CN116769734A - Construction and application of anoxic microenvironment response oncolytic virus strain - Google Patents
Construction and application of anoxic microenvironment response oncolytic virus strain Download PDFInfo
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Abstract
The application belongs to the technical field of genetic engineering, relates to a construction method of an anoxic micro-environment response oncolytic virus strain and application thereof, in particular to a construction method of a parainfluenza virus PIV5-L oncolytic virus strain, and the application carries out genetic modification of carrying and expressing exogenous genes TNF-alpha and Leptin and tumor-targeted anoxic micro-environment oncolytic characteristics of a novel parainfluenza virus strain PIV5-L derived from human B cells, and comprises the following steps: isolation and identification of PIV5-L virus strains, carrying exogenous genes and genetic modification responsive to oncolytic properties of anoxic microenvironments, and anti-tumor efficacy alone or in combination. The application also includes therapeutic applications of the engineered oncolytic strains described above for different types of tumors.
Description
Technical Field
The application belongs to the technical field of genetic engineering, relates to a construction method of an anoxic micro-environment response oncolytic virus strain and application thereof, in particular to a construction method of an oncolytic virus strain based on parainfluenza virus PIV5-L, and especially relates to gene construction of an oncolytic virus PIV5-L strain with tumor micro-environment response oncolytic characteristics and application thereof in anti-tumor curative effects used singly or in combination.
Background
The prior art discloses parainfluenza virus type 5 (PIV 5) as a non-segmented, single-stranded negative strand RNA virus, belonging to the genus mumps, family paramyxoviridae. Studies have reported that PIV5 is widely found in nature and humans and a variety of animals can be infected, PIV5 does not induce human disease although it infects humans, and its specific neutralizing antibodies can be detected in human blood without clinical symptoms. The full length 15246nt of the PIV5 genome accords with the six-base principle of paramyxovirus and totally comprises 7 sections of genes. The 5 'end of each gene is provided with a gene initial region, the 3' end of each gene is provided with a gene termination region, and gene interval regions with different lengths are arranged between adjacent genes. The gene spacers separate the individual genes so that they are expressed as separate mRNAs and are involved in the initiation and transcription of mRNAs. The PIV5 virus genes are expressed independently, and the insertion of exogenous genes does not have great influence on the transcription of the virus. PIV5 has been widely used as a vaccine vector for influenza virus, MERS-CoV, etc. due to its characteristics of convenience for reverse genetics manipulation, genome stability, and non-causation of human diseases, etc.
It has been reported that PIV5 can proliferate in primary kidney cells of various genera including humans (e.g., hamster kidney cell BHK-21, african green monkey kidney cell Vero, etc.), wherein infection of cells with PIV5 is most susceptible to cytopathy rarely occurs. The PIV5-L strain (GenBank: MT 160087) is separated from B lymphocytes preserved in a laboratory by the research team, and the sequencing identification shows that the PIV5-L strain has the most similar sequence with the PIV5-AGS strain (GenBank: KX 060176) and the homology reaches 99 percent. To further understand the pathogenicity of the PIV5-L virus strain, infection of cells other than kidney cells with PIV5-L was found to infect B lymphoma cells and cause a partial B lymphoma cell shrinkage lytic lesion. In addition, PIV5-L was found to infect and kill tumor cells such as melanoma and colorectal cancer, but not normal human cells such as HUVEC.
In recent years, oncolytic virus therapy has attracted considerable attention in the field of tumor therapy, in which oncolytic viruses can selectively infect tumor cells, replicate in large amounts therein and eventually lyse the tumor cells, while releasing tumor antigens in large amounts, improving the tumor microenvironment. The tumor microenvironment is a small ecological environment formed by tumor tissues and surrounding tissues, and comprises tumor cells, other various cells surrounding the tumor cells, biomolecules infiltrating the tumor cells and surrounding physical and chemical environments. The characteristic of rapid growth of tumor cells causes malignant proliferation of tumor cells faster than blood vessel formation, causes insufficient blood supply in the tumor to cause a certain degree or regional hypoxia phenomenon, and finally forms a local hypoxia microenvironment.
The existing research of oncolytic viruses is focused on searching suitable tumor types of different oncolytic viruses and research related to tumor immunosuppression microenvironment reactivation, but oncolytic viruses targeted by tumor hypoxia microenvironment have not been reported, and certain oncolytic viruses have limited replication capacity in the hypoxia environment, so that the hypoxia microenvironment is one of the problems to be solved in oncolytic virus therapy.
Hypoxia inducible factors (hypoxia inducible factors, HIFs), a superfamily of transcription factors that are sensitive to oxygen. HIFs are widely found in mammals and humans, stably express and activate expression of a variety of downstream hypoxia-inducible related genes under hypoxic conditions [1]. Proline at positions 402 and 564 of HIF 1. Alpha. Protein molecules may be modified by prolyl hydroxylase hydroxylation in normoxic conditions, and hydroxylated HIF 1. Alpha. Binds to E3 ubiquitin ligase VHL and is thereby degraded. However, under hypoxic conditions, HIF1α cannot be modified by hydroxylation and cannot be recognized by VHL to accumulate for expression [2]. The ODD domain of HIF1α (amino acids 401 to 603) has been shown to have features that are degraded under normoxic conditions and stably expressed under hypoxia [3]. The strategy of fusion expression of ODD domains with certain exogenous genes, which can make the exogenous genes highly expressed only in anoxic environments, has been used for anoxic microenvironment targeted therapies [3-5].
TNF-alpha is a multifunctional pro-inflammatory cytokine belonging to the Tumor Necrosis Factor (TNF) superfamily. The cytokine is secreted mainly by macrophages and binds to its receptors TNFRSF1A/TNFR1 and TNFRSF 1B/TNFBR. TNF- α can be involved in regulating immune cells, cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. TNF- α can exist as a multimer of two, three or five non-covalent linking units, which has a role in the immune response to bacterial, viral, parasitic and certain fungal infections as well as in specific tumor necrosis. TNF- α, when injected into tumor bearing mice, causes tumor necrosis. The cytokine is associated with a variety of diseases including autoimmune diseases, insulin resistance, and cancer. The TNF-alpha gene is often chosen as the model therapeutic gene because it can exert a cytotoxic effect by inducing apoptosis in many types of cancer cells.
Leptin (Leptin) is a protein hormone consisting of 167 amino acids and has an important role in regulating body weight, metabolism and development of tumorigenesis. Leptin has a molecular mass of about 16kDa and is encoded by the obesity (ob) gene. Leptin is secreted primarily by adipocytes, and small amounts of leptin are also secreted by gastric and placental epithelial cells. Leptin receptors are highly expressed in the hypothalamic region of the body weight regulatory center, but Leptin receptors are also present on T lymphocytes and vascular endothelial cell surfaces. Leptin is involved in regulating food intake, energy expenditure and obesity through hypothalamic leptin receptor. Leptin has powerful metabolic reprogramming functions, such as promoting oxidation of glucose and fat and mitochondrial biogenesis. It has been shown that Leptin induces local autocrine/paracrine cascades within the tumor microenvironment, affecting tumor growth and progression through multiple processes such as inflammation and oxidative stress, cell proliferation, inhibition of apoptosis, angiogenesis, immunomodulation [6]. However, recent studies have found that Leptin stimulation can enhance T cell proliferation and synthesize more cytokines due to the expression of Leptin receptor by T cells, and have reported that Leptin, a feature, has been successfully used for tumor therapy [7].
Based on the current state and basis of the prior art, the inventor aims to provide a construction method of an anoxic micro-environment response oncolytic virus strain and application thereof, in particular to a construction method of an anti-tumor micro-environment response oncolytic virus strain based on parainfluenza virus PIV5-L, and particularly relates to gene construction of the parainfluenza virus PIV5-L with tumor micro-environment response oncolytic characteristics and application thereof in single or combined use of the anti-tumor therapeutic effect.
The references relevant to the present application are,
1.Jiang,B.H.,et al.,Transactivation and inhibitory domains of hypoxia-inducible factor 1alpha.Modulation of transcriptional activity by oxygen tension.J Biol Chem,1997.272(31):p.19253-60.
2.Masson,N.,et al.,Independent function of two destruction domains in hypoxia-inducible factor-alpha chains activated by prolyl hydroxylation.Embo j,2001.20(18):p.5197-206.
3.Huang,L.E.,et al.,Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway.Proc Natl Acad Sci U S A,1998.95(14):p.7987-92.
4.Kim,H.A.,et al.,Transcriptional and post-translational regulatory system for hypoxia specific gene expression using the erythropoietin enhancer and the oxygen-dependent degradation domain.J Control Release,2007.121(3):p.218-24.
5.Koshikawa,N.and K.Takenaga,Hypoxia-regulated expression of attenuated diphtheria toxin A fused with hypoxia-inducible factor-1alpha oxygen-dependent degradation domain preferentially induces apoptosis of hypoxic cells in solid tumor.Cancer Res,2005.65(24):p.11622-30.
6.Ray,A.and M.P.Cleary,The potential role of leptin in tumor invasion and metastasis.Cytokine Growth Factor Rev,2017.38:p.80-97.
7.Rivadeneira,D.B.,et al.,Oncolytic Viruses Engineered to Enforce Leptin Expression Reprogram Tumor-Infiltrating T Cell Metabolism and Promote Tumor Clearance.Immunity,2019.51(3):p.548-560.e4.。
disclosure of Invention
The application aims to provide a construction method and application of an anoxic micro-environment response oncolytic virus strain based on the current state and basis of the prior art, in particular to a construction method of an anoxic micro-environment response oncolytic virus strain based on parainfluenza virus PIV5-L, and particularly relates to gene construction of an anoxic micro-environment response oncolytic virus PIV5-L strain of tumor micro-environment response oncolytic characteristics and application of the anoxic micro-environment response oncolytic virus strain in anti-tumor curative effects used singly or in combination.
The application is used for rescuing parainfluenza virus type 5L strain (PIV 5-L) by establishing a reverse genetics operation system, and modifying the PIV5-L into the oncolytic virus with response to the specificity of the anoxic microenvironment and targeting killing tumor by using the system.
The application separates an unknown virus strain from a human B lymphocyte line, and identifies the virus genome sequence by a second generation sequencing and reverse transcription PCR method. After sequence analysis, it was found that the sequence was most similar to that of PIV5 virus, and although the homology to PIV5-AGS strain (GenBank: KX 060176) was 99%, there were some differences in the sequence, and it was named PIV5-L (GenBank: MT 160087).
According to the application, the exogenous gene eGFP is embedded into the full-length genome cDNA of the PIV5-L virus, so that the PIV5-L-eGFP carrying the eGFP fluorescent gene is saved, and through continuous passage, the result shows that the recombinant virus PIV5-L-eGFP carrying the exogenous gene eGFP can be stably passed on Vero cells for more than 10 generations.
The application is to embed human gene TNF-alpha (NM_ 000594.4) into full-length genome cDNA of PIV5-L virus, and successfully obtain modified strain PIV 5-L-TNF-alpha capable of expressing TNF-alpha, and the recombinant virus PIV 5-L-TNF-alpha carrying exogenous gene TNF-alpha can be stably passaged on Vero cells for more than 10 generations through continuous passage.
The human gene Leptin (NM_ 000230.3) is embedded into the full-length genome cDNA of the PIV5-L virus, so that the PIV5-L-Leptin capable of expressing the Leptin is successfully obtained, and the recombinant virus PIV5-L-Leptin carrying the exogenous gene Leptin can be stably passaged on Vero cells for more than 10 generations through continuous passage.
The application couples the oxygen-dependent degradation structural domain ODD (527 aa-603 aa) region of HIF-1 alpha with TNF-alpha or Leptin through a segment of GGGSGGGSGGGS connecting sequence and then embeds the coupled domain ODD into full-length genome cDNA of PIV5-L virus, thus successfully obtaining PIV 5-L-ODD-TNF-alpha and PIV5-L-ODD-Leptin which selectively express exogenous TNF-alpha or Leptin under anoxic environment.
The application verifies that the recombinant virus genome contains exogenous genes through sequencing and reverse transcription PCR, and verifies that the exogenous genes can be normally expressed in host cells through experiments such as immunoblotting, immunofluorescence and the like.
The application detects the activity of adherent cells by CCK-8 method, the activity of suspension cells by a living cell counter, and the killing activity of different oncolytic modified virus strains to different types of tumor cells and normal cells by observing cytopathic status by combining with microscopic imaging.
The application uses C57BL/6 mice to carry out B16 melanoma subcutaneous tumor formation experiment until the tumor volume reaches 50mm 3 Later, the modified oncolytic virus was pressed at 2X10 6 PFU/100 μl/dose is injected into tumor three times every day to attack toxin. The oncolytic effect of the engineered oncolytic virus strain was assessed by detecting mouse tumor growth and mouse survival.
The application uses C57BL/6 mice to carry out B16 melanoma subcutaneous tumor formation experiment until the tumor volume reaches 50mm 3 Later, the modified oncolytic virus was pressed at 2X10 6 PFU/100 μl/dose is injected into tumor three times every day to attack toxin. The method is characterized in that the tumor-dissolving virus is detected by in-vivo bioluminescence of a mouse and fluorescence imaging technology, the tumor-dissolving virus is positioned in the mouse after intratumoral injection, and meanwhile, whether the main viscera are infected by the virus is judged by immunohistochemical staining, so that the tumor-dissolving virus is estimatedSafety of the therapy.
Drawings
FIG. 1. Growth curves of PIV5-L in Vero or MDCK cells.
FIG. 2 PIV5-L is capable of infecting a variety of B lymphocytes.
FIG. 3 shows the viral full-length cDNA in 6-segment PCR.
FIG. 4 modification of the viral full length genomic vector pB-BSM.
FIG. 56 fragments of viral genome were sequentially ligated into pBS-BSM by the method of In-fusion.
FIG. 6 shows the successful construction of plasmid pB-PIV5-L-WT containing the full-length genome of the virus by digestion.
FIGS. 7 and 8 construction of a full-length cDNA cloning vector for a viral genome containing the GFP gene.
FIG. 9 construction of helper plasmids.
Construction of a BHK-optT7 stable cell line.
FIG. 11 successful rescue of PIV 5-L-GFP.
FIG. 12 successful rescue of PIV5-TNF- α, PIV5-Leptin, PIV5-ODD-TNF- α and PIV 5-ODD-Leptin.
