CN105802899A - Genetically engineered bacterium for suppressing tumor growth and construction method and application thereof - Google Patents

Genetically engineered bacterium for suppressing tumor growth and construction method and application thereof Download PDF

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CN105802899A
CN105802899A CN201610145045.7A CN201610145045A CN105802899A CN 105802899 A CN105802899 A CN 105802899A CN 201610145045 A CN201610145045 A CN 201610145045A CN 105802899 A CN105802899 A CN 105802899A
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engineering bacterium
tumor growth
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CN105802899B (en
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徐寒梅
赵长宏
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China Pharmaceutical University
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Abstract

The invention discloses a genetically engineered bacterium for suppressing tumor growth and a construction method and application thereof and belongs to the technical field of biology.The genetically engineered bacterium is obtained by connecting a DNA sequence coded with Sox2shRNA to a carrier vector pGPU6 to construct recombinant plasmids and then transforming the recombinant plasmids into attenuated salmonella typhimurium VNP20009.The preparation method of the genetically engineered bacterium is simple and easy to operate.The invention further provides the construction method of the genetically engineered bacterium and the application of the genetically engineered bacterium in tumor treatment medicine.The medicine prepared through the genetically engineered bacterium and used for treating tumors has the advantages of being safe, free of toxin, strong in tumor targeting and the like.When the genetically engineered bacterium and vasoinhibitor medicine are combined to treat the tumors, the antitumous effect of combined medicine administration is remarkable.

Description

A kind of genetic engineering bacterium suppressing tumor growth and construction method thereof and application
Technical field
The invention belongs to biological technical field, more particularly, it relates to a kind of genetic engineering bacterium suppressing tumor growth and construction method thereof and application.
Background technology
Tumor stem cell is tumor sustainable growth, the basic reason that medicine produces resistance, tumor recurrence.Tumor stem cell is characterized by: become the ability of the ability of tumor, medicine resistance ability serum-free culture balling-up (sphereformation) strong, external, differentiation in the cyton that the ability of continuous proliferation, quantity are little.A lot of albumen playing critical function in embryonic stem cell also high expressed and state of being activated in tumor stem cell.Sox2 (sex-determiningregionY-box2) is the gene maintaining the multifarious key of stem cell, and it is high expressed in kinds of tumor cells and cells of resistant tumors.ShRNA (shorthairpinRNA) perturbation technique is by expressing the expression that the RNA interfering fragment of short hairpin structure can reduce the gene of targeting efficiently.
The Salmonella typhimurium of attenuation is a kind of facultative anaerobe, has a lot of anoxic zones in tumor tissues, and Salmonella can be enriched with in tumor hypoxia region, and the clump count in tumor is far longer than clump count in other tissue.Although in preclinical experiment, attenuation salmonella VNP20009 has good antitumor action, but the result of study of a clinical phase shows VNP20009 antineoplastic poor effect in human body, main manifestations is: can not effectively in tumor tissues be enriched with, antitumous effect not notable.
The speed of growth of tumor is faster than normal cell, it is necessary to consume substantial amounts of nutrition and oxygen.Tumor cell can promote that the formation of periphery blood vessel is to meet demand.Vasoinhibitor suppresses the growth of tumor to migrate by suppressing the formation of tumor periphery blood vessel.Recent study finds when treating breast carcinoma with vasoinhibitor class medicine Sunitinib, suppressing the inside tumor anoxia that angiogenesis causes, oxygen deficient induction factor 1 (Hypoxia-induciblefactor1) can promote the rising of the ratio of tumor stem cell in tumor.In order to reach better therapeutic effect, also need to combine other medicine that can be simultaneously targeting tumor cell when using vasoinhibitor class medicine.When vasoinhibitor and chemotherapeutic drugs used time, play a role owing to chemotherapeutics to pass through blood capillary arrival tumor cell, order and dosage that conservative control is administered, in-convenience in use, chemotherapeutics is very big to the injury of human body simultaneously, and targeting is poor, also has side effect.
Summary of the invention
1. the problem solved
There is the problems such as accumulation ability difference, antitumous effect be not notable for existing antineoplastic genetic engineering bacterium, the present invention provides a kind of genetic engineering bacterium suppressing tumor growth and construction method thereof and application, by the DNA sequence of coding Sox2shRNA is connected to construction recombination plasmid in vector plasmid pGPU6, then this recombinant plasmid transformed to attenuated salmonella typhimurium VNP20009 is obtained genetic engineering bacterium.The genetic engineering bacterium safety of the present invention, toxic and side effects is little, has the activity better suppressing tumor growth, and after associating angiogenesis inhibitors use, antitumous effect is more preferably, meets clinical demand.
