CN108220251B

CN108220251B - Recombinant infectious pustulosis virus, and preparation method and application thereof

Info

Publication number: CN108220251B
Application number: CN201810142968.6A
Authority: CN
Inventors: 郝文波; 罗树红; 陈达香; 陈瑜
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2018-02-11
Filing date: 2018-02-11
Publication date: 2022-03-04
Anticipated expiration: 2038-02-11
Also published as: CN108220251A

Abstract

The invention discloses an ORFV oncolytic virus, a preparation method and application thereof, and the preparation method of the recombinant infectious pustular oncolytic virus comprises the following steps: (1) performing overlap PCR amplification on a gene p53 of the protein according to claim 7; (2) connecting to a pSPV-EGFP vector, inserting a p53 gene to obtain a recombinant plasmid p53-pSPV-EGFP, and then inserting the recombinant plasmid into the left side and the right side of an ORFV132 gene to construct a shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132 RF; (3) transforming host bacteria TOP10 to obtain positive recombinant bacteria, (4) carrying out fluorescence identification on fusion expression of p53 and EGFP after OFTu cell transfection; and preparing the purified recombinant infectious pustulosis virus. (5) The prepared recombinant infectious pustular oncolytic virus can replicate and proliferate in OFTu and various tumor cells, and effectively inhibit the growth of various tumor cells. The invention can be observed in a microscope, is easy to operate, convenient and quick, and greatly shortens the screening time.

Description

Recombinant infectious pustulosis virus, and preparation method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a recombinant infectious pustular oncolytic virus and a preparation method and application thereof.

Background

Oncolytic viruses are a class of viruses that specifically infect and kill tumor cells. In recent decades, oncolytic virus therapy has attracted much attention and related research has made tremendous progress. The most deeply studied oncolytic viruses at present comprise adenovirus, herpes simplex virus type I (HSV), vaccinia virus and the like, which specifically recognize and infect tumor cells, finally cause the cells to swell and destroy the tumor cells, but cannot replicate in normal body cells without killing effect, and theoretically have higher antitumor effect and lower side effect. The oncolytic virus has the capacity of targeted infection and killing tumor cells, and different virus particles can be used as nano-scale drugs for treating different tumors based on the difference of the susceptibility of different viruses to the tumor cells. Selective infection of tumor cells is an inherent property of viruses, and appropriate modification of the capsid structure of a virus can enhance the susceptibility of the virus to tumor cells. The modification of the viral genome by insertion or deletion of specific genes enables the virus to carry exogenous therapeutic molecules into tumor cells to kill them and attenuate their effect on normal cells. The tumor cells can be effectively killed and killed by in vivo and in vitro experiments to research the oncolytic vaccine carrying the genes for enhancing the immune response and/or promoting apoptosis and the like. A variety of oncolytic virus vaccines have been used in tumor therapy, and their safety and efficacy have also been well characterized. Imlygic (talimogen laherparepvec; T-VEC) by Adam (Amgen) was the first oncolytic viral therapy approved by the U.S. FDA. The medicine is HSV1 which is genetically modified and is used for treating melanoma with lesion on skin and lymph node and can not be completely removed through operation.

The aphtha is also called contagious ecthyma of sheep, is an acute and skin-prone infectious disease caused by contagious ecthyma virus (ORFV), mainly infects sheep and goats, and can infect people, and is a typical zoonosis. The contagious ecthyma virus (ORFV) is one of the main members of parapoxvirus of Poxviridae, and has brick shape or oval shape, cord-shaped crisscross arrangement on the surface, complex structure, core, flank body and envelope. With the progress of research on the molecular biological and immunological properties of ORFV, there is a growing body of evidence that ORFV is likely to be an ideal oncolytic viral vector. Compared to other poxvirus or adenoviral vectors, ORFV has the following unique biological properties that make it more suitable for the development of oncolytic viruses: firstly, the ORFV gene is replicated and transcribed in cytoplasm and is not integrated into a host genome, so that the safety is high; secondly, the host range is limited, the caused injury has skin limitation, rapid healing and low toxicity, and no evidence of systemic transmission of the virus exists; thirdly, the humoral and adaptive immune response of the body is rapidly mediated, and the non-infectious host is particularly obvious; fourthly, neutralizing serum antibodies are not generated after immunization, multiple times of immunization can be supported in a short time, and the specific immune reaction of the carrier can be mediated even in the presence of the antibodies; fifthly, an attenuated strain of the virus can be constructed by targeted knockout of virulence genes of the virus; sixthly, the gene used as a virus vaccine vector has high compatibility and can replicate and express an exogenous gene; and seventhly, after the ORFV is infected, the secretion of immune regulatory molecules is caused, including granulocyte stimulating factor (CSF), Interferon (IFN), IL-2, tumor necrosis factor (TNF-), and the like. Thus, ORFV may be a highly desirable oncolytic viral vector. The ORFV genome is 138kb in length and rich in GC content, and is presumed to contain 132 genes. The genes at two ends of the genome are relatively not conserved and are ORF001-ORF008 and ORF112-ORF134 respectively. These genes are often associated with immune evasion, immune regulation and virulence of the virus in the host. The results of our preliminary laboratory studies show that the transfection level of ORFV132 gene is significantly increased after infecting human dermal fibroblasts with ORFV HFF-18h by RNA sequencing analysis. Relatively few reports on the function studies of ORFV132 gene are available, ORFV132 encodes vascular endothelial growth factor VEGF-B, mediating the VEGF-B receptor 2 signaling pathway, and further studies indicate that ORFV132 is involved in the cell cycle. In conclusion, ORFV132 is closely related to the development and development of inflammation. From the idea of transforming oncolytic virus, the ORFV132 gene is knocked out, so that the toxicity of orf virus is reduced, and the orf virus is safer for human. Therefore, the present invention determines to knock out the ORFV132 gene to construct a highly attenuated strain. Meanwhile, the position of the ORFV132 gene is found to be very suitable for inserting foreign antigen. Recently, there have been many reports of successful foreign antigen expression by ovine impetigo as a viral vector: ORFV recombinant virion ORFV D1701 expresses pseudorabies virus (PRV) glycoproteins gC and gD, useful for preventing PRV infection in swine, recombinant virion expressing classical swine fever virus protein (CSFV) to immunize swine, epidemic diarrhea virus (PEDV); rabbit hemorrhagic fever virus protein (RHDV); h5 hemagglutinin, etc., to exert a protective immune response. These studies indicate that ORFV is expected to be an ideal viral vaccine delivery system. Therefore, p53, a foreign antigen, can be inserted into the ORFV132 gene, and ORFV can be used as a vaccine delivery vector, thereby functioning as p 53.

p53 is an important cancer suppressor gene, which exerts many biological functions, such as regulating cell cycle, inducing apoptosis, inhibiting tumor angiogenesis, repairing DNA damage, controlling aging and metabolism. Because p53 has irreplaceable effect in tumor treatment, research on p 53-based anti-cancer drug development and treatment is always a hotspot at the leading edge of life science. However, p53 is neither a cell surface protein nor an enzyme, so recently, neither antibodies for targeted molecular therapy nor low molecular weight enzyme inhibitors are the best candidates for p53 anticancer drugs. Due to the unique advantages of the contagious ecthyma virus as a vector, p53 becomes possible to be used as an anti-cancer vaccine, and a new intervention method is provided for preventing and treating tumors.

