CN110699380A

CN110699380A - EV71 virus nucleic acid plasmid and construction method and application thereof

Info

Publication number: CN110699380A
Application number: CN201910832157.3A
Authority: CN
Inventors: 王长兵; 田新贵; 游爱萍; 刘文宽; 周志超; 李潇; 周荣
Original assignee: First Affiliated Hospital of Guangzhou Medical University
Current assignee: First Affiliated Hospital of Guangzhou Medical University
Priority date: 2019-09-04
Filing date: 2019-09-04
Publication date: 2020-01-17
Anticipated expiration: 2039-09-04
Also published as: CN110699380B

Abstract

The invention discloses an EV71 virus nucleic acid plasmid, wherein the EV71 virus nucleic acid plasmid comprises an EV71 virus full-length cDNA sequence and a eukaryotic expression plasmid sequence. The invention also provides a construction method of the EV71 virus nucleic acid plasmid, and the virus nucleic acid plasmid can package live viruses in tumor cells and trigger apoptosis of the tumor cells. The invention also provides application of the EV71 virus nucleic acid plasmid in preparing a solid tumor inhibiting medicine. The EV71 virus nucleic acid plasmid can enter tumor cells through a delivery vehicle to package live viruses, so that apoptosis of the tumor cells is triggered, and the virus oncolytic function is effectively improved.

Description

EV71 virus nucleic acid plasmid and construction method and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to an EV71 virus nucleic acid plasmid, a construction method of the EV71 virus nucleic acid plasmid and application of the EV71 virus nucleic acid plasmid in preparation of a solid tumor inhibiting drug.

Background

At present, most of cancer treatments adopt traditional methods of operation, radiotherapy and chemotherapy, and the traditional cancer treatment technology has the defect of great side effect and seriously influences the life quality of cancer patients. Especially when the patients develop to the middle and late stage of cancer, the traditional treatment technology has insignificant effect and poorer prognosis, and serious side effects also influence the life quality of the patients. At present, the immune node technology is adopted for treating cancer, and a certain treatment effect is achieved. The side effect is small, the effective rate can reach 50% in tumor patients with high expression of PD-1 and PD-L1 protein, and the survival quality of the patients is effectively improved. However, in recent years, it has been found that many cancer patients develop drug resistance to PD-1 mab drugs, especially for more than 1 year. In general, cancer disease is an evolutionarily complex disease that cannot be addressed by one treatment method alone.

Oncolytic viruses have been developed for many years and can be regarded as a novel therapeutic approach to the treatment of tumors, which exploits the direct apoptotic tumor cell function of viruses and the role of immunostimulation. However, the traditional oncolytic virus treatment method has some bottleneck problems, such as immune barrier, drug resistance and the like. Due to the natural immune barrier of the human body, the oncolytic virus is recognized by immune substances of the body once entering the body, such as neutralizing antibodies against virus and various immune cells, the virus is combined by the neutralizing antibodies or phagocytized by the immune cells before reaching tumor cells, and only a very small amount of the virus can enter the tumor cells to exert the effect. The human immune system greatly reduces the efficacy of oncolytic viruses. In addition, oncolytic viruses require entry into tumor cells, necessarily via receptor proteins present on the tumor cells. Tumor cells are naturally evolving variant cells, which soon evolve tumor cells lacking the oncolytic virus receptor protein, at which point the oncolytic virus cannot invade the variant tumor cells, thereby generating drug resistance.

Disclosure of Invention

An object of the present invention is to provide an EV71 virus nucleic acid plasmid capable of apoptosis of tumor cells, which addresses the above technical problems.

The invention also aims to provide a construction method of the EV71 virus nucleic acid plasmid.

Still another object of the present invention is to provide the use of the EV71 virus nucleic acid plasmid.

In order to achieve the above object, the present invention provides an EV71 virus nucleic acid plasmid, wherein the EV71 virus nucleic acid plasmid comprises an EV71 virus full-length cDNA sequence and a eukaryotic expression plasmid sequence. In addition, the invention also provides a construction method of the EV71 virus nucleic acid plasmid, which comprises the following steps: extracting EV71 virus RNA nucleic acid, reversely transcribing the EV71 virus RNA into cDNA, and connecting the full-length sequence of EV71 virus cDNA to a eukaryotic plasmid vector by using a homologous recombination method so as to construct the EV71 virus nucleic acid plasmid.