FIG. 13 PIV5-L-GFP P0 supernatant was able to infect and replicate in Vero cells.
FIG. 14 immunofluorescence staining method for detecting expression of recombinant viral P/V protein in Vero cells.
FIG. 15 RT-PCR to detect the integrity of recombinant PIV5-L-GFP viral genome.
FIG. 16 RT-PCR detection of the presence of foreign genes in the recombinant oncolytic viral genome.
FIG. 17 immunoblotting method for detecting the expression of foreign genes in recombinant oncolytic viruses.
FIG. 18.Focus forming assay assay for PIV5-L-GFP P0-P2 third generation viral titers.
FIG. 19 killing of B16 tumor cells by different engineered strains under normoxic and hypoxic conditions, respectively.
FIG. 20 killing of different tumor cells by different modified strains under normoxic and hypoxic conditions, respectively.
FIGS. 21 and 22 show the oncolytic effect of the oncolytic modified strain in the C57BL/6 mouse melanoma model.
FIGS. 23 and 24, safety and targeting of oncolytic modified strains in C57BL/6 mouse melanoma models.
Detailed Description
The present application will be described in detail with reference to the accompanying drawings.
EXAMPLE 1 isolation and identification of PIV5-L Virus
PIV5-L strain is derived from B lymphocyte of a certain strain (named BCBL-LK) stored in a laboratory, after centrifugation for 15min at the temperature of 200 rmp and 4 ℃, B lymphocyte culture supernatant is collected, RNA in the supernatant is extracted by EasyPure Viral RNA Kit (Transgen: ER 201-01), and after RNA-seq sequencing, comparison and analysis results are carried out on the strain with NCBI database, and the PIV5-L strain has the most similar sequence with PIV5-AGS strain (GenBank: KX 060176), and the homology is up to 99%. To understand the growth characteristics of the newly isolated virus, PIV5-L growth curves were determined using Vero and MDCK cells, respectively, as shown in FIG. 1, for PIV5-L to reach the viral growth plateau 3.5d after infection, with a maximum viral titer of about 2X10 7 PFU/ml. Since PIV5-L could not be assayed for viral titer by the conventional method of viral plaque on both Vero and MDCK cells, the application uses the method of focus forming assay for the assay of viral titer. In addition, a plurality of B lymphocyte lines were also individually infected with the same MOI, 1d and 3d cells after infection were collected, and uninfected cells were collected as negative controls, and immunoblotting was performed to examine whether PIV5 virus proteins were expressed in the cells using V5 antibody (CST: 13202S), and the results showed that P protein and V protein expression could be detected in each of the B lymphocyte lines infected with PIV5-L (FIG. 2), indicating that the isolated PIV5-L had infectivity and could infect a variety of B lymphocytes.
EXAMPLE 2 establishment of PIV5-L reverse genetics operating System
Construction of 2.1PIV5-L viral genome full-length cDNA and expression vector
Establishing a PIV5-L reverse genetics operating system requires the construction of BHK21 cells stably expressing T7RNA polymerase and four vectors: the full-length cDNA cloning vector of the viral genome and the expression vectors of proteins NP, P, and L. First, cell Total RNA was extracted from host cell BCBL-LK of PIV5-L (Tirol method), and reverse transcribed into cDNA (reverse transcription kit: transcript first-strand cDNA synthesis supermix, transgen: AT 301)
The reverse transcription system is as follows:
since the full length 15246nt of PIV5-L genome, we constructed the full length cDNA of the genome by using a segmented cloning method. The application adopts Primerstar high-fidelity enzyme of Takara to divide the whole length into 6 sections of PCR, the sequences of 6 pairs of primers are shown in table 1, and after agarose gel electrophoresis (figure 3) of PCR products, tapping rubber is recovered.
TABLE 1 viral specific primer sequences
PCR system:
to ensure the accuracy of the 3 'end of the viral genome, a hepatitis D ribozyme sequence (HDVRz) is introduced downstream of the 3' end, which is a self-cleaving RNA sequence in the HDV precursor RNA molecule, has a unique secondary structure, is essential for HDV double rolling circle replication, and has intermolecular cleavage activity, which is used to cleave viral sequences from other heterologous RNA molecules in RNA virus rescue.
In the application, the ribozyme sequence is synthesized by "Optimus" of the family Praeparata: HDVRz-T7terminal
ATCTTGGTTTTCCCCTTGGT
GGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGC
AACATTCCGAGGGGACCGTCCCCTCGGTAATGGCGAATGGGAC
CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG
The F6 fragment from the previous step was ligated to the synthetic sequence HDVRz-T7terminal by Overlap PCR, and the Overlap PCR primers were as follows:
F6-F：TCGCCAGCAACAATTACTACCTGACC
PIV5-F6-R：TATGACCATGACGCGTCAAAAAACCCCTCAAGAC
overlay PCR conditions:
after F6-HDVRz-T7terminal was obtained, the above 6 fragments were: F1-F5, F6-HDVRz-T7terminal, partially for subsequent ligation into pBS-BSM and partially into pMD20-Tvector (Takara: 6019) to facilitate stable preservation of the gene fragment and subsequent engineering.
2.2 engineering of the full-Length viral genome vector pBluScript IISK (+)
The polyclonal site region of pBluScript IISK (+) is modified to only contain BssHII, snaBI, MIUI restriction sites by a PCR method, and is named pBS-BSM, so that the subsequent introduction of viral genome fragments is facilitated.
Vector modification primer sequence:
Pbs-F:5’GATCTACGTAACGCGTCATGGTCATAGCTGTTTCCTGTGTG 3’
Pbs-R:5’CCAATACGTAGCGCGCTCACTGGCCGTCGTTTTACAACGTC3’
PCR conditions:
and (3) after the rubber cutting of the PCR product is recovered, carrying out enzyme digestion by using SnaBI, and connecting the enzyme digestion products by using T4 ligase after the enzyme digestion products pass through PCR-clean. The ligation product transformation competence is obtained as a successfully transformed vector, and as shown in FIG. 4, the transformed vector can be digested by SnaBI and MIuI, but not by the original digestion site KpnI.
Enzyme cutting system
T4 ligase ligation system
2.3 6 fragments of 2.1 were sequentially ligated into pBS-BSM by the method of In-fusion (FIG. 5)
The first step: construction of vector pB-PIV5-L-1
Digestion of pBS-BSM to obtain linearized vector
And (3) enzyme cutting system:
the upstream primer of the PCR amplified fragments F1, F2 and F1 is introduced with a T7 promoter sequence and a 20bp base sequence homologous to the left side of the carrier restriction enzyme cutting site SnaBI, the downstream of the F2 is introduced with a 20bp base sequence homologous to the right side of the carrier restriction enzyme cutting site MIUI, and the specific sequence of the fragment amplified primer is shown in Table 1.
Ligation of F1, F2 fragments into vectors by seamless cloning (homologous recombination)
According to pBS-BSM (Vector): f1: f2 Molar ratio =1:2:2 configures In-fusion system:
connection system of pB-PIV 5-L-1:
the linked system was transformed with Stbl3 competent cells as follows:
mu.l of the ligation product sample from the previous step, 20. Mu.l, was added to 100. Mu.l of Stbl3 competent cells, gently mixed, and the mixture was placed on ice for 30min.