The contagious ecthyma virus strain NA1/11 is a low virulent strain isolated from the goat with aphtha in the agricultural and security county of Jilin province at the early stage of the subject group. The invention aims to knock out the ORFV132 gene of NA1/11, reduce the pathogenicity of the ORFV132 gene and construct a highly attenuated NA1/11 defective strain. Meanwhile, the foreign gene p53 is inserted into the position, and a foundation is laid for constructing a recombinant virus oncolytic vector vaccine. At present, no relevant report about the insertion of the broad-spectrum cancer suppressor gene p53 in the genome of the contagious ecthyma virus exists.

Disclosure of Invention

The invention aims to provideCombining the oncolytic effects of oncolytic viruses with the anti-tumor effects of p53 Produce high-efficiency anticancer effectThe recombinant infectious pustular oncolytic virus and a preparation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme: the invention provides a novel recombinant infectious pustular oncolytic virus, which is an oncolytic virus carrying a P53-EGFP fusion protein gene.

Further, the fusion protein has the following amino acid sequence:

(1) a protein consisting of an amino acid sequence shown as SEQ ID No. 7.

(2) And the amino acid sequence homology defined by the sequence SEQ ID No.7 is 80% to 100% and encodes the same functional protein.

(3) And (2) the protein which is derived from the protein (1) and has the same activity after one or more amino acids are added, deleted or replaced by the amino acid sequence shown as SEQ ID No. 7.

A nucleic acid molecule of the invention encoding the fusion protein of

claim

1 or 2.

Further, the nucleotide sequence is shown as SEQ ID No. 8.

The preparation method of the recombinant infectious pustular oncolytic virus is characterized by comprising the following steps of:

(1) performing overlap PCR amplification on a gene p53 of the protein according to claim 7;

(2) connecting to a pSPV-EGFP vector, inserting a p53 gene into the vector to obtain a recombinant plasmid p53-pSPV-EGFP, and then inserting the recombinant plasmid into the left side and the right side of an ORFV132 gene to construct a shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132 RF; the ORFV132 gene is knocked out by inserting p53 and EGFP genes into the position of the viral genome 132 by using a homologous recombination technology;

(3) transforming the strain into a host bacterium TOP10 to obtain a positive recombinant bacterium;

(4) fluorescence after transfection of OFTu cells identified fusion expression of p53 and EGFP; screening recombinant contagious ecthyma virus defective strain NA1/11 delta 132-GFP and NA1/11 delta 132-p53-GFP. (ii) a After 6-8 rounds of screening, purified recombinant infectious pustulosis virus is prepared.

Further, the air conditioner is provided with a fan,

in step (2), PCR and double digestion of ORFV132LF-p53-pSPV-EGFP-ORFV132 RF: the recombinant plasmid band is at 6500bp, the PCR amplification band is at 700bp, and the two bands are 5600bp and 700bp respectively after enzyme digestion; the predicted ORFV132LF-p53-pSPV-EGFP-ORFV132RF is 6392bp, ORFV132RF is 676bp, and the sizes of the two bands after double enzyme digestion are 5649bp and 676bp respectively; sequencing shows that the plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF is successfully constructed.

Further, the air conditioner is provided with a fan,

in step (1), PCR primers were designed using Primer Premier5 software:

the upstream primer Hp53-Fw has the nucleotide sequence of SEQ ID NO. 1;

the downstream primer Hp53-Rv has the nucleotide sequence of SEQ ID NO. 2.

Further, the air conditioner is provided with a fan,

in step (2), the upstream and downstream primers of recombinant plasmid ORFV132LF-p53-pSPV-EGFP were constructed:

the upstream primer NA1/11-ORFV132L-Fw has the nucleotide sequence of SEQ ID NO. 3;

the downstream primer NA1/11-ORFV132L-Rv has the nucleotide sequence of SEQ ID NO. 4;

further, in the step (2),

constructing an upstream primer and a downstream primer of a shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132 RF:

the upstream primer NA1/11-ORFV132R-Fw has the nucleotide sequence of SEQ ID NO. 5;

the downstream primer NA1/11-ORFV132R-Rv has the nucleotide sequence of SEQ ID NO. 6.

The kit of the recombinant infectious pustulosis virus and the application thereof in targeted killing of tumor cells are provided.

Has the advantages that: the traditional screening method of the recombinant virosome utilizes Neo/glu screening, and the recombinant virosome can be observed in a microscope by adopting EGFP screening, so that the method is easy to operate, convenient and quick, and greatly shortens the screening time.

Compared with the prior art, the invention has the following advantages:

(1) the recombined sheep infective pustule virus defective strain is used as a vector and is inserted into a p53 gene, so that ORFV becomes possible to be oncolytic virus further.

(2) The wild ORFV strain used in the invention is NA1/11 low virulent strain, and then ORFV132 gene is knocked out to become highly attenuated recombinant ORFV, which is closer to the step that the recombinant contagious ecthyma virus is used as a vaccine delivery vector;

(3) the foreign p53 gene is inserted into the 132 gene position, which can fully play the role of p53 by utilizing the strong and unique immunoregulation function of ORFV, and has great significance for constructing oncolytic vaccine. The construction of the defective strain lays a foundation for further preparing the recombinant ORFV vector vaccine. Meanwhile, the inserted exogenous gene p53 has multiple biological functions, such as inhibiting cell proliferation, inducing tumor cell apoptosis and the like. In the invention, p53 can exert its biological function by virtue of the advantages of recombinant ORFV, provides some ideas for p53 to induce apoptosis to treat tumors, and is helpful for developing drugs or vaccines with potential therapeutic value on tumors.

(4) The shuttle plasmid is successfully constructed, and the contagious ecthyma virus defective strain NA1/11 delta 132-GFP and NA1/11 delta 132-p53-GFP are screened out after transfection and homologous recombination. The pathological degree of OFTu cell infected by the defective strain is as follows: NA1/11> NA1/11 delta 132-GFP > NA1/11 delta 132-p53-GFP, but the replication capacity after infection of OFTu cells in vitro growth is not obviously different from that of the wild strain NA 1/11. Because NA1/11 is a low virulent strain and ORFV132 gene is a main pathogenic gene, ORFV132 gene is knocked out, and p53 gene is inserted into the position, the recombinant contagious ecthyma virus with high attenuation is successfully constructed, and the recombinant contagious ecthyma virus is expected to be a potential oncolytic virus vector, and lays a foundation for constructing an oncolytic vaccine.

Drawings

FIG. 1 shows PCR amplification of target genes p53, ORFV132LF and ORFV132RF according to the invention;

FIG. 2 is a diagram showing the PCR and restriction enzyme identification of the recombinant plasmid p53-pSPV-EGFP according to the present invention;

FIG. 3 is the PCR and restriction enzyme identification of recombinant plasmid ORFV132LF-p53-pSPV-EGFP according to the present invention;

FIG. 4 shows the PCR and restriction enzyme identification of the recombinant plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF of the present invention;

FIG. 5 is a graph of fluorescence identification of p53 and EGFP expression following transfection of OFTu cells of the invention;

FIG. 6 is a representation of p53 and EGFP expression after transfection of a Westernblot identification plasmid of the invention;

FIG. 7 is a graph showing the results of fluorescence screening for NA 1/11. delta.132-GFP and NA 1/11. delta.132-p 53-GFP according to the present invention;

FIG. 8 is a PCR verified profile of recombinant sheep infective pustule virions in accordance with the present invention;

FIG. 9 is a graph showing the expression of GFP in an OFTu cell of the present invention after infection with three viral strains NA1/11, NA 1/11. delta. 132-GFP, and NA 1/11. delta. 132-p53-GFP by a fluorescence microscope; figure 9A shows normal OFTu cells; FIG. 9B shows that after OFTu cells were infected with NA1/11, the cell morphology changed from long spindle to oblate without GFP expression; FIG. 9C shows that after OFTu cells are infected with NA 1/11. delta.132-GFP, the cell morphology changes from long spindle to oblate, with GFP expression and most of the GFP localized in the cytoplasm; FIG. 9D shows that after OFTu cells are infected with NA 1/11. delta. 132-p53-GFP, the morphology of the cells changes from long spindle to oblate, and GFP is expressed and localized mostly to the nucleus;

FIG. 10 is a graph showing the expression of p53 and EGFP proteins in OFTu cells of the present invention after stimulation with NA1/11, NA 1/11. delta.132-GFP, NA 1/11. delta.132-p 53-GFP;

FIG. 11 is a graph showing the dynamic expression of p53 protein at different time points after OFTu cells are infected with NA 1/11. delta.132-p 53-GFP of the present invention;

FIG. 12 is a graph showing the expression of ORFV086 of the present invention in three viruses;

FIG. 13 is a graph showing the analysis of cell growth curves after OFTu infection with three viruses of the present invention;

FIG. 14 is a graph showing the analysis of growth curves of three viruses of the present invention after infection of OFTu and various tumor cells;

FIG. 15 is a graph of the effect of three viruses of the present invention on the proliferative activity of various colorectal cancer cells;

FIG. 16 is a schematic diagram of the construction of the recombinant contagious pustular oviral strain of the present invention. (1) - (3) is a schematic drawing of shuttle plasmid construction. The empty vector is pSPV-EGFP, and then the p53 gene is inserted into the recombinant plasmid p 53-pSPV-EGFP. Then the right and left sides of ORFV132 gene are inserted to construct shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132 RF. The ORFV132 gene is knocked out by inserting p53 and EGFP genes into the viral genome 132 by homologous recombination.

Detailed Description

The present invention is further illustrated in detail by the following examples, but it should be noted that the scope of the present invention is not limited by these examples at all.

The recombinant infectious pustulosis virus is a fusion protein, and the fusion protein comprises a P53 protein and an EGFP protein.

The fusion protein has the following amino acid sequence:

(1) a protein consisting of an amino acid sequence shown as SEQ ID No. 7.

A nucleic acid molecule of the invention encoding the fusion protein of

claim

1 or 2.

The nucleotide sequence is shown as SEQ ID No. 8.

The preparation method of the recombinant infectious pustular oncolytic virus comprises the following steps:

(2) connecting to a pSPV-EGFP vector, inserting a p53 gene into the vector to obtain a recombinant plasmid p53-pSPV-EGFP, and then inserting the recombinant plasmid into the left side and the right side of an ORFV132 gene to construct a shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132 RF; the ORFV132 gene is knocked out by inserting p53 and EGFP genes into the position of the viral genome 132 by using a homologous recombination technology; PCR and double digestion of ORFV132LF-p53-pSPV-EGFP-ORFV132 RF: the recombinant plasmid band is at 6500bp, the PCR amplification band is at 700bp, and the two bands are 5600bp and 700bp respectively after enzyme digestion; the predicted ORFV132LF-p53-pSPV-EGFP-ORFV132RF is 6392bp, ORFV132RF is 676bp, and the sizes of the two bands after double enzyme digestion are 5649bp and 676bp respectively; sequencing shows that the plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF is successfully constructed.

PCR primers were designed using Primer Premier5 software:

the upstream primer Hp53-Fw has the nucleotide sequence of SEQ ID NO. 1;

the downstream primer Hp53-Rv has the nucleotide sequence of SEQ ID NO. 2.

Constructing upstream and downstream primers of a recombinant plasmid ORFV132LF-p 53-pSPV-EGFP:

construction of shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF

The upstream and downstream primers of (1):

(3) Transforming the strain into a host strain TOP10 to obtain a positive recombinant strain.

(4) Fluorescence after transfection of OFTu cells identified fusion expression of p53 and EGFP; screening recombinant contagious ecthyma virus defective strain NA1/11 delta 132-GFP and NA1/11 delta 132-p 53-GFP; after 6-8 rounds of screening, purified recombinant infectious pustulosis virus is prepared.

Example 1

Construction of recombinant plasmid p53-pSPV-EGFP

(1) Amplification and identification of target gene p53

PCR primers were designed using p53 as template and Primer Premier5 software (underlined as the cleavage site):

Hp53-Fw:

5’-CAGTGACGCCTCGAGGAATTCATGGAGGAGCCGCAGTCAGA-3’(EcoRⅠ)；SEQ ID No.1

Hp53-Rv:

5’-GCTCACCATGGTGGCGAATTCGTCTGAGTCAGGCCCTTCTGTCTT-3’(EcoRⅠ)。SEQ ID No.2

PCR reactions (50. mu.L) were performed according to the following system as shown in Table 1:

TABLE 1

Reaction conditions are as follows:

and (5) carrying out agarose electrophoresis identification on the PCR product, and tapping and recovering.

(2) pSPV-EGFP digestion and tapping recovery are shown in Table 2:

TABLE 2

The enzyme is cut for 2h at 37 ℃ according to the system, and the rubber is cut and recovered.

(3) The target gene is linked to a vector

The gene of interest was ligated to the vector according to the standard protocol of the seamless cloning kit, as indicated in Table 3 (10. mu.l):

TABLE 3

Note: the fusion efficiency is highest when the molar ratio of the inserted fragment of interest to the vector is 2: 1. Therefore, the optimal dosage is as follows: pSPV-EGFP tapping recovery: [0.01 × 3737bp (vector length) ] ng ═ 37.27ngP53 tapping recovery: [0.02x 1179bp (insert length) ] ng ═ 23.58 ng; the positive control system is shown in table 4:

TABLE 4

The ligation product was transformed into competent E.coli TOP10, and after about 24h of transformation, a monoclonal colony of moderate size was visible. Taking 3 shake bacteria tubes, adding 5mL LB culture medium and 5 μ L ampicillin, picking single clone in the shake bacteria tubes, placing the shake bacteria tubes in shake culture overnight. Extracting recombinant plasmid by conventional method, and performing PCR, double digestion and sequencing identification.

