CN105802899B - A genetically engineered bacterium that inhibits tumor growth and its construction method and application - Google Patents

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

A genetically engineered bacterium that inhibits tumor growth and its construction method and application

technical field

The invention belongs to the field of biotechnology, and more particularly, relates to a genetically engineered bacterium for inhibiting tumor growth and a construction method and application thereof.

Background technique

Cancer stem cells are the root cause of tumor growth, drug resistance, and tumor recurrence. The characteristics of cancer stem cells are: the ability to continue to proliferate, the ability of a small number of cells to form tumors in vivo, the ability to resist drugs, the ability to be cultured in vitro without serum into spheres (sphere formation), and the ability to differentiate. Many proteins that play important functions in embryonic stem cells are also highly expressed and activated in cancer stem cells. Sox2 (sex-determining region Y-box2) is a key gene for maintaining stem cell diversity, and it is highly expressed in a variety of tumor cells and drug-resistant tumor cells. shRNA (shorthairpin RNA) interference technology can efficiently reduce the expression level of targeted genes by expressing interfering RNA fragments with short hairpin structures.

Attenuated Salmonella typhimurium is a facultative anaerobic bacteria. There are many hypoxic areas in tumor tissue. Salmonella can be enriched in the hypoxic areas of tumors, and the number of colonies in the tumor is much larger than that in other tissues. Although in preclinical experiments, attenuated Salmonella VNP20009 has a good anti-tumor effect, the results of a clinical phase I study show that VNP20009 has a poor anti-tumor effect in humans, mainly as follows: it cannot effectively enrich the tumor tissue. Collective, anti-tumor effect is not significant.

Tumors grow faster than normal cells and consume large amounts of nutrients and oxygen. Tumor cells can promote the formation of peripheral blood vessels to meet the demand. Vascular inhibitors inhibit tumor growth and migration by inhibiting the formation of blood vessels around the tumor. Recent studies have found that when the vascular inhibitor Sunitinib is used to treat breast cancer, it inhibits the internal hypoxia caused by angiogenesis, and the hypoxia-inducible factor 1 (Hypoxia-inducible factor 1) promotes the increase of the proportion of cancer stem cells in the tumor. . In order to achieve better therapeutic effect, it is necessary to combine other drugs that can simultaneously target tumor cells when using vascular inhibitor drugs. When a vascular inhibitor is used in combination with a chemotherapeutic drug, since the chemotherapeutic drug has to reach the tumor cells through the microvessels, it is necessary to control the order and dose of administration reasonably, which is inconvenient to use. There are also side effects.

SUMMARY OF THE INVENTION

1. The problem to be solved

Aiming at the problems of poor enrichment ability and insignificant anti-tumor effect of the existing anti-tumor genetically engineered bacteria, the present invention provides a genetically engineered bacteria that inhibits tumor growth and a construction method and application thereof. A recombinant plasmid was constructed in the vector plasmid pGPU6, and then the recombinant plasmid was transformed into attenuated Salmonella typhimurium VNP20009 to obtain genetically engineered bacteria. The genetically engineered bacteria of the invention are safe, have less toxic and side effects, have better activity for inhibiting tumor growth, have better anti-tumor effects after being used in combination with an angiogenesis inhibitor, and meet clinical needs.

2. Technical solutions

For solving the above problems, the technical scheme of the present invention is as follows:

A genetically engineered bacterium for inhibiting tumor growth includes a host strain and a target gene transferred into the host strain, wherein the target gene is an shRNA expression gene, and the expressed shRNA sequence targets the gene Sox2.

Preferably, the host strain is attenuated Salmonella typhimurium VNP20009.

The above-mentioned method for constructing a genetically engineered bacteria that inhibits tumor growth includes the following steps: connecting the shRNA expression gene to the carrier plasmid pGPU6 to obtain a recombinant expression plasmid, and then electro-transforming the recombinant expression plasmid into attenuated Salmonella typhimurium VNP20009.

Preferably, the conditions for electrical conversion are: voltage 2500V, resistance 200Ω, and voltage 25μF.

