CN108220251A

CN108220251A - A kind of recombination contagious ecthyma oncolytic virus and preparation method and application

Info

Publication number: CN108220251A
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: 2018-06-29
Anticipated expiration: 2038-02-11
Also published as: CN108220251B

Abstract

The invention discloses a kind of ORFV oncolytic virus and its preparation method and application, the preparation method of recombination contagious ecthyma oncolytic virus of the invention includes the following steps：(1) gene type Ⅶ of albumen described in overlap PCR amplifications claim 7；(2) pSPV EGFP carriers are connected to, p53 genes is inserted into and obtains recombinant plasmid p53 pSPV EGFP, are then inserted into the left and right sides of ORFV132 genes, structure shuttle plasmid ORFV132LF p53 pSPV EGFP ORFV132RF；(3) host strain TOP10 is converted, obtains positive restructuring bacterium, (4) transfect the amalgamation and expression of Fluorescence Identification p53 and EGFP after OFTu cells；The recombination contagious ecthyma oncolytic virus of purifying is made.(5) recombination contagious ecthyma oncolytic virus obtained can replicate proliferation in OFTu and kinds of tumor cells, and effectively inhibit kinds of tumor cells growth.The present invention is observable in microscope, easily operated, convenient and efficient, substantially reduces screening time.

Description

A kind of recombination contagious ecthyma oncolytic virus and preparation method and application

Technical field

The present invention relates to gene engineering technology fields, and in particular to a kind of recombination contagious ecthyma oncolytic virus and its preparation Method and application.

Background technology

Oncolytic virus is the virus of a kind of energy specific infection and killing tumor cell.In recent decades, oncolytic virus is controlled Treatment attracts wide attention, and correlative study achieves huge progress.The most deep oncolytic virus of research includes adenovirus, I at present Herpes simplex virus type (herpes simplex virus, HSV), vaccinia virus etc., their specific recognitions and infected tumor Cell eventually leads to cell and is swollen and destroyed tumor cell, but can not into the cell replicate in normal body and make without killing With theoretically with higher anti-tumor effect and lower side effect.Oncolytic virus has that targeting is infected and to kill tumour thin The ability of born of the same parents has differences the neurological susceptibility of tumour cell based on different virus, and different virions can be used as nanoscale medicine Object is used for the treatment of different tumours.Selective infected tumor's cell is the build-in attribute of virus, the capsid of appropriate transformation virus Structure can enhanced virus to the neurological susceptibility of tumour cell.Viral genome virus is transformed by being inserted into or lacking specific gene Exogenous therapeutic molecule can be carried and enter tumour cell so as to killing tumor cell, and weaken its influence to normal cell. It can effectively be killed by internal and external test research carrying enhancing immune response and/or the rush isogenic oncolytic vaccine of apoptosis swollen Oncocyte.A variety of oncolytic virus vaccines have been used to oncotherapy, and safety and validity are also identified well.An Jin (Amgen) Imlygic (talimogene laherparepvec；T-VEC) it is first oncolytic being approved by the FDA in the United States disease Malicious therapy.The drug is the HSV1 by genetic modification, could not be complete by performing the operation for treating lesion in skin and lymph node The melanoma of removing.

Sore mouth virus is also known as sheep infective pustule, is caused by sheep infective pustule virus (Orf virus, ORFV) Acute, skin tendency sexually transmitted disease, infection object is mainly sheep and goat, can also infect people, be a kind of typical people beast Illness altogether.Sheep infective pustule virus (ORFV) is one of Major Members that Poxviridae parapoxvirus belongs to, in brick shape or oval Shape, surface is in cord-like criss-cross arrangement, complicated, there is core, lateral body and coating.With to its molecular biology and exempting from Epidemiology characteristic research is gradually goed deep into, and more and more evidences show that ORFV is likely to become a kind of ideal oncolytic virus and carries Body.Compared with other poxvirus or adenovirus vector, the following unique biological characteristics that ORFV has make it be more suitable for developing Oncolytic virus：First, ORFV genes replicate transcription in cytoplasm, safe in unconformity to host genome；2nd, host Range is than relatively limited, and with skin limitation, quick recovery from illness, toxicity is low for caused damage, has no that virus system spreads through sex intercourse evidence； 3rd, quickly the body fluid of mediation body and adaptive immunity reaction, non-infectious host are particularly evident；4th, in not generated after being immunized With property serum antibody, multiple immunity inoculation is supported in a short time, even if can still mediate carrier special in the presence of antibody Immune response；5th, the attenuated strain of virus can be built by the virulence gene of targeting knock out virus；6th, as viral vaccine The gene compatibility of carrier is big, reproducible and expression allogenic gene；7th, cause point of immune modulatory molecules after ORFV infection It secretes, including granulocyte stimulating factor (CSF), interferon (IFN), IL-2, tumor necrosis factor (TNF-) etc..Therefore, ORFV can It can be a kind of ideal oncolytic virus carrier.ORFV genome lengths are 138kb, and G/C content enriches, thus it is speculated that contains 132 Gene.The both ends gene of wherein genome is not guarded relatively, respectively ORF001-ORF008, ORF112-ORF134.These bases Because usually with the immune evasion of host, immunological regulation and viral virulence are related.Our laboratory early-stage studies the result shows that, lead to RNA sequencing analysis is crossed, after with ORFV infection human skin fibroblasts HFF-18h, the transfection level of ORFV132 genes is apparent Rise.Report at present about the functional study of ORFV132 genes is relatively fewer, ORFV132 encoding VEGFs 2 signal path of VEGF-B, mediation VEGF-B receptor, also some researches show that ORFV132 is related with the cell cycle.In short, ORFV132 It is closely related with the occurrence and development of inflammation.From the thinking of transformation oncolytic virus, knockout ORFV132 genes have been selected so that The virulence of sheep of virus declines, safer to the mankind.Therefore the present invention determines that knockout ORFV132 genes are highly attenuated to build Strain.Simultaneously also studies have found that, the positions of ORFV132 genes is very suitable for being inserted into exotic antigen.Recently, have and much passed about sheep Report of the metachromia pustule virus as viral vectors successful expression exotic antigen：ORFV recombinant virions ORFV D1701 expression is pseudo- Hydrophobin (PRV) glycoprotein gC and gD, available for preventing the PRV infection of pig, the typical pig pyreticosis disease of recombinant virions expression It is immune that toxalbumin (CSFV) generates pig, epidemic diarrhea virus (PEDV)；Rabbit hemorrhagic fever viruse albumen (RHDV)；H5 haemocytes Agglutinin etc., so as to play protective immune response effect.These are research shows that ORFV is expected to become a kind of ideal virus Vaccine delivery system.Therefore, this exotic antigen of p53 can be inserted into the position of ORFV132 genes, using ORFV as vaccine Delivery vector, and then play the function of p53.

