CN113943752A

CN113943752A - Constructs, oncolytic viruses with improved sensitivity and uses thereof

Info

Publication number: CN113943752A
Application number: CN202010680925.0A
Authority: CN
Inventors: 张旭辉; 罗显麟; 何亮亮; 宁维瑜; 陈小锋; 李文佳
Original assignee: Dongguan Dongyangguang Biopharmaceutical Research And Development Co ltd; Sunshine Lake Pharma Co Ltd
Current assignee: Dongguan Dongyangguang Biopharmaceutical Research And Development Co ltd; Sunshine Lake Pharma Co Ltd
Priority date: 2020-07-15
Filing date: 2020-07-15
Publication date: 2022-01-18
Also published as: WO2022012028A1

Abstract

The invention relates to a construct, an oncolytic virus with improved sensitivity and application thereof. According to the invention, ICP34.5 gene and ICP47 gene of the I-type herpes simplex virus are knocked out, and an immunostimulating factor expression sequence is inserted into UL23 gene to construct the obtained recombinant virus, so that the neurotoxicity and the selectