CN108342366B - Recombinant oncolytic gene-adenovirus of targeted cancer and construction method and application thereof - Google Patents
Recombinant oncolytic gene-adenovirus of targeted cancer and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a group of cancer-targeted recombinant oncolytic genes-adenovirus OncoAd，OncoAdRGD, and OncoAdShrkm2. The invention also discloses the recombinant oncolytic gene-adenovirus OncoAdA method for constructing RGD. shPKM2 and application thereof in preparing a medicament for treating pancreatic cancer. Recombinant oncolytic gene-adenovirus Onco of the inventionAdshPKM2, which realizes the expression silencing of a rate-limiting enzyme gene PKM2 by means of the replication of an adenovirus vector, and can play an effective role in killing pancreatic cancer.
Description
Technical Field
The invention relates to the technical field of oncolytic adenovirus, in particular to a recombinant oncolytic gene-adenovirus targeting cancer and a construction method and application thereof.
Background
Pancreatic cancer is the most malignant solid tumor, and according to global cancer ranking statistics, the incidence rate of pancreatic cancer in men ranks tenth, the incidence rate of pancreatic cancer in women ranks ninth, but the mortality rate of pancreatic cancer is third. The incidence and mortality of pancreatic cancer increases year by year, and by the year 2030, the mortality of pancreatic cancer will be the second of all cancers. The five-year survival rate of pancreatic cancer patients is less than 5%, the pancreatic cancer patients have hidden diseases due to the reasons, effective diagnosis means are lacked in the early stage, and the patients in the later stage often lose the opportunity of effective surgical treatment due to the particularity of the anatomical structure and position of pancreas. Even patients who undergo surgical resection are very easy to relapse and transfer. At present, the treatment means of pancreatic cancer mainly comprises traditional surgical treatment, radiotherapy, chemotherapy, chemoradiotherapy and emerging targeted treatment. Regardless of the regimen, improvements in patient survival are limited, and the search for new targets and methods for treating pancreatic cancer is urgent.
The targeting drug can provide a bright road for treating pancreatic cancer due to its targeting property and low toxicity. Currently, the targeted drugs for pancreatic cancer mainly comprise an EGFR (epidermal growth factor receptor) monoclonal antibody, a Her2 monoclonal antibody, a MAPK monoclonal antibody, an mTOR inhibitor, an IGF-I R inhibitor, a JAK inhibitor, a VEGF (vascular endothelial growth factor) inhibitor for inhibiting angiogenesis, endostatin, an MMPs (tumor necrosis factor) inhibitor, a DNA topoisomerase inhibitor, a PD-1 inhibitor for influencing tumor immunity and the like. However, the results show that these targeted drugs still do not have significant efficacy against pancreatic cancer, but it is encouraging that these drugs exhibit lower toxicity and better tolerability than traditional therapies.
Currently, targeted drugs aimed at altering tumor metabolism have not been reported in pancreatic cancer. Changes in tumor metabolism are considered one of ten major features and have been a focus of research. Even with sufficient oxygen content, tumors still undergo glycolysis, and the result of this energy-producing approach is the consumption of large amounts of glucose and the production of large amounts of lactic acid. This metabolic mode is known as the warburg effect. The important role of PKM2 (pyruvate kinase subtype 2) as the rate-limiting enzyme in glycolysis process in cancer development and development is also a hotspot in cancer metabolism research, and the role thereof in pancreatic cancer is rarely reported. We found that PKM2 is significantly higher in tumor tissues than in paraneoplastic tissues in pancreatic cancer patients and appears to be negatively correlated with patient prognosis. This result is consistent with the conclusions of S Calabretta, Goran Hamid Mohammad, abroad.
CTGVT is constructed by adding an anti-cancer gene to an Oncolytic Virus (OV), and is therefore an OV-gene therapy. CTGVT, as our guiding strategy, shows considerable anticancer effects in cancer therapy. The therapy can simultaneously exert the tumor killing ability of the gene and the cell replication and lysis ability of the virus. The idea of this strategy is to utilize the ability of oncolytic virus to specifically target tumor cells, introduce foreign genes (tumor killing genes) into tumor cells, and express a large amount of killing proteins along with the amplification of the virus, thereby achieving the purpose of killing tumor cells.
