CN105802899B - Genetically engineered bacterium for inhibiting tumor growth and construction method and application thereof - Google Patents

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

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CN105802899B
CN105802899B CN201610145045.7A CN201610145045A CN105802899B CN 105802899 B CN105802899 B CN 105802899B CN 201610145045 A CN201610145045 A CN 201610145045A CN 105802899 B CN105802899 B CN 105802899B
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徐寒梅
赵长宏
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Abstract

The invention discloses a genetic engineering bacterium for inhibiting tumor growth and a construction method and application thereof, belonging to the technical field of biology, wherein the genetic engineering bacterium is obtained by connecting a DNA sequence for coding Sox2shRNA to a vector plasmid pGPU6 to construct a recombinant plasmid, and then transforming the recombinant plasmid to attenuated salmonella typhimurium VNP 20009. The preparation method of the genetic engineering bacteria is simple and easy to operate. The invention also provides a construction method of the gene engineering bacterium and application of the gene engineering bacterium in medicaments for treating tumors, and medicaments for treating tumors prepared by the gene engineering bacterium have the advantages of safety, no toxicity, strong tumor targeting property and the like. When the genetically engineered bacterium is combined with the vascular inhibitor drugs to treat tumors simultaneously, the combined administration has obvious anti-tumor effect.

Description

Genetically engineered bacterium for inhibiting tumor growth and construction method and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a genetic engineering bacterium for inhibiting tumor growth, and a construction method and application thereof.
Background
Tumor stem cells are the root cause of the continuous growth of tumors, the generation of resistance to drugs and the recurrence of tumors. Tumor stem cells are characterized by: the capacity of continuous proliferation, the capacity of in vivo nodulation of a few cells, strong drug resistance, the capacity of in vitro serum-free culture balling (sphere formation) and differentiation. Many proteins that play important functions in embryonic stem cells are also highly expressed and activated in tumor stem cells. Sox2(sex-determining region Y-box2) is a key gene for maintaining stem cell diversity, which is highly expressed in a variety of tumor cells and drug-resistant tumor cells. shRNA (short hairpin RNA) interference technology can efficiently reduce the expression level of a targeted gene by expressing an interfering RNA fragment with a short hairpin structure.
The attenuated salmonella typhimurium is a facultative anaerobe, a plurality of anoxic areas exist in tumor tissues, the salmonella can be enriched in the tumor anoxic areas, and the colony number in the tumor is far greater than that in other tissues. Although the attenuated salmonella VNP20009 has a good anti-tumor effect in preclinical experiments, the first-stage clinical research results show that VNP20009 has a poor anti-tumor effect in human body, and mainly shows that: can not be effectively enriched in tumor tissues and has insignificant anti-tumor effect.
Tumors grow faster than normal cells and require a large consumption of nutrients and oxygen. Tumor cells can promote the formation of peripheral blood vessels to meet demand. Angiogenesis inhibitors inhibit the growth and migration of tumors by inhibiting the formation of blood vessels around the tumor. Recent studies found that when a angiogenesis-induced Hypoxia inside a tumor is inhibited when a breast cancer is treated with a vasculostatic drug, Sunitinib, Hypoxia-inducible factor 1 (Hypoxia-inducer factor 1) promotes an increase in the proportion of tumor stem cells in the tumor. In order to achieve better treatment effect, the combination of other medicines capable of simultaneously targeting tumor cells is also needed when the angiogenesis inhibitor medicines are used. When the blood vessel inhibitor is combined with the chemotherapeutic drug, the chemotherapeutic drug reaches tumor cells through microvessels to play a role, the order and dosage of administration need to be reasonably controlled, the use is inconvenient, and meanwhile, the chemotherapeutic drug has great harm to human bodies, poor targeting property and side effects.
Disclosure of Invention
1. Problems to be solved
Aiming at the problems of poor enrichment capability, insignificant anti-tumor effect and the like of the existing anti-tumor genetic engineering bacteria, the invention provides the genetic engineering bacteria for inhibiting tumor growth and a construction method and application thereof, wherein a DNA sequence for coding Sox2shRNA is connected into a vector plasmid pGPU6 to construct a recombinant plasmid, and then the recombinant plasmid is transformed into attenuated salmonella typhimurium VNP20009 to obtain the genetic engineering bacteria. The genetically engineered bacterium is safe, has small toxic and side effects, has good activity of inhibiting tumor growth, has better anti-tumor effect after being used together with the angiogenesis inhibitor, and meets clinical requirements.
2. Technical scheme
In order to solve the problems, the technical scheme of the invention is as follows:
a genetic engineering bacterium for inhibiting tumor growth comprises a host strain and a target gene transferred into the host strain, wherein the target gene is shRNA expression gene, and an expressed shRNA sequence target gene Sox 2.
Preferably, the host strain is attenuated salmonella typhimurium VNP 20009.
The construction method of the genetic engineering bacteria for inhibiting the growth of the tumor comprises the following steps: the shRNA expression gene is connected to a vector plasmid pGPU6 to obtain a recombinant expression plasmid, and the recombinant expression plasmid is electrically transformed into the attenuated salmonella typhimurium VNP 20009.
Preferably, the conditions for the electrotransformation are: voltage 2500V, resistance 200 Ω, voltage 25 μ F.
The application of the genetic engineering bacteria for inhibiting the growth of the tumor in preparing the tumor treatment medicine.
Preferably, the genetically engineered bacteria are used in combination with angiogenesis inhibitors in the preparation of a medicament for the treatment of tumors.
Preferably, the tumor is primary or secondary cancer, melanoma, hemangioma and sarcoma originating from the head and neck, brain, thyroid, esophagus, pancreas, lung, liver, stomach, breast, kidney, gall bladder, colon or rectum, ovary, cervix, uterus, prostate, bladder or testis of a human.
Preferably, the angiogenesis inhibitor is an inhibitor for inhibiting tumor peripheral angiogenesis, and comprises an antibody inhibitor and a protein kinase inhibitor.
Preferably, the antibody inhibitor is bevacizumab; the protein kinase inhibitor is sunitinib.
The genetic engineering bacteria of the invention are attenuated salmonella VNP20009 carrying shRNA expression plasmids, and the expressed shRNA sequence targets gene Sox 2.
Wherein, the genetic engineering bacteria VNP20009 contains pGPU6 plasmid, and the shRNA coding sequence of the target Sox2 expressed by the plasmid is cloned on the pGPU6 plasmid.
The method for constructing the gene engineering bacteria comprises the following steps: firstly, designing a plurality of shRNAs with different target point sequences, obtaining shRNA with the best Sox2 inhibition expression effect through experimental screening, then synthesizing a DNA primer of the shRNA sequence, carrying out annealing reaction on an upstream primer and a downstream primer to obtain a double-stranded DNA fragment with a viscous end, connecting the viscous end into an expression plasmid pGPU6 to obtain a shRNA recombinant expression plasmid pGPU6-shSox2, electrically converting the pGPU6-shSox2 recombinant expression plasmid into attenuated Salmonella typhimurium VNP20009, and screening by utilizing Ampicillin (Ampicillin) carried by the recombinant plasmid to obtain genetically engineered bacterium VNP 20009.
The genetic engineering bacteria for inhibiting the growth of the tumor overcome the defects that the existing genetic engineering bacteria cannot effectively enrich in tumor tissues, have insignificant anti-tumor effect and the like. The target gene Sox2 is an important factor for maintaining the growth of embryonic stem cells, and Sox2 is positively correlated with the malignancy degree of various tumor cells. In order to find an effective vector system to reduce the expression level of the Sox2 gene in a tumor tissue, the invention designs a plurality of shRNA expression plasmids aiming at the Sox2 gene, and obtains an expression plasmid with the best effect of targeted inhibition of the Sox2 gene by screening. After obtaining an ideal expression plasmid, how to deliver the expression plasmid to tumor tissues in a targeted way becomes a further problem for the research of the inventor, and although the research on plasmid vectors is numerous at present, in view of the particularity of the biomedical field, it is quite possible that the expression plasmid with good effect indicated in the existing report cannot achieve the expected effect after being applied to the invention, and even the opposite effect may occur. Therefore, the inventor carries out theoretical analysis and experimental verification for numerous times, finally screens and determines the attenuated salmonella VNP20009 strain, and the strain can deliver the expression plasmid of the invention to tumor tissues in a targeted manner, and has the advantages of good targeting and low side effect. After the shRNA expression plasmid targeting Sox2 is transferred into VNP20009, a good anti-tumor effect can be obtained only by a small number of colonies. In the experimental process, the inventor unexpectedly discovers that when the genetically engineered bacteria are combined with vascular inhibitor drugs, on one hand, the vascular inhibitor can inhibit the neogenesis of blood vessels around tumors and inhibit the growth of the tumors, and on the other hand, the vascular inhibitor enables the tumors to generate an anoxic environment.
The gene engineering bacteria constructed by the invention have good tumor targeting property, can infect tumor cells in tumor tissues, release shRNA recombinant expression plasmid pGPU6-shSox2 into the cells, target the shRNA expression of Sox2, and reduce the expression level of gene Sox 2. Sox2 is a factor playing an important role in the growth of tumor stem cells, and researches show that when the expression level of Sox2 is reduced, the growth and migration of tumor cells are inhibited, so that host tumor cells are subjected to apoptosis, and therefore the genetically engineered bacteria can be used for treating human tumors.