Heat shock in a water bath at 42 ℃ for 90 seconds, then rapidly putting back into an ice bath, and standing for 3-5 min.
Adding lml LB culture solution without antibiotics, gently mixing, and shake culturing at 30deg.C for 1 hr.
The bacterial liquid was centrifuged at 13000rpm for 1min to precipitate bacterial cells. Most of the culture solution was aspirated, and about 50-100. Mu.l of the culture solution remained to resuspend the cells. All were then spread evenly onto LB plates containing amp+ antibiotics and incubated overnight in a 30℃incubator.
The following day (16-20 h) the single clone on the plate was picked for colony PCR, and 3-4 clones from the PCR positive clones were selected for Sanger sequencing identification.
And a second step of: construction of vector pB-PIV5-L-2
Cutting pB-PIV5-L-1 to obtain linearization vector, and cutting with the same system; and (3) performing PCR amplification on F3 and F4, and performing seamless cloning connection, wherein the steps are the same as the above, so as to form a vector pB-PIV5-L-2.
And a third step of: construction of vector PB-PIV5-L-WT
Cutting pB-PIV5-L-2 to obtain linearization vector, and cutting with the same system; f5 and F6 are amplified by PCR, wherein F6, HDVRz and T7terminal sequences are connected together through overlap PCR to form F6-HDVRz-T7terminal, F5, F6-HDVRz-T7terminal is connected into pB-PIV5-L-2 by seamless cloning, the steps are the same, the vector pB-PIV5-L-WT is formed, the whole length of the vector is verified again by sequencing and then is subjected to XbaI digestion identification, and as shown in FIG. 6, the PB-PIV5-L-WT has two XbaI digestion sites, the vector can be digested into 10504bp and 7776bp fragments by the XbaI.
2.4 construction of full-Length cDNA cloning vector of viral genome containing GFP Gene
Inserting exogenous genes between HN genes and L genes, and respectively using V/P transcription initiation site for gene transcription initiation and termination sequences of the exogenous genes: AGGCCCGGACGGGT and NP transcription termination site: TTTTAAGAAAAAAG.
Introduction of foreign Gene GFP: the pB-PIV5-L-WT plasmid obtained in the previous step was digested with NcoI and AvrII, and the small fragment was excised to include a region between the 588 th base of the HN gene and the 2577 th base of the L gene. The excised portions of HN gene, GFP gene and L gene were then subjected to three-segment PCR, the schematic diagrams of the three fragments are shown in FIG. 7, and the PCR primer sequences of the three fragments are shown in Table 2. The three fragments are connected back to the cut linearization vector through an In-fusion technology to obtain a pB-PIV5-L-GFP plasmid, meanwhile, salI and NotI cleavage sites are respectively introduced from the head to the tail of the GFP gene, so that the subsequent insertion of any exogenous genes is facilitated, the construction of the pB-PIV5-L-GFP plasmid is successful through Sanger sequencing and enzyme digestion identification, as shown In FIG. 8, after the SalI and NotI double cleavage of the pB-PIV5-L-GFP plasmid, a fragment with the same size as the GFP gene is visible near 700bp, and the pB-PIV5-L-WT plasmid cannot be digested by SalI and NotI double cleavage.
Table 2: primer sequences
2.5 construction of full-Length cDNA cloning vector of viral genome containing other exogenous genes
Restriction enzyme plasmid pB-PIV5-L-GFP
After the completion of the cleavage, a large band of 18Kb was separated by 0.8% agarose gel, and the rubber cuts were recovered.
Constructing recombinant plasmids pBs-PIV 5-TNF-alpha, pBs-PIV5-leptin, pBs-PIV5-BH3, pBs-PIV 5-ODD-TNF-alpha, pBs-PIV5-ODD-leptin, pBs-PIV5-ODD-BH3;
after the ligation is completed, stbl3 competence is transformed, and the transformation steps are the same as before.
After 18-20h, 10 colonies are arbitrarily selected to be used as colony PCR, the colony PCR positive clones are sent to Sanger for sequencing, the full-length genome of the virus is detected through sequencing, and the constructed plasmid is amplified and then stored for a long time at minus 20 ℃.
Plasmid large pumping step: the recombinant plasmid is transformed in Stbl3 competent cells, single clone is selected after culturing for 18h at30 ℃, activation is carried out for 10h at 220rpm at30 ℃, and the activated bacterial liquid is inoculated into fresh LB culture medium (for example, 400 μl:200 ml) at 1:500, and the temperature is 220rpm at30 ℃ for 12-14h. Subsequent plasmid large pumping experiments can be performed.
2.6 helper plasmid construction
The helper plasmid uses pTM1 (pTM 1 vector is derived from pTM1-VP30, addgene: 69119) as vector, and three genes NP, P, and L are inserted respectively.
Construction of pTM1-NP and pTM 1-P: the plasmid pTM1-VP30 is linearized by EcoRI and SalI double digestion, the gene NP and P are subjected to PCR by taking PB-PIV5-L-WT as templates, the PCR product is digested by EcoRI and SalI after tapping recovery, then the digested pTM1-VP30 is connected with the digested pTM1-VP30 by a T4 ligase method (the digested products are purified by a PCR-clean mode), the connection products are converted into Stbl3, and positive clones are verified by sequencing (the steps are the same as before).
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Construction of pTM 1-L: plasmid pTM1-VP30 is linearized by double cleavage of NcoI and SalI, and gene L is PCR with PB-PIV5-L-WT as template, and since the L gene is large, the L gene is amplified in two segments, and the amplification primers are as follows:
the PCR is recovered by tapping and then the linearized plasmid is connected by an In-fusion method, and the connection system is the same as before.
2.7 Construction of BHK-optT7 stable cell line
The BHK-optT7 stable cell line for stably expressing T7RNA polymerase is constructed by adopting a pLenti-CMV-GFP-Hygro lentiviral transduction system, and the specific construction process is as follows:
1) Construction of lentiviral packaging plasmid pLVX-optT7RNAP-hygro
The plasmid pLenti-CMV-GFP-Hygro was first digested with XbaI and SalI to remove its GFP gene and then ligated by In-fusion with the PCR amplified optT7RNAP gene sequence, which was a eukaryotic codon optimized T7RNA polymerase gene amplified from plasmid pCAGGS-optT7 (available from Addgene: 65974).
optT7RNAP amplification primer:
F：CTCCATAGAAGACACCGACTCTAGAATGGGCGGGGAGGGCCTT
R：TCCGATTTCGCATTTGCCTGAGTCGACAATCAACCTCTGGATTACAA
after the plasmid is successfully constructed by sequencing verification, the plasmid is preserved at the temperature of minus 20 ℃ and is used for subsequent lentivirus packaging.
2) Packaging of lentiviruses
20 hours prior to transfection, 293T cells, which were well grown and free of mycoplasma contamination, were plated at 0.75 million/well/ml in 12-well plates.
Transfection system:
| pLVX-optT7RNAP | 400ng |
| PSPA | 400ng |
| VSVG | 200ng |
| Fugene HD | 4μl |
| Opti-MEM | make up to a total volume of 75. Mu.l |
Incubate at room temperature for 15min.
Mu.l of the medium was aspirated from the original medium, and the transfection solution was slowly added dropwise and mixed well.
48 hours after transfection, culture supernatant was collected, split charging supernatant at 1500 rpm for 10min, 50. Mu.l/branch, and standing at-80℃for further use.