Preferably, the specific steps of the construction method are as follows:

step 1: extracting RNA of EV71 virus;

step 2: reverse transcribing RNA from EV71 virus into cDNA;

and step 3: amplifying a cDNA fragment of EV71 virus by using EV71 virus primers, carrying out electrophoresis on the amplified product, and then recovering an amplified EV71 virus cDNA nucleic acid fragment;

and 4, step 4: amplifying DNA segments of eukaryotic expression plasmids by using primers of the eukaryotic expression plasmids, electrophoretically amplifying products, and then recovering amplified eukaryotic expression plasmid nucleic acid segments;

and 5: connecting the EV71 virus cDNA nucleic acid fragment obtained in the step 3 with the eukaryotic expression plasmid nucleic acid fragment obtained in the step 4, and transforming the connection product into competent bacteria to obtain transformed bacteria;

step 6: the cultured transformed bacteria were plated on LB agar plates containing ampicillin, monoclonal bacteria were selected, and EV71 virus nucleic acid plasmid bacteria that were successfully ligated were selected and identified.

And 7: culturing a large amount of successfully connected EV71 virus nucleic acid plasmid bacteria, extracting EV71 virus nucleic acid plasmids, performing transfection and identification on the extracted EV71 virus nucleic acid plasmids, adding a preservation solution, and preserving at-20 ℃.

Preferably, the EV71 virus of the invention comprises subtype A, B, C, most preferably EV71 virus type C.

Preferably, the eukaryotic expression plasmid of the present invention comprises pcDNA-3.1, pcDNA-3.1-E, pcDNA3.1/Zeo (+/-), pcDNA4, pcDNA4/HisMAX, pRc/RSV, and most preferably pcDNA-3.1.

The invention also provides application of the EV71 virus nucleic acid plasmid in preparing a solid tumor inhibiting medicine.

The invention also provides application of the EV71 virus nucleic acid plasmid in preparing a medicament for preventing and/or treating tumors.

Preferably, the tumor is one or more of cervical cancer, lung cancer, esophageal cancer, breast cancer, brain glioma, melanoma, myosarcoma, gastric cancer, colon cancer, head and neck cancer, prostate cancer, ovarian cancer.

The invention also provides a delivery method for transfecting the EV71 virus nucleic acid plasmid into cells, wherein the EV71 virus nucleic acid plasmid is transfected into the cells in vitro, and the LIP3000 transfection method is mainly adopted; the EV71 virus nucleic acid plasmid is transfected to tumor cells in vivo, and liposome, nano material methods and the like are mainly adopted. Among them, the method of transfecting viral nucleic acid plasmid in vitro is preferably a liposome method.

The invention also designs a cell model of in vitro neutralizing antibody according to the characteristic that the conventional oncolytic virus is easily blocked by the in vivo neutralizing antibody, adds the EV71 virus neutralizing antibody into a cell culture solution, then respectively transfects the EV71 virus nucleic acid plasmid or adds the EV71 virus, and observes the effect difference of the two. The results show that the EV71 virus nucleic acid plasmid can package live virus in Hela cells added with EV71 virus neutralizing antibodies, and can rapidly cause apoptosis of the Hela cells. The EV71 virus cannot infect Hela cells added with neutralizing antibodies of the EV71 virus.

Taking cervical cancer as an example, the cervical cancer solid tumor animal model established by the invention has the following specific operation steps: hela cells were cultured, cells were digested, and centrifuged Hela cells were diluted to 2 x 10 with PBS solution⁷Individual/ml concentration, stored on ice. Hela cells were injected to the outside of hind limbs of Balb/c nude mice in an amount of 0.2 mL. Typically, after 2 weeks, tumor tissue in nude mice grew to 500mm³(Length and Width)²And/2) injection of the EV71 virus nucleic acid plasmid drug can be started.

The EV71 virus is a main pathogen causing hand-foot-and-mouth disease of children under 5 years old, and rarely can cause diseases in adults. The EV71 virus gene is small, the live virus can be easily replicated in tumor cells, the virus propagation speed is high, and the tumor cell apoptosis efficiency is high. Therefore, the EV71 virus is selected as a research object for constructing the oncolytic virus nucleic acid plasmid. In addition, most adults have virus neutralizing antibodies of EV71, and the EV71 virus nucleic acid plasmid packages virus in tumor cells, can display EV71 virus proteins on the surfaces of the tumor cells, and can also trigger an in vivo immune system to attack the tumor cells infected by the virus, so that secondary attack of the tumor cells is formed.