P53 is a kind of important tumor suppressor gene, it plays various biological function, as cell cycle regulation, induction are thin Born of the same parents' apoptosis inhibits Tumor angiogenesis, DNA damage reparation, control aging and metabolism etc..Because p53 is in terms of oncotherapy There is irreplaceable role, the research of anticancer drug exploitation and treatment based on p53 is the hot spot of Front Fields of Life Sciences always. But p53 is neither cell surface protein, nor enzyme, so being recently used for the antibody and low molecular weight of targeted molecular treatment Enzyme inhibitor be not p53 anticancer drugs optimal selection.And because of unique advantage of the sheep infective pustule virus as carrier, So that as a kind of anti-cancer vaccine, new interference method is provided for tumor prevention treatment by p53.

Sheep infective pustule virus strain NA1/11 is to suffer from sore mouth chimera in Jilin Province Nongan County this seminar early period The low virulent strain separated.The present invention is directed to knock out the ORFV132 genes of NA1/11, make its pathogenic decrease, structure height subtracts The NA1/11 defect strains of poison.At the same time, it is inserted into foreign gene p53 on the position, to build recombinant virus oncolytic vectors epidemic disease Seedling lays the foundation.There is presently no about the correlation that wide spectrum Suppressor p53 is inserted into sheep infective pustule virus genome Report.

Invention content

The purpose of the present invention is to provide one kindThe anti-tumor effect of the oncolysis of oncolytic virus and p53 is joined together Generate efficient anticancer effectRecombination contagious ecthyma oncolytic virus and preparation method and application.

In order to achieve the above objectives, present invention employs following technical proposals：The present invention provides a kind of novel recombinations to infect Property warts oncolytic virus, the recombination oncolytic virus be carry P53-EGFP antigen-4 fusion protein genes oncolytic virus.

Further, the fusion protein has following amino acid sequence：

(1) protein being made of the amino acid sequence shown in SEQ ID No.7.

(2) with the amino acid sequence homology that sequence SEQ ID No.7 are limited identical function egg is encoded 80% to 100% The amino acid sequence of white matter.

(3) amino acid sequence shown in SEQ ID No.7 has together through increasing, lacking or replacing one or more amino acid The albumen as derived from (1) of isoreactivity.

A kind of nucleic acid molecules of the present invention encode the fusion protein described in claims 1 or 2.

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

The preparation method of recombination contagious ecthyma oncolytic virus of the present invention, it is characterised in that include the following steps：

(1) gene type Ⅶ of albumen described in overlap PCR amplifications claim 7；

(2) pSPV-EGFP carriers are connected to, p53 genes is inserted into and obtains recombinant plasmid p53-pSPV- as recombinant plasmid EGFP is then inserted into the left and right sides of ORFV132 genes, structure shuttle plasmid ORFV132LF-p53-pSPV-EGFP- ORFV132RF；P53 and EGFP gene are inserted into the position of viral genome 132 using the technology of homologous recombination, thus will ORFV132 gene knockouts；

(3) host strain TOP10 is converted, obtains positive restructuring bacterium；

(4) transfect OFTu cells after Fluorescence Identification p53 and EGFP amalgamation and expression；Screening recombination sheep infective pustule virus Defect strain NA1/11 Δ 132-GFP and NA1/11 Δs 132-p53-GFP.；After 6-8 wheel screenings, the recombination that purifying is made passes Metachromia warts oncolytic virus.

Further,

In step (2), the PCR and double digestion of ORFV132LF-p53-pSPV-EGFP-ORFV132RF：Recombinant plasmid item Band is at 6500bp, and PCR amplification band is at 700bp, latter two band of digestion is respectively in 5600bp and 700bp；Prediction ORFV132LF-p53-pSPV-EGFP-ORFV132RF is 6392bp, ORFV132RF 676bp, latter two band of double digestion is big Small is respectively 5649bp and 676bp；Through sequencing, it is found that plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF is built into Work(.

Further,

In step (1), 5 Software for Design PCR primers of Primer Premier are utilized：

Sense primer Hp53-Fw:Nucleotide sequence with SEQID NO.1；

Downstream primer Hp53-Rv has the nucleotide sequence of SEQID NO.2.

Further,

In step (2), the upstream and downstream primer of construction recombination plasmid ORFV132LF-p53-pSPV-EGFP:

Sense primer NA1/11-ORFV132L-Fw has the nucleotide sequence of SEQID NO.3；

Downstream primer NA1/11-ORFV132L-Rv has the nucleotide sequence of SEQID NO.4；

Further, in step (2),

Build the upstream and downstream primer of shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF:

Sense primer NA1/11-ORFV132R-Fw has the nucleotide sequence of SEQID NO.5；

Downstream primer NA1/11-ORFV132R-Rv has the nucleotide sequence of SEQID NO.6.

The kit of recombination contagious ecthyma oncolytic virus of the present invention and its in target killing tumor cell Using.

Advantageous effect：The conventional screening methods of recombinant virions of the present invention are screened using Neo/glu, and the present invention then adopts It is screened with EGFP, is observable in microscope, it is easily operated, it is convenient and efficient, substantially reduce screening time.

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

(1) by the sheep infective pustule virus defect strain of recombination be carrier, be inserted into p53 genes so that ORFV further into It is possibly realized for oncolytic virus.

(2) the wild ORFV strain that the present invention uses is NA1/11 low virulent strains, then knocks out ORFV132 genes, becomes height and subtracts Poison recombination ORFV, from recombination sheep infective pustule virus as vaccine delivery vector more further；

(3) the p53 genes of external source are inserted in the position of 132 genes, the powerful and unique immune tune of ORFV can be utilized Section function gives full play to the effect of p53, is of great importance to structure oncolytic vaccine.Defect strain is configured to further Prepare restructuring ORFV carrier bacterins lay the foundation.Meanwhile there are many biological functions by the foreign gene p53 of insertion, such as inhibit cell Proliferation, lure Lead apoptosis of tumor cells etc..In the present invention, p53 can play its biological function by the advantage of recombination ORFV, be lured for p53 It leads apoptosis therapy tumour and provides some thinkings, help to develop the drug or vaccine for thering is tumour potential treatment to be worth.