Example 2

Construction of recombinant plasmid ORFV132LF-p53-pSPV-EGFP

The method for constructing the recombinant plasmid ORFV132LF-p53-pSPV-EGFP is the same as 1. Wherein the upstream and downstream primers are respectively (the underlined part is the enzyme cutting site):

NA1/11-ORFV132L-Fw：5’-AGTAGGCCTGCGCGCAAGCTTCGTCTTCTCCCGCTGGATAAA-3’(HindⅢ)SEQ ID No.3

NA1/11-ORFV132L-Rv：5’-GACCTGCAGGCATGCAAGCTTGCCTCACCCTTAAAAGTTGG-3’(HindⅢ)SEQ ID No.4；

example 3

Construction of shuttle plasmids ORFV132LF-p53-pSPV-EGFP-ORFV132RF and ORFV132LF-pSPV-EGFP-ORFV132RF

The method for constructing the shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF is the same as 1. Wherein the upstream and downstream primers are respectively (the underlined part is the enzyme cutting site):

NA1/11-ORFV132R-Fw：5’-CGCATGCATCCGCGGAGATCTCAACTTTTTATGGACCGCAG-3’(BglⅡ)SEQ ID No.5；

NA1/11-ORFV132R-Rv：5’-TATAGGGAGACCGGCAGATCTGCGGGATGCTGGTCTAATCAC TA-3’(BGLⅡ)SEQ ID No.6；

in addition, the shuttle plasmid ORFV132LF-pSPV-EGFP-ORFV132RF is obtained by carrying out EcoR I enzyme digestion on ORFV132LF-p53-pSPV-EGFP-ORFV132 RF.

Schematic diagrams of the construction of the shuttle plasmid are shown in fig. 16 (1) to (3), and sequencing identification is performed after the shuttle plasmid construction is completed. A schematic representation of the recombinant contagious pustular strain in sheep is shown in FIG. 16:

(1) - (3) is a schematic drawing of shuttle plasmid construction.

The empty vector is pSPV-EGFP, and then the p53 gene is inserted into the recombinant plasmid p 53-pSPV-EGFP. Then the right and left sides of ORFV132 gene are inserted to construct shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132 RF. The ORFV132 gene is knocked out by inserting p53 and EGFP genes into the viral genome 132 by homologous recombination.

Example 4

Transfection of OFTu cells and homologous recombination

The OFTu cells were recovered, added to a culture dish (10cm2) containing a medium (10% MEM), shaken up and cultured in an incubator (37 ℃ C., 5% CO 2). Cells were counted and added to six well plates at 5X 105/well. The OFTu cells were transfected when they grew to 70-80% confluence (four wells in a six-well plate were transfected).

2h before transfection, OFTu cells were infected by adding NA1/11(MOI ═ 1) per well. The method comprises the steps of taking 8 EP tubes, adding 100 mu L of opti-MEM into each EP tube, then adding 2 mu g of plasmids pSPV-EGFP, p53-pSPV-EGFP, ORFV132LF-p53-pSPV-EGFP-ORFV132RF and ORFV132LF-pSPV-EGFP-ORFV132RF into the opti-MEM in 4 EP tubes, fully mixing, and standing for 5 min. 4 tubes of the plasmid-containing opti-MEN were mixed with 4 tubes of opti-MEM, and left for 15 min. The medium in the six-well plate was aspirated off, and 1mL of opti-MEM was added. The plasmid-containing liquid in the EP tube was added drop by drop slowly to a six-well plate, 1mL of serum-containing medium was added after 3h, and the solution was changed after 6 h. After 24h of transfection, the expression of p53 was preliminarily judged by observing the presence or absence of green fluorescence in a fluorescence microscope. And collecting samples after 48h, removing the culture medium, washing with PBS, adding 100 mu L of 1x SDS-Loading buffer, fully cracking, scraping cells with a cell scraper, collecting the cells in an EP tube, boiling the samples for 15min, centrifuging at 14000rpm, and storing at-20 ℃ for subsequent Westernblot detection and identification of p53 expression.

Example 5

Screening recombinant contagious ecthyma virus defective strain NA1/11 delta 132-GFP and NA1/11 delta 132-p53-GFP.

(1) Preliminary screening of 96-well plates

1) The plasmids were ORFV132LF-pSPV-EGFP-ORFV132RF and ORFV132LF-p53-pSPV-EGFP-ORFV132RF, respectively, transfected as described above.

2) Cell supernatants (virus fluid) were collected after 48 h.

3) After repeated freeze thawing for 3 times, centrifuging at 800rpm for 5 min.

4) The day before the addition of the virus 96-well plates were plated at 10000 OFTu cells/well.

5) 100 μ L of the supernatant was added to an EP tube containing 900 μ L and diluted from 10-1 to 10-11 in a gradient.

6) The first 7 wells of the 1-10 columns of the 96-well plate were filled with 100. mu.L of diluted virus solution, and 100. mu.L of serum-containing medium was added to each of the 12 th and 8 th rows as blanks.

7) After 48h, the cells were photographed by observation under a fluorescence microscope, and the strongly fluorescent cells with a high dilution ratio were scraped with a small gun head into an EP tube containing 150. mu.L of medium, freeze-thawed repeatedly for 3 times, and then the next round of screening was performed.

(2) Screening of plaques by six-hole plate

1) The OFTu cells were plated in six-well plates at 3x 105 cells per well.

2) And (3) repeatedly freezing and thawing the virus liquid screened by 3-4 rounds for 3 times, centrifuging at 800rpm for 5min, diluting according to a gradient of 10 < -1 > -10 < -5 >, and adding the virus liquid into a six-hole plate to achieve the effect of gradient dilution.

3) After 2h, the culture supernatant was removed and 2mL of MEM medium containing 5% FBS and 0.5% low melting agarose was added to each well.

4) After 48h, observing under a fluorescence microscope, marking a place with green fluorescence by using a Marker pen, picking a monoclonal from a middle gun head, placing the monoclonal in an EP tube containing 150 mu L, and storing the monoclonal at the temperature of-80 ℃ for the next round of screening.

5) After 6-8 rounds of screening, the purified recombinant virus can be obtained.

6) And adding the screened virus into a six-well plate for amplification culture, extracting virus DNA and carrying out PCR verification.

Example 6

Observation of expression of GFP, p53 and ORFV086 after OFTu cells are infected with NA1/11 delta 132-GFP and NA1/11 delta 132-p53-GFP

(1) Culturing and concentrating the virus

1)75cm²Culturing 30 bottles of OFTu cells in a culture bottle, and adding 500 mu L of virus solution NA1/11, NA1/11 delta 132-GFP and NA1/11 delta 132-p53-GFP into every ten bottles;

2) after 5d, scraping cells by using a cell scraper, collecting the cells in a 50mL centrifuge tube, and repeatedly freezing and thawing for 3 times at 37 ℃;

3) centrifuging at 4000rpm for 10 min;

4) taking the supernatant, and centrifuging at 14000rpm for 1 h;

5) discarding the supernatant, and resuspending with 5ml PBS;

6) centrifuging at 14000rpm for 1 h;

7) resuspending 7mL of PBS, and subpackaging in 7 EP tubes, wherein each EP tube contains 1mL of PBS and resuspending at-80 ℃;

(2) determination of viral titre

1) Laying 3 96 pore plates, wherein each pore is 10000 OFTus;

2) taking 100 mu L of virus supernatant after repeated freeze thawing in an EP tube filled with 900 mu of LMEM on the next day, and performing gradient dilution (10-1-10-11);

3) the first 7 wells of 1-10 columns of 3 96-well plates were filled with diluted virus solution (100. mu.L each of NA1/11, NA 1/11. delta. 132-GFP and NA 1/11. delta. 132-p53-GFP, and 100. mu.L each of serum-containing medium in column 12 and row 8 as blanks;

4) after 7d, the number of lesions per column of cells was observed microscopically and the titer of the virus was calculated from the median lethal dose (TCID 50).