The EV71 virus nucleic acid plasmid is carried into a tumor cell by a carrier (such as liposome or nano material) and can enter the tumor cell to package a living EV71 virus, and the death of solid tumor tissues is triggered by directly cracking the tumor cell, destroying the enzyme function of the tumor cell and releasing virus protein on the surface of the tumor cell, rather than by the action of receptor protein on the tumor cell. The EV71 virus nucleic acid plasmid provided by the invention can package live viruses in most tumor cells to trigger tumor cell apoptosis, and a neutralizing antibody of the virus nucleic acid plasmid does not exist in a human body, so that the virus nucleic acid plasmid cannot be recognized and attacked by an immune system in the human body, and the oncolytic effect is ensured. The virus nucleic acid plasmid enters tumor cells by means of liposome or nanometer material, and mainly utilizes the characteristic of negative charge existing in cell membranes. The negatively charged nature of the tumor cell membrane is unlikely to be altered, and thus it cannot be evolutionarily protected against the entry of viral nucleic acid plasmids into the cell membrane. Therefore, the EV71 virus nucleic acid plasmid provided by the invention can overcome the problem of tumor tissue heterogeneity, and partially solves the problem of drug resistance caused by tumor variation.

Drawings

FIG. 1 is a schematic representation of the viability of Hela cells in the EV71 virus nucleic acid plasmid group and EV71 virus group under neutralizing antibody interference.

FIG. 2 is a graphical representation of the change in tumor volume following treatment of tumor-bearing mice with the EV71 virus nucleic acid plasmid group and the EV71 virus group.

FIG. 3 is a schematic representation of tumor weight comparison under treatment with EV71 virus nucleic acid plasmid group and EV71 virus group.

FIG. 4 is a graphical comparison of tumor inhibition rates for the EV71 virion group and the EV71 virion group.

Detailed Description

The technical solution of the present invention is further described below with reference to the following specific embodiments and the accompanying drawings, but the scope of the present invention is not limited to the following embodiments. It is noted that, unless otherwise specified, the materials, reagents and equipment used in the following examples are available or commercially available, and although some experimental procedures are not described in detail, those skilled in the art are familiar with such procedures.

Example 1: construction of oncolytic virus plasmids

Experimental materials: EV71 virus culture medium (genebank number, virus concentration: 107TCID50), PCDNA3.1 plasmid (Roche, plasmid concentration 300 ng/. mu.l), DNA/RNA extraction kit (Takara), RT kit, high-fidelity PCR enzyme Prime STAR MAX (Takara, 2X), homologous recombination kit

HD cloning (Takara Co., Ltd.), competent TOP10(Takara Co., Ltd.), primers.

The test procedure was as follows:

1.1 viral RNA extraction: RNA of EV71 virus was extracted according to the protocol of DNA/RNA extraction kit (Takara Co.).

1.2 two-step RT reaction: a denaturation reaction solution (Oligo dT primer (50. mu.M); dNTP 1. mu.l; EV71 viral RNA 2. mu.l; RNase free dH2O 6. mu.l) was prepared, incubated at 65 ℃ for 5min, and then rapidly cooled on ice. Then preparing RT reaction liquid (10 mul of the denatured reaction liquid; 4 mul of 5 XPrimerscript II Buffer; 0.5 mul of RNase inhibitor; 1 mul of Primerscript II RTase; 4.5 mul of RNase free dH2O 4.5), slowly mixing the above solutions uniformly at 42 ℃ for 60 min; and carrying out long-fragment cDNA amplification at 70 ℃ for 15min to obtain an EV71 virus cDNA template.

1.3PCR reaction, namely respectively carrying out PCR amplification on the EV71 virus cDNA and pcDNA3.1 plasmid, wherein primers for amplifying the EV71 virus cDNA are respectively a primer 1(SEQ ID NO.1) and a primer 2(SEQ ID NO. 2); the primers for amplifying the PCDNA3.1 plasmid are primer 3(SEQ ID NO.3) and primer 4(SEQ ID NO.4), respectively.

Preparation of PCR reaction solution for viral cDNA: 2. mu.l of viral cDNA; prime STAR Max (2 ×)25 μ l; 11 μ l of primer; 21 μ l of primer; dH2O 21 μ l.

Preparation of PCR reaction solution of plasmid pCDNA3.1 reaction solution: PCDNA3.1 (100-fold dilution) 2. mu.l; prime STARMax (2 ×)25 μ l; 31 μ l of primer; 41 μ l of primer; dH2O 21 μ l.