(4) present invention successfully constructs shuttle plasmid, and sheep infective pustule virus is filtered out after transfection and homologous recombination Defect strain NA1/11 Δ 132-GFP and NA1/11 Δs 132-p53-GFP.OFTu cytopathy degree is infected in the defect strain： NA1/11>NA1/11Δ132-GFP>NA1/11 Δs 132-p53-GFP. and infect after OFTu cells growth in vitro replication capacity with Wild strain NA1/11 no significant differences.Because NA1/11 is low virulent strain and ORFV132 genes are main Disease-causing gene, therefore knock out ORFV132 genes, and it is inserted into p53 genes on the position, highly attenuated recombination sheep infective pustule virus is successfully constructed, It is expected to become a kind of potential oncolytic virus carrier, lay the foundation for structure oncolytic vaccine.

Description of the drawings

Fig. 1 schemes for PCR amplification target gene p53, ORFV132LF, ORFV132RF of the present invention；

Fig. 2 is the PCR of recombinant plasmid p53-pSPV-EGFP of the present invention and digestion qualification figure；

Fig. 3 is the PCR of recombinant plasmid ORFV132LF-p53-pSPV-EGFP of the present invention and digestion qualification figure；

The PCR and digestion identification that Fig. 4 is the recombinant plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF of the present invention Figure；

Fig. 5 is Fluorescence Identification p53 and EGFP expression figure after transfection OFTu cells of the invention；

The Westernblot that Fig. 6 is the present invention identifies p53 and EGFP expression figures after plasmid transfection；

Fig. 7 is NA1/11 Δ 132-GFP and NA1/11 the Δ 132-p53-GFP fluorescent screening result figures of the present invention；

Fig. 8 is the collection of illustrative plates of the PCR verification recombination sheep infective pustule virus bodies of the present invention；

Fig. 9 is the OFTu cells of the present invention through three kinds of Strain NA1/11, NA1/11 Δ 132-GFP, NA1/11 Δ 132- After p53-GFP infection, the expression figure of fluorescence microscope GFP；Fig. 9 A illustrate normal OFTu cells；Fig. 9 B are NA1/11 After infecting OFTu cells, cellular morphology becomes oblate from spindle shape, without the expression of GFP；Fig. 9 C are NA1/11 Δs 132-GFP After infecting OFTu cells, cellular morphology becomes oblate from spindle shape, and GFP is expressed and major part is positioned at cytoplasm；Fig. 9 D are After NA1/11 Δs 132-p53-GFP infection OFTu cells, cellular morphology becomes oblate from spindle shape, GFP expression and major part It is positioned at nucleus；

The OFTu cells that Figure 10 is the present invention are pierced through NA1/11, NA1/11 Δs 132-GFP, NA1/11 Δ 132-p53-GFP P53 and EGFP protein expression figures after swashing；

Figure 11 is moved for the p53 albumen of different time points after the NA1/11 Δs 132-p53-GFP infection OFTu cells of the present invention State expression figure；

Figure 12 is expression figures of the ORFV086 of the present invention in three kinds of virus；

Three kinds of viruses that Figure 13 is the present invention infect cell growth curve analysis chart after OFTu；

Three kinds of viruses that Figure 14 is the present invention infect growth curve analysis chart after OFTu and kinds of tumor cells；

Figure 15 is influence collection of illustrative plates of three kinds of viruses to a variety of colorectal cancer cell proliferation activities of the present invention；

The recombination sheep infective pustule virus strain that Figure 16 is the present invention builds schematic diagram.(1)-(3) are built for shuttle plasmid Schematic diagram.Zero load is pSPV-EGFP, is inserted into p53 genes as recombinant plasmid p53-pSPV-EGFP.Then it is inserted into The left and right sides of ORFV132 genes, structure shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF.Using homologous heavy P53 and EGFP gene are inserted into the position of viral genome 132 by the technology of group, thus by ORFV132 gene knockouts.

Specific embodiment

The present invention is further described by following embodiment, but should be noted that the scope of the present invention is not implemented by these Any restrictions of example.

A kind of recombination contagious ecthyma oncolytic virus of the present invention, is fusion protein, and the fusion protein includes P53 eggs White and EGFP albumen.

The fusion protein has following amino acid sequence：

(1) protein being made of the amino acid sequence shown in SEQ ID No.7.

Its nucleotide sequence is as shown in SEQ ID No.8.

The preparation method of recombination contagious ecthyma oncolytic virus of the present invention, includes the following steps：

(1) gene type Ⅶ of albumen described in overlap PCR amplifications claim 7；

(2) pSPV-EGFP carriers are connected to, p53 genes is inserted into and obtains recombinant plasmid p53-pSPV- as recombinant plasmid EGFP is then inserted into the left and right sides of ORFV132 genes, structure shuttle plasmid ORFV132LF-p53-pSPV-EGFP- ORFV132RF；P53 and EGFP gene are inserted into the position of viral genome 132 using the technology of homologous recombination, thus will ORFV132 gene knockouts；The PCR and double digestion of ORFV132LF-p53-pSPV-EGFP-ORFV132RF：Recombinant plasmid band exists At 6500bp, PCR amplification band is at 700bp, latter two band of digestion is respectively in 5600bp and 700bp；Prediction ORFV132LF-p53-pSPV-EGFP-ORFV132RF is 6392bp, ORFV132RF 676bp, latter two band of double digestion is big Small is respectively 5649bp and 676bp；Through sequencing, it is found that plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF is built into Work(.

Utilize 5 Software for Design PCR primers of Primer Premier：

Sense primer Hp53-Fw:Nucleotide sequence with SEQID NO.1；

Downstream primer Hp53-Rv has the nucleotide sequence of SEQID NO.2.

The upstream and downstream primer of construction recombination plasmid ORFV132LF-p53-pSPV-EGFP:

Sense primer NA1/11-ORFV132L-Fw has the nucleotide sequence of SEQID NO.3；

Build shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF

Upstream and downstream primer:

Sense primer NA1/11-ORFV132R-Fw has the nucleotide sequence of SEQID NO.5；

Downstream primer NA1/11-ORFV132R-Rv has the nucleotide sequence of SEQID NO.6.

(3) host strain TOP10 is converted, obtains positive restructuring bacterium.

(4) transfect OFTu cells after Fluorescence Identification p53 and EGFP amalgamation and expression；Screening recombination sheep infective pustule virus Defect strain NA1/11 Δ 132-GFP and NA1/11 Δs 132-p53-GFP；After 6-8 wheel screenings, the recombination that purifying is made infects Property warts oncolytic virus.

Embodiment 1

Construction recombination plasmid p53-pSPV-EGFP

(1) amplification and identification of target gene p53

According to the requirement of seamless Cloning Kit, using p53 as template, drawn using Primer Premier5 Software for Design PCR Object (underscore part is restriction enzyme site)：

Hp53-Fw:

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

Hp53-Rv:

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

It is as shown in table 1 that PCR reactions (50 μ L) are carried out according to following system：

Table 1

Reaction condition：

PCR product carries out agarose electrophoresis identification, and is tapped and recovered.

