell is E.coli Top10.
Step 1) the enzyme action mode is Jing Age I and Bsiw I double digestions, connected using T4 to digestion products after purification
Enzyme is connected in Abvec-Igk plasmids.
Step 3) the competent Salmonella typhimurium VNP20009 strains, it is prepared by the following method：
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).
A kind of method that application above-mentioned bacterial strains prepare DNase I, AIF or are integrated with the recombinant single chain antibody of the toxin, bag
Include following steps：HEK293-T cells and bacterial strain described in claim 1 are taken, with the cell concentration of the two as 1:120 ratio is thin
Mix in born of the same parents' culture plate, cultivate 16h, then the culture medium in Tissue Culture Plate is replaced by containing for Salmonella typhimurium
Antibiotic cell culture medium, cultivate 56h, then smudge cellses collect supernatant.
The addition of HEK293-T cells is 12000/hole；HEK293-T cells are with bacterial strain described in claim 1 thin
Total liquid volume is 420 μ L in every hole after mixing in born of the same parents' culture plate.
Embodiment 4
Express DNase I, AIF or be integrated with the carrier bacterial strain of the recombinant single chain antibody of the toxin, the bacterial strain is by following
Prepared by method：
1) T18-DNase I expressing gene, sequence of the sequence as shown in SEQ ID No.1 is taken as shown in SEQ ID No.2
T18-AIF expressing genes, sequence such as SEQ ID No.4 institutes as shown in SEQ ID No.3 of DNase I expressing genes, sequence
The AIF expressing genes for showing, are connected respectively by enzyme action mode with Abvec-Igk plasmids, respectively obtain recombiant plasmid Abvec-
Igk-T18-DNase I、Abvec-Igk-DNase I、Abvec-Igk-T18-Aif、Abvec-Igk-Aif；
2) take step 1 respectively) gained recombiant plasmid Abvec-Igk-T18-DNase I, Abvec-Igk-DNase I,
Abvec-Igk-T18-Aif, Abvec-Igk-Aif, carry out plasmid amplification in host cell；
3) respectively collection step 2) recombiant plasmid Abvec-Igk-T18-DNase I, Abvec-Igk-DNase after amplification
I, Abvec-Igk-T18-Aif, Abvec-Igk-Aif, import competent Salmonella typhimurium by electric robin respectively
In bacterium VNP20009 strains, that is, obtain the carrier bacterium of described expression DNase I, AIF or the recombinant single chain antibody for being integrated with the toxin
Strain.
A kind of method that application above-mentioned bacterial strains prepare DNase I, AIF or are integrated with the recombinant single chain antibody of the toxin, bag
Include following steps：HEK293-T cells and bacterial strain described in claim 1 are taken, with the cell concentration of the two as 1:100 ratio is thin
Mix in born of the same parents' culture plate, cultivate 8h, then the culture medium in Tissue Culture Plate is replaced by containing for Salmonella typhimurium
Antibiotic cell culture medium, cultivate 48h, then smudge cellses collect supernatant.
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.