The application of the above-mentioned genetically engineered bacteria for inhibiting tumor growth in the preparation of a tumor-treating drug.

Preferably, the genetically engineered bacteria are used in combination with an angiogenesis inhibitor when preparing a tumor drug.

Preferably, the tumor originates from human head and neck, brain, thyroid, esophagus, pancreas, lung, liver, stomach, breast, kidney, gallbladder, colon or rectum, ovary, cervix, uterus, prostate, Primary or secondary cancers of the bladder or testis, melanomas, hemangiomas, and sarcomas.

Preferably, the angiogenesis inhibitor is an inhibitor of tumor peripheral angiogenesis, including antibody inhibitors and protein kinase inhibitors.

Preferably, the antibody inhibitor is bevacizumab; the protein kinase inhibitor is sunitinib.

The genetically engineered bacteria of the present invention is an attenuated Salmonella VNP20009 carrying a shRNA expression plasmid, and the expressed shRNA sequence targets the gene Sox2.

Wherein, the genetically engineered bacteria VNP20009 contains pGPU6 plasmid, and the shRNA coding sequence targeting Sox2 expressed by the plasmid is cloned on the pGPU6 plasmid.

The method for constructing the genetically engineered bacteria of the present invention includes the following steps: first, a plurality of shRNAs with different target sequences are designed, the shRNA with the best effect of inhibiting the expression of Sox2 is obtained through experimental screening, then the DNA primers of the shRNA sequences are synthesized, and the upstream primers are Annealing reaction with the downstream primer to obtain a double-stranded DNA fragment with cohesive ends, and connecting the cohesive end into the expression plasmid pGPU6 to obtain the shRNA recombinant expression plasmid pGPU6-shSox2, and the pGPU6-shSox2 recombinant expression plasmid was electroporated into attenuated Salmonella typhimurium VNP20009, The genetically engineered strain VNP20009 was obtained by screening the resistance of Ampicillin carried by the recombinant plasmid.

The genetically engineered bacteria for inhibiting tumor growth of the present invention overcomes the shortcomings of the existing genetically engineered bacteria that cannot be effectively enriched in tumor tissue and have insignificant anti-tumor effects. The target gene Sox2 of the present invention is an important factor for maintaining the growth of embryonic stem cells, and Sox2 is positively correlated with the malignancy of various tumor cells. In order to find an effective vector system to reduce the expression level of the Sox2 gene in the tumor tissue, the present invention designed a variety of shRNA expression plasmids for the Sox2 gene, and obtained an expression plasmid with the best effect of targeting the Sox2 gene through screening. After obtaining an ideal expression plasmid, how to deliver the expression plasmid to tumor tissue in a targeted manner has become another difficult problem for the inventor. Although there are many studies on plasmid vectors, in view of the particularity of the biomedical field , it is very likely that the expression plasmid with good effect pointed out in the existing report cannot have the expected effect after being applied to the present invention, and may even have the opposite effect. For this reason, the inventor has carried out numerous theoretical analyses and experimental verifications, and finally screened and determined the attenuated Salmonella VNP20009 strain, which can deliver the expression plasmid of the present invention to tumor tissue in a targeted manner, and has good targeting properties. The advantage of low side effects. By transferring the shRNA expression plasmid targeting Sox2 into VNP20009, only a small number of colonies can be obtained to achieve good anti-tumor effect. During the experiment, the inventor unexpectedly found that when the genetically engineered bacteria of the present invention is used in combination with vascular inhibitor drugs, on the one hand, the vascular inhibitor can inhibit the angiogenesis around the tumor, inhibit the growth of the tumor, and on the other hand, the vascular inhibitor can inhibit the growth of blood vessels. The agent can generate a hypoxic environment in the tumor. At this time, the genetically engineered bacteria of the present invention can better survive in the tumor tissue, infect the tumor cells, and inhibit the growth of the tumor. Breakthroughs are important.