3) Transduction of lentiviruses
The day before transduction cells were plated onto 12-well plates, grown to about 70% confluency, 100. Mu.l of virus was added to medium containing 6ug/ml polybrene, centrifuged at30℃for 30min at 1000g, and after 1 hour at 37℃the virus-containing medium was discarded and replaced with fresh medium. Screening was performed 24 hours after viral transduction by adding 750ug/ml Hygromycin B.
4) Verification of BHK-optT7 cell line
To verify whether the BHK-optT7 cell line exerts T7RNA polymerase activity, it was first verified whether pTM1-P and pTM1-NP-2flag-c driven by the T7 promoter were expressed, 293T cells were co-transfected with plasmid pCAGGS-optT7 and pTM1-P or pTM1-NP-2flag-c, and after 24 hours, western detection was performed on the harvested cells, as shown in FIG. 9, in cells co-transfected with pCAGGS-optT7, both pTM1-P and pTM1-NP-2flag-c plasmids were expressed.
The plasmid pTM1-NP-2flag-c, which was confirmed to be expressed under the drive of T7RNA polymerase, was then transfected into BHK-optT7 cells alone, and the protein was recovered 24 hours after transfection for Western detection, and the result was shown in FIG. 10, in which the pTM1-NP-2flag-c plasmid was expressed in BHK-optT7 cell line. The experiments prove that the constructed BHK-optT7 cell line can express T7RNA polymerase and exert the polymerase activity thereof.
2.8 Rescue of PIV5-L
1) PIV5-L rescue procedure (rPIV 5-L-GFP is taken as an example)
BHK-optT7 cells were plated at 4X10 one day in advance 5 Well was plated in 6-well plates, at approximately 70% confluency, and fresh 6% FBS DMEM was used prior to transfection, without anti-media, the transfection system was as follows:
| pB-PIV5-L-GFP | 3ug |
| PTM1-NP-2flag-c | 1ug |
| PTM1-P | 0.2ug |
| PTM1-L | 1.5ug |
| Fugene HD(1:4) | 23μl |
| Opti-MEM | 200μl total |
mixing, standing at room temperature for 15min, dropwise adding the transfection system into a 6-well plate, then placing back into a 37 ℃ cell incubator for culturing, observing every day, wherein 3d after transfection, as shown in fig. 11, the rPIV5-L-GFP group can see weak fluorophores, 4d and 5d after transfection, the number of fluorophores is gradually increased, the brightness is gradually increased, and a plurality of nuclei are visible as multinuclear giant cells wrapped by the same layer of cell membrane under white light, namely syncytia. Syncytia (syncytia) is a phenomenon of viral infection. Some viruses, when they infect host cells, promote fusion of the host cells with surrounding like cells and eventually form multinucleated megacells. Since the rPIV 5-TNF-alpha, rPIV5-Leptin, rPIV 5-ODD-TNF-alpha, rPIV5-ODD-Leptin and other viruses do not contain GFP gene, it is shown that whether or not viruses are produced can be judged by observing syncytia, and as shown in FIG. 12, syncytia production can be seen.
5d after transfection, the supernatant was collected at 3500rpm (1800 g), centrifuged at 4℃for 15min, and the supernatant was sub-packed at 500. Mu.l/min, designated as P0, and stored at-80 ℃.
2) Virus rescue validation
a. Verifying whether the virus supernatant P0 collected in the previous step contains viruses
Mu.l of the virus supernatant collected in the previous step was taken and infected with Vero cells (Vero cells were infected with the virus according to 0.2X10 a day in advance) 6 Well,24-well plate) at 37℃and 2h after 500. Mu.l fresh 6% FBS DMEM in medium with the three antibodies. After infection, vero cell culture media was observed daily for yellowing, lesions and fluorescence. As shown in fig. 13, the green fluorescence was gradually increased 1d, 2d, and 3d after infection, reflecting the replication of the virus in Vero cells (GFP channel on the left and merger on the right), indicating that there was virus in the collected P0 supernatant and that the virus was able to replicate normally.
After 5d, at 3500rpm, centrifugation at 4℃for 15min, the supernatant was collected, 200. Mu.l/branch was dispensed, and stored at-80℃and designated as P1.
b. Virus-specific antibody immunofluorescent staining
For rPIV 5-ODD-TNF-alpha and rPIV5-ODD-Leptin, immunofluorescence staining method was used to determine whether the rescued recombinant viruses could infect Vero cells, as shown in FIG. 14, all three recombinant viruses could be detected by V5 antibody (red light) after Vero infection, representing the expression of virus V/P gene. Immunofluorescence staining steps were as follows: vero cells were washed three times with PBS, fixed with 4% fpa for 15min at room temperature, and immunofluorescent staining was started after three washes with PBS. The primary antibody, V5 antibody (CST: anti-V5) was incubated in a 1:500 dilution into a blocking buffer (blocking buffer: PBS with 0.02% Tixton-100,1% FBS) and 150. Mu.l of diluted antibody was added to each well for incubation for 2h at room temperature. After three times of Blocking buffer washing, incubating the secondary antibody, diluting the coat-anti-Rabbit 594 into the Blocking buffer according to the ratio of 1:1000, and adding 150 μl of diluted antibody into each well to incubate for 1h at room temperature. After three Blocking buffer washes, 150 μl of DAPI diluted 1:1000 in PBS was added to each well, washed three times with PBS and photographed with a fluorescence microscope at room temperature in the dark for 5min.
c. RT-PCR method for detecting integrity of viral genome in viral supernatant P1
The kit Transgen is adopted: ER201-01 extracts viral genome RNA from supernatant, and specifically comprises the following steps:
mu.l of Proteinase K was added to a sterile 1.5ml EP tube. 200 μl BB5 was added and vortexed for 15s. 200 μl of the viral supernatant was added to the EP tube, vortexed for 15s and incubated at 56℃for 15min. 250 μl of absolute ethanol (where flocculent precipitate may occur) was added, vortexed for 15s, and left at room temperature for 5min. The solution was added to a centrifuge column together with the precipitate, and 12000g was centrifuged for 1min, and the effluent was discarded. 500. Mu.l WB5 was added and 12000g centrifuged for 1min and the effluent was discarded. This step is repeated once. Centrifuge at 12000g for 1min at room temperature to thoroughly remove residual ethanol. The column was transferred to a new 1.5ml RNase-free EP tube and 20. Mu.l RNase-free ddH was added to the center of the column 2 O, standing at room temperature for 1min. The RNA was eluted by centrifugation at 12000g for 1min at room temperature. RNA was either stored at-80℃or immediately reverse transcribed. By assist in the holy lifeThe II 1st Strand cDNA Synthesis SuperMix for qPCR kit (Yeasen: 11123ES 60) carries out reverse transcription, and the specific operation steps are as follows:
the following mixture was prepared in an RNase free centrifuge tube, and gently mixed by pipetting. Incubate at 42℃for 2min to remove residual genomic DNA.
10 μl was directly added to the reaction tubeII Supermix plus, mix gently with a pipette. According to 25 ℃ for 5min; 30min at 42 ℃; incubation was performed at 85℃for 5min.
The reverse transcription product can be used immediately for PCR reaction or stored at-20 ℃ for short term.