PCR reaction procedure: pre-denaturation at 98 ℃ for 3 min; then 30 circulating reactions (95 ℃ for 10s, 58 ℃ for 10s, 72 ℃ for 60s) are carried out; finally 5min at 72 ℃. The primer sequences are shown in Table 1.

TABLE 1 PCR reaction primer sequence Listing

1.4 electrophoresis: gel electrophoresis (1% agarose, 130V voltage), electrophoresis for 30 minutes, in the target position for gel cutting.

1.5 glue recovery: and (3) performing gel recovery on the PCR amplification product of the virus and the PCR amplification product of the PCDNA3.1 plasmid according to the operation instruction of the gel recovery kit.

1.6 homologous recombination:according to Takara

The HD cloning kit has the operation instruction that the product recovered from the gel is mixed with homologous recombinase according to a certain proportion and reacts for 15min at 50 ℃.

1.7 transformation: standing on ice for 30min, sucking and adding the homologous recombination product into the competence, and performing heat shock at 42 ℃ for 45 s; adding 900 μ l LB medium (without antibiotics), shaking (37 deg.C, 250rpm) and culturing for 45 min; centrifuging at 5000rpm for 5min, discarding 850 μ l of supernatant, resuspending the precipitate in the remaining liquid, plating, A + mp agar plate, and standing in 37 deg.C incubator for 12-16 h.

1.8, plasmid identification: selecting single colony, and shaking at 37 deg.C and 250rpm in 800 μ lLB culture medium for 6 hr; the plasmid was extracted using a small sample DNA extraction kit, subjected to electrophoresis test, and sequenced.

Example 2: anti-tumor cell effects of oncolytic virus plasmids.

Experimental materials: 12 tumor cells (see table 1), EV71 virus nucleic acid plasmid, lip3000 transfection reagent, DMEM medium, F12 medium, fetal bovine serum and CCK-8.

The experimental procedure was as follows:

2.1 cell culture: culturing cells, selecting the cells with good growth state (plating effect up to 90%), digesting the cells to obtain cell suspension with cell concentration of 10%⁵One/ml of the cells was added to a 96-well cell culture plate at 100. mu.l/well and cultured for 16 to 18 hours.

2.2 cell transfection: when the plating rate of the cells was 85-90%, 0.1ug of EV71 virus nucleic acid plasmid was added per well according to the protocol of lip3000 reagent instructions. Negative control group for experimental setup: only medium was added. Each group of cells was provided with 3 multiple wells. After transfection, the cell plates were placed in a 37 ℃ carbon dioxide incubator for culture, and the cells were observed daily for pathological changes.

2.3 apoptosis of test cells: and (3) culturing the cells for 72 hours, adding a CCK-8 reagent, adding 10 mu l of the reagent into each hole, gently mixing the reagent and the hole uniformly, taking out the cell culture plate after 1 hour of action, and testing the cell culture plate by using an enzyme-labeling instrument (the wavelength is 450 nm). Cell viability as OD value of transfection group/OD value of negative control group × 100%.

The results are shown in table 2, the EV71 virus nucleic acid plasmid was able to efficiently apoptosis 12 tumor cells.

TABLE 2.12 cell viability of human tumor cells after transfection of the EV71 viral plasmid

Cell line name	Tumor type	Survival rate (%)
			Hela	Human cervical cancer cell	2.5
A549	Human lung cancer cell	12.6
			U-87MG	Human astrocytoma	1.8
HGC-27	Human gastric cancer cell	8.9
			RD	Human rhabdomyosarcoma	2.3
BT-474	Human milkDuctal adenocarcinoma cell	12.3
			LoVo	Human colorectal cancer cell	16.8
A-375	Human malignant melanoma cells	17.2
			SK-MES-1	Human squamous cell lung carcinoma	9.2
MDA-MB-231	Human breast cancer cell	12.1
			TE-1	Human esophageal cancer cell	8.3
DU 145	Human prostate cancer cell	3.5

Example 3: safety testing of oncolytic virus plasmids

Experimental materials: MRC-5 cells (human embryonic lung cells), EV71 virus nucleic acid plasmids, lip3000 transfection reagent, DMEM medium and fetal bovine serum.