Clinical data indicate that many patients develop resistance to drugs gradually when treated with one anti-tumor drug for a long period of time. The simultaneous use of multiple antitumor drugs can reduce the possibility of drug resistance of tumor cells and obtain better treatment effect. However, when a plurality of antitumor drugs are simultaneously administered, the disadvantages of great side effects and insignificant antitumor effects may exist. When the genetic engineering bacteria constructed by the invention and the angiogenesis inhibitor are used together to treat tumors, on one hand, angiogenesis around the tumors is inhibited through the angiogenesis inhibitor, and the supply of oxygen and nutrition of tumor cells is inhibited; on one hand, by the tumor targeting of the genetic engineering bacteria, a large number of genetic engineering bacteria are specifically proliferated in tumor tissues to infect tumor cells, the carried shRNA recombinant expression plasmid pGPU6-shSox2 is released, the shRNA of the targeted Sox2 is expressed, the expression of the Sox2 gene is inhibited, and the growth of the tumor is inhibited. Because the gene engineering bacteria of the invention have high-efficiency tumor targeting, and have no side effect when being combined with the vascular inhibitor for simultaneous administration. The gene engineering bacteria and the blood vessel inhibitor constructed by the invention respectively target cells in the tumor and blood vessels around the tumor, and simultaneously obtain better effect of inhibiting the growth of human solid tumor.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) the gene engineering bacteria for inhibiting the growth of the tumor has strong tumor targeting property, is a novel safe and nontoxic biological medicament with anti-tumor activity, transports shRNA recombinant expression plasmid pGPU6-shSox2 to tumor cells by taking attenuated salmonella typhimurium VNP20009 as a vector, inhibits the expression of gene Sox2, and inhibits the growth of the tumor;
(2) the shRNA sequence of the targeting Sox2 carried by the genetically engineered bacterium can efficiently reduce the expression of Sox2, and the genetically engineered bacterium can obtain a better anti-tumor effect when being singly administered, and can obtain a remarkable anti-tumor effect when being used together with a vascular inhibitor;
(3) the genetically engineered bacterium is easy to culture, can obtain a large amount of thalli in a short time, and has high safety;
(4) the invention combines the vascular inhibitor and the genetically engineered bacteria for resisting tumor, avoids the defect of combination of the vascular inhibitor and the chemotherapeutic drugs, does not need strict administration sequence and dosage, has no toxic action on human body, can enrich attenuated salmonella in tumor within a short time after entering the body, and can not detect the attenuated salmonella in blood after 2 days.
Drawings
FIG. 1 is a map of expression plasmid pGPU6-shSox2 expressing shRNA targeting Sox 2;
FIG. 2 is a map of plasmid-carrying E.coli colonies screened on Ampicillin-resistant agar plates;
FIG. 3 is a drawing of agarose gel electrophoresis of the extracted shRNA expression plasmid;
FIG. 4 is a diagram showing the effect of the genetically engineered bacterium of the present invention on various tumors.
Detailed Description
The invention will be better understood from the following examples of embodiment. The matters described in the examples are illustrative of the invention only and should not be construed as limiting the invention as described in detail in the claims.
Example 1:
construction of genetically engineered bacteria
(1) shRNA recombinant expression plasmid pGPU6-shSox2 for constructing targeted gene Sox2
First, shRNA (shSox2, SEQ ID NO: 1) targeting Sox2 was designed based on the sequence of Sox2 Gene (Gene ID:6657), and upstream (SEQ ID NO: 2) and downstream (SEQ ID NO: 3) primer DNA fragments of shSox2 sequence were synthesized by Gene synthesis company (Shanghai Jima pharmaceutical technology Co., Ltd.). Meanwhile, as a negative control group of an experiment, a control shRNA (shCon, SEQ ID NO: 4) expression sequence which can not target any gene is designed, and an upper (SEQ ID NO: 5) primer DNA segment and a lower (SEQ ID NO: 6) primer DNA segment are synthesized. According to the OD value of the synthesized DNA fragment, deionized water was added to dilute the DNA concentration to 20. mu.M. The annealing reaction process comprises: mu.L of the forward primer and 5. mu.L of the reverse primer were mixed, and 5. mu.L of 10 XNEB buffer 2(10mM Bis Tris Propane-HCl, 10mM MgCl. RTM.) was added 2 1mM dithiothreitol, pH 7.0), 35. mu.L of deionized water was added to a final volume of 50. mu.L, and the mixed system was incubated in boiling water at 100 ℃ for 4min and then cooled naturally to room temperature. The upstream and downstream primers were annealed as described above to obtain a DNA fragment of shRNA template with sticky ends.
The shRNA template DNA fragment is connected to an expression vector pGPU6 (figure 1), and the connection reaction process is as follows: mu.L of the product of the annealing reaction (cohesive-end shRNA template DNA fragment), 20ng of linearized pGPU6 plasmid (purchased from Shanghai Jima pharmaceutical technology Co., Ltd.), 2. mu.L of 10 XNEB T4DNA ligase reaction mixture (New England Biolabs Inc.), 1. mu.L of NEB T4DNA ligase, and deionized water were added to a final volume of 20. mu.L, and the reaction was allowed to proceed overnight at 16 ℃.