(3) The degree of cytopathic effect after infection with the three viruses was compared and expression of GFP, p53 and ORFV086 was observed.

1) Spreading 4 six-well OFTu cells (3 × 10 cells per well)⁵；

2) OFTu cells were infected with three viruses (NA1/11, NA1/11 Δ 132-GFP and NA1/11 Δ 132-p53-GFP, respectively, at MOI ═ 1, and GFP expression was visualized by photographing under a fluorescent microscope after 48h, and another well served as a negative control;

3) collecting samples by 150 mu L of 1x SDS loading buffer after photographing, and detecting the expression of GFP, p53 and ORFV086 by Western blot;

example 7

Determination of in vitro replication and growth capacity of OFTu cells infected with NA1/11, NA1/11 delta 132-GFP and NA1/11 delta 132-p 53-GFP;

1) spreading 3 six-well OFTu cells (3 × 10 cells per well)⁵；

2) Three viruses (NA1/11, NA1/11 Δ 132-GFP and NA1/11 Δ 132-p53-GFP were used at MOI ═ 1. ) Respectively infecting OFTu cells, and changing the cell liquid after 1 h;

3) collecting samples at 1h, 24h, 36h, 48h, 72h and 96h respectively;

4) TCID50 was measured from each time point harvest (as above) to compare whether there was a difference in replication capacity between the three viruses in vitro.

Example 8

Observe the effect of NA1/11, NA1/11 delta 132-GFP and NA1/11 delta 132-p53-GFP on the growth and proliferation of tumor cells

(1) Detection of the ability of the Virus to replicate and proliferate in tumor cells (TCID50)

1) Spreading 8x105 tumor cells per well in 6-well plate, spreading 7 wells for each cell;

2) after 24 hours of cell spreading, three viruses (MOI 1) were added to stimulate tumor cells;

3) after 2 hours of cell exchange, each tumor cell and supernatant from one well were collected (first time point);

4) cells and supernatants from additional wells were then collected at various time points (12, 24, 36, 48, 60, 72 hours) after cell exchange;

5) repeatedly freezing and thawing the collected cells and the supernatant in water bath at 37 ℃ and at-80 ℃ for three times, and centrifuging for 10 minutes at 4000rcf to collect the supernatant;

6) the virus concentration of each supernatant in step 5 was examined and the growth curve of the virus in different tumor cells was plotted (TCID 50).

(2) CCK-8 examination of the Effect of three viruses on tumor cell growth

1) Spreading 104 tumor cells/well in 96-well plate, spreading 12 cells each;

2) after 24 hours of cell spreading, three viruses are respectively added according to different titers (MOI is 0, 1, 10 and 100) to stimulate tumor cells, and each titer is provided with 3 multiple wells;

3) after 24 hours of virus stimulation, CCK-8 detection reagent (CCK-8 reagent was diluted to 100ul of working solution per well of 10ul of stock solution using medium containing 10% FBS) was added and incubated for 2 hours in a cell incubator at 37 ℃ with 5% CO 2;

4) the values of the wells are detected and recorded using a microplate reader at a wavelength equal to 450 nm.

Test 1

FIG. 1PCR successfully amplified target genes p53, ORFV132LF, ORFV132RF

1:DNAmarkerDL2000

2&3:the PCRproducts ofp53

4&5:the PCRproduct ofORFV132LF

6&7:the PCRproduct ofORFV132RF

The results of PCR amplification of the target gene are shown in FIG. 1: p53 gene and NA1/11 are used as templates to design p53, ORFV132LF and ORFV132RF primers, and a target fragment is amplified. As can be seen in FIG. 1, clear bands were observed at 1200bp, 800bp, and 700bp, and the predicted p53 was 1179bp, 132LF was 743bp, and 132RF was 676bp, which were consistent in size. Therefore, the target genes p53, ORFV132LF and ORFV132RF have been successfully amplified by PCR.

Test 2

FIG. 2 PCR and enzyme digestion identification of recombinant plasmid p53-pSPV-EGFP

1: the molecular weight of the DNA is measured by a standard,

2&3 plasmid pSPV-EGFP

4&5 PCR products of recombinant plasmid p53-pSPV-EGFP

6&7 restriction enzyme digestion product of recombinant plasmid p53-pSPV-EGFP

The PCR and double digestion results of p53-pSPV-EGFP are shown in FIG. 2, the recombinant plasmid band is at 5000bp, the PCR amplification band is at 1200bp, and the two bands after digestion are respectively about 1200bp and 4000 bp. The predicted p53-pSPV-EGFP is 4896bp, p53 is 1179bp, and the sizes of the two bands after double digestion are 3727bp and 1179bp respectively. The plasmid p53-pSPV-EGFP is successfully constructed by sequencing.

Test 3

FIG. 3 PCR and restriction identification of recombinant plasmid ORFV132LF-p53-pSPV-EGFP 1: DNA molecular weight standards

2&3 recombinant plasmid p53-pSPV-EGFP

4&5 PCR products of recombinant plasmid ORFV132LF-p53-pSPV-EGFP

6&7, enzyme digestion product of recombinant plasmid ORFV132LF-p53-pSPV-EGFP

The results of PCR and double digestion of ORFV132LF-p53-pSPV-EGFP are shown in FIG. 3, the recombinant plasmid band is 5600bp, the PCR amplified band is 800bp, and the two bands after digestion are respectively about 5000bp and 800 bp. The predicted ORFV132LF-p53-pSPV-EGFP is 5649bp, ORFV132LF is 743bp, and the sizes of the two bands after double digestion are 4896bp and 743bp respectively. Sequencing shows that the plasmid ORFV132LF-p53-pSPV-EGFP is successfully constructed.

Test 4

FIG. 4 shows the PCR and restriction enzyme identification of the recombinant plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF of the present invention

1: DNA molecular weight standards

2&3 recombinant plasmid ORFV132LF-p53-pSPV-EGFP

4&5 PCR product of recombinant plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF

6&7, enzyme digestion product of recombinant plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF

The results of PCR and double digestion of ORFV132LF-p53-pSPV-EGFP-ORFV132RF are shown in FIG. 4, the band of the recombinant plasmid is 6500bp, the band of the PCR amplification is 700bp, and the two bands after digestion are about 5600bp and 700bp, respectively. The predicted ORFV132LF-p53-pSPV-EGFP-ORFV132RF is 6392bp, ORFV132RF is 676bp, and the sizes of the two bands after double enzyme digestion are 5649bp and 676bp respectively. Sequencing shows that the plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF is successfully constructed.