Disclosure of Invention
After further exploring the role of PKM2 in pancreatic cancer, the invention discovers that after the expression of PKM2 is knocked out in cells, the growth of the cells is inhibited, and the metastasis of the cells is greatly reduced. Next, subcutaneous tumorigenic experiments in nude mice using cells expressing PKM 2-low showed that knockdown PKM2 inhibited the growth of subcutaneous transplanted tumors in nude mice and inhibited liver metastasis, consistent with in vitro experiments. It is shown that PKM2 plays an important role in the growth, metastasis, and tumor formation in pancreatic cancer. Therefore, the inventor tries to take PKM2 as a target to develop a new targeted drug so as to obtain more efficient and stable curative effect and find new direction and suggestion for treating pancreatic cancer.
In view of the above, in a first aspect of the present invention, there is provided a set of cancer-targeting recombinant oncolytic gene-adenoviruses.
Recombinant oncolytic gene-adenovirus Onco for targeting cancerAdThe recombinant oncolytic gene-adenovirus OncoAdThe viral E1A gene was driven by the survivin promoter, while the E1B gene region was knocked out of the viral genome.
Recombinant oncolytic gene-adenovirus Onco for targeting cancerAdRGD, the recombinant oncolytic gene-adenovirus OncoAdRGD is in the recombinant oncolytic-gene adenovirus OncoAdCarrying the fiber region of the modified RGD.
Recombinant oncolytic gene-adenovirus Onco for targeting cancerAdRGD, shPKM2, the recombinant oncolytic gene-adenovirus OncoAdThe recombinant oncolytic gene-adenovirus Onco is RGD.shPKM2AdOn the basis of RGD, siRNA interference sequence interfering with the expression of rate-limiting enzyme gene is inserted.
According to the invention, the rate-limiting enzyme is PKM 2.
According to the invention, the siRNA interference sequence for interfering the expression of the rate-limiting enzyme gene is a PKM2shRNA expression cassette.
In a second aspect of the invention, the recombinant oncolytic gene-adenovirus Onco is providedAdThe construction method of the RGD.shPKM2 comprises the following steps:
step one, construction of pShuttle-pSur-E1A-delta E1B: pShuttle plasmid in which survivin min is used as a promoter and E1B is knocked out. Carrying out XhoI on pZd 55-AFP-E1A-delta E1B, carrying out double enzyme digestion on SnaBI and recovering a large fragment; designing a primer pssp, carrying out PCR amplification by using pAd. SP-E1A (delta 24) -delta E1B as a template to obtain a survivin promoter, carrying out XhoI and SnaBI double enzyme digestion on a PCR product, recovering fragments, connecting with a large fragment, converting, and cloning to obtain pZd 55-pSur-E1A-delta E1B; carrying out XhoI and MfeI enzyme digestion on the obtained pZd 55-pSur-E1A-delta E1B to recover a small fragment, and connecting the small fragment with a large fragment which is obtained by carrying out XhoI and MfeI enzyme digestion on pSW-X to obtain pShuttle-pSur-E1A-delta E1B;
step two, construction of pShuttle-pSur-E1A-delta E1B-shPKM 2: connecting the pShuttle-pSur-E1A-delta E1B obtained in the step one with a PKM2shRNA expression cassette with a BglII enzyme cutting site to obtain pShuttle-pSur-E1A-delta E1B-shPKM 2;
step three, constructing a plasmid pAdEasy-RGD-pSur-E1A-delta E1B-shPKM 2: an oncolytic adenovirus is constructed by a pAdEasy-1-RGD system, a shuttle plasmid pShuttle-pSur-E1A-delta E1B-shPKM2 containing a therapeutic gene expression frame is cut by PmeI, a BJ5183 competent cell containing the pAdEasy-1-RGD plasmid is transformed, and the plasmid pAdEasy-RGD-pSur-E1A-delta E1B-shPKM2 is obtained by screening and identifying kanamycin;
step four, recombinant oncolytic adenovirus OncoAdConstruction of shrpkm 2: PacI enzyme digestion step three plasmid pAdEasy-RGD-pSur-E1A-delta E1B-shPKM2, and transfection of the fragment into human embryonic kidney cell HEK-293 by using Effectene transfection reagent to generate recombinant oncolytic gene-adenovirus Onco targeted to cancersAd.RGD.shPKM2。
In a third aspect of the invention, the recombinant oncolytic gene-adenovirus Onco is providedAd，OncoAdRGD, and OncoAdApplication of shPKM2 in preparing a medicament for treating pancreatic cancer.