The experimental procedure was as follows:

3.1 cell culture: culturing MRC-5 cells, selecting the cells with good growth state (the plating effect reaches 90%), digesting the cells to prepare cell suspension with the cell concentration of 10%⁵Each cell was added to a 24-well cell culture plate at 1000. mu.l/well and cultured for 16 to 18 hours.

3.2 cell transfection: when the plating rate of the cells was 85-90%, 0.5ug of EV71 virus nucleic acid plasmid was added per well according to the protocol of lip3000 reagent instructions. Negative control group for experimental setup: only medium was added. After transfection, the cell plates were placed in a 37 carbon dioxide incubator for culture, and the cells were observed daily for pathological changes.

3.3 cytopathic and viral culture: after 72 hours of cell culture, no cytopathic effect was observed. And repeatedly freezing and thawing the cell plate for 3 times, centrifugally sucking cell culture supernatant, and transferring the cell culture supernatant to Vero cells at 37 ℃ for acting for 2 hours. Removing cell culture supernatant, adding DMEM medium, placing in a carbon dioxide incubator for 3 days, and observing cytopathic condition every day. After three days, the cell culture supernatant was aspirated, and the viral nucleic acid was extracted to test the EV71 viral nucleic acid.

The results show that: the EV71 virus nucleic acid plasmid failed to apoptosis MRC-5 cells. Cell supernatants from MRC-5 cells transfected with the EV71 viral nucleic acid plasmid did not contain live EV71 virus, demonstrating that the EV71 viral nucleic acid plasmid failed to package EV71 virus in MRC-5 cells. MRC-5 cells belong to a human normal cell system, and the EV71 virus nucleic acid plasmid has no toxicity to MRC-5.

Example 4: experimental materials for in vitro neutralizing antibody interference assay of oncolytic virus plasmid: hela cells, EV71 virus nucleic acid plasmid, EV71 virus (10)⁷TCID50), EV71 virus neutralizing antibody serum (neutralizing titer 1:512), lip3000 transfection reagent, DMEM medium, fetal bovine serum.

The experimental procedure was as follows:

4.1 cell culture: culturing Hela cells, digesting Hela cells to obtain cell suspension with cell concentration of 10⁵One/ml of the cells was added to a 96-well cell culture plate at 100. mu.l/well and cultured for 16 to 18 hours.

4.2 infection comparison experiment: when the plating rate of Hela cells is 85-90%, DMEM medium is replaced, incomplete DMEM medium is added to 100. mu.l/well, and 10. mu. lEV71 virus neutralizing antibody serum is added simultaneously (serum neutralizing titer is 1: 256). According to the method of the lip3000 reagent instructions, each0.1ug of EV71 virus nucleic acid plasmid was added to the wells. Another group of experiments, dilution of EV71 virus to 10⁶TCID50 was added to Hela cells, and 10. mu.l of virus dilution was added to each well. The experimental group was designed as a negative control. Each group of cells was provided with 3 multiple wells. After transfection, the cell plates were placed in a 37 ℃ carbon dioxide incubator for 2 hours, then all the wells were deprived of medium, DMEM medium containing 2% fetal bovine serum was added, and the cells were observed daily for pathological changes.

4.3 apoptosis of test cells: after the cells are cultured for 72 hours and the CCK-8 reagent is added to act for 1 hour, the cell culture plate is taken out to be tested by a microplate reader (the wavelength is 450 nm). Cell viability as OD value of transfection group/OD value of negative control group × 100%. The results of the experiment are shown in FIG. 1.

The test results show that: the EV71 virus nucleic acid plasmid can apoptosis Hela cells under the action of neutralizing antibodies of EV71 virus, and the natural EV71 virus cannot infect the Hela cells under the action of neutralizing antibodies and cannot cause the Hela cells to apoptosis.

Example 5: solid tumor effect test of oncolytic virus plasmid

Experimental materials: EV71 virus nucleic acid plasmid, Balb/c nude mouse, Hela cell, DMEM culture medium, fetal bovine serum, pancreatin-EDTA, TurboFect in vivo Transfection reagent, electronic balance, vernier caliper and syringe.

The experimental procedure was as follows:

5.1 establishing an animal model: culturing Hela cells, trypsinizing Hela cells, centrifuging Hela cells at 1500rpm, adding PBS to dilute Hela cells, and controlling Hela cell concentration to be 2 × 10⁷One per ml. Hela cells were injected into the hind limb side of Balb/c nude mice, 0.1ml each. A total of 15 Balb/c nude mice were injected and observed 1 time per week, and the weight and tumor size of the mice were measured each time.