Test 5

FIG. 5 is a graph showing the fluorescent identification of p53 and EGFP expression after transfection of OFTu cells of the present invention

After plasmid transfection, fluorescence signals were observed for 48h and photographed as in FIG. 5: FIG. 5A shows that after transfection of pSPV-EGFP, green fluorescence is distributed throughout the cytoplasm; FIG. 5B shows that after p53-pSPV-EGFP transfection, green fluorescence is distributed in the nucleus, and p53 is localized in the nucleus, and the expression of p53 is preliminarily judged; FIG. 5C shows that after ORFV132LF-pSPV-EGFP-ORFV132RF was transfected, green fluorescence was distributed throughout the cytoplasm; FIG. 5D shows that ORFV132LF-p53-pSPV-EGFP-ORFV132RF has green fluorescence distributed in the nucleus, and p53 is localized in the nucleus, and it is preliminarily determined that p53 is expressed.

Test 6

FIG. 6 shows the expression of p53 and EGFP after transfection of the Westernblot identification plasmid of the present invention

After plasmid transfection, expression of p53 and EGFP was characterized using a Westernblot. As shown in fig. 6: after transfection of the first well with pSPV-EGFP, the primary antibody was anti-GFP and the band size was 27kDa, indicating GFP expression; the second hole is p53-pSPV-EGFP transfection, the primary antibody is anti-GFP, the size of the strip is 80kDa, and the fusion expression of GFP and p53 is indicated; the third hole is ORFV132LF-pSPV-EGFP-ORFV132RF transfection, the primary antibody is anti-GFP, the size of the band is 27kDa, which indicates GFP expression; the fourth hole is the anti-GFP of the primary antibody after ORFV132LF-p53-pSPV-EGFP-ORFV132RF transfection, the size of the strip is 80kDa, which indicates that GFP and p53 are fused and expressed; the fifth well is ORFV132LF-pSPV-EGFP-ORFV132RF, after transfection, the primary antibody is anti-p 53, no band, indicating no p53 expression; the sixth hole is the anti-p 53 after ORFV132LF-p53-pSPV-EGFP-ORFV132RF is transfected, the size of the band is 80kDa, which indicates that GFP and p53 are fused and expressed; therefore, the recombinant plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF is identified by Westernblot, and p53 and EGFP are successfully expressed after the transfection.

Test 7

FIG. 7 shows the fluorescence screening of recombinant viruses NA 1/11. delta. 132-GFP and NA 1/11. delta. 132-p53-GFP carrying EGFP and p53 according to the invention

The fluorescence screening results are shown in FIG. 7, and green fluorescence is observed after OFTu infection by NA 1/11. delta. 132-GFP and NA 1/11. delta. 132-p53-GFP, wherein the green fluorescence is localized in cytoplasm after NA 1/11. delta. 132-GFP infection, and NA 1/11. delta. 132-p53-GFP is localized in nucleus. After Merge, the green fluorescence was found to coincide with the cells.

Test 8

FIG. 8 shows the purity of the purified rTPV verified by PCR in accordance with the present invention

1: the template is NA1/11, the primer is ORFV132, a band appears at about 400bp, 132 gene amplification succeeds

2: the template is NA1/11 delta 132-GFP, the primer is ORFV132, and no band appears

3: the template is NA1/11 delta 132-p53-GFP, the primer is ORFV132, and no band appears

4: the template is NA1/11, the primer is EGFP, and no band appears

5: the template is NA1/11 delta 132-GFP, the primer is EGFP, and a band is arranged at about 700bp, which indicates that the EGFP gene amplification is successful

6: the template is NA1/11 delta 132-p53-GFP, the primer is EGFP, and a band is arranged at about 700bp, which indicates that EGFP gene amplification is successful

7: template is NA1/11, primer is p53, band does not appear

8: the template is NA1/11 delta 132-GFP, the primer is p53, and no band appears

9: the template is NA1/11 delta 132-p53-GFP, the primer is p53, and a band is arranged around 1200, which indicates that the p53 gene is successfully amplified

After the NA 1/11. delta.132-GFP and NA 1/11. delta.132-p 53-GFP were purified, the purity was verified by PCR. As can be seen from FIG. 8, the NA 1/11. delta.132-GFP and NA 1/11. delta.132-p 53-GFP deficient strains were successfully purified.

Test 9

FIG. 9 shows the expression of GFP identified by fluorescence microscopy after OFTu cells are infected with three viruses of the present invention, NA1/11, NA 1/11. delta.132-GFP and NA 1/11. delta.132-p 53-GFP

FIG. 9 shows the expression of GFP in OFTu cells stimulated by three viral strains NA1/11, NA 1/11. delta. 132-GFP, NA 1/11. delta. 132-p53-GFP. Figure 9A shows normal OFTu cells; FIG. 9B shows that after OFTu cells were infected with NA1/11, the cell morphology changed from long spindle to oblate without GFP expression; FIG. 9C shows that after OFTu cells are infected with NA 1/11. delta.132-GFP, the cell morphology changes from long spindle to oblate, with GFP expression and most of the GFP localized in the cytoplasm; FIG. 9D shows that after OFTu cells are infected with NA 1/11. delta. 132-p53-GFP, the morphology of the cells changed from long spindle to oblate, and GFP was expressed and localized mostly to the nucleus.

Test 10

FIG. 10 is a Western blot for identifying the expression patterns of p53 and EGFP proteins of OFTu cells stimulated by NA1/11, NA1/11 delta 132-GFP and NA1/11 delta 132-p53-GFP (MOI: 5) according to the invention

1: OFTu cells, without viral stimulation

2: OFTu cells were stimulated by NA1/11

3: OFTu cells were stimulated by NA 1/11. delta.132-GFP

4: OFTu cells were stimulated by NA 1/11. delta.132-p 53-GFP

GFP and p53 expression is shown in FIG. 10,

(1) with the anti-p 53 antibody as a primary antibody, the OFTu cells were found to express GFP and p53 in a fusion manner after stimulation by NA1/11 delta 132-p53-GFP, and the size of the OFTu cells was 80 kDa;

(2) using an anti-GFP antibody as a primary antibody, it was found that after OFTu cells were stimulated by NA1/11 Δ 132-GFP, GFP was expressed and the size was 27 kDa;

(3) using an antibody against GFP as a primary antibody, it was found that upon stimulation of OFTu cells with NA 1/11. delta. 132-p53-GFP, GFP and p53 were expressed as a fusion, and the size was 80 kDa.

Test 11

FIG. 11 is a graph showing the dynamic expression of p53 protein at different time points after OFTu cells were infected with NA 1/11. delta.132-p 53-GFP of the present invention

FIG. 11 shows the expression of p53 at 0h, 12h, 24h, 36h, 48h and 72h after infection of OFTu cells of the present invention with NA 1/11. delta.132-p 53-GFP, showing that fusion expression of p53 and GFP protein is observed at 12h and later, and the size is 80 kDa. GAPDH is a reference balance protein.