In a fourth aspect of the invention, a pharmaceutical composition for treating pancreatic cancer is provided, and the pharmaceutical composition comprises the cancer-targeted recombinant oncolytic gene-adenovirus OncoAd，OncoAdRGD, and OncoAdAny one or more of rgd.
The invention has the beneficial effects that: firstly, the successful interference of the rate-limiting enzyme PKM2 is realized by inserting the interference rate-limiting enzyme gene shPKM2 into a replication type oncolytic adenovirus and replicating an adenovirus vector. Secondly, in the preparation of pancreatic cancer drugs, a targeted drug aiming at changing pancreatic cancer metabolism is realized. Thirdly, the obtained recombinant oncolytic adenovirus OncoAdThe shPKM2 has low toxicity and good pancreatic cancer resistance.
Drawings
FIG. 1 is OncoAd(abbreviated as O in the figure)Ad)，OncoAdRGD (abbreviated as O in the figure)AdR), recombinant oncolytic Gene-adenovirus OncoAdRgd. shpkm2 (abbreviated as O in the figure)AdA schematic representation of the construction of r.shpkm2).
FIG. 2 is a schematic representation of the crystal violet cytotoxicity assay of example 2.
FIG. 3 is another schematic representation of the crystal violet cytotoxicity assay of example 2.
Figure 4 is a schematic of the cck-8 cytotoxicity assay of example 2.
Figure 5 is another schematic of the cck-8 cytotoxicity assay of example 2.
FIG. 6 is a schematic representation of the experiment for inhibiting the growth of PANC-1 cell transplanted tumors in nude mice according to example 3.
FIG. 7 is a schematic representation of the apoptosis experiment of example 4.
FIG. 8 is a schematic representation of the autophagy assay of example 5.
Detailed Description
The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. Unless otherwise indicated, the techniques employed in the present invention are conventional in the art, such as molecular cloning techniques, microbiological techniques, cell biology techniques, and the like.
Example 1
Recombinant oncolytic gene-adenovirus Onco of the inventionAdThe construction method of the RGD.shPKM2 comprises the following steps:
step one, construction of pShuttle-pSur-E1A-delta E1B
The plasmid pShuttle which takes survivin min as a promoter and E1B for knockout carries out XhoI on pZd 55-AFP-E1A-delta E1B (Complete operation of hepatomas using an on-bacterial adenovirus controlling AFP promoter controlling E1A and an E1B deletion to drive IL-24 expressor cancer Gene Ther 2012,19(9):619-629), and large fragments are recovered by double digestion of SnaBI; designing a primer pssp, carrying out PCR amplification by taking pShuttle-pSur-delta 24-delta E1B (Xu HN, Huang WD, Cai Y, Ding M, Gu JF, Wei N, Sun LY, Cao X, Li HG, Zhang KJ, Liu XY (2011) HCCS1-arm, square-regulated on-colytic enzymatic antigen for laser targeting gene a Cancer targeting gene-heavy-thermal targeting. mol Cancer 10,133) as a template to obtain a survivin promoter, carrying out XhoI and SnaBI double digestion on a PCR product, recovering a fragment, connecting a large fragment, transforming, and cloning to obtain pZd 55-pSur-E1A-delta E1B; carrying out XhoI and MfeI enzymatic cleavage on the obtained pZd 55-pSur-E1A-delta E1B to recover small fragments, and connecting the small fragments with large fragments which are obtained by carrying out XhoI and MfeI enzymatic cleavage on pSW-X (obtained according to a pSW-X construction method disclosed by Chinese patent CN 106190992A) to obtain pShuttle-pSur-E1A-delta E1B;
(1) the primer sequences for pssp are as follows:
f:5’-3’TAATACTGGTACCGCGGCCGCCTCGAGCGCGTTCTTTGA(SEQ ID NO：1)
r:5’-3’GTCTCATTTTCAGTACGTATGCCGCCGCCGC(SEQ ID NO：2)
(2) the PCR reaction system is as follows:
the kod-plus neo PCR enzyme was used, and three-step PCR was performed at 94 ℃, 2min, 98 ℃, 10s, 65 ℃, 30s, 68 ℃, 15s, 68 ℃, 5min, 30 cycles.