(2) pSPV-EGFP digestions and be tapped and recovered as shown in table 2：

Table 2

By above-mentioned system in 37 DEG C of digestion 2h, it is tapped and recovered.

(3) target gene is connect with carrier

Target gene is connect with carrier according to the standard operating procedure of seamless Cloning Kit, system (10 μ l) such as table 3 It is shown：

Table 3

Note：When the molar ratio for being inserted into target fragment and carrier is 2:When 1, fusion efficiencies highest.Therefore optimum amount is： PSPV-EGFP is tapped and recovered：[0.01x 3737bp (carrier lengths)] ng=37.27ngP53 is tapped and recovered：[0.02x 1179bp (Insert Fragment length)] ng=23.58ng；Positive reaction control systems are as shown in table 4：

Table 4

Connection product transformed competence colibacillus Escherichia coli TOP10, conversion about for 24 hours after, it is seen that the monoclonal bacterium colony being of moderate size. It takes 3 to shake tube, adds in 5mL LB culture mediums and 5 μ L ammonia benzyl mycins, choose monoclonal in shaking in tube, tube will be shaken and be placed in shaking table Overnight incubation.Recombinant plasmid is extracted according to a conventional method, and carries out PCR, double digestion and sequencing identification.

Embodiment 2

Construction recombination plasmid ORFV132LF-p53-pSPV-EGFP

The method of construction recombination plasmid ORFV132LF-p53-pSPV-EGFP is the same as 1.Wherein upstream and downstream primer be respectively (under Dashed part is restriction enzyme 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；

Embodiment 3

Build shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF and ORFV132LF-pSPV-EGFP- ORFV132RF

The method of shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF is built with 1.Wherein upstream and downstream primer Respectively (underscore part is restriction enzyme 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 shuttle plasmid ORFV132LF-pSPV-EGFP-ORFV132RF is according to ORFV132LF-p53-pSPV-EGFP- ORFV132RF is i.e. available after I digestions of EcoR.

(1)-(3) in the schematic diagram below figure 16 of shuttle plasmid are built, identification is sequenced after the completion of shuttle plasmid structure.Weight It is as shown in figure 16 that schematic diagram is built in group sheep infective pustule virus strain：

(1)-(3) build schematic diagram for shuttle plasmid.

Zero load is pSPV-EGFP, is inserted into p53 genes as recombinant plasmid p53-pSPV-EGFP.Then it is inserted into The left and right sides of ORFV132 genes, structure shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF.Using homologous heavy P53 and EGFP gene are inserted into the position of viral genome 132 by the technology of group, thus by ORFV132 gene knockouts.

Embodiment 4

Transfect OFTu cells and homologous recombination

Recovery OFTu cells are added in the culture dish containing culture medium (10%MEM) (10cm2), shake up after incubator It is cultivated in (37 DEG C, 5%CO2).Cell count is added to by the quantity in 105/ holes of 5x in six orifice plates.Treat that OFTu cells are grown to It can be transfected during the degrees of fusion of 70-80% in (four holes in six orifice plates of transfection).

2h before transfection adds in NA1/11 (MOI=1) per hole and infects OFTu cells.8 EP pipes are taken, it is each in each EP pipes 100 μ L opti-MEM are added in, 2 μ g plasmids pSPV-EGFP, p53- are then separately added into the opti-MEM in 4 EP pipes PSPV-EGFP, ORFV132LF-p53-pSPV-EGFP-ORFV132RF and ORFV132LF-pSPV-EGFP-ORFV132RF fill Divide mixing, place 5min.By opti-MEN of 4 pipes containing plasmid respectively with the 4 abundant mixings of pipe opti-MEM, 15min is placed.It sops up Culture medium in six orifice plates adds 1mL opti-MEM.Liquid containing plasmid in EP pipes is drop by drop slowly added to six holes 1mL is added in plate, after 3h has blood serum medium, and liquid is changed after 6h.Transfection is for 24 hours after having seen whether green in fluorescence microscope Fluorescence, so as to tentatively judge whether p53 expresses.Sample is received after 48h, first removes culture medium, is washed one time with PBS, adds 100 μ L 1x SDS-Loading buffer are fully cracked, and with cell scraping cell scraping, are then closed in EP pipes, are boiled sample 15min, The expression that 14000rpm centrifugations are preserved after -20 DEG C for follow-up Westernblot Testing and appraisals p53.

Embodiment 5

Screening recombination sheep infective pustule virus defect strain NA1/11 Δ 132-GFP and NA1/11 Δs 132-p53-GFP.

(1) 96 orifice plate tentatively screens

1) it transfects as stated above, plasmid is respectively ORFV132LF-pSPV-EGFP-ORFV132RF and ORFV132LF- p53-pSPV-EGFP-ORFV132RF。

2) cell conditioned medium (virus liquid) is collected after 48h.

3) after multigelation 3 times, 800rpm centrifugations 5min.

4) plus the previous day of virus spreads 96 orifice plates by the amount of 10000 OFTu cells/wells.

5) 100 μ L supernatants is taken to add in the EP pipes containing 900 μ L, gradient dilution is carried out from 10-1-10-11.

6) preceding 7 holes of the 1-10 row of 96 orifice plates add the 100 μ L of virus liquid diluted, and the 12nd row and eighth row respectively add 100 μ L's has blood serum medium to make blank control.

7) it after 48h, takes pictures in fluorescence microscopy Microscopic observation, with the higher hyperfluorescence cell of small pipette tips scraping extension rate In the EP pipes equipped with 150 μ L culture mediums, next round screening is carried out after multigelation 3 times.

(2) six orifice plates carry out plaque screening

1) paving OFTu cells are 3x 105 per hole cell number in six orifice plates.

2) by after the virus liquid multigelation 3 times by 3-4 wheel screenings, 800rpm centrifugation 5min, by 10-1-10-5 gradients Dilution, is added in six orifice plates and achievees the effect that gradient dilution.

3) after 2h, culture supernatant is removed, the MEM cultures that 2mL contains 5%FBS and 0.5% low melting-point agarose are added in per hole Base.

4) after 48h, in fluorescence microscopy Microscopic observation, have and made marks at green fluorescence with Marker, middle pipette tips picking list Clone is placed in -80 DEG C of preservations in the EP pipes containing 150 μ L, for the next round of screening.

5) after 6-8 wheel screenings, you can the recombinant virus purified.

6) virus after screening is added in six orifice plates and is enlarged culture, extracted viral DNA and carry out PCR and test Card.