Test 12

FIG. 12 shows the expression patterns of ORFV086 protein after OFTu infection with NA1/11, NA 1/11. delta.132-GFP and NA 1/11. delta.132-p 53-GFP of the present invention

1: OFTu cells, without viral stimulation

2: OFTu cells after NA1/11 stimulation

3: OFTu cells after stimulation with NA 1/11. delta.132-GFP

4: OFTu cells after stimulation with NA 1/11. delta.132-p 53-GFP

ORFV086 protein is a virus structural protein and is one of the markers for mature release of virus particles. The expression of ORFV086 in three viruses is shown in FIG. 12. After incubation with antibodies against ORFV086, all three virus-stimulated OFTu cells expressed ORFV086 protein, with sizes of 20kDa and 100kDa, respectively. Western blot shows that the expression level of ORFV086 protein in NA1/11 Delta132-GFP is higher than that in other two viruses, while the expression level of ORFV086 in NA1/11 and NA1/11 Delta132-p 53-GFP is similar. Wherein GAPDH is internal reference.

Test No. 13

FIG. 13 is a diagram showing the analysis of cell growth curves of OFTu infected with three viruses of the present invention, NA1/11, NA 1/11. delta.132-GFP and NA 1/11. delta.132-p 53-GFP

Comparison of the replication capacity of the three viruses after in vitro growth is shown in FIG. 13, the replication cycle of poxviruses is about 20 hours, so that mature virus particles are released to infect new OFTu cells in about 0-24 hours. 24-48h is a new replication cycle, so at 0-48h, the virus replication is in logarithmic growth phase. The replication of the virus tends to be stable from 48h to 96 h. The TCID50 of each time point is introduced into GraphPad Prism 6 software to draw a growth curve, and the in vitro growth replication capacity of the three viruses of NA1/11, NA1/11 delta 132-GFP and NA1/11 delta 132-p53-GFP is not statistically different.

Test 14

FIG. 14 is a graph showing the analysis of growth curves of the host cell OFTu infected with three viruses and various tumor cells of the present invention

a. After Na1/11 is respectively infected with various colorectal cancer cells/lung cancer cells/host cells, a virus proliferation curve is drawn

b. Respectively infecting various colorectal cancer cells/lung cancer cells/host cells by NA1/11 delta 132-GFP, and then drawing a virus proliferation curve

c. Respectively infecting various colorectal cancer cells/lung cancer cells/host cells with NA1/11 delta 132-p53-GFP and then drawing a virus proliferation curve

d. The host cell OFTu cells are respectively infected with Na1/11, NA1/11 delta 132-GFP and NA1/11 delta 132-p53-GFP, and virus propagation curves are drawn

e. Viral proliferation curves were plotted for colorectal cancer cells LoVo cells infected with Na1/11, NA 1/11. delta. 132-GFP, NA 1/11. delta. 132-p53-GFP, respectively

f. Respectively infecting the lung cancer cell A549 cell with Na1/11, NA1/11 delta 132-GFP and NA1/11 delta 132-p53-GFP, and drawing a virus proliferation curve

Na1/11, NA1/11 delta 132-GFP and NA1/11 delta 132-p53-GFP can proliferate and replicate in host cells OFTu and various tumor cells (LoVo, HCT116, CT26, A549 and H1299) (a-c); there was no significant difference in the proliferative replication capacity of the three viruses in cells (d-f). The result shows that the deletion of ORFV132 gene and the insertion of P53 gene do not affect the virus' ability to multiply and replicate.

Test 15

FIG. 15 is a graph showing the effect of three viruses of the present invention on the proliferative activity of various colorectal cancer cells

a. After various colorectal cancer cells were infected with Na1/11 (MOI: 10), the survival rate of the cells was examined at different time points

b. The survival rate of cells was measured at different time points after NA 1/11. delta. 132-GFP (MOI: 10) was infected with various colorectal cancer cells, respectively

c. After NA1/11 delta 132-p53-GFP (MOI: 10) is respectively infected with various colorectal cancer cells, the survival rate of the cells is detected at different time points

d. HCT116 cells were infected with Na1/11, NA 1/11. delta. 132-GFP, NA 1/11. delta. 132-p53-GFP (MOI: 10), respectively, and the survival rate of the cells was examined at different time points

e. RKO cells were infected with Na1/11, NA 1/11. delta. 132-GFP, NA 1/11. delta. 132-p53-GFP (MOI: 10) respectively, and the proportion of cell survival was examined at different time points

f. SW480 cells are respectively infected with Na1/11, NA1/11 delta 132-GFP and NA1/11 delta 132-p53-GFP (MOI: 10), and the survival rate of the cells is detected at different time points;

the wild strain virus Na1/11 can inhibit the proliferation of various colorectal cancer cells (HCT116, RKO, Caco-2, SW480, SW1116 and LoVo), has the most obvious effect on the LoVo cells and has the weakest effect on the SW480 cells (a); the inhibition effect of NA1/11 delta 132-GFP on the proliferation of colorectal cancer cells is weaker than that of the wild strain Na1/11 (b); NA1/11 delta 132-p53-GFP can also inhibit the proliferation of colorectal cancer cells, and the inhibition effect is obviously enhanced compared with that of a wild strain and an ORFV132 deletion strain (c); in the HCT116 and RKO cell lines which are more sensitive to viruses, the inhibition effects of the three viruses on the proliferation of the HCT116 and RKO cells are statistically different 24h after the viruses are infected, wherein the NA1/11 delta 132-p53-GFP is strongest, the Na1/11 times is lower, and the NA1/11 delta 132-GFP is weakest (d, e); in the virus-insensitive SW480 cell line, after the virus is infected for 24 hours, the inhibition capacity of the wild strain Na1/11 and the ORFV132 deletion strain NA1/11 delta 132-GFP on the proliferation of the tumor cells has no statistical difference, the inhibition capacity of the NA1/11 delta 132-p53-GFP and the NA1/11 delta 132-GFP on the proliferation of the tumor cells has statistical difference, after the virus is infected for 72 hours, the inhibition capacity of the three viruses on the proliferation of the tumor cells has statistical difference, the NA1/11 delta 132-p53-GFP is strongest, the Na1/11 times is high, and the NA1/11 delta 132-p53-GFP is weakest (f).

FIG. 16 is a schematic diagram of the construction of the recombinant contagious pustular oviral strain of the present invention.

(1) - (3) is a schematic drawing of shuttle plasmid construction. The empty vector is pSPV-EGFP, and then the p53 gene is inserted into the recombinant plasmid p 53-pSPV-EGFP. Then the right and left sides of ORFV132 gene are inserted to construct shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132 RF. The ORFV132 gene is knocked out by inserting p53 and EGFP genes into the viral genome 132 by homologous recombination.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification, and equivalents thereof.