Step two, construction of pShuttle-pSur-E1A-delta E1B-shPKM2
A PKM 2-specific siRNA target sequence (CATCTACCACTTGCAATTA (SEQ ID NO: 3)) with confirmed knockout efficiency was ligated into pLVX-shRNA1 vector (Yang Y, Yin X, Yang H, Xu, Y. (2015) Histon Demethyl LSD2 acids as an E3Ubiquitin ligand and inhibitors Cancer Cell Growth through protein Degradation of OGT. mol. cell.58,47-59) using BamHI and EcoRI, inserted into U6 promoter (Yang Y, Yin X, Yang H, Xu, Y. (2015) Histon Demethyl LSD2 acids an E3Ubiquitin ligand and inhibitors Cell Degradation of Growth G.19. cell.47-59). And then obtaining a PKM2shRNA expression frame with a BglII enzyme cutting site by PCR amplification and addition of BglII enzyme. Wherein, the PCR reaction system is as follows: KOD-Plus-Neo PCR enzyme was used for 30 cycles using three-step PCR at 94 ℃, 2min, 98 ℃, 10s, 60 ℃, 30s, 68 ℃, 15s, 68 ℃, 5 min. And then, connecting the PKM2shRNA expression cassette with the BglII enzyme cutting site with the pShuttle-pSur-E1A-delta E1B obtained in the first step to obtain pShuttle-pSur-E1A-delta E1B-shPKM 2.
Step three, constructing a plasmid pAdEasy-RGD-pSur-E1A-delta E1B-shPKM2
Oncolytic adenovirus is constructed by pAdEasy-1-RGD system, a shuttle plasmid pShuttle-pSur-E1A-delta E1B-shpKM2 containing a therapeutic gene expression frame is cut by PmeI, a BJ5183 competent cell (yu y. RGD-modified oncotic adenovirus ex inhibited cell cytotoxic effect on-negative blank holder-inhibitor cells) containing pAdEasy-1-RGD plasmid is transformed, and plasmid pAdEasy-RGD-E1A-delta E1B-shpMM 2 is obtained by kanamycin screening and identification.
Step four, recombinant oncolytic gene-adenovirus Onco for targeting cancerAdConstruction of shPKM2 under RGD
PacI restriction step three plasmid pAdEasy-RGD-pSur-E1A-delta E1B-shPKM2, and the fragment was transfected into human embryonic kidney cell HEK-293 (purchased from ATCC in USA) by using Effectene transfection reagent to generate recombinant oncolytic gene-adenovirus Onco targeted to cancerAdRgd. shpkm2 (hereinafter referred to as O for short)AdShpkm2), gene-virus structure diagram see figure 1.