Embodiment 6

NA1/11 Δ 132-GFP and NA1/11 Δs 132-p53-GFP infection OFTu cells after observation GFP, p53 and ORFV086 expressions

(1) culture and concentrating virus

1)75cm²30 bottles of OFTu cells of culture bottle culture, every ten bottles of each addition virus liquid NA1/11, NA1/11 Δ 132- 500 μ L of GFP and NA1/11 Δs 132-p53-GFP；

2) it after 5d, with cell scraping scraping cells, is collected in 50mL centrifuge tubes, 37 DEG C of multigelations 3 times；

3) 4000rpm centrifuges 10min；

4) supernatant, 14000rpm centrifugations 1h are taken；

5) supernatant is abandoned, is resuspended with 5mLPBS；

6) 14000rpm centrifuges 1h；

7) 7mL PBS are resuspended, and are sub-packed in 7 EP pipes, each EP pipes 1mL, -80 DEG C of resuspensions；

(2) virus titer is measured

1) 3 96 orifice plates are spread, per 10000, hole OFTu；

2) viral supernatants after 100 μ L multigelations are taken within second day in the EP pipes equipped with 900 μ LMEM, it is dilute to carry out gradient It releases (10-1-10-11)；

3) preceding 7 holes of the 1-10 row of 3 96 orifice plates add virus liquid (NA1/11, NA1/11 the Δ 132- diluted Each 100 μ L of GFP and NA1/11 Δs 132-p53-GFP, the 12nd row and eighth row respectively add 100 μ L's to have blood serum medium to make blank pair According to；

4) after 7d micro- sem observation each column cell lesion number, according to median lethal dose (TCID50) come calculate virus drop Degree.

(3) compare the metainfective cytopathy degree of three kinds of viruses and observe the expression of GFP, p53 and ORFV086.

1) the OFTu cells of 4 six orifice plates are spread, are 3x 10 per hole cell number⁵；

2) by MOI=1, with three kinds of viruses, (NA1/11, NA1/11 Δ 132-GFP and NA1/11 Δ 132-p53-GFP distinguish OFTu cells are infected, observation GFP expressions of taking pictures under fluorescence microscope after 48h, another hole is as negative control；

3) after taking pictures with 150 μ L 1x SDS loading buffer receive sample, Western blot detection GFP, p53 and The expression of ORFV086；

Embodiment 7

Replication in vitro is grown after NA1/11, NA1/11 Δ 132-GFP and NA1/11 Δ 132-p53-GFP infection OFTu cells Ability measures；

1) the OFTu cells of 3 six orifice plates are spread, are 3x 10 per hole cell number⁵；

2) by MOI=1, with three kinds of viruses (NA1/11, NA1/11 Δ 132-GFP and NA1/11 Δ 132-p53-GFP.) point Not Gan Ran OFTu cells, change liquid to cell after 1h；

3) respectively in 1h, for 24 hours, 36h, 48h, 72h, 96h receive sample；

4) sample is received according to Each point in time to survey the TCID50 (method is same as above) of virus, it is viral external so as to compare three kinds Whether replication capacity has difference.

Embodiment 8

NA1/11 is observed, NA1/11 Δs 132-GFP and NA1/11 Δ 132-p53-GFP is to growth of tumour cell and proliferation It influences

(1) detection (TCID50) of duplication proliferative capacity of the virus in tumour cell

1) add various tumour cell 8x105/hole in 6 orifice plate middle berths, each cell spreads 7 holes；

2) paving cell is separately added into three kinds of virus (MOI=1) stimulation tumour cells after 24 hours；

3) after cell changes liquid after 2 hours, each tumour cell and supernatant (first time point) in a hole are collected；

4) then the different time points after cell changes liquid collect the thin of other each hole for (12,24,36,48,60,72 hours) Born of the same parents and supernatant；

5) by the cell being collected into and supernatant, three times, 4000rcf centrifuges 10 points to multigelation in 37 DEG C of water-baths and -80 DEG C Clock collects supernatant；

6) virus concentration of each supernatant and growth curve of the virus in different tumour cells is drawn in detecting step 5 (TCID50)。

(2) CCK-8 checks influence of three kinds of viruses to growth of tumour cell

1) add various 104/holes of tumour cell in 96 orifice plate middle berths, each cell spreads 12 holes；

2) paving cell is separately added into three kinds of virus stimulation tumours after 24 hours by different titers (MOI=0,1,10,100) Cell, each titre set 3 multiple holes；

3) after virus stimulates 24 hours, CCK-8 detection reagents is added in and (are pressed using the culture medium containing 10%FBS per hole 10ul Stoste by CCK-8 reagent dilutions to 100ul working solutions), be incubated 2 hours in 5%CO2,37 DEG C of cell incubator；

4) it using microplate reader, in the case where wavelength is equal to 450nm, detects and records each hole count value.

Experiment 1

Fig. 1 PCR Successful amplification target gene p53, ORFV132LF, ORFV132RF

1:DNAmarkerDL2000

2&3:the PCRproducts ofp53

4&5:the PCRproduct ofORFV132LF

6&7:the PCRproduct ofORFV132RF

PCR amplification target gene result such as Fig. 1：Using p53 genes, NA1/11 as template, design p53, ORFV132LF, ORFV132RF primers expand target fragment.Fig. 1 is as it can be seen that a visible clear band, prediction at 1200bp, 800bp, 700bp P53 be 1179bp, 132LF 743bp, 132RF 676bp, size is consistent.So PCR successfully amplifies target gene p53、ORFV132LF、ORFV132RF。

Experiment 2

The PCR of Fig. 2 recombinant plasmids p53-pSPV-EGFP and digestion identification

1：DNA molecular amount standard,

2&3:Plasmid pSPV-EGFP

4&5:The PCR product of recombinant plasmid p53-pSPV-EGFP

6&7:The digestion products of recombinant plasmid p53-pSPV-EGFP

The PCR of p53-pSPV-EGFP and double digestion result such as Fig. 2:Recombinant plasmid band is at 5000bp, PCR amplification item Band is at 1200bp, latter two band of digestion is respectively in 1200bp and 4000bp or so.The p53-pSPV-EGFP of prediction is 4896bp, p53 1179bp, latter two stripe size of double digestion is respectively 3727bp and 1179bp.Through sequencing, plasmid is found P53-pSPV-EGFP is built successfully.