SEQUENCE LISTING
<110>University Of Nanchang
<120>Express DNase I, AIF or be integrated with carrier bacterial strain and the application of the bacterial strain of the recombinant single chain antibody of the toxin
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 1620
<212> DNA
<213>Artificial sequence
<400> 1
ctgaagatcg cagccttcaa catccagaca tttggggaga ccaagatgtc caatgccacc 60
ctcgtcagct acattgtgca gatcctgagc cgctatgaca tcgccctggt ccaggaggtc 120
agagacagcc acctgactgc cgtggggaag ctgctggaca acctcaatca ggatgcacca 180
gacacctatc actacgtggt cagtgagcca ctgggacgga acagctataa ggagcgctac 240
ctgttcgtgt acaggcctga ccaggtgtct gcggtggaca gctactacta cgatgatggc 300
tgcgagccct gcgggaacga caccttcaac cgagagccag ccattgtcag gttcttctcc 360
cggttcacag aggtcaggga gtttgccatt gttcccctgc atgcggcccc gggggacgca 420
gtagccgaga tcgacgctct ctatgacgtc tacctggatg tccaagagaa atggggcttg 480
gaggacgtca tgttgatggg cgacttcaat gcgggctgca gctatgtgag accctcccag 540
tggtcatcca tccgcctgtg gacaagcccc accttccagt ggctgatccc cgacagcgct 600
gacaccacag ctacacccac gcactgtgcc tatgacagga tcgtggttgc agggatgctg 660
ctccgaggcg ccgttgttcc cgactcggct cttcccttta acttccaggc tgcctatggc 720
ctgagtgacc aactggccca agccatcagt gaccactatc cagtggaggt gatgctgaag 780
gaattcgcgg gtaatcgtgt gcgtcgctct gttggtctta agggtggtgg aggtggttct 840
ggtggtggag gttctggtgg tggtggatct ggccagcttg tgctgactca gccaccttcc 900
ctctctgcat ctcctggagc atcagccagt ctcacctgca ccttacgcag tgacatcaat 960
gttggttcct acaggatatc ctggtaccag cagaagccag ggagtcctcc ccagtatctc 1020
ctgagctaca aatcagactc agataagcag aagggctctg gagtccccag ccgcttctct 1080
ggatccaaag atgcttcggc caatgcaggg attttactca tctctgggct ccagtctgag 1140
gatgaggctg actattattg tatgatttgg cacaacagcg ctggggtgtt cggcgggggc 1200
accaagctga ccgtcctagg cggtggttcc tctagatctt cctcctctgg tggcggtggc 1260
tcgggcggtg gtgggcaggt gcagctgcag gagtcgggag gaaccttggt acagcctggg 1320
gggtccctga gactctcttg tgaagcctct ggattcacct ttagcaacta tgccatgggc 1380
tgggtccgcc agactccagg aaaggggctg gagtggctgt cggctattcg taaaagtggc 1440
actaccacat actacgcgga ctccgtgaag ggccggttca tcatctccag agacaattcc 1500
aagaacaccc tgtatctgca aatgaatagg ctgagagtcg gcgacacggc cacttattac 1560
tgtgcgactc accccatcgc gggctactgg ggccagggaa gcctggtcac tgtctcctcc 1620
<210> 2
<211> 780
<212> DNA
<213>Artificial sequence
<400> 2
ctgaagatcg cagccttcaa catccagaca tttggggaga ccaagatgtc caatgccacc 60
ctcgtcagct acattgtgca gatcctgagc cgctatgaca tcgccctggt ccaggaggtc 120
agagacagcc acctgactgc cgtggggaag ctgctggaca acctcaatca ggatgcacca 180
gacacctatc actacgtggt cagtgagcca ctgggacgga acagctataa ggagcgctac 240
ctgttcgtgt acaggcctga ccaggtgtct gcggtggaca gctactacta cgatgatggc 300
tgcgagccct gcgggaacga caccttcaac cgagagccag ccattgtcag gttcttctcc 360
cggttcacag aggtcaggga gtttgccatt gttcccctgc atgcggcccc gggggacgca 420
gtagccgaga tcgacgctct ctatgacgtc tacctggatg tccaagagaa atggggcttg 480
gaggacgtca tgttgatggg cgacttcaat gcgggctgca gctatgtgag accctcccag 540
tggtcatcca tccgcctgtg gacaagcccc accttccagt ggctgatccc cgacagcgct 600
gacaccacag ctacacccac gcactgtgcc tatgacagga tcgtggttgc agggatgctg 660
ctccgaggcg ccgttgttcc cgactcggct cttcccttta acttccaggc tgcctatggc 720
ctgagtgacc aactggccca agccatcagt gaccactatc cagtggaggt gatgctgaag 780
<210> 3
<211> 2376
<212> DNA
<213>Artificial sequence
<400> 3
ttagggctga caccagaaca gaaacagaaa aaggccgcgt tatctgcttc agaaggagag 60
gaagttcctc aagacaaggc gccaagtcat gttcctttcc tgctaattgg tggaggcaca 120
gctgcttttg ctgcagccag atccatccgg gctcgggatc ctggggccag ggtactgatt 180
gtatctgaag atcctgagct gccgtacatg cgacctcctc tttcaaaaga actgtggttt 240
tcagatgacc caaatgtcac aaagacactg cgattcaaac agtggaatgg aaaagagaga 300
agcatatatt tccagccacc ttctttctat gtctctgctc aggacctgcc tcatattgag 360
aatggtggtg tggctgtcct cactgggaag aaggtagtac agctggatgt gagagacaac 420
atggtgaaac ttaatgatgg ctctcaaata acctatgaaa agtgcttgat tgcaacagga 480
ggtactccaa gaagtctgtc tgccattgat agggctggag cagaggtgaa gagtagaaca 540
acgcttttca gaaagattgg agactttaga agcttggaga agatttcacg ggaagtcaaa 600
tcaattacga ttatcggtgg gggcttcctt ggtagcgaac tggcctgtgc tcttggcaga 660