The integrity of the genome of the virus is detected by using 10 pairs of primers with reverse transcription as a template, the two-step PCR procedure is adopted, the primer sequences are shown in table 3, the detection results are shown in fig. 15, and 10 sections can be detected, so that the genome of the recombinant virus is proved to have no deletion phenomenon.
Table 3:10 pairs of primer sequences
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For three recombinant viruses containing exogenous genes, the primers for the exogenous genes are used for detecting whether the exogenous genes are contained in viral genome RNA in the supernatant, and the corresponding exogenous genes can be detected to exist in viral genomes as shown in FIG. 16.
d. Immunoblotting (WB) to detect expression of foreign genes in Vero cells infected with recombinant viruses
The RT-PCR method only can judge whether the virus genome contains the sequence of the exogenous gene, but the exogenous gene is expressed or not, and needs to verify from the protein level, as shown in figure 17, after virus infection, the cells are respectively cultured in an normoxic incubator and an anoxic incubator, and after 48 hours, the expression of the exogenous protein is detected, and the result shows that the expression level of the exogenous protein ODD-TNF-alpha, ODD-Leptin is higher in an anoxic environment.
The specific steps of immunoblotting are as follows:
vero cells plated in 6-well plates were infected with 400. Mu. l P1 virus/well, two wells per virus, two wells distributed on two 6-well plates, and the infection procedure was the same as before. After 2h of infection, one of the 6-well plates was placed in a chamber containing 1%O 2 ,5％CO 2 The other plate is placed in the original incubator (i.e. containing 21% O) 2 ,5％CO 2 ) Culturing.
After 48 hours, the 6-well plate was removed from the incubator, the supernatant was rapidly discarded, washed twice with PBS, 1ml of PBS was added to each well, the cells were scraped off with a cell scraper, and collected into a 1.5ml EP tube together with PBS, centrifuged at 1500g for 5min, and the supernatant was aspirated.
Mu.l of RIPA protein lysate (1% NP-40,2mM EDTA,150mM NaCl,50mM Tris,1Mm PMSF,1g/mL Aprotin, 1g/mL leupeptin and 1g/mL pepstatin) was added to the EP tube, the cells were resuspended, lysed on ice for 30min, after every 5min vortex shaking to allow full lysis, centrifuged at 14500rpm at 4℃for 5min, the supernatant was transferred to a fresh pre-chilled 1.5mL EP tube, placed on ice and 1. Mu.l was taken for detection of protein concentration.
200ug of total protein was taken for each sample, and the protein was denatured by adding DDT-containing 6X SDS loading Buffer,100 degree metal bath heating for 10min.
Adding the denatured sample into a 10% SDS-PAGE gel loading hole for gel electrophoresis, and keeping the constant pressure at 80V for 40min;100V, about 90min, until the bromophenol blue indicator band runs to the bottom of the separation gel, stop electrophoresis.
NC membrane (6 cm x 9 cm) was immersed in 1 Xmembrane balance solution for 30s, and the rotating membrane clamps were taken out, and were installed in the order of positive electrode-dry foam-NC membrane-gel-dry foam-negative electrode.
The films were transferred using an eBlotTM L1 fast wet transfer apparatus for 10min according to the set procedure.
Closing: NC membrane after membrane transfer was incubated in 5% skimmed milk (PBS) for 0.5-1h at room temperature, and then washed 3 times with 1 XTBST for 5 min/time.
Incubating primary antibodies: murine TNF- α (Ag 11413) and murine Flag (M2) primary antibodies were prepared using 1 XTBS buffer at a dilution ratio of 1:1000, NC membrane was placed in the prepared antibodies and incubated overnight at 4 ℃. Washing the film: 1 XTBST was washed 3 times, 5 min/time.
Incubating a secondary antibody: after incubation with goat anti-rabbit fluorescent secondary antibody 800 (PBS dilution 1:10000) for 1h at room temperature in the dark, washing with 1 XTBST 3 times, 5 min/time.
Finally, an odyssey bicolor infrared laser imaging system is used for imaging and analysis.
2.9 viral titre was measured using Focus forming assay
For subsequent quantification experiments, the application adopts Focus forming assay method to measure the virus titer, and FIG. 18 is a graph showing the results of titer measurement of PIV5-L-GFP in different generations. The method comprises the following specific steps:
and (3) paving: advance 2d Vero cells in 96 well plates at 2X10 4 Well plating, infection was done after two days when confluence reached 100%.
Infection: 180 μl of DMEM medium containing 2% FBS was added to the sterile 96-well U-plate, then 20 μl of virons/well was added to the first row of wells, two auxiliary wells were set, and mixed well. Then performing multiple dilution, sequentially taking 20 μl of virus dilution liquid of the previous row to the next row by using a row gun, and diluting to 10 -6 。
The cell culture solution in the 96-well cell culture plate is thrown off in a biosafety cabinet, 100 mu l of virus dilution solution is taken from each row of the U-shaped plate and added into the cell culture plate, and the dilution times of the viruses are marked.
The cell culture plates were placed in a 37℃cell incubator, and after 2 hours, 125. Mu.l of methylcellulose (2 XDMEM,2% FBS) was added to each well to cover the cells, and the methylcellulose was preheated at 37℃in advance.
Dyeing: 3d after infection, 100. Mu.l 2% PFA/well was added, and the mixture was fixed at room temperature for 40min, washed three times with PBS (Blocking buffer) containing 0.02% Txiton-100 and 1% FBS, 150. Mu.l/well/time. The Blocking buffer residual liquid is dried as much as possible by throwing the plate each time, but the cells cannot be dried.
Incubating primary antibodies: the primary antibody (Anti-V5) was diluted 1:1000 with Blocking buffer, then 40. Mu.l of diluted primary antibody was added to each well and incubated at room temperature for more than 2h or overnight at 4 ℃. The primary antibody was thrown off and Blocking buffer washed three times, 150. Mu.l/well/time.
Incubating a secondary antibody: the secondary antibody (Goat anti Rabbit HRP) was diluted 1:2500 with Blocking buffer, then 50 μl of diluted secondary antibody was added per well and incubated for more than 2h at room temperature. The secondary antibody was thrown off and Blocking buffer washed three times, 150. Mu.l/well/time.
The HRP substrate Trueblue,30 μl/well, was added and left at room temperature for 20-40min, and blue spots were seen to appear (immediately stopping the development when the background of the plate became blue).
Terminating the color development: after washing three times with ddH2O, the wells were dried, and the number of spots in each well was counted under a microscope to complete a few spots as much as possible within 24 hours. And (5) keeping the board in a dark place.
Viral titer was calculated: the holes are counted as Kong Weizhun, typically within 20-50 spots. Mean value of several sub-wells of this well x fold of dilution of this well x10 = PFU/ml.