Sequence listing

<110> southern medical university

<120> recombinant infectious pustulosolsis virus, and preparation method and application thereof

<130> 2018

<141> 2018-02-11

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 41

<212> DNA

<213> Artificial sequence (Hp53-Fw primer sequence)

<400> 1

cagtgacgcc tcgaggaatt catggaggag ccgcagtcag a 41

<210> 2

<211> 45

<212> DNA

<213> Artificial sequence (Hp53-Rv primer sequence)

<400> 2

gctcaccatg gtggcgaatt cgtctgagtc aggcccttct gtctt 45

<210> 3

<211> 42

<212> DNA

<213> Artificial sequence (NA1/11-ORFV132L-Fw primer sequence)

<400> 3

agtaggcctg cgcgcaagct tcgtcttctc ccgctggata aa 42

<210> 4

<211> 41

<212> DNA

<213> Artificial sequence (NA1/11-ORFV132R-Rv primer sequence)

<400> 4

gacctgcagg catgcaagct tgcctcaccc ttaaaagttg g 41

<210> 5

<211> 41

<212> DNA

<213> Artificial sequence (NA1/11-ORFV132L-Fw primer sequence)

<400> 5

cgcatgcatc cgcggagatc tcaacttttt atggaccgca g 41

<210> 6

<211> 44

<212> DNA

<213> Artificial sequence (NA1/11-ORFV132R-Rv primer sequence)

<400> 6

tatagggaga ccggcagatc tgcgggatgc tggtctaatc acta 44

<210> 7

<211> 636

<212> PRT

<213> Artificial sequence (describing amino acid sequence)

<400> 7

Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln

1 5 10 15

Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu

20 25 30

Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp

35 40 45

Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro

50 55 60

Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala Pro Ala Ala Pro

65 70 75 80

Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser

85 90 95

Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly

100 105 110

Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro

115 120 125

Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln

130 135 140

Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met

145 150 155 160

Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys

165 170 175

Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln

180 185 190

His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp

195 200 205

Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu

210 215 220

Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser

225 230 235 240

Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr

245 250 255

Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val

260 265 270

His Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn

275 280 285

Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr

290 295 300

Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys

305 310 315 320

Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu

325 330 335

Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp

340 345 350

Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His

355 360 365

Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met

370 375 380

Phe Lys Thr Glu Gly Pro Asp Ser Asp Gly Lys Ala Thr Met Val Ser

385 390 395 400

Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu

405 410 415

Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu

420 425 430

Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr

435 440 445

Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr

450 455 460

Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp

465 470 475 480

Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile

485 490 495

Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe

500 505 510

Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe

515 520 525

Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn

530 535 540

Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys

545 550 555 560

Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu

565 570 575

Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu

580 585 590

Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp

595 600 605

Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala

610 615 620

Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

625 630 635

<210> 8

<211> 1911

<212> DNA

<213> Artificial sequence (descriptive nucleotide sequence)

<400> 8

atggaggagc cgcagtcaga tcctagcgtc gagccccctc tgagtcagga aacattttca 60

gacctatgga aactacttcc tgaaaacaac gttctgtccc ccttgccgtc ccaagcaatg 120

gatgatttga tgctgtcccc ggacgatatt gaacaatggt tcactgaaga cccaggtcca 180

gatgaagctc ccagaatgcc agaggctgct ccccccgtgg cccctgcacc agcagctcct 240

acaccggcgg cccctgcacc agccccctcc tggcccctgt catcttctgt cccttcccag 300

aaaacctacc agggcagcta cggtttccgt ctgggcttct tgcattctgg gacagccaag 360

tctgtgactt gcacgtactc ccctgccctc aacaagatgt tttgccaact ggccaagacc 420

tgccctgtgc agctgtgggt tgattccaca cccccgcccg gcacccgcgt ccgcgccatg 480

gccatctaca agcagtcaca gcacatgacg gaggttgtga ggcgctgccc ccaccatgag 540

cgctgctcag atagcgatgg tctggcccct cctcagcatc ttatccgagt ggaaggaaat 600

ttgcgtgtgg agtatttgga tgacagaaac acttttcgac atagtgtggt ggtgccctat 660

gagccgcctg aggttggctc tgactgtacc accatccact acaactacat gtgtaacagt 720

tcctgcatgg gcggcatgaa ccggaggccc atcctcacca tcatcacact ggaagactcc 780

agtggtaatc tactgggacg gaacagcttt gaggtgcatg tttgtgcctg tcctgggaga 840

gaccggcgca cagaggaaga gaatctccgc aagaaagggg agcctcacca cgagctgccc 900

ccagggagca ctaagcgagc actgcccaac aacaccagct cctctcccca gccaaagaag 960

aaaccactgg atggagaata tttcaccctt cagatccgtg ggcgtgagcg cttcgagatg 1020

ttccgagagc tgaatgaggc cttggaactc aaggatgccc aggctgggaa ggagccaggg 1080

gggagcaggg ctcactccag ccacctgaag tccaaaaagg gtcagtctac ctcccgccat 1140

aaaaaactca tgttcaagac agaagggcct gactcagacg aattcgccac catggtgagc 1200

aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 1260

aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg 1320

accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 1380

accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac 1440

ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 1500

gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 1560

atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag 1620

tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag 1680

gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac 1740

cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc 1800

acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag 1860

ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta a 1911

Claims

1. A preparation method of recombinant infectious pustulosis virus is characterized by comprising the following steps: the recombinant oncolytic virus is an oncolytic virus carrying a P53-EGFP fusion protein gene, and the fusion protein comprises a P53 protein and an EGFP protein; the fusion protein has the following amino acid sequence:

a protein consisting of an amino acid sequence shown in SEQ ID No. 7;

the preparation method comprises the following steps:

(1) amplifying a gene p53 of a protein consisting of an amino acid sequence shown in SEQ ID No.7 by overlap PCR;

2. A nucleic acid molecule encoding the fusion protein of claim 1, having a nucleotide sequence as set forth in SEQ ID No. 8.

3. The method for producing a recombinant infectious pustulosolvirus according to claim 1, characterized in that: in step (2), PCR and double digestion of ORFV132LF-p53-pSPV-EGFP-ORFV132 RF: the recombinant plasmid band is at 6500bp, the PCR amplification band is at 700bp, and the two bands are 5600bp and 700bp respectively after enzyme digestion; the predicted ORFV132LF-p53-pSPV-EGFP-ORFV132RF is 6392bp, ORFV132RF is 676bp, and the sizes of the two bands after double enzyme digestion are 5649bp and 676bp respectively; sequencing shows that the plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF is successfully constructed.

4. The method for producing a recombinant infectious pustulosolvirus according to claim 1, characterized in that: in step (1), PCR primers were designed using Primer Premier5 software:

the upstream primer Hp53-Fw has the nucleotide sequence of SEQ ID NO. 1;

the downstream primer Hp53-Rv has the nucleotide sequence of SEQ ID NO. 2.

5. The method for producing a recombinant infectious pustulosolvirus according to claim 1, characterized in that: in step (2), the upstream and downstream primers of recombinant plasmid ORFV132LF-p53-pSPV-EGFP were constructed:

the downstream primer NA1/11-ORFV132L-Rv has the nucleotide sequence of SEQ ID NO. 4.

6. The method for producing a recombinant infectious pustulosolvirus according to claim 1, characterized in that: in step (2), the upstream and downstream primers of shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF were constructed:

7. A kit for preparing a targeted killer of colorectal cancer cells, comprising the recombinant infectious pustular oncolytic virus of any one of claims 1-6.

8. Use of the recombinant infectious pustulosolvirus of any one of claims 1-6 for the preparation of a medicament for targeted killing of colorectal cancer cells.