Experiment 3

The PCR of Fig. 3 recombinant plasmids ORFV132LF-p53-pSPV-EGFP and digestion identification 1：DNA molecular amount standard

2&3:Recombinant plasmid p53-pSPV-EGFP

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

6&7:The digestion products of recombinant plasmid ORFV132LF-p53-pSPV-EGFP

The PCR of ORFV132LF-p53-pSPV-EGFP and double digestion result such as Fig. 3, recombinant plasmid band at 5600bp, PCR amplification band is at 800bp, latter two band of digestion is respectively in 5000bp and 800bp or so.The ORFV132LF- of prediction P53-pSPV-EGFP is 5649bp, ORFV132LF 743bp, latter two stripe size of double digestion be respectively 4896bp and 743bp.Through sequencing, it is found that plasmid ORFV132LF-p53-pSPV-EGFP is built successfully.

Experiment 4

The PCR and digestion identification that Fig. 4 is the recombinant plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF of the present invention

1：DNA molecular amount standard

2&3:Recombinant plasmid ORFV132LF-p53-pSPV-EGFP

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

6&7:The digestion products of recombinant plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF

The PCR of ORFV132LF-p53-pSPV-EGFP-ORFV132RF and double digestion result such as Fig. 4, recombinant plasmid band At 6500bp, PCR amplification band is at 700bp, latter two band of digestion is respectively in 5600bp and 700bp or so.Prediction ORFV132LF-p53-pSPV-EGFP-ORFV132RF is 6392bp, ORFV132RF 676bp, latter two band of double digestion is big Small is respectively 5649bp and 676bp.Through sequencing, it is found that plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF is built into Work(.

Experiment 5

Fig. 5 is Fluorescence Identification p53 and EGFP expression figure after transfection OFTu cells of the invention

After plasmid transfection, 48h observation fluorescence signals are taken pictures such as Fig. 5：After Fig. 5 A is pSPV-EGFP transfections, it is seen that have green Fluorescence distribution is in entire cytoplasm；After Fig. 5 B is p53-pSPV-EGFP transfections, it is seen that there is green fluorescence to be distributed in nucleus, and P53 is positioned at nucleus, the preliminary expression for judging to have p53；Fig. 5 C are transfected for ORFV132LF-pSPV-EGFP-ORFV132RF Afterwards, it is seen that there is green fluorescence to be distributed in entire cytoplasm；Fig. 5 D are visible for ORFV132LF-p53-pSPV-EGFP-ORFV132RF There is green fluorescence to be distributed in nucleus, and p53 is positioned at nucleus, the preliminary expression for judging to have p53.

Experiment 6

The Westernblot that Fig. 6 is the present invention identifies p53 and EGFP expression after plasmid transfection

After plasmid transfection, with the expression of Westernblot identifications p53 and EGFP.As shown in Figure 6：First hole is pSPV- After EGFP transfections, primary antibody is anti-GFP, stripe size 27kDa, illustrates that GFP is expressed；Second hole turns for p53-pSPV-EGFP After dye, primary antibody is anti-GFP, and stripe size 80kDa illustrates GFP and p53 amalgamation and expressions；Third hole is ORFV132LF- After pSPV-EGFP-ORFV132RF transfections, primary antibody is anti-GFP, stripe size 27kDa, illustrates that GFP is expressed；4th hole is ORFV132LF-p53-pSPV-EGFP-ORFV132RF transfection after, primary antibody be anti-GFP, stripe size 80kDa, illustrate GFP and P53 amalgamation and expressions；After ORFV132LF-pSPV-EGFP-ORFV132RF transfections is in 5th hole, primary antibody is anti-p53, without item Band illustrates no p53 expression；After ORFV132LF-p53-pSPV-EGFP-ORFV132RF transfections is in 6th hole, primary antibody is anti- P53, stripe size 80kDa illustrate GFP and p53 amalgamation and expressions；So it is identified through Westernblot, recombinant plasmid P53 and EGFP successful expressions after ORFV132LF-p53-pSPV-EGFP-ORFV132RF transfections.

Experiment 7

Fig. 7 is that the fluorescent screening of the present invention carries recombinant virus NA1/11 Δ 132-GFP and the NA1/11 Δs of EGFP and p53 132-p53-GFP

After fluorescent screening result such as Fig. 7, NA1/11 Δ 132-GFP and NA1/11 Δ 132-p53-GFP infection OFTu, observation To there is green fluorescence, green fluorescence is positioned at cytoplasm, NA1/11 Δs 132-p53- after wherein NA1/11 Δs 132-GFP infection GFP is positioned at nucleus.After Merge, it is found that green fluorescence and cell overlap.

Experiment 8

Fig. 8 is the recombination sheep infective pustule virus purity of the PCR verification purifying of the present invention

1：Template is NA1/11, primer ORFV132, occurs a band, 132 gene magnifications success at about 400bp

2：Template does not occur band for NA1/11 Δ 132-GFP, primer ORFV132

3：Template does not occur band for NA1/11 Δ 132-p53-GFP, primer ORFV132

4：Template is NA1/11, and primer EGFP does not occur band

5：Template has a band for NA1/11 Δ 132-GFP, primer EGFP at about 700bp, illustrates that EGFP gene expands Increase successfully

6：Template has a band for NA1/11 Δ 132-p53-GFP, primer EGFP at about 700bp, illustrates EGFP bases Gene-amplification success

7：Template is NA1/11, and primer p53 does not occur band

8：Template does not occur band for NA1/11 Δ 132-GFP, primer p53

9：Template is NA1/11 Δ 132-p53-GFP, primer p53, has a band about 1200 or so, illustrates p53 bases Gene-amplification success

After NA1/11 Δ 132-GFP and NA1/11 Δs 132-p53-GFP is purified, purity is verified with PCR.As shown in Figure 8, NA1/11 Δ 132-GFP and NA1/11 Δ 132-p53-GFP defect strains purify successfully.

Experiment 9

Fig. 9 is three kinds of virus N A1/11, NA1/11 Δ 132-GFP, NA1/11 Δ 132-p53-GFP infection of the present invention After OFTu cells, fluorescence microscope identifies the expression of GFP

Fig. 9 illustrates OFTu cells through three kinds of Strain NA1/11, NA1/11 Δ 132-GFP, NA1/11 Δ 132-p53- After GFP stimulations, the expression of GFP.Fig. 9 A illustrate normal OFTu cells；Fig. 9 B be NA1/11 infection OFTu cells after, Cellular morphology becomes oblate from spindle shape, without the expression of GFP；Fig. 9 C be NA1/11 Δs 132-GFP infection OFTu cells after, Cellular morphology becomes oblate from spindle shape, and GFP is expressed and major part is positioned at cytoplasm；Fig. 9 D are NA1/11 Δs 132-p53- After GFP infection OFTu cells, cellular morphology becomes oblate from spindle shape, and GFP is expressed and major part is positioned at nucleus.