2. technical scheme
For solving the problems referred to above, technical scheme is as follows:
A kind of genetic engineering bacterium suppressing tumor growth, including host strain and the genes of interest proceeding to host strain, described genes of interest is shRNA expressing gene, the shRNA sequence target gene Sox2 of expression.
Preferably, described host strain is attenuated salmonella typhimurium VNP20009.
The construction method of above-mentioned a kind of genetic engineering bacterium suppressing tumor growth, its step includes: shRNA expressing gene is connected to vector plasmid pGPU6 and obtains recombinant expression plasmid, then convert recombinant expression plasmid electricity to attenuated salmonella typhimurium VNP20009.
Preferably, the condition that electricity converts is: voltage 2500V, resistance 200 Ω, voltage 25 μ F.
The application in preparation tumor of the above-mentioned a kind of genetic engineering bacterium suppressing tumor growth.
Preferably, genetic engineering bacterium is used in combination with angiogenesis inhibitors when preparing tumor.
Preferably, described tumor is the constitutional or secondary carcinoma, melanoma, hemangioma and the sarcoma that originate from the incidence of people, brain, thyroid, esophagus, pancreas, lung, liver, stomach, mammary gland, kidney, gallbladder, colon or rectum, ovary, cervix uteri, uterus, prostate, bladder or testis.
Preferably, described angiogenesis inhibitors is the inhibitor suppressing tumor periphery angiogenesis, including antibody class inhibitor and kinases inhibitor.
Preferably, described antibody class inhibitor is bevacizumab；Described protein kinases inhibitor is Sutent.
The genetic engineering bacterium of the present invention is the attenuation salmonella VNP20009 carrying shRNA expression plasmid, the shRNA sequence target gene Sox2 of expression.
Wherein, including pGPU6 plasmid in described genetic engineering bacterium VNP20009, the shRNA coding sequence of the targeting Sox2 of plasmid expression is on pGPU6 plasmid.
The method building genetic engineering bacterium of the present invention comprises the following steps: first devise the shRNA that a plurality of target sequence is different, the shRNA suppressing Sox2 expression effect best is obtained through experiment screening, then the DNA primer of this shRNA sequence is synthesized, forward primer and downstream primer annealing reaction are obtained the double chain DNA fragment of toughness end, it is connected into expression plasmid pGPU6 by sticky end and obtains shRNA recombinant expression plasmid pGPU6-shSox2, pGPU6-shSox2 recombinant expression plasmid electricity is converted the Salmonella typhimurium VNP20009 to attenuation, ampicillin (Ampicillin) resistance screening that recombiant plasmid carries is utilized to obtain genetic engineering bacterium VNP20009.
The genetic engineering bacterium suppressing tumor growth of the present invention then overcomes existing genetic engineering bacterium and there is weak points such as can not being effectively enriched with in tumor tissues, antitumous effect is not notable.The target gene Sox2 of the present invention is the grade malignancy positive correlation of the important factor maintaining embryonic stem cell growth, Sox2 and kinds of tumor cells.In order to find a kind of effective carrier system to be reduced by the Sox2 gene expression dose in tumor tissues, the present invention is directed to the multiple shRNA expression plasmid of Sox2 gene design, obtain a best expression plasmid of the targeted inhibition Sox2 potency of gene through screening.After obtaining desirable expression plasmid, how being transported to expression plasmid targeting in tumor tissues and becoming an another difficult problem for inventor's research, although the research about plasmid vector has a lot at present, but the particularity in view of biomedicine field, the good expression plasmid of effect pointed out in very possible existing report can not play the effect of anticipation after being applied to the present invention, in some instances it may even be possible to there will be reverse effect.Theory analysis many times and experimental verification is inventors performed for this, final screening determines attenuation salmonella VNP20009 bacterial strain, the expression plasmid targeting of the present invention can be transported in tumor tissues by this bacterial strain, has targeting good, the advantage that side effect is low.After the shRNA expression plasmid of targeting Sox2 is proceeded to VNP20009, it is only necessary to the antitumous effect that little colony counts just can obtain.Experimentation inventors discovered unexpectedly that, when using the genetic engineering bacterium associating vasoinhibitor class drug combination of the present invention, vasoinhibitor can suppress the new life of tumor periphery blood vessel on the one hand, suppress the growth of tumor, vasoinhibitor makes to produce in tumor anaerobic environment on the other hand, now the genetic engineering bacterium of the present invention can be survived better in tumor tissues, infected tumor's cell, suppress the growth of tumor, research high efficiency anti-tumor drug world is an important breakthrough by this, significant.