The genetically engineered bacteria constructed in the present invention have good tumor targeting, and can infect tumor cells in tumor tissues, release the shRNA recombinant expression plasmid pGPU6-shSox2 into the cells, target the shRNA expression of Sox2, and reduce the expression of the gene Sox2 Level. Sox2 is a factor that plays an important role in the growth of tumor stem cells. Studies have found that when the expression level of Sox2 is reduced, the growth and migration of tumor cells are inhibited, resulting in apoptosis of host tumor cells. Therefore, the genetically engineered bacteria of the present invention can be used in in the treatment of human tumors.

Clinical data show that many patients develop drug resistance gradually when treated with an antitumor drug for a long time. The simultaneous use of multiple anti-tumor drugs can reduce the possibility of tumor cells developing drug resistance and achieve better therapeutic effects. However, when multiple antitumor drugs are administered at the same time, there may be disadvantages of large side effects and insignificant antitumor effect. When the genetically engineered bacteria constructed by the present invention are used in combination with an angiogenesis inhibitor to treat tumors, on the one hand, the angiogenesis around the tumor is inhibited by the vascular inhibitor, and the supply of oxygen and nutrients to tumor cells is inhibited; Targeting, a large number of genetically engineered bacteria specifically proliferate in tumor tissue, infect tumor cells, release the shRNA recombinant expression plasmid pGPU6-shSox2, express shRNA targeting Sox2, inhibit the expression of Sox2 gene, and inhibit tumor growth . Since the genetically engineered bacteria of the present invention have high tumor targeting properties, there is no side effect when combined with an vascular inhibitor for simultaneous administration. The genetically engineered bacteria and the vascular inhibitor constructed in the present invention respectively target the cells in the tumor and the blood vessels around the tumor, and at the same time use to achieve better effect of inhibiting the growth of human solid tumors.

3. Beneficial effects

Compared with the prior art, the beneficial effects of the present invention are:

(1) The genetically engineered bacteria for inhibiting tumor growth of the present invention has strong tumor targeting, and is a novel biological drug that is safe, non-toxic and has anti-tumor activity. By using attenuated Salmonella typhimurium VNP20009 as a carrier, the shRNA recombinant expression plasmid pGPU6-shSox2 is transported to tumor cells, inhibiting the expression of gene Sox2 and inhibiting tumor growth;

(2) The shRNA sequence targeting Sox2 carried by the genetically engineered bacteria of the present invention can efficiently reduce the expression of Sox2. When the genetically engineered bacteria of the present invention are administered alone, a better antitumor effect can be obtained. When used in combination with vascular inhibitors, significant anti-tumor effects can be obtained;

(3) the genetically engineered bacteria of the present invention is easy to cultivate, and a large amount of cells can be obtained in a short time, and the genetically engineered bacteria of the present invention is high in safety;

(4) The present invention combines vascular inhibitor and genetically engineered bacteria to fight tumors, avoids the defect of combining vascular inhibitor and chemotherapeutic drugs, does not require strict administration order and dosage, has no toxic effect on human body, and attenuated Salmonella Once in the body, the tumor was enriched within a short period of time, and the attenuated Salmonella was undetectable in the blood after 2 days.

Description of drawings

Fig. 1 is the map of the expression plasmid pGPU6-shSox2 expressing the shRNA targeting Sox2;

Fig. 2 is a picture of Escherichia coli colonies carrying plasmids screened by Ampicillin (ampicillin) resistant agar plates;

Fig. 3 is the figure of the agarose gel electrophoresis of the extracted shRNA expression plasmid;

Figure 4 is a diagram showing the effect of the genetically engineered bacteria of the present invention against various tumors.

Detailed ways

The present invention can be well understood from the following examples. What is described in the examples is only used to illustrate the invention, and should not and should not limit the invention described in detail in the claims.