aaggctcgag ccttgggcac agaagtgatt caactcttcc ccgagaaagg aaatatggga 720
aagatcctcc ccgaatacct cagcaactgg accatggaaa aagtcagacg agagggggtt 780
aaggtgatgc ccaatgctat tgtgcaatcc gttggagtca gcagtggcaa gttacttatc 840
aagctgaaag acggcaggaa ggtagaaact gaccacatag tggcagctgt gggcctggag 900
cccaatgttg agttggccaa gactggtggc ctggaaatag actcagattt tggtggcttc 960
cgggtaaatg cagagctaca agcacgctct aacatctggg tggcaggaga tgctgcatgc 1020
ttctacgata taaagttggg aaggaggcgg gtagagcacc atgatcacgc tgttgtgagt 1080
ggaagattgg ctggagaaaa tatgactgga gctgctaagc cgtactggca tcagtcaatg 1140
ttctggagtg atttgggccc cgatgttggc tatgaagcta ttggtcttgt ggacagtagt 1200
ttgcccacag ttggtgtttt tgcaaaagca actgcacaag acaaccccaa atctgccaca 1260
gagcagtcag gaactggtat ccgatcagag agtgagacag agtccgaggc ctcagaaatt 1320
actattcctc ccagcacccc ggcagttcca caggctcccg tccaggggga ggactacggc 1380
aaaggtgtca tcttctacct cagggacaaa gtggtcgtgg ggattgtgct atggaacatc 1440
tttaaccgaa tgccaatagc aaggaagatc attaaggacg gtgagcagca tgaagatctc 1500
aatgaagtag ccaaactatt caacattcat gaagacgaat tcgcgggtaa tcgtgtgcgt 1560
cgctctgttg gtcttaaggg tggtggaggt ggttctggtg gtggaggttc tggtggtggt 1620
ggatctggcc agcttgtgct gactcagcca ccttccctct ctgcatctcc tggagcatca 1680
gccagtctca cctgcacctt acgcagtgac atcaatgttg gttcctacag gatatcctgg 1740
taccagcaga agccagggag tcctccccag tatctcctga gctacaaatc agactcagat 1800
aagcagaagg gctctggagt ccccagccgc ttctctggat ccaaagatgc ttcggccaat 1860
gcagggattt tactcatctc tgggctccag tctgaggatg aggctgacta ttattgtatg 1920
atttggcaca acagcgctgg ggtgttcggc gggggcacca agctgaccgt cctaggcggt 1980
ggttcctcta gatcttcctc ctctggtggc ggtggctcgg gcggtggtgg gcaggtgcag 2040
ctgcaggagt cgggaggaac cttggtacag cctggggggt ccctgagact ctcttgtgaa 2100
gcctctggat tcacctttag caactatgcc atgggctggg tccgccagac tccaggaaag 2160
gggctggagt ggctgtcggc tattcgtaaa agtggcacta ccacatacta cgcggactcc 2220
gtgaagggcc ggttcatcat ctccagagac aattccaaga acaccctgta tctgcaaatg 2280
aataggctga gagtcggcga cacggccact tattactgtg cgactcaccc catcgcgggc 2340
tactggggcc agggaagcct ggtcactgtc tcctcc 2376
<210> 4
<211> 1536
<212> DNA
<213>Artificial sequence
<400> 4
ttagggctga caccagaaca gaaacagaaa aaggccgcgt tatctgcttc agaaggagag 60
gaagttcctc aagacaaggc gccaagtcat gttcctttcc tgctaattgg tggaggcaca 120
gctgcttttg ctgcagccag atccatccgg gctcgggatc ctggggccag ggtactgatt 180
gtatctgaag atcctgagct gccgtacatg cgacctcctc tttcaaaaga actgtggttt 240
tcagatgacc caaatgtcac aaagacactg cgattcaaac agtggaatgg aaaagagaga 300
agcatatatt tccagccacc ttctttctat gtctctgctc aggacctgcc tcatattgag 360
aatggtggtg tggctgtcct cactgggaag aaggtagtac agctggatgt gagagacaac 420
atggtgaaac ttaatgatgg ctctcaaata acctatgaaa agtgcttgat tgcaacagga 480
ggtactccaa gaagtctgtc tgccattgat agggctggag cagaggtgaa gagtagaaca 540
acgcttttca gaaagattgg agactttaga agcttggaga agatttcacg ggaagtcaaa 600
tcaattacga ttatcggtgg gggcttcctt ggtagcgaac tggcctgtgc tcttggcaga 660
aaggctcgag ccttgggcac agaagtgatt caactcttcc ccgagaaagg aaatatggga 720
aagatcctcc ccgaatacct cagcaactgg accatggaaa aagtcagacg agagggggtt 780
aaggtgatgc ccaatgctat tgtgcaatcc gttggagtca gcagtggcaa gttacttatc 840
aagctgaaag acggcaggaa ggtagaaact gaccacatag tggcagctgt gggcctggag 900
cccaatgttg agttggccaa gactggtggc ctggaaatag actcagattt tggtggcttc 960
cgggtaaatg cagagctaca agcacgctct aacatctggg tggcaggaga tgctgcatgc 1020
ttctacgata taaagttggg aaggaggcgg gtagagcacc atgatcacgc tgttgtgagt 1080
ggaagattgg ctggagaaaa tatgactgga gctgctaagc cgtactggca tcagtcaatg 1140
ttctggagtg atttgggccc cgatgttggc tatgaagcta ttggtcttgt ggacagtagt 1200
ttgcccacag ttggtgtttt tgcaaaagca actgcacaag acaaccccaa atctgccaca 1260
gagcagtcag gaactggtat ccgatcagag agtgagacag agtccgaggc ctcagaaatt 1320
actattcctc ccagcacccc ggcagttcca caggctcccg tccaggggga ggactacggc 1380
aaaggtgtca tcttctacct cagggacaaa gtggtcgtgg ggattgtgct atggaacatc 1440
tttaaccgaa tgccaatagc aaggaagatc attaaggacg gtgagcagca tgaagatctc 1500
aatgaagtag ccaaactatt caacattcat gaagac 1536
<210> 5
<211> 4756
<212> DNA
<213>Artificial sequence
<400> 5
ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca 60
ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 120
ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 180
acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 240
ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 300
aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 360
tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 420
gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 480
gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 540
ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt 600
ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga 660
caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt 720
gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta 780
gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac 840
tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc 900
acctcggttc tatcgattga attccaccat gggatggtca tgtatcatcc tttttctagt 960
agcaactgca accggtgtac actcgagcgt acgaagcttg gccgccatgg cccaacttgt 1020
ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 1080
catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 1140
tctggatcga tcgggaatta attcggcgca gcaccatggc ctgaaataac ctctgaaaga 1200
ggaacttggt taggtacctt ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta 1260
gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat 1320
tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc 1380
atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta 1440
actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca 1500
gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga 1560
ggcctaggct tttgcaaaaa gctgttaaca gcttggcact ggccgtcgtt ttacaacgtc 1620
gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccccttcg 1680
ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgtagcc 1740
tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 1800
accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg 1860
tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 1920
cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 1980
ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 2040
tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 2100
gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 2160
tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 2220
aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat 2280
tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc aactccgcta 2340
tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc 2400
tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 2460
tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagta ttcttgaaga 2520
cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 2580
tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 2640
taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 2700
tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 2760
gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 2820
gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 2880
cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 2940
tgtggcgcgg tattatcccg tgatgacgcc gggcaagagc aactcggtcg ccgcatacac 3000
tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 3060
atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 3120
ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 3180
gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 3240
gagcgtgaca ccacgatgcc agcagcaatg gcaacaacgt tgcgcaaact attaactggc 3300
gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 3360
gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 3420
gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 3480
cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 3540
atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 3600
tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 3660
ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 3720
gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 3780
tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 3840
ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 3900
ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 3960
gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 4020
ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 4080
tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 4140
cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 4200
agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 4260
agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 4320
gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 4380
tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 4440
accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 4500
gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 4560
attcattaat ccagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 4620
gcaattaatg tgagttacct cactcattag gcaccccagg ctttacactt tatgcttccg 4680
gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 4740
catgattacg aattaa 4756
<210> 6
<211> 34
<212> DNA
<213>Artificial sequence
<400> 6
acgtcgagtc gaaccggttg ggaagcggca ctgg 34
<210> 7
<211> 38
<212> DNA
<213>Artificial sequence
<400> 7
gtcgtgctca ccgtacgtta ggaggagacg gtgaccag 38
<210> 8
<211> 41
<212> DNA
<213>Artificial sequence
<400> 8
gtcgtgctca ccgtacgtta gtcttcatga atgttgaata g 41
<210> 9
<211> 41
<212> DNA
<213>Artificial sequence
<400> 9
gtcgtgctca ccgtacgtta cttcagcatc acctccactg g 41
Claims (10)
1. express DNase I, AIF or be integrated with the carrier bacterial strain of the recombinant single chain antibody of the toxin, it is characterised in that the bacterial strain
It is to be prepared by following methods：
1) T18-DNase I expressing gene, sequence of the sequence as shown in SEQ ID No.1 is taken as shown in SEQ ID No.2
T18-AIF expressing genes, the sequence of DNase I expressing genes, sequence as shown in SEQ ID No.3 is as shown in SEQ ID No.4
AIF expressing genes, be connected with Abvec-Igk plasmids by enzyme action mode respectively, respectively obtain recombiant plasmid Abvec-Igk-
T18-DNase I、Abvec-Igk-DNase I、Abvec-Igk-T18-Aif、Abvec-Igk-Aif；
2) take step 1 respectively) gained recombiant plasmid Abvec-Igk-T18-DNase I, Abvec-Igk-DNase I, Abvec-
Igk-T18-Aif, Abvec-Igk-Aif, carry out plasmid amplification in host cell；
3) respectively collection step 2) recombiant plasmid Abvec-Igk-T18-DNase I after amplification, Abvec-Igk-DNase I,
Abvec-Igk-T18-Aif, Abvec-Igk-Aif, import competent Salmonella typhimurium by electric robin respectively
In VNP20009 strains, that is, obtain the carrier bacterial strain of described expression DNase I, AIF or the recombinant single chain antibody for being integrated with the toxin.
2. express DNase I, AIF according to claim 1 or be integrated with the carrier bacterial strain of the recombinant single chain antibody of the toxin,
It is characterized in that：
The T18-DNase I expressing genes be with pET302-T18-DNAse I as template, with sequence such as SEQ ID No.6,
DNA fragmentation shown in SEQ ID No.7 is primer, and Jing PCR amplifications are obtained；
The DNase I expressing genes are with pET302-T18-DNAse I as template, with sequence such as SEQ ID No.6, SEQ
DNA fragmentation shown in ID No.9 is primer, and Jing PCR amplifications are obtained；
The T18-AIF expressing genes are with pCDNA3.3C-T18-Aif as template, with sequence such as SEQ ID No.6, SEQ ID
DNA fragmentation shown in No.7 is primer, and Jing PCR amplifications are obtained；
The AIF expressing genes are with pCDNA3.3C-T18-Aif as template, with sequence such as SEQ ID No.6, SEQ ID
DNA fragmentation shown in No.8 is primer, and Jing PCR amplifications are obtained.
3. express DNase I, AIF according to claim 1 or be integrated with the carrier bacterial strain of the recombinant single chain antibody of the toxin,
It is characterized in that step 2) host cell is E.coli Top10.
4. express DNase I, AIF according to claim 1 or be integrated with the carrier bacterial strain of the recombinant single chain antibody of the toxin,
It is characterized in that step 1) the enzyme action mode is Jing Age I and Bsiw I double digestions, connected using T4 to digestion products after purification
Connect enzyme to be connected in Abvec-Igk plasmids.