In order to screen single colonies of E.coli carrying pGPU6-shSox2 and pGPU6-shCon plasmids, the products of the ligation reaction of shSox2 and shCon fragments with expression vectors were transformed into E.coli DH5 alpha competent cells, respectively. The operation steps are as follows: placing 25 μ L DH5 α competent cells on ice, adding 2 μ L ligation product after thawing, mixing, and ice-cooling on ice for 30 min; hot shocking at 45 deg.C for 45s, ice-cooling for 2 min; adding 1mL of non-resistant LB liquid medium, and performing shake culture at 37 ℃ for 45 min; centrifuging at 3000rpm for 5min, reserving 80 μ L of culture medium, uniformly blowing cell sediment by using a pipette, and coating a coating rod on an LB agar plate containing Ampicillin resistance; the plates were incubated overnight in an incubator at 37 ℃.
After the clone of the agar plate grows out (figure 2), a single colony of escherichia coli carrying pGPU6-shSox2 and pGPU6-shCon plasmids is selected to be cultured in 1mL of LB liquid culture medium containing Ampicillin resistance by shaking table at 37 ℃ overnight, thalli are collected, plasmid DNA is extracted by using a plasmid column type extraction kit (purchased from Shanghai Biotechnology engineering Co., Ltd.) (figure 3), and the plasmid is sent out for sequencing to further confirm that the sequence of shRNA on the plasmid is completely correct.
(2) Construction and identification of VNP20009 strain carrying pGPU6-shSox2 plasmid
The extracted pGPU6-shCon plasmid expressing the control shRNA and the pGPU6-shSox2 plasmid expressing the Sox2shRNA are respectively transformed into a VNP20009 strain and are respectively named as ConshRNA-VNP and Sox2 shRNA-VNP. The specific construction process is as follows:
competent bacteria VNP20009 were placed on ice, and after melting, transferred to a pre-cooled electric cuvette, to which 2. mu.L of plasmid was added, mixed well and ice-cooled for 1 min. The electric rotor was placed in an electric rotor under conditions set to 2500V, 200. omega. resistance, and 25. mu.F capacitance. Immediately adding 1mL of LB culture medium after electric shock is finished, gently mixing uniformly, and performing shake culture at 37 ℃ for 1 h; centrifuging at 3000rpm for 5min, reserving 80 μ L of culture medium, uniformly blowing and beating the bacterial precipitate by a pipette, and then coating the bacterial precipitate on an LB agar culture medium plate containing Ampicillin; the plates were incubated overnight in a 37 ℃ incubator.
Selecting a single colony, culturing Sox2shRNA-VNP and ConshRNA-VNP by using an LB liquid culture medium, extracting plasmids, and sending the plasmids to Shanghai Bioengineering Co., Ltd for sequencing to determine that shRNA coding sequences on the plasmids are correct. Through comparison, the shRNA expression plasmid coding sequence carried by the genetic engineering bacteria is correct.
Example 2:
MTT method for detecting inhibition effect of gene engineering bacteria on growth of various human tumor cells
The MTT method is used for detecting the inhibition effect of the genetically engineered bacteria on the growth of various human tumor cells. At 37 deg.C, 5% CO 2 The tumor cells were cultured in the incubator of (1) until the density reached 90%, then the cells were digested and collected with 0.25% trypsin, the cells were resuspended in a cell culture medium and counted, and the cell concentration was adjusted to 2.0X 10 4 Cell suspension was seeded into 96-well plates at 100. mu.L/well in 37 ℃ with 5% CO 2 The culture was carried out overnight in an incubator. After the cells adhere to the wall, the genetically engineered bacteria (1.0X 10) of the invention are added 6 cfu), the genetically engineered bacteria and the blood vessel inhibitor bevacizumab are used as a combined administration group (2.5 mug), the genetically engineered bacteria, the sunitinib (4ng) combined administration group and the docetaxel (500ng) are used as a positive drug control group, a culture solution without any drug is used as a blank control group, and the drug is diluted to a corresponding preset concentration by the culture solution. Each dilution was added to a 96-well plate at 100. mu.L/well at 37 ℃ in 5% CO 2 Incubate for 48 h. mu.L of 5mg/mL MTT was added to each well of the 96-well plate and incubation was continued for 4 h. The medium was aspirated off and dissolved by adding 100. mu.L DMSO per well. Measuring the light absorption value at the detection wavelength of 570nm by using a microplate reader, and calculating the growth inhibition rate, wherein the formula is as follows: the tumor growth inhibition (%) was (1-absorbance of administration group/absorbance of negative group) × 100%, and the experiment was independently repeated 3 times, and the obtained results were expressed as mean ± standard deviation (fig. 4).
TABLE 1 inhibition ratio (%)
Figure BDA0000941331950000061
Table 1 the results show that: when the salmonella is singly administrated, the salmonella VNP20009 has better effect of inhibiting tumor proliferation than the unmodified salmonella VNP 20009; when the salmonella and the blood vessel inhibitor bevacizumab or sunitinib are simultaneously administrated, the combined administration group has better anti-tumor effect than the salmonella alone, and the anti-tumor effect of the combined administration group is better than that of the positive drug docetaxel.
Figure IDA0000941332030000011
Figure IDA0000941332030000021