Example 1:

Construction of genetically engineered bacteria

(1) Construction of shRNA recombinant expression plasmid pGPU6-shSox2 targeting gene Sox2

Firstly, shRNA targeting Sox2 (shSox2, SEQ ID NO: 1) was designed according to the sequence of Sox2 gene (Gene ID: 6657), and the upstream of the shSox2 sequence (SEQ ID NO: 2) was synthesized in Gene Synthesis Company (Shanghai Gema Pharmaceutical Technology Co., Ltd.). ) and downstream (SEQ ID NO: 3) primer DNA fragments. At the same time, as the negative control group of the experiment, a control shRNA (shCon, SEQ ID NO: 4) expression sequence that cannot target any gene was designed, and the upper (SEQ ID NO: 5) and downstream (SEQ ID NO: 6) primer DNA fragments were synthesized . According to the OD value of the synthesized DNA fragments, add deionized water to dilute the DNA concentration to 20 μM. The annealing reaction process is as follows: after mixing 5 μL of upstream primer and 5 μL of downstream primer, add 5 μL of 10×NEB buffer 2 (10 mM Bis Tris Propane-HCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH 7.0), add 35 μL of deionized The final volume of water was 50 μL, and the mixed system was incubated in boiling water at 100° C. for 4 min, and then cooled to room temperature naturally. The shRNA template DNA fragments with sticky ends were obtained by annealing the upstream and downstream primers according to the above method.

The shRNA template DNA fragment was ligated to the expression vector pGPU6 (Figure 1). The process of the ligation reaction was as follows: 2 μL of the product obtained from the annealing reaction (the shRNA template DNA fragment with sticky ends) was added to the ligation reaction system, and 20 ng of linearized pGPU6 plasmid (purchased from Shanghai Gema Pharmaceutical Technology Co., Ltd.), 2 μL of 10×NEB T4 DNA ligase reaction solution (New England Biolabs Inc.), 1 μL of NEB T4 DNA ligase, supplemented with deionized water to a final volume of 20 μL, 16 °C reaction overnight.

In order to screen single colonies of E. coli carrying pGPU6-shSox2 and pGPU6-shCon plasmids, the products of ligation reaction between shSox2 and shCon fragments and expression vectors were transformed into E. coli DH5α competent cells, respectively. The operation steps are as follows: take a tube of 25 μL of DH5α competent cells and place it on ice. After it is thawed, add 2 μL of the above ligation product to it, mix well and place it in an ice bath for 30 min; Bath for 2 min; add 1 mL of non-resistant LB liquid medium, incubate at 37°C for 45 min; centrifuge at 3000 rpm for 5 min, retain 80 μL of the medium, use a pipette to pipette the cell pellet evenly, and coat with a coating rod containing Ampicillin antibody on a LB agar plate; place the plate in a 37°C incubator overnight.

After the clones grew out of the agar plates (Figure 2), single colonies of E. coli carrying pGPU6-shSox2 and pGPU6-shCon plasmids were selected and placed in 1 mL of LB liquid medium containing Ampicillin resistance, incubated at 37°C with shaking overnight, and the cells were collected. , using a plasmid column extraction kit (purchased from Shanghai Sangon Bioengineering Co., Ltd.) to extract plasmid DNA (Figure 3), and the plasmid was sent for sequencing to further confirm that the shRNA sequence on the plasmid was completely correct.

(2) Construction and identification of VNP20009 strain carrying pGPU6-shSox2 plasmid

The extracted pGPU6-shCon plasmid expressing control shRNA and pGPU6-shSox2 plasmid expressing Sox2 shRNA were electroporated into VNP20009 strain, named ConshRNA-VNP and Sox2shRNA-VNP, respectively. The specific construction process is as follows:

The competent bacteria VNP20009 was placed on ice, and after it was dissolved, it was transferred to a pre-cooled electroporation cup, 2 μL of plasmid was added to it, and the mixture was ice-bathed for 1 min. Put the electroporation cup into the electroporator, and set the conditions as voltage 2500V, resistance 200Ω, and capacitance 25μF. Immediately after the electric shock, 1 mL of LB medium was added, mixed gently, shaken at 37°C for 1 h; centrifuged at 3000 rpm for 5 min, 80 μL of medium was retained, and the bacterial pellet was uniformly pipetted and spread on LB agar containing Ampicillin culture medium plate; the plate was placed in a constant temperature incubator at 37°C for overnight incubation.

A single colony was selected, Sox2shRNA-VNP and ConshRNA-VNP were cultured in LB liquid medium, and the plasmid was extracted and sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing to confirm that the shRNA coding sequence on the plasmid was correct. After comparison, the coding sequence of the shRNA expression plasmid carried by the genetically engineered bacteria of the present invention is correct.