By a similar method, the plasmid pShuttle-pSur-E1A-delta E1B obtained in the step one is respectively recombined with pAdEasy-E3 and pAdEasy-1-RGD system (obtained according to the construction method of pSW-X disclosed by Chinese patent CN 106190992A), and the control virus Onco is obtained after the control virus Onco is packaged by human embryonic kidney cells HEK-293Ad(all: OncoAd. pSur-E1A-. DELTA.E 1B, which recombinant gene-adenovirus drives the viral E1A gene by survivin promoter while removing the E1B gene region from the viral genome) and OncoAdRGD (collectively: Oncoad. pSur-E1A- Δ E1B-fiber (RGD), the recombinant gene-adenovirus carrying the fiber region modifying RGD). The gene-virus structure is shown in FIG. 1.
Example 2 recombinant oncolytic Gene-adenovirus OncoAdCytotoxicity assay for rgd. shpkm2
The recombinant oncolytic Gene-adenovirus O obtained in example 1AdshPKM2 and unloaded virus OncoAd(hereinafter referred to as "O")Ad)，OncoAdRGD (hereinafter referred to as O)AdR), cytotoxicity experiments were performed. O isAd，OAdR is an empty virus, as a control, its construction method and O of example 1AdThe same procedure was used for constructing the r.shpkm2 (as shown in fig. 1), which has an anti-cancer effect only by oncolytic virus without therapeutic gene, in order to demonstrate that gene-loaded oncolytic virus has a better anti-cancer effect than unloaded virus.
Two experimental methods of crystal violet and cck-8 are adopted to verify the cytotoxic and toxic effects of the recombinant oncolytic gene-adenovirus on pancreatic normal cells, pancreatic cancer cells, breast cancer cells MCF-7 and bladder cancer cells T24. The procedures for the handling of crystal violet and cck-8 are conventional in the art, and reference is made specifically to the following documents:
the crystal violet test method is described in Zhang KJ, Zhang J, Wu YM, Qian J, Liu XJ, Yan LC, et al, complete operation of hepatomas using an on-polar endothermic alpha P promoter controlling E1A and an E1B deletion to drive IL-24 expression cancer Gene Ther 2012,19(9):619 and 629. The results are shown in FIG. 2.
Cck-8 method refer to Yuan SJ, Fang XL, Xu YN, Ni AM, Liu XY, Chu L. an oncotic adenvirus which express the HAb18 and interleukin 24 genes expressed antibody activity in a cytolytic cell. oncotarget, 2016. The results are shown in FIG. 3.
All experiments were repeated three times.
As can be seen from FIG. 2, the cell survival rate of pancreatic normal cells hTERT-HPNE does not change much with the increase of multiplicity of infection (MOI), OAd，OAd.R，OAdShpkm2 has little toxicity to pancreatic normal cells hTERT-HPNE; the cell survival rate of pancreatic cancer cells PANC-1, BxPC-3 and AsPC-1 is lower and lower.
As can be seen from FIG. 3, the cell survival rates of the breast cancer cell MCF-7 and the bladder cancer cell T24 decreased with the increase of the multiplicity of infection (MOI), but were much lower than those of the pancreatic cancer cell in FIG. 2, OAd.R，OAdShpkm2 did not differentiate in the extent of killing of breast cancer cells MCF-7 and bladder cancer cells T24.
As can be seen from FIG. 4, O increases with the number of daysAdThe killing capacity of the shPKM2 to pancreatic cancer cells is obviously greater than that of unloaded virus OAdR and OAdIn which O isAdThe killing ability of R to pancreatic cancer cells is significantly greater than OAd；OAd，OAd.R，OAdshPKM2 has little toxicity to normal cell hTERT-HPNE.
As can be seen from FIG. 5, O increases with the number of daysAdshPKM2 killing ability against breast and bladder cancer cells and empty-loading virus OAdR has no significant difference, OAd.R.Killing effect of shPKM2 on breast cancer cells and bladder cancer cells other than pancreatic cancer cells and unloaded virus OAdR is in agreement.
And (4) experimental conclusion:
1、OAdthe killing capacity of the shPKM2 to pancreatic cancer cells is obviously greater than that of unloaded virus OAdR and OAdBut has no toxicity to normal cells hTERT-HPNE;
2、OAdshpkm2 killing effect in cancer cells other than pancreatic cancer and unloaded virus OAdR is in agreement.