5.2 tumor treatment: after 10 days, the tumor volume of the mice reached 500cm3 (length X width)²And/2), the treatment is carried out by injecting the medicament. The experimental design is as follows: an EV71 virus nucleic acid plasmid intratumoral injection group, an EV71 virus intratumoral injection group and a PBS intratumoral injection control group. EV71 viral nucleic acid plasmid according to TurboFect in vivo Transfection reagent instructions, 50ugThe EV71 virus nucleic acid plasmid was dissolved in 400. mu.l of a 5% glucose solution, and 6. mu.l of TurboFect in vivo transfection reagent was added thereto and mixed, followed by incubation at room temperature for 20 minutes. The EV71 virus nucleic acid plasmid was then injected in a volume of 50. mu.l intratumorally. Dilution of EV71 Virus to 10⁷TCID50, 50. mu.l/mouse intratumorally. PBS was injected intratumorally at 50. mu.l/mouse. Once weekly for 4 weeks. Mice were observed 1 time per week, and the body weight and tumor size of the mice were each weighed. Tumor volume is length x width²/2. The body weight and tumor volume size of the mice are shown in figure 2.

After 5.331 days, the mice were sacrificed, the subcutaneous tumors were removed, and the tumors were weighed in grams, as shown in FIG. 3. The tumor inhibition rate is calculated by the formula: tumor inhibition rate is (C-T)/C × 100%, wherein C is the average weight of the tumor in the control group (PBS control group), and T is the average weight of the tumor in other experimental groups. The tumor inhibition rates of the EV71 virus nucleic acid plasmid group and the EV71 virus group are calculated according to a tumor inhibition rate calculation formula, and are shown in figure 4.

The experimental results show that: when the tumor of the mouse is treated by inoculating the EV71 virus nucleic acid plasmid, the growth of the tumor is obviously inhibited, and the growth speed of the tumor is obviously smaller than that of a PBS control group and is obviously smaller than that of an EV71 virus experimental group. The tumor inhibition rate of the EV71 virus nucleic acid plasmid treatment group can reach 59 percent, and is obviously higher than that of the EV71 virus treatment group (12 percent).

Sequence listing

<110> Guangzhou medical university affiliated first hospital (Guangzhou respiratory center)

<120> EV71 virus nucleic acid plasmid and construction method and application thereof

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ctacccagtt agtttttcga aacccagcct agtctatgta gatgctagcg agtattaccc 3600