Example 2:

MTT assay detects the inhibitory effect of the genetically engineered bacteria of the present invention on the growth of various human tumor cells

The MTT method was used to detect the inhibitory effect of the genetically engineered bacteria of the present invention on the growth of various human tumor cells. After culturing the tumor cells to a density of 90% in a 37°C, 5% _CO2 incubator, they were harvested by 0.25% trypsinization, and the cells were resuspended in cell culture medium and counted, and the cell concentration was adjusted to 2.0× ¹⁰⁴ cells /mL, inoculate the cell suspension into 96-well plates at 100 μL per well and culture overnight in a 37°C, 5% _CO2 incubator. After the cells adhered, the genetically engineered bacteria of the present invention (1.0×10 ⁶ cfu), the genetically engineered bacteria of the present invention and the vascular inhibitor bevacizumab (2.5 μg) were combined to administer the group. The engineering bacteria and sunitinib (4ng) combined administration group and docetaxel (500ng) were used as the positive drug control group, the culture solution without any drug was used as the blank control group, and the drug was diluted with the culture solution to the corresponding predetermined concentration . Each dilution was added to a 96-well plate, 100 μL per well, and incubated at 37°C in a 5% CO ₂ incubator for 48 h. 20 μL of 5 mg/mL MTT was added to each well of the 96-well plate, and the culture was continued for 4 h. Aspirate the medium and add 100 μL of DMSO to each well to dissolve. The absorbance value was measured with a microplate reader at the detection wavelength of 570 nm, and the growth inhibition rate was calculated. The formula is as follows: tumor growth inhibition rate (%) = (1-absorbance value of administration group/absorbance value of negative group) × 100%, experimental Three independent replicates were performed and the obtained results were expressed as mean ± standard deviation (Figure 4).

Table 1 Inhibition rate (%) of the genetically engineered bacteria of the present invention on the growth of various tumors alone or in combination with vascular inhibitors

The results in Table 1 show that: when the Salmonella of the present invention is administered alone, it has a better inhibitory effect on tumor proliferation than the unmodified Salmonella VNP20009; when the Salmonella of the present invention is combined with the vascular inhibitor bevacizumab or sunitinib When administered at the same time, the antitumor effect of the combined administration group is better than that of the Salmonella of the present invention administered alone, and the antitumor effect of the combined administration group is better than that of the positive drug docetaxel.

Claims (6)

1. a construction method of the genetically engineered bacteria that suppresses tumor growth, its step comprises: the shRNA expression gene of targeting gene Sox2 is connected to carrier plasmid pGPU6 and obtains recombinant expression plasmid, then the recombinant expression plasmid is transformed into attenuated Salmonella typhimurium VNP20009, wherein the upstream primer for expression of shRNA targeting Sox2 is SEQ ID NO: 2, and the downstream primer is SEQ ID NO: 3. 2 . A tumor therapeutic drug, characterized in that its active ingredient comprises a genetically engineered bacteria that inhibits tumor growth constructed by the method of claim 1 . 3 . A tumor therapeutic drug, characterized in that its active ingredients comprise genetically engineered bacteria and angiogenesis inhibitors constructed by the method of claim 1 for inhibiting tumor growth. 4. a kind of tumor treatment medicine according to claim 2 or 3, is characterized in that, described tumor is originated from human head and neck, brain, thyroid, esophagus, pancreas, lung, liver, stomach, mammary gland , primary or secondary cancers of the kidney, gallbladder, colon or rectum, ovary, cervix, uterus, prostate, bladder or testis, melanoma, hemangioma, and sarcoma. 5 . The tumor therapeutic drug according to claim 3 , wherein the angiogenesis inhibitor is an inhibitor for inhibiting angiogenesis around the tumor, including antibody inhibitors and protein kinase inhibitors. 6 . 6 . The tumor therapeutic drug according to claim 5 , wherein the antibody inhibitor is bevacizumab; and the protein kinase inhibitor is sunitinib. 7 .

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