Example 3 oncolytic Effect of different engineered oncolytic Virus strains
3.1 CCK8 method for detecting cytocidal effect of PIV 5-ODD-TNF-alpha and PIV5-ODD-Leptin
The application detects murine or human cells of melanoma, colorectal cancer and lymphoma types. One day in advance by 3x10 4 Tumor cells were plated into 96-well plates 24 hours later, and after cells were grown to a confluency of 70% or more, oncolytic virus strains were added to different tumor cells at MOI = 0.1,1, 10, each MOI being provided with three secondary wells. Before the infection step, after the infection, the same cell line is respectively put into normal oxygen and anoxic culture, after the culture is carried out for 48 hours, the original culture medium is thrown away, the CCK8 stock solution is diluted into the DMEM without blood and resistance according to the volume ratio of 1:10, and 100 mu l of diluted CCK8 solution is added into each hole. After incubation for 30min-1h at 37 DEG CThe absorbance value is detected at the wavelength of 450nm, and the cell viability is calculated according to the formula. The results are shown in FIG. 19, and CCK8 experiment results show that all recombinant virus strains have no obvious difference in killing effect on the mouse melanoma cells B16 under normoxic conditions; when the treatment is performed in the absence of oxygen, the cytocidal effect of the PIV 5-TNF-alpha and the PIV5-Leptin is weakened, and the cytocidal effect of the PIV 5-ODD-TNF-alpha and the PIV5-ODD-Leptin is unchanged or even slightly enhanced. It is demonstrated that the incorporation of the ODD region can stabilize the expression of the foreign protein in the hypoxic environment, thereby enhancing the cytocidal effect of rPIV 5s. The application also detects cytotoxicity of PIV 5-ODD-TNF-alpha and PIV5-ODD-Leptin and other tumor cells combined with the two. As shown in FIG. 20, neither rPIV5-ODD-TNF- α nor PIV5-ODD-Leptin induced cytotoxicity in HCT116, RKO or SW480 even under hypoxic conditions; whereas when PIV 5-ODD-TNF-alpha/PIV 5-ODD-Leptin was co-infected, about 30% of HTC116 was killed at moi=10. Under the anoxic state, PIV5-ODD-Leptin can target Hela and obviously inhibit the activity of Hela. In addition, the PIV 5-ODD-TNF-alpha/PIV 5-ODD-Leptin combined infection can inhibit the activity of SIHA in the anoxic state to a certain extent. For the mouse colorectal cancer cell MC38, the strain was sensitive to PIV5 modified strain under normoxic conditions, and only to PIV 5-ODD-TNF-alpha and PIV5-ODD-Leptin under hypoxia conditions. The results show that the engineered oncolytic strains have different degrees of selectivity for different tumor types and have hypoxic microenvironment sensitivity.
3.2 modification of the therapeutic Effect of oncolytic Virus strains on the B16 melanoma model
In order to study the anti-tumor effect of the recombinant oncolytic virus, the application establishes a B16 melanoma subcutaneous oncological model when the tumor volume is more than 50mm 3 At the time, tumor-bearing mice are treated by injecting oncolytic viruses into the tumors. The modified oncolytic virus is pressed by 2x10 6 PFU/100 μl/dose is injected into tumor three times every day to attack toxin. The recombinant virus PIV 5-ODD-TNF-alpha, PIV5-ODD-Leptin, the independent treatment group, and the PIV 5-ODD-TNF-alpha/PIV 5-ODD-Leptin combination group were set up to 3 experimental groups, and the DMEM treatment group and the PIV5-L-GFP group were set up as control groups. As shown in fig. 21, after intratumoral injection of the virus, the experimental group had tumor growth compared to the control groupThe growth is inhibited to a certain extent, wherein the PIV 5-ODD-TNF-alpha+PIV 5-ODD-Leptin combined group is most obvious, the tumor of two mice completely disappears, and the survival rate of the mice reaches 40% after 60 days. HE staining found a decrease in tumor cell/stromal cell ratio in the experimental group, with the PIV5-ODD-TNF- α+PIV5-ODD-Leptin combination group most significantly decreased (FIG. 22). In addition, the application also detects the positioning condition of oncolytic virus after intratumoral injection, and on the 7 th day after the injection of the virus, the GFP positioning of PIV5-L-GFP is detected by an IVIS spectral imaging system, and GFP fluorescence is only found at the tumor part (FIG. 23A); on day 15 after virus injection, P/V protein expression in the major viscera was detected by immunohistochemical staining, and P/V expression was detected only in tumor tissue (FIG. 23B), demonstrating that virus can effectively target tumor sites when rPIV5s was injected intratumorally. Meanwhile, the expression of foreign proteins and viral P/V proteins in tumor tissues was detected by immunohistochemistry and immunoblotting experiments (FIGS. 23C, 23D). In addition, by immunofluorescent staining of tumor tissue, the results showed that Leptin from the PIV5-ODD-Leptin treatment group was more easily recruited to hif2α expression region than PIV5-Leptin (fig. 24), indicating that coupling of ODD region makes foreign protein more stable in hypoxic environment.
In summary, experimental data indicate that when mice are treated by intratumoral injection, the PIV5 modified strain expresses exogenous proteins only in tumor tissues and does not diffuse to other organs, and the coupling of the ODD region enables the exogenous proteins to have anoxic microenvironment targeting.
Sequence listing
<110> university of double denier
<120> construction of anoxic micro-environment responsive oncolytic virus strain and application thereof
<160> 58
<170> SIPOSequenceListing 1.0
<210> 1
<211> 55
<212> DNA
<213> F1 F-ends
<400> 1
acggccagtg agcgcgctac taatacgact cactataggg accaggggga aaacg 55
<210> 2
<211> 32
<212> DNA
<213> F1 R-ends
<400> 2
cagtaggatc cattgtggat tgtggattgc gc 32
<210> 3
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<212> DNA
<213> F2 F-ends
<400> 3
atccacaatg gatcctactg gtctgagctt ctc 33
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<212> DNA
<213> F2 R-ends
<400> 4
ctatgaccat gacgcgttac gtatggacct tcataagtgg cg 42
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<211> 34
<212> DNA
<213> F3 F-ends
<400> 5
atgaaggtcc atacccctga caaggaggga agag 34
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<211> 45
<212> DNA