5. express DNase I, AIF according to claim 1 or be integrated with the carrier bacterial strain of the recombinant single chain antibody of the toxin,
It is characterized in that step 3) described in electric robin comprise the following steps：Take recombiant plasmid respectively to hinder with the competent Mus
Cold Salmonella VNP20009 strain mixes in electric revolving cup, then electric with the condition of voltage 1.8kv, the Ω of resistance 200, electric capacity 25uF
Turn 4.7ms.
6. express DNase I, AIF according to claim 1 or be integrated with the carrier bacterial strain of the recombinant single chain antibody of the toxin,
It is characterized in that step 3) the competent Salmonella typhimurium VNP20009 strains are to be prepared by the following method
's：
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.
7. the method for preparing DNase I, AIF using bacterial strain described in claim 1 or being integrated with the recombinant single chain antibody of the toxin,
It is characterized in that comprising the following steps：HEK293-T cells and bacterial strain described in claim 1 are taken, with the cell concentration of the two as 1:80
～1:120 ratio mixes in Tissue Culture Plate, cultivates 4～16h, is then replaced by the culture medium in Tissue Culture Plate and contains
There is the cell culture medium of the antibiotic for Salmonella typhimurium, cultivate 40～56h, then smudge cellses collect supernatant.
8. method according to claim 7, it is characterised in that the addition of HEK293-T cells is 8000～12000/
Hole；HEK293-T cells mix in Tissue Culture Plate with bacterial strain described in claim 1 after be 380 per total liquid volume in hole～
420μL。
9. method according to claim 7, it is characterised in that the antibiotic for Salmonella typhimurium is penicillium sp
Element and streptomycin.
10. method according to claim 7, it is characterised in that after the collection supernatant, continue executing with following steps：Take
TEM1 positive cells SJSA-1 and TEM1 negative cells HOS is inoculated in Tissue Culture Plate with the inoculum concentration of 6000/ hole cell, extremely
Supernatant described in adding 100 μ L during cell concentration 30% or so, cultivate to cell occur broken or dead when, examined using MTT colorimetrys
Survey the specific killing action of protein on cells in the supernatant.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109295083A (en) * | 2018-11-13 | 2019-02-01 | 南昌大学 | A kind of engineered strain embolism of contraception and the preparation method of capsule |
| WO2020028973A1 (en) * | 2018-08-07 | 2020-02-13 | Pulmomed Inc. | Methods for regulating endogenous production of dnase1 |
| CN110938647A (en) * | 2019-12-26 | 2020-03-31 | 深圳市前海金卓生物技术有限公司 | Recombinant expression vector, recombinant attenuated salmonella typhimurium and construction method and application thereof |
| CN111088204A (en) * | 2019-12-26 | 2020-05-01 | 南昌大学 | Recombinant escherichia coli expressing Caspase-3 recombinant scFv78 and functional verification method thereof |
| CN114249828A (en) * | 2021-12-20 | 2022-03-29 | 南京诺唯赞生物科技股份有限公司 | Monoclonal antibody of DNase I and preparation method thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006017759A2 (en) * | 2004-08-05 | 2006-02-16 | Kirin Brewery Co., Ltd. | Tumor endothelial marker-1 (tem1) binding antibodies and uses thereof |
| CN1974759A (en) * | 2006-07-26 | 2007-06-06 | 吉林大学 | Attenuated salmonella transporting recombinant plasmid and its application in treating tumor |
| CN101144081A (en) * | 2006-09-14 | 2008-03-19 | 复旦大学 | Nucleic acid molecule TRAIL and application in preparation of anti-tumour pharmaceutical |
| CN102168058A (en) * | 2010-11-30 | 2011-08-31 | 湖南师范大学 | Anti-tumor targeting engineering bacteria and bacterial agent and method for preparing bacterial agent |
| WO2013162748A1 (en) * | 2012-04-27 | 2013-10-31 | The Trustees Of The University Of Pennsylvania | Anti-tumor endothelial marker-1 (tem1) antibody variants and uses thereof |