Experiment 10

The Western blot that Figure 10 is the present invention identify OFTu cells through NA1/11, NA1/11 Δs 132-GFP, NA1/11 Δ132-p53-GFP(MOI：5) p53 and EGFP protein expression figures after stimulating

1：OFTu cells are stimulated without virus

2：OFTu cells are stimulated through NA1/11

3：OFTu cells are stimulated through NA1/11 Δs 132-GFP

4：OFTu cells are stimulated through NA1/11 Δs 132-p53-GFP

GFP and p53 expressions are as shown in Figure 10,

(1) by the use of anti-p53 antibody as primary antibody, OFTu cells are found after NA1/11 Δs 132-p53-GFP stimulations, GFP and P53 amalgamation and expressions, size 80kDa；

(2) by the use of the antibody of anti-GFP as primary antibody, discovery OFTu cells are after NA1/11 Δs 132-GFP stimulations, GFP expression, Size is 27kDa；

(3) by the use of the antibody of anti-GFP as primary antibody, OFTu cells are found after NA1/11 Δs 132-p53-GFP stimulations, GFP With p53 amalgamation and expressions, size 80kDa.

Experiment 11

Figure 11 is moved for the p53 albumen of different time points after the NA1/11 Δs 132-p53-GFP infection OFTu cells of the present invention State expression figure

Figure 11 be the present invention OFTu cells through NA1/11 Δs 132-p53-GFP infection after in 0h, 12h, for 24 hours, 36h, The p53 expressions of 48h, 72h, the visible amalgamation and expression for having p53 and GFP albumen at 12 hours and later, size are in figure 80kDa.GAPDH balances albumen for internal reference.

Experiment 12

After Figure 12 is NA1/11, NA1/11 Δ 132-GFP and NA1/11 Δ 132-p53-GFP infection OFTu of the present invention, ORFV086 protein expression figures

1：OFTu cells, not plus virus stimulates

2：OFTu cells are after NA1/11 is stimulated

3：OFTu cells are after NA1/11 Δs 132-GFP stimulations

4：OFTu cells are after NA1/11 Δs 132-p53-GFP stimulations

ORFV086 albumen is virus structural protein, is one of the mark of virion maturation release.ORFV086 is at three kinds Expression such as Figure 12 in virus.After antibody incubation with anti-ORFV086, the OFTu cells of three kinds of virus stimulations have The expression of ORFV086 albumen, size are respectively 20kDa, 100kDa.And by Western blot it is found that ORFV086 albumen exists Expression quantity is high compared with other two kinds viruses in NA1/11 Δs 132-GFP, and in NA1/11 and NA1/11 Δs 132-p53-GFP ORFV086 expression quantity is close.Wherein GAPDH is internal reference.

Experiment 13

Figure 13 is three kinds of virus N A1/11, NA1/11 Δ 132-GFP, NA1/11 Δ 132-p53-GFP infection of the present invention Cell growth curve analysis chart after OFTu

The metainfective growth in vitro replication capacity of three kinds of viruses is more as shown in figure 13, the replicative cycle about 20 of poxvirus Or so a hour, therefore in 0-24h or so, there is ripe virion release to go to infect new OFTu cells.24-48h is new for one Replicative cycle, therefore in 0-48h, virus replication is in exponential phase.In 48h-96h, viral duplication tends towards stability.It will be each The TCID50 at a time point, which is imported into 6 softwares of GraphPad Prism, draws growth curve discovery, NA1/11, NA1/11 Δs Growth in vitro replication capacity viral tri- kinds of 132-GFP, NA1/11 Δ 132-p53-GFP does not have significant difference.

Experiment 14

Three kinds of viruses that Figure 14 is the present invention infect growth curve analysis chart after host cell OFTu and kinds of tumor cells

A. after infecting various colorectal cancer cell/lung carcinoma cell/host cells respectively for Na1/11, it is bent to draw virus multiplication Line

B. it after infecting various colorectal cancer cell/lung carcinoma cell/host cells respectively for NA1/11 Δs 132-GFP, draws Virus multiplication curve

C. after infecting various colorectal cancer cell/lung carcinoma cell/host cells respectively for NA1/11 Δs 132-p53-GFP, Draw virus multiplication curve

D. Na1/11, NA1/11 Δ 132-GFP, NA1/11 Δ 132-p53- are infected respectively for host cell OFTu cells GFP draws virus multiplication curve

E. Na1/11, NA1/11 Δ 132-GFP, NA1/11 Δ 132- are infected respectively for colorectal cancer cell LoVo cells P53-GFP draws virus multiplication curve

F. Na1/11, NA1/11 Δ 132-GFP, NA1/11 Δ 132-p53- are infected respectively for lung cell A549 cell GFP draws virus multiplication curve

Na1/11, NA1/11 Δ 132-GFP, NA1/11 Δ 132-p53-GFP can be in host cell OFTu and a variety of swollen Proliferation replicates (a-c) in oncocyte (LoVo, HCT116, CT26, A549, H1299)；Proliferation of three kinds of viruses in cell replicates Ability does not have notable difference (d-f).The result shows that it knocks out ORFV132 genes and is inserted into the proliferation that P53 genes have no effect on virus Replication capacity.

Experiment 15

Figure 15 is influence of the three kinds of viruses of the present invention to a variety of colorectal cancer cell proliferation activities

A. it is Na1/11 (MOI：10) after infecting various colorectal cancer cells respectively, different time points detection cell survival ratio Example

B. it is NA1/11 Δ 132-GFP (MOI：10) after infecting various colorectal cancer cells respectively, different time points detection Cell survival ratio

C. it is NA1/11 Δ 132-p53-GFP (MOI：10) after infecting various colorectal cancer cells respectively, different time points Detect cell survival ratio

D. Na1/11, NA1/11 Δ 132-GFP, NA1/11 Δ 132-p53-GFP (MOI are infected respectively for HCT116 cells： 10), different time points detection cell survival ratio

E. Na1/11, NA1/11 Δ 132-GFP, NA1/11 Δ 132-p53-GFP (MOI are infected respectively for RKO cells： 10), different time points detection cell survival ratio

F. Na1/11, NA1/11 Δ 132-GFP, NA1/11 Δ 132-p53-GFP (MOI are infected respectively for SW480 cells： 10), different time points detection cell survival ratio；