The genetic engineering bacterium that the present invention builds has a good tumor-targeting, and in tumor tissues infected tumor's cell, shRNA recombinant expression plasmid pGPU6-shSox2 is discharged in cell, the shRNA of targeting Sox2 expresses, and reduces the expression of gene Sox2.Sox2 is the factor playing critical function in tumor stem cell growth, research finds that the growth of tumor cell after the expression of Sox2 is lowered and migration are suppressed, causing host's tumor cell generation apoptosis, therefore the genetic engineering bacterium of the present invention may be used for the treatment of people source tumor.
Clinical data shows, during the treatment of life-time service one antitumor drug, a lot of patients can develop immunity to drugs gradually.Multiple antitumor drug uses simultaneously and can reduce the probability that tumor cell develops immunity to drugs, and obtains better therapeutic effect.But multiple antitumor drug would be likely to occur when being administered, and side effect is big, the inapparent shortcoming of antitumous effect simultaneously.When the genetic engineering bacterium present invention built and angiogenesis inhibitors are used in combination treatment tumor, suppressed the angiogenesis of tumor periphery on the one hand by vasoinhibitor, it is suppressed that the oxygen of tumor cell and the supply of nutrition；On the one hand by the tumor-targeting of genetic engineering bacterium, substantial amounts of genetic engineering bacterium is specific breeds in tumor tissues, infected tumor's cell, the shRNA recombinant expression plasmid pGPU6-shSox2 that release is carried, express the shRNA of targeting Sox2, suppress the expression of Sox2 gene, it is suppressed that the growth of tumor.Owing to the genetic engineering bacterium of the present invention has efficient tumor-targeting, associating vasoinhibitor is free from side effects when being administered simultaneously.Cell in genetic engineering bacterium that the present invention builds and vasoinhibitor respectively target tumor and the blood vessel of tumor periphery, use simultaneously obtains the effect better suppressing people's entity tumor growth.
3. beneficial effect
Compared to prior art, the invention have the benefit that
(1) genetic engineering bacterium suppressing tumor growth of the present invention has strong tumor-targeting, it is a kind of safety non-toxic the new bio medicine possessing anti-tumor activity, by with the Salmonella typhimurium VNP20009 of attenuation for carrier, shRNA recombinant expression plasmid pGPU6-shSox2 is transported to tumor cell, the expression of suppressor gene Sox2, it is suppressed that the growth of tumor；
(2) the shRNA sequence of the targeting Sox2 that the genetic engineering bacterium of the present invention carries can reduce the expression of Sox2 efficiently, when the genetic engineering bacterium of the present invention is individually dosed, good antitumous effect can be obtained, when said gene engineering bacteria and vasoinhibitor are used in combination, it is possible to obtain significant antitumous effect；
(3) genetic engineering bacterium of the present invention is prone to cultivate, it is possible to obtain substantial amounts of thalline at short notice, and the genetic engineering bacterium safety of the present invention is high simultaneously；
(4) vasoinhibitor and genetic engineering bacterium are combined antitumor by the present invention, avoid the defect of vasoinhibitor and chemotherapeutics coupling, the order that need not be strictly administered and dosage, to human non-toxic's evil effect, the Salmonella of attenuation enter internal after, just can be enriched with in tumor in a short period of time, blood just can't detect after 2 days the Salmonella of attenuation.
Accompanying drawing explanation
Fig. 1 is the collection of illustrative plates of the expression plasmid pGPU6-shSox2 of the shRNA expressing targeting Sox2；
Fig. 2 is the E. coli clones figure carrying plasmid of Ampicillin (ampicillin) resistance agar plate screening；
Fig. 3 is the figure of the agarose gel electrophoresis of the shRNA expression plasmid extracted；
Fig. 4 is the design sketch of the anti-kinds of tumors of genetic engineering bacterium of the present invention.
Detailed description of the invention
According to following embodiment, it is possible to be well understood by the present invention.Content described in example is merely to illustrate the present invention, and should without the present invention of detailed description in restriction claims.