| CN103656684A (en) * | 2013-12-03 | 2014-03-26 | 南京华贞生物医药科技有限公司 | Attenuated salmonella typhimurium and application of genetically engineered bacterium of attenuated salmonella typhimurium to preparation of medicines for treating pancreatic cancer |
| CN105925598A (en) * | 2016-05-13 | 2016-09-07 | 南昌大学 | Preparation method and application of attenuated salmonella typhimurium for secretory expression of GLP-1 (glucagon-like peptide 1) |
| CN105983103A (en) * | 2015-03-17 | 2016-10-05 | 广州华津医药科技有限公司 | Application of genetically engineered bacterium VNP-20009-M in preparation of medicines for preventing and treating cancer metastasis |
-
2016
- 2016-12-07 CN CN201611112222.8A patent/CN106591208A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006017759A2 (en) * | 2004-08-05 | 2006-02-16 | Kirin Brewery Co., Ltd. | Tumor endothelial marker-1 (tem1) binding antibodies and uses thereof |
| CN1974759A (en) * | 2006-07-26 | 2007-06-06 | 吉林大学 | Attenuated salmonella transporting recombinant plasmid and its application in treating tumor |
| WO2008014668A1 (en) * | 2006-07-26 | 2008-02-07 | Jilin University | Attenuated salmonella carrying effective plasmid and its antitumorigenic use |
| CN101144081A (en) * | 2006-09-14 | 2008-03-19 | 复旦大学 | Nucleic acid molecule TRAIL and application in preparation of anti-tumour pharmaceutical |
| CN102168058A (en) * | 2010-11-30 | 2011-08-31 | 湖南师范大学 | Anti-tumor targeting engineering bacteria and bacterial agent and method for preparing bacterial agent |
| WO2013162748A1 (en) * | 2012-04-27 | 2013-10-31 | The Trustees Of The University Of Pennsylvania | Anti-tumor endothelial marker-1 (tem1) antibody variants and uses thereof |
| CN103656684A (en) * | 2013-12-03 | 2014-03-26 | 南京华贞生物医药科技有限公司 | Attenuated salmonella typhimurium and application of genetically engineered bacterium of attenuated salmonella typhimurium to preparation of medicines for treating pancreatic cancer |
| CN105983103A (en) * | 2015-03-17 | 2016-10-05 | 广州华津医药科技有限公司 | Application of genetically engineered bacterium VNP-20009-M in preparation of medicines for preventing and treating cancer metastasis |
| CN105925598A (en) * | 2016-05-13 | 2016-09-07 | 南昌大学 | Preparation method and application of attenuated salmonella typhimurium for secretory expression of GLP-1 (glucagon-like peptide 1) |
Non-Patent Citations (3)
| Title |
|---|
| CHUNSHENG LI ET AL.: "" Antibody-based tumor vascular theranostics targeting endosialin/TEM1 in a new mouse tumor vascular model"", 《CANCER BIOLOGY&THERAPY》 * |
| CHUNSHENG LI ET AL.: ""Immunotargeting of tumor vasculature:preclinical development of novel antibody-based imaging and therapy against TEM1/CD248"", 《JOURNAL FOR IMMUNOTHERAPY OF CANCER》 * |
| CORNELIA MARTY ET AL.: ""Isolation and characterization of a scFv antibody specific for tumor endothelial marker 1 (TEM1), a new reagent for targeted tumor therapy"", 《CANCER LETTERS》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020028973A1 (en) * | 2018-08-07 | 2020-02-13 | Pulmomed Inc. | Methods for regulating endogenous production of dnase1 |
| CN109295083A (en) * | 2018-11-13 | 2019-02-01 | 南昌大学 | A kind of engineered strain embolism of contraception and the preparation method of capsule |
| CN110938647A (en) * | 2019-12-26 | 2020-03-31 | 深圳市前海金卓生物技术有限公司 | Recombinant expression vector, recombinant attenuated salmonella typhimurium and construction method and application thereof |
| CN111088204A (en) * | 2019-12-26 | 2020-05-01 | 南昌大学 | Recombinant escherichia coli expressing Caspase-3 recombinant scFv78 and functional verification method thereof |
| CN114249828A (en) * | 2021-12-20 | 2022-03-29 | 南京诺唯赞生物科技股份有限公司 | Monoclonal antibody of DNase I and preparation method thereof |
| CN114249828B (en) * | 2021-12-20 | 2022-10-18 | 南京诺唯赞生物科技股份有限公司 | Monoclonal antibody of DNase I and preparation method thereof |
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