Claims (6)

1. A construction method of a genetic engineering bacterium for inhibiting tumor growth comprises the following steps: the shRNA expression gene of the target gene Sox2 is connected to a vector plasmid pGPU6 to obtain a recombinant expression plasmid, and the recombinant expression plasmid is transformed to attenuated salmonella typhimurium VNP20009, wherein the upstream primer of the shRNA expression of the target gene Sox2 is SEQ ID NO: 2, the downstream primer is SEQ ID NO: 3.
2. a tumor therapeutic drug, characterized in that the active ingredient comprises the genetically engineered bacterium which is constructed by the method of claim 1 and inhibits the growth of tumors.
3. A tumor therapeutic drug, characterized in that the active ingredients thereof comprise the genetically engineered bacterium for inhibiting tumor growth and the angiogenesis inhibitor constructed by the method of claim 1.
4. A medicament as claimed in claim 2 or 3, wherein the neoplasm is primary or secondary cancer, melanoma, hemangioma or sarcoma derived from the head and neck, brain, thyroid, oesophagus, pancreas, lung, liver, stomach, breast, kidney, gall bladder, colon or rectum, ovary, cervix, uterus, prostate, bladder or testis of a human.
5. The drug for treating tumor according to claim 3, wherein the angiogenesis inhibitor is an inhibitor for inhibiting angiogenesis around tumor, and comprises an antibody inhibitor and a protein kinase inhibitor.
6. The drug for treating tumor according to claim 5, wherein the antibody inhibitor is bevacizumab; the protein kinase inhibitor is sunitinib.
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Publication number Priority date Publication date Assignee Title
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
CN103961721A (en) * 2014-04-30 2014-08-06 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 in preparation of medicine for treating prostatic cancer
WO2016025582A2 (en) * 2014-08-12 2016-02-18 Forbes Neil S Targeting epigenetic regulators using a bacterial delivery system

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN103961721A (en) * 2014-04-30 2014-08-06 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 in preparation of medicine for treating prostatic cancer
WO2016025582A2 (en) * 2014-08-12 2016-02-18 Forbes Neil S Targeting epigenetic regulators using a bacterial delivery system

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SOX2下调表达对MDA-MB-231乳腺癌细胞β-catenin与转录中介因子γ的影响;李雄武 等;《第三军医大学学报》;20151124;第38卷;摘要、第60页左栏第2段 *

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