<213> F3 R-ends
<400> 6
ctatgaccat gacgcgttac gtactggtag acgaagggtt ggtca 45
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<211> 31
<212> DNA
<213> F4 F-ends
<400> 7
aacccttcgt ctaccagtac atttggatca g 31
<210> 8
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<212> DNA
<213> F4 R-ends
<400> 8
gtactgagtt ccatcagagc ttgttgagtg tgcct 35
<210> 9
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<212> DNA
<213> F5 F-ends
<400> 9
gctctgatgg aactcagtac gaatccaatg ttacg 35
<210> 10
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<212> DNA
<213> F5 R-ends
<400> 10
ctatgaccat gacgcgttac gtagtaattg ttgctggcga atg 43
<210> 11
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<212> DNA
<213> F6 F-ends
<400> 11
tcgccagcaa caattactac ctgacc 26
<210> 12
<211> 25
<212> DNA
<213> F6 R-ends
<400> 12
accaagggga aaaccaagat taatc 25
<210> 13
<211> 20
<212> DNA
<213> HDVRz-T7 terminal 1
<400> 13
atcttggttt tccccttggt 20
<210> 14
<211> 41
<212> DNA
<213> HDVRz-T7 terminal 2
<400> 14
ggccggcatg gtcccagcct cctcgctggc gccggctggg c 41
<210> 15
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<212> DNA
<213> HDVRz-T7 terminal 3
<400> 15
aacattccga ggggaccgtc ccctcggtaa tggcgaatgg gac 43
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<212> DNA
<213> HDVRz-T7 terminal 4
<400> 16
ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttg 48
<210> 17
<211> 26
<212> DNA
<213> F6-F
<400> 17
tcgccagcaa caattactac ctgacc 26
<210> 18
<211> 34
<212> DNA
<213> PIV5-F6-R
<400> 18
tatgaccatg acgcgtcaaa aaacccctca agac 34
<210> 19
<211> 41
<212> DNA
<213> Pbs-F
<400> 19
gatctacgta acgcgtcatg gtcatagctg tttcctgtgt g 41
<210> 20
<211> 41
<212> DNA
<213> Pbs-R
<400> 20
ccaatacgta gcgcgctcac tggccgtcgt tttacaacgt c 41
<210> 21
<211> 14
<212> DNA
<213> V/P transcription initiation site
<400> 21
aggcccggac gggt 14
<210> 22
<211> 14
<212> DNA
<213> NP transcription termination site
<400> 22
ttttaagaaa aaag 14
<210> 23
<211> 40
<212> DNA
<213> NCOI-F
<400> 23
tcctcaaatc aatttgtttc catgggaatc attgaaccca 40
<210> 24
<211> 20
<212> DNA
<213> HN-GFP-R
<400> 24
taaaaccaat attgtgtgtc 20
<210> 25
<211> 53
<212> DNA
<213> HN-GFP-F
<400> 25
gacacacaat attggtttta agaaaaaagt aggcccggac gggtcgacgc cac 53
<210> 26
<211> 27
<212> DNA
<213> GFP-L-R
<400> 26
cttaaaagcg gccgcattac ttgtaca 27
<210> 27
<211> 31
<212> DNA
<213> GFP-L-F
<400> 27
gtaatgcggc cgcttttaag aaaaaaccaa g 31
<210> 28
<211> 39
<212> DNA
<213> AVrII-R
<400> 28
tgatgattgg gtgcattctc ctaggacatc cgctgtcaa 39
<210> 29
<211> 40
<212> DNA
<213> NP F
<400> 29
ataataccac gaattctggg aatgtcatcc gtgcttaaag 40
<210> 30
<211> 27
<212> DNA
<213> NP R
<400> 30
gtcgacctag atgtcgagat cacccag 27
<210> 31
<211> 41
<212> DNA
<213> P F
<400> 31
ataataccac gaattctggg aatggatcct actggtctga g 41
<210> 32
<211> 25
<212> DNA
<213> P R
<400> 32
gtcgactcaa attgcactgc ggatg 25
<210> 33
<211> 35
<212> DNA
<213> L1 F
<400> 33
tgaaaaacac gataatacca tggctgggtc tcggg 35
<210> 34
<211> 35
<212> DNA
<213> L1 R
<400> 34
gtactgagtt ccatcagagc ttgttgagtg tgcct 35
<210> 35
<211> 35
<212> DNA
<213> L2 F
<400> 35
gctctgatgg aactcagtac gaatccaatg ttacg 35
<210> 36
<211> 39
<212> DNA
<213> L2 R
<400> 36
ttagcagccg gatcgtcgac ttagatttcc tcgccatcg 39
<210> 37
<211> 43
<212> DNA
<213> optT7RNAP F
<400> 37
ctccatagaa gacaccgact ctagaatggg cggggagggc ctt 43
<210> 38
<211> 47
<212> DNA
<213> optT7RNAP R
<400> 38
tccgatttcg catttgcctg agtcgacaat caacctctgg attacaa 47
<210> 39
<211> 47
<212> DNA
<213> F1 F
<400> 39
cagtgagcgc gctactaata cgactcacta taaccagggg gaaaacg 47
<210> 40
<211> 32
<212> DNA
<213> F1 R
<400> 40
cagtaggatc cattgtggat tgtggattgc gc 32
<210> 41
<211> 33
<212> DNA
<213> F2 F
<400> 41
atccacaatg gatcctactg gtctgagctt ctc 33
<210> 42
<211> 34
<212> DNA
<213> F2 R
<400> 42
gactgagatg agagaatgcg atctactttt gtct 34
<210> 43
<211> 21
<212> DNA
<213> F3 F
<400> 43
tctctcatct cagtctctaa c 21
<210> 44
<211> 33
<212> DNA
<213> F3 R
<400> 44
tcaggggtat ggaccttcat aagtggcgaa tcg 33
<210> 45
<211> 34
<212> DNA
<213> F4 F
<400> 45
atgaaggtcc atacccctga caaggaggga agag 34
<210> 46
<211> 25
<212> DNA
<213> F4 R
<400> 46
gtagtcgtag ctgatgtgat tgcag 25
<210> 47
<211> 27
<212> DNA
<213> F5 F
<400> 47
atcacatcag ctacgactac aagtgta 27
<210> 48
<211> 30
<212> DNA
<213> F5 R
<400> 48
gtactggtag acgaagggtt ggtcagtaac 30
<210> 49
<211> 31
<212> DNA
<213> F6 F
<400> 49
aacccttcgt ctaccagtac atttggatca g 31
<210> 50
<211> 27
<212> DNA
<213> F6 R
<400> 50
cttaaaagcg gccgcattac ttgtaca 27
<210> 51
<211> 31
<212> DNA
<213> F7 F
<400> 51
gtaatgcggc cgcttttaag aaaaaaccaa g 31
<210> 52
<211> 21
<212> DNA
<213> F7 R
<400> 52
gaaagtttag caacagccgt c 21
<210> 53
<211> 22
<212> DNA
<213> F8 F
<400> 53
ctgttgctaa actttctcga ag 22
<210> 54
<211> 35
<212> DNA
<213> F8 R
<400> 54
gtactgagtt ccatcagagc ttgttgagtg tgcct 35
<210> 55
<211> 35
<212> DNA
<213> F9 F
<400> 55
gctctgatgg aactcagtac gaatccaatg ttacg 35
<210> 56
<211> 19
<212> DNA
<213> F9 R
<400> 56
tgggcttgga acgataacg 19
<210> 57
<211> 21
<212> DNA
<213> F10 F
<400> 57
cgttatcgtt ccaagcccaa c 21
<210> 58
<211> 25
<212> DNA
<213> F10 R
<400> 58
accaagggga aaaccaagat taatc 25
Claims (6)
1. A method of constructing an anoxic microenvironment responsive oncolytic virus strain, wherein the oncolytic virus strain is a parainfluenza virus PIV5-L oncolytic virus strain, the method comprising: isolation and identification of PIV5-L strains, carrying exogenous genes and genetic engineering in response to oncolytic properties of anoxic microenvironments.
2. A method according to claim 1, further comprising the construction of oncolytic virus strains PIV5-TNF- α and PIV5-Leptin carrying exogenous genes expressing TNF- α and Leptin, respectively.
3. The method of claim 1, further comprising constructing the exogenous gene oncolytic virus strains PIV5-ODD-TNF- α and PIV5-ODD-Leptin carrying the expression of ODD-TNF- α and ODD-Leptin, respectively, in response to hypoxia stress.
4. The method of claims 2 and 3, wherein the engineered oncolytic strain is constructed to have killing activity against different types of tumor cells and normal cells.
5. Use of an oncolytic modified strain constructed according to the method of claims 2 and 3 for the preparation of an anti-tumor therapeutic.
6. The use according to claim 5, wherein the constructed oncolytic altered strains are used alone or in combination.
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