Embodiment 1:
Build genetic engineering bacterium
(1) the shRNA recombinant expression plasmid pGPU6-shSox2 of target gene Sox2 is built
First shRNA (the shSox2 according to Sox2 gene (GeneID:6657) sequential design targeting Sox2, SEQIDNO:1), upstream (SEQIDNO:2) and downstream (SEQIDNO:3) primer/DNA fragment of shSox2 sequence is synthesized in gene chemical synthesis company (Shanghai JiMa pharmacy Technology Co., Ltd).Simultaneously as the negative control group of experiment, devise can not comparison shRNA (shCon, the SEQIDNO:4) expressed sequence of any gene of targeting, (SEQIDNO:5) and downstream (SEQIDNO:6) primer/DNA fragment in synthesis.According to the OD value of the DNA fragmentation of synthesis, add the concentration of deionized water dilution DNA to 20 μMs.Annealing reaction process is: after being mixed by the downstream primer of the forward primer of 5 μ L and 5 μ L, add 10 × NEBbuffer2 (10mMBisTrisPropane-HCl, the 10mMMgCl of 5 μ L2, 1mMdithiothreitol, pH7.0), deionized water to the final volume adding 35 μ L is 50 μ L, after the system of mixing is hatched 4min in 100 DEG C of boiling water, naturally cools to room temperature.According to above method, upstream and downstream primer annealing obtained the shRNA template DNA fragment with sticky end.
ShRNA template DNA fragment is connected on expression vector pGPU6 (Fig. 1), the process of coupled reaction is: add the product (the shRNA template DNA fragment with sticky end) that 2 μ L annealing reactions obtain in coupled reaction system, the linearizing pGPU6 plasmid (purchase producer is Shanghai JiMa pharmacy Technology Co., Ltd) of 20ng, 10 × NEBT4DNA ligase reactant liquor (NewEnglandBiolabsInc.) of 2 μ L, the NEBT4DNA ligase of 1 μ L, supplementing deionized water to final volume 20 μ L, 16 DEG C of reactions are overnight.
In order to screen the escherichia coli list bacterium colony carrying pGPU6-shSox2 and pGPU6-shCon plasmid, the product of shSox2 and shCon fragment Yu expression vector coupled reaction is transformed in bacillus coli DH 5 alpha competent cell respectively.Operating procedure is: the DH5 α competent cell taking a pipe 25 μ L is placed on ice, after it melts, is added thereto to the above-mentioned connection product of 2 μ L, and mixing is placed on ice bath 30min on ice；45 DEG C of thermal shock 45s, ice bath 2min；Adding the nonresistant LB fluid medium of 1mL, 45min cultivated by 37 DEG C of shaking tables；3000rpm is centrifuged 5min, retains the culture medium of 80 μ L, uses pipettor by cell precipitation piping and druming uniformly, and spreading rod is coated on the LB agar plate containing Ampicillin resistance；Flat board is placed in the incubator overnight incubation of 37 DEG C.
Agar plate clone grows rear (Fig. 2), select the escherichia coli list bacterium colony carrying pGPU6-shSox2 and pGPU6-shCon plasmid in the 1mL LB fluid medium containing Ampicillin resistance, 37 DEG C of shaking table concussion overnight incubation, collect thalline, use plasmid pillar to extract test kit (purchased from Shanghai Sheng Gong biological engineering company limited) and extract plasmid DNA (Fig. 3), plasmid is sent order-checking and further determines that on plasmid, shRNA sequence is completely correct.
(2) VNP20009 bacterial strain and the qualification of carrying pGPU6-shSox2 plasmid are built
The pGPU6-shCon plasmid expressing comparison shRNA extracted and the pGPU6-shSox2 plasmid electricity respectively expressing Sox2shRNA are converted to VNP20009 bacterial strain, is respectively designated as ConshRNA-VNP and Sox2shRNA-VNP.Concrete building process is as follows:
Competence antibacterial VNP20009 is placed on ice, is transferred to after it dissolves in the electric revolving cup of pre-cooling, be added thereto to the plasmid of 2 μ L, ice bath 1min after mixing.Putting in electroporation by electricity revolving cup, condition setting is voltage 2500V, resistance 200 Ω, and electric capacity is 25 μ F.Adding the LB culture medium of 1mL after having shocked by electricity immediately, mix gently, 1h cultivated by 37 DEG C of shaking tables；3000rpm is centrifuged 5min, retains 80 μ L culture medium, coats on the LB agar culture medium flat board containing Ampicillin after bacterial precipitation piping and druming uniformly with pipettor；Flat board is placed in overnight incubation in the constant incubator of 37 DEG C.