Due to the recombinant oncolytic gene-adenovirus OncoAdThe killing effect of shpkm2 on cancer cells other than pancreatic cancer cells is not significant, and therefore subsequent experiments are only performed on pancreatic cancer cells.
Example 3 recombinant oncolytic Gene-adenovirus OncoAdGrowth experiment of PANC-1 cell-inhibiting nude mouse transplanted tumor by RGD.shPKM2
The recombinant oncolytic Gene-adenovirus O obtained in example 1AdshPKM2 and empty virus OAd，OAdR for experiments inhibiting the growth of nude mouse transplantable tumors.
The procedures for experiments for inhibiting the growth of transplanted tumor in nude mice are conventional in the art, and are described in detail in Yang Y, Xu H, Shen J, Yang Y, Wu S, Xiao J, Xu Y, Liu XY and Chu L.RGD-modified oncogenic Adenoviral expressed potential effector on CAR-negative blade holder-initiating cells, cell death & disease.2015; e 1760. All experiments were repeated three times.
Wherein: FIG. 6(A) shows OAdThe growth degree of the transplanted tumor of the PANC-1 cell nude mouse of the R.shPKM2 is obviously higher than that of the unloaded virus OAd，OAdR; FIG. 6(B) is a photograph of a tumor; FIG. 6(C) shows a diagram of OAdshPKM2-treated tumors weigh significantly less than unloaded virus OAd，OAdR; FIG. 6(D) shows detection of OAdEffect of shpkm2 on silencing of PKM2 expression.
And (4) conclusion: intratumoral injection of OAdshPKM2 can effectively inhibit the growth of pancreatic cancer cells PANC-1 nude mouse transplanted tumors.
Example 4 recombinant oncolytic Gene-adenovirus OncoAdApoptosis assay of RGD. shPKM2
The recombinant oncolytic Gene-adenovirus O obtained in example 1AdshPKM2 and empty virus OAd.R、OAdApoptosis experiments were performed. All experiments were repeated three times.
Wherein: FIG. 7(A) shows OAdThe percentage of apoptotic cells in shPKM2-treated cells was higher than that of unloaded virus OAd.R、OAdA treatment group; FIG. 7(B) is a view showing OAdShpkm 2-treated cells activated caspase apoptotic signaling pathway to a greater extent than unloaded virus OAd.R、OAdTreatment group intensity; FIGS. 7(C) and (D) show intratumoral injection of OAdShpkm2 is capable of inducing apoptosis in pancreatic cancer cells PANC-1 nude mouse transplantable tumors, and OAdThe degree of apoptosis in the r.shpkm2 injected tumors was more significant.
Flow and western blot and tunel staining conclusions: o isAdThe shPKM2 can induce the apoptosis of pancreatic cancer cells PANC-1, and also induce the apoptosis in the subcutaneous transplantation tumor of pancreatic cancer cells PANC-1 nude mice. O isAdThe ability of shPKM2 to induce pancreatic cancer cell PANC-1 to undergo apoptosis in vitro and in vivo is obviously greater than that of unloaded virus OAd.R、OAd。
Example 5 recombinant oncolytic Gene-adenovirus OncoAdAutophagy assay for rgd. shpkm2
The recombinant oncolytic Gene-adenovirus O obtained in example 1AdshPKM2 and empty virus OAd.R、OAdDetection of key proteins in the autophagy-related signaling pathway was performed.
FIG. 8(A) shows the detection of a marker protein for autophagy, and the results show that OAdShpkm 2-treated cells autophagy to a lesser extent than the unloaded virus OAd.R、OAdA treatment group; FIG. 8(B) shows intratumoral injection of OAdThe shPKM2 can effectively inhibit the autophagy degree of pancreatic cancer cell PANC-1 nude mouse transplantation tumor.