Wild strain virus N a1/11 can inhibit a variety of colorectal cancer cells (HCT116, RKO, Caco-2, SW480, SW1116, LoVo) proliferation, it is the most apparent to LoVo cytosiies, it is most weak (a) to SW480 cytosiies effect；NA1/11Δ The inhibiting effect that 132-GFP is proliferated colorectal cancer cell is weaker than wild strain Na1/11 (b)；NA1/11 Δs 132-p53-GFP It can inhibit colorectal cancer cell proliferation function, relative to wild strain and ORFV132 gene-deleted strains, inhibiting effect is remarkably reinforced (c)；In to the more sensitive HCT116 of virus and RKO cell lines, after virus infection for 24 hours, three kinds of viruses are thin with RKO to HCT116 Has there is significant difference in the inhibiting effect of born of the same parents' proliferation, and wherein NA1/11 Δs 132-p53-GFP is most strong, and Na1/11 takes second place, NA1/11 Δs 132-GFP most weak (d, e)；And in the insensitive SW480 cell lines of virus, after virus infection for 24 hours, wild strain Na1/ 11 and ORFV132 gene-deleted strains NA1/11 Δs 132-GFP is to the rejection ability no difference of science of statistics of tumor cell proliferation, NA1/11 Δs To the rejection ability of tumor cell proliferation there are significant difference between 132-p53-GFP and NA1/11 Δs 132-GFP, in virus After infecting 72h, there is significant difference to the rejection ability of tumor cell proliferation between three kinds of viruses, be still NA1/11 Δs 132-p53-GFP is most strong, and Na1/11 takes second place, and NA1/11 Δs 132-p53-GFP is most weak (f).

The recombination sheep infective pustule virus strain that Figure 16 is the present invention builds schematic diagram.

(1)-(3) build schematic diagram for shuttle plasmid.Zero load is pSPV-EGFP, and being inserted into p53 genes becomes recombination matter Grain p53-pSPV-EGFP.Then it is inserted into the left and right sides of ORFV132 genes, structure shuttle plasmid ORFV132LF-p53-pSPV- EGFP-ORFV132RF.P53 and EGFP gene are inserted into the position of viral genome 132 using the technology of homologous recombination, from And by ORFV132 gene knockouts.

Basic principle, main feature and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, the present invention Claimed range is delineated by the appended claims, the specification and equivalents thereof from the appended claims.

Sequence table

<110>Nanfang Medical Univ

<120>A kind of recombination contagious ecthyma oncolytic virus and preparation method and application

<130> 2018

<141> 2018-02-11

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<170> SIPOSequenceListing 1.0

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<210> 2

<211> 45

<212> DNA

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

<400> 2

gctcaccatg gtggcgaatt cgtctgagtc aggcccttct gtctt 45

<210> 3

<211> 42

<212> DNA

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

<400> 3

agtaggcctg cgcgcaagct tcgtcttctc ccgctggata aa 42

<210> 4

<211> 41

<212> DNA

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

<400> 4

gacctgcagg catgcaagct tgcctcaccc ttaaaagttg g 41

<210> 5

<211> 41

<212> DNA

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

<400> 5

cgcatgcatc cgcggagatc tcaacttttt atggaccgca g 41

<210> 6

<211> 44

<212> DNA

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

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

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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

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<213>Artificial sequence (described nucleotide sequence)

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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 kind of recombination contagious ecthyma oncolytic virus, it is characterised in that：The recombination oncolytic virus is melted to carry P53-EGFP The oncolytic virus of hop protein gene, the fusion protein include P53 albumen and EGFP albumen.

2. recombination contagious ecthyma oncolytic virus according to claim 1, it is characterised in that：The fusion protein has such as Lower amino acid sequence：

(1) protein being made of the amino acid sequence shown in SEQ ID No.7.

(2) with the amino acid sequence homology that sequence SEQ ID No.7 are limited identical function protein is encoded 80% to 100% Amino acid sequence.

(3) amino acid sequence shown in SEQ ID No.7 has equal live through increasing, lacking or replacing one or more amino acid The albumen as derived from (1) of property.

3. a kind of nucleic acid molecules encode the fusion protein described in claims 1 or 2.

4. nucleic acid molecules according to claim 3, nucleotide sequence is as shown in SEQ ID No.8.

5. the preparation method of recombination contagious ecthyma oncolytic virus described in claim 1, it is characterised in that include the following steps：

(1) gene type Ⅶ of albumen described in overlap PCR amplifications claim 7；

(2) pSPV-EGFP carriers are connected to, p53 genes is inserted into and obtains recombinant plasmid p53-pSPV-EGFP as recombinant plasmid, Then it is inserted into the left and right sides of ORFV132 genes, structure shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF； P53 and EGFP gene are inserted into the position of viral genome 132 using the technology of homologous recombination, thus by ORFV132 genes It knocks out；

(3) host strain TOP10 is converted, obtains positive restructuring bacterium；

(4) transfect OFTu cells after Fluorescence Identification p53 and EGFP amalgamation and expression；Screening recombination sheep infective pustule virus defect Strain NA1/11 Δ 132-GFP and NA1/11 Δs 132-p53-GFP；After 6-8 wheel screenings, the recombination infectiousness purulence of purifying is made Blister oncolytic virus.

6. the preparation method of recombination contagious ecthyma oncolytic virus according to claim 5, it is characterised in that：

In step (2), the PCR and double digestion of ORFV132LF-p53-pSPV-EGFP-ORFV132RF：Recombinant plasmid band exists At 6500bp, PCR amplification band is at 700bp, latter two band of digestion is respectively in 5600bp and 700bp；Prediction ORFV132LF-p53-pSPV-EGFP-ORFV132RF is 6392bp, ORFV132RF 676bp, latter two band of double digestion is big Small is respectively 5649bp and 676bp；Through sequencing, it is found that plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF is built into Work(.

7. the preparation method of recombination contagious ecthyma oncolytic virus according to claim 5, it is characterised in that：In step (1) in, 5 Software for Design PCR primers of Primer Premier are utilized：

Sense primer Hp53-Fw:Nucleotide sequence with SEQID NO.1；

Downstream primer Hp53-Rv has the nucleotide sequence of SEQID NO.2.

8. the preparation method of recombination contagious ecthyma oncolytic virus according to claim 5, it is characterised in that：In step (2) in, the upstream and downstream primer of construction recombination plasmid ORFV132LF-p53-pSPV-EGFP:

Sense primer NA1/11-ORFV132L-Fw has the nucleotide sequence of SEQID NO.3；

9. the preparation method of recombination contagious ecthyma oncolytic virus according to claim 5, it is characterised in that：In step (2) in, the upstream and downstream primer of structure shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF:

Sense primer NA1/11-ORFV132R-Fw has the nucleotide sequence of SEQID NO.5；

Downstream primer NA1/11-ORFV132R-Rv has the nucleotide sequence of SEQID NO.6.

10. claim 1-9 any one of them recombination contagious ecthyma oncolytic virus kit and its in target killing The application of tumour cell.