agccaggtac caatcacatc tcatgctcgc acagggtcac tcagaacctg gtgactgtgg 3660

tggcatcctt agatgccaac atggcgttgt cggcatagtg tctactggtg gtaatgggct 3720

cgttggcttt gctgacgtca gagacctctt gtggttagat gaagaagcta tggaacaggg 3780

cgtgtccgat tacatcaagg gtcttggaga tgcttttgga acaggcttca ctgacgcagt 3840

ctcgagggag gttgaagctc tcaagaacta tcttataggg tctgaaggag cagttgagaa 3900

aattttaaaa aatcttatta aactaatctc tgcactagtg attgtgatca ggagtgatta 3960

cgacatggtt accctcactg caaccttagc gctgataggt tgtcatggca gtccttgggc 4020

ttggattaag gccaaaacag cctccatctt aggtatccct atcgctcaaa agcagagcgc 4080

ttcctggctc aagaagttca atgacatggc caacgccgct aagggattag agtgggtttc 4140

caacaagatc agcaagttta ttgattggct taaggagaaa atagtaccag cagccaggga 4200

gaaggttgaa tttctaaaca acttgaaaca gctgccactg ctagagaatc aaatctcaaa 4260

cttggaacaa tctgctgcct cacaagagga ccttgaagtc atgtttggga atgtgtcgta 4320

tctagctcac ttctgtcgca agttccaacc gctgtacgcc acggaagcta aaagagtcta 4380

tgccctggag aagagaatga ataactatat gcagttcaag agcaaacacc gaattgaacc 4440

tgtatgtctc attattaggg gctcaccagg cactgggaag tctctagcca ctggtattat 4500

tgcccgggca atcgctgata agtaccactc tagcgtgtac tcgctcccac cagacccgga 4560

tcattttgat ggttacaagc aacaggtggt tacagtaatg gatgatttgt gtcaaaaccc 4620

cgatggtaag gatatgtctt tattctgtca gatggtatcc accgtagact tcattccacc 4680

aatggcttct ctcgaggaga agggagtttc cttcacctct aagttcgtca tcgcatccac 4740

taatgccagt aacatcatag taccaacagt gtctgattct gacgctattc gccgcaggtt 4800

ctacatggac tgtgatattg aagtgacaga ctcatacaaa acagatctag gtagactgga 4860

tgcagggcga gccgctaaac tgtgctctga aaacaacact gcaaatttta aacgttgcag 4920

cccattggtg tgtgggaagg ccatccaact tagagatagg aagtctaaag tcagatacag 4980

tgtggacaca gtggtttcag aactcattag ggagtacagc aataggtccg ccattggcaa 5040

cacaatcgag gctcttttcc aaggtccacc caagttcaga ccaattagga ttagtcttga 5100

agaaaaacca gccccagacg ctattagcga cctccttgct agcgtagata gtgaagaagt 5160

gcgccagtac tgcagggatc aaggctggat tattcctgaa gctcccacca atgtagagcg 5220

gcatcttaac agagcggtgc tcgttatgca atccatcgcc acaatagtgg cagttgtctc 5280

gttggtgtac gtcatctaca agctctttgc agggtttcag ggtgcgtatt ctggtgctcc 5340

caaacaaatg ctcaagaaac ccgctcttcg aacagcgaca gtgcagggcc cgagccttga 5400

ctttgctctc tccctactga gaaggaacat caggcaagtc caaacagacc aggggcattt 5460

caccatgttg ggtgttaggg atcgcttagc agtcctcccg cgccactcac aacctggcaa 5520

aactatttgg attgagcaca aactcgtgaa cgtccttgat gcagttgaac tggtggatga 5580

gcagggagtc aacctggaat taaccctcat cactcttgac accaacgaaa aatttaggga 5640

tatcaccaaa ttcatcccag aaaatatcag cactgctagt gatgccaccc tagtgatcaa 5700

cacggagcac atgccatcaa tgtttgtccc ggtgggtgat gttgtgcagt atggcttttt 5760

gaatctcagt ggtaagccta cccatcgtac catgatgtac aactttccta ctaaagcagg 5820

acagtgtgga ggagtggtga catctgttgg gaagattgtc ggcattcaca ttggcggcaa 5880

tggcagacaa ggtttttgcg caggtctcaa gaggagttac tttgctagtg aacaaggaga 5940

gatccagtgg gttaagccca ataaagaaac tggaagactc aacatcaatg gaccaacccg 6000

tactaagttg gaacctagtg tattccatga catcttcgag ggaagtaaag aaccagctgt 6060

cttgcacagt aaagaccccc gacttgaagt agatttcgaa caggccctgt tctctaagta 6120

tgtgggaaac acactacatg agcctgacga gtacatcaag gaagcagctc ttcattatgc 6180

aaaccaatta aagcaattag aaattaatac ctctcagatg agcatggagg aggcctgcta 6240

cggtactgag aatcttgagg ctattgatct tcacactagt gcaggttacc cctatagtgc 6300

cctggggata aagaaaagag acatcttaga ccctaccacc agggacgtga gtaaaatgaa 6360

gttttacatg gacaagtatg gtcttgatct tccttactcc acttatgtca aggatgagct 6420

gcgctcgatt gacaaaatca agaaagggaa gtcccgcctg atcgaggcca gtagtctaaa 6480

tgattcagtg tacctcagaa tggctttcgg acatttgtat gaggctttcc atgcaaatcc 6540

tgggacgata actggatcgg ctgtggggtg taaccctgac acattttgga gcaagctgcc 6600

aattttgctc cctggttcac tctttgcctt tgactactca ggttatgacg ccagccttag 6660

ccctgtctgg ttcagagcat tagaattggt tcttagggag gtgggttata gtgaagaggc 6720

aatttcactc attgagggaa tcaaccacac acatcatgtg tatcgtaata agacctattg 6780

cgtgcttggt gggatgccct ctggttgttc aggaacatcc atcttcaact caatgatcaa 6840

caacattatt atcagagcac tgctcataaa aacatttaag ggcattgatt tggatgaact 6900

caatatggtt gcttatggag acgatgtgct cgctagctat cccttcccaa ttgattgctt 6960

ggaactagca aagactggta aggagtatgg tctaactatg actcctgctg ataaatctcc 7020

ttgctttaat gaggtcaatt ggggtaatgc gaccttcctc aaaaggggct tcttgcccga 7080

tgaacagttt ccatttttga ttcaccccac tatgccaatg agagagatcc atgagtctat 7140

tcgatggacc aaggacgcac gaaacactca agatcatgta cgatccttgt gcctcttagc 7200

atggcataat ggtaagcaag aatatgagaa gtttgtgagc acaattaggt ctgtcccagt 7260

aggaagagcg ctggctattc caaattatga aaatcttaga cgcaattggc tcgagttatt 7320

ttagaggtta cacatacctc aaccccacca gaaatctggt cgtgaatatg actggtgggg 7380

gtaaatttgt tataaccaga atagc 7405

<210>7

<211>6138

<212>DNA

<213>pcdna3.1 vector

<400>7

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtttaaactt aagcttggta ccgagctcgg atccaccggt cgccaccatg gtgagcaagg 960

gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg 1020

gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc 1080

tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc 1140

tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct 1200

tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg 1260

gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg 1320

agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca 1380

actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga 1440

acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc 1500

agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc 1560

agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg 1620

tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaagc ggccgcatcg 1680

ataagcttgt cgacgatatc tctagagggc ccgtttaaac ccgctgatca gcctcgactg 1740

tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 1800

aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 1860

gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 1920

aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 1980

ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg 2040

gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 2100

tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 2160

gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 2220

attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 2280

cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 2340

ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 2400

aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt 2460

agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 2520

ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 2580

catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct 2640

aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 2700

agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg 2760

aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca 2820

agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc 2880

ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc 2940

tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga 3000

cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac 3060

gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct 3120

gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa 3180

agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc 3240

attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct 3300

tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc 3360

caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg 3420

cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct 3480

gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct 3540

tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca 3600

gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa 3660

atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc 3720

tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 3780

ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 3840

tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 3900

agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 3960

agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 4020

acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 4080

gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 4140

tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 4200

cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 4260

gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 4320

aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 4380

gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 4440

aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 4500

gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 4560

ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 4620

cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 4680

ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 4740

actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4800

tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 4860

gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 4920

ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 4980

ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 5040

gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 5100

aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 5160

gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 5220

gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 5280

cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 5340

gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 5400

gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 5460

ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 5520

tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 5580

ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 5640

cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 5700

accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 5760

cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 5820

tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 5880

cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 5940

acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 6000

atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 6060

tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 6120

aaagtgccac ctgacgtc 6138

Claims

1. An EV71 viral nucleic acid plasmid, characterized by: the EV71 virus nucleic acid plasmid comprises an EV71 virus full-length cDNA sequence and a eukaryotic expression plasmid sequence.

2. The EV71 viral nucleic acid plasmid according to claim 1, characterized in that: the EV71 virus is any one of A-type, B-type or C-type EV71 viruses.

3. The EV71 viral nucleic acid plasmid according to claim 1 or 2, characterized in that: the eukaryotic expression plasmid is any one of pcDNA-3.1, pcDNA-3.1-E, pcDNA3.1/Zeo (+/-), pcDNA4, pcDNA4/HisMAX and pRc/RSV.

4. The EV71 viral nucleic acid plasmid according to claim 1 or 2, characterized in that: the eukaryotic expression plasmid is pcDNA-3.1.

5. The method of constructing the EV71 viral nucleic acid plasmid according to claims 1 to 4, the method comprising the steps of: extracting EV71 virus RNA nucleic acid, reversely transcribing the EV71 virus RNA into cDNA, and connecting the full-length sequence of EV71 virus cDNA to a eukaryotic plasmid vector by using a homologous recombination method so as to construct the EV71 virus nucleic acid plasmid.

6. The method according to claim 5, wherein the steps of the construction method are specifically as follows:

step 1: extracting RNA of EV71 virus;

step 2: reverse transcribing RNA from EV71 virus into cDNA;

7. Use of the EV71 virus nucleic acid plasmid according to claims 1-4 for the preparation of a medicament for inhibiting solid tumors.

8. Use of the EV71 virus nucleic acid plasmid according to claims 1 to 4 for the preparation of a medicament for the prophylaxis and/or treatment of tumors.

9. Use according to claim 8, characterized in that: the tumor is one or more of cervical cancer, lung cancer, esophageal cancer, breast cancer, brain glioma, melanoma, myosarcoma, gastric cancer, colon cancer, head and neck cancer, prostatic cancer and ovarian cancer.