Conclusion of autophagy experiments: o isAdshPKM2 can inhibit pro-kinaseThe autophagy phenomenon of pancreatic cancer cell PANC-1 growth can also inhibit the autophagy degree of pancreatic cancer cell PANC-1 nude mouse subcutaneous transplantation tumor, and the autophagy inhibition capability of the pancreatic cancer cell PANC-1 nude mouse is obviously greater than that of the unloaded virus OAd.R、OAd。
SEQUENCE LISTING
<110> Shanghai Xiyuan Biotechnology Co., Ltd
<120> cancer-targeted recombinant oncolytic gene-adenovirus and construction method and application thereof
<130> 171001
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 39
<212> DNA
<213> Artificial Synthesis
<400> 1
taatactggt accgcggccg cctcgagcgc gttctttga 39
<210> 2
<211> 31
<212> DNA
<213> Artificial Synthesis
<400> 2
gtctcatttt cagtacgtat gccgccgccg c 31
<210> 3
<211> 19
<212> DNA
<213> PKM 2-specific siRNA target sequence with knockout efficiency
<400> 3
catctaccac ttgcaatta 19
Claims (1)
1. Recombinant oncolytic gene-adenovirus Onco for targeting cancerAdThe application of the shPKM2 in preparing the medicine for inhibiting autophagy of pancreatic cancer cells is characterized in that the recombinant oncolytic gene-adenovirus OncoAdThe construction method of the RGD. shPKM2 comprises the following steps:
step one, construction of pShuttle-pSur-E1A-delta E1B: carrying out XhoI on pZd 55-AFP-E1A-delta E1B by using survivin min as a promoter and E1B to knock out pShuttle plasmid, and carrying out double enzyme digestion by SnaBI to recover a large fragment; designing a primer, carrying out PCR amplification by taking pAd, SP-E1A (Δ 24) and Δ E1B as templates to obtain a survivin promoter, carrying out XhoI and SnaBI double enzyme digestion on a PCR product, recovering fragments, connecting the fragments with a large fragment, and carrying out transformation and cloning to obtain pZd55-pSur-E1A- Δ E1B; carrying out XhoI and MfeI enzyme digestion on the obtained pZd 55-pSur-E1A-delta E1B to recover a small fragment, and connecting the small fragment with a large fragment which is obtained by carrying out XhoI and MfeI enzyme digestion on pSW-X to obtain pShuttle-pSur-E1A-delta E1B;
step two, construction of pShuttle-pSur-E1A-delta E1B-shPKM 2: connecting the pShuttle-pSur-E1A-delta E1B obtained in the first step with a PKM2shRNA expression frame with a BglII enzyme cutting site to obtain pShuttle-pSur-E1A-delta E1B-shPKM2, wherein the PKM2shRNA expression frame is a target sequence shown in SEQ ID NO: 3, an interference fragment;
step three, constructing a plasmid pAdEasy-RGD-pSur-E1A-delta E1B-shPKM 2: an oncolytic adenovirus is constructed by a pAdEasy-1-RGD system, a shuttle plasmid pShuttle-pSur-E1A-delta E1B-shPKM2 containing a therapeutic gene expression frame is cut by PmeI, a BJ5183 competent cell containing the pAdEasy-1-RGD plasmid is transformed, and the plasmid pAdEasy-RGD-pSur-E1A-delta E1B-shPKM2 is obtained by screening and identifying kanamycin;
step four, recombinant oncolytic adenovirus OncoAdConstruction of shrpkm 2: PacI enzyme digestion step III plasmid pAdEasy-RGD-pSur-E1A-delta E1B-shPKM2, and the enzyme digestion fragment is transfected into human embryonic kidney cells HEK-293 by using Effectene transfection reagent to generate the recombinant oncolytic gene-adenovirus Onco targeted to cancersAd.RGD.shPKM2。
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| CN111500632B (en) * | 2020-04-28 | 2023-09-15 | 浙江理工大学绍兴生物医药研究院有限公司 | Oncolytic adenovirus construction for expressing ST13 and TRAIL and application thereof |
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