Picking individual colonies, after being cultivated with LB fluid medium by Sox2shRNA-VNP and ConshRNA-VNP, extracts plasmid, delivers to Shanghai Sheng Gong biological engineering company limited and check order, it is determined that the shRNA coded sequence on plasmid is correct.Through comparison, the shRNA expression plasmid coded sequence that the genetic engineering bacterium of the present invention carries is correct.
Embodiment 2:
The genetic engineering bacterium of the mtt assay detection present invention inhibitory action to various human source growth of tumour cell
Mtt assay is used to detect the genetic engineering bacterium of the present invention inhibitory action to various human source growth of tumour cell.At 37 DEG C, 5%CO2Incubator in culture of tumor cell to density 90%, the trypsinization with 0.25% is collected, and with cell culture fluid re-suspended cell and count, cell concentration is adjusted to 2.0 × 104Individual/mL, by cell suspension inoculation to 96 orifice plates, every hole 100 μ L, and in 37 DEG C, 5%CO2Overnight incubation in incubator.After cell attachment, to add the genetic engineering bacterium (1.0 × 10 of the present invention6Cfu), the genetic engineering bacterium of the present invention and vasoinhibitor bevacizumab make (2.5 μ g) administering drug combinations group, the genetic engineering bacterium of the present invention and Sutent (4ng) administering drug combinations group, docetaxel (500ng) are as positive drug control group, to be not added with the culture fluid of any medicine as blank group, dilute medicine to corresponding predetermined concentration with culture fluid.Each diluent is separately added in 96 orifice plates, every hole 100 μ L, at 37 DEG C, 5%CO2Incubator hatches 48h.In 96 orifice plates, every hole adds the MTT of 20 μ L5mg/mL, continues to cultivate 4h.Sucking culture medium, every hole adds 100 μ LDMSO and dissolves.With microplate reader detection wavelength be 570nm place mensuration light absorption value, and calculate growth inhibition ratio, formula is as follows: inhibition rate of tumor growth (%)=(1-administration group light absorption value/feminine gender group light absorption value) × 100%, experiment is independent to be repeated 3 times, and the result obtained represents (Fig. 4) with mean+SD.
Suppression ratio (%) to kinds of tumors growth during the genetic engineering bacterium vasoinhibitor alone or in combination of table 1 present invention
Table 1 result shows: when the Salmonella of the present invention is individually dosed, and ratio has the effect better suppressing tumor proliferation without the Salmonella VNP20009 of transformation；When combining vasoinhibitor bevacizumab by the Salmonella of the present invention or Sutent is administered simultaneously, administering drug combinations group is better than the individually dosed antitumous effect of the Salmonella of the present invention, and the antitumous effect of administering drug combinations group is better than positive drug docetaxel.

Claims (8)

1. suppress a genetic engineering bacterium for tumor growth, including host strain and the genes of interest proceeding to host strain, it is characterised in that described genes of interest is shRNA expressing gene, the shRNA sequence target gene Sox2 of expression.
2. a kind of genetic engineering bacterium suppressing tumor growth according to claim 1, it is characterised in that described host strain is attenuated salmonella typhimurium VNP20009.
3. the construction method of a kind of genetic engineering bacterium suppressing tumor growth described in claim 1, its step includes: shRNA expressing gene is connected to vector plasmid pGPU6 and obtains recombinant expression plasmid, then convert recombinant expression plasmid to attenuated salmonella typhimurium VNP20009.
4. a kind of described in claim 1 suppresses the application in preparation tumor of the genetic engineering bacterium of tumor growth.
5. a kind of genetic engineering bacterium suppressing tumor growth according to claim 4 application in preparation tumor, it is characterised in that genetic engineering bacterium is used in combination with angiogenesis inhibitors when preparing tumor.
6. a kind of according to claim 4 or 5 suppresses the application in preparation tumor of the genetic engineering bacterium of tumor growth, it is characterized in that, described tumor is the constitutional or secondary carcinoma, melanoma, hemangioma and the sarcoma that originate from the incidence of people, brain, thyroid, esophagus, pancreas, lung, liver, stomach, mammary gland, kidney, gallbladder, colon or rectum, ovary, cervix uteri, uterus, prostate, bladder or testis.
7. a kind of genetic engineering bacterium suppressing tumor growth according to claim 5 application in preparation tumor, it is characterized in that, described angiogenesis inhibitors is the inhibitor suppressing tumor periphery angiogenesis, including antibody class inhibitor and kinases inhibitor.
8. a kind of genetic engineering bacterium suppressing tumor growth according to claim 7 application in preparation tumor, it is characterised in that described antibody class inhibitor is bevacizumab；Described kinases inhibitor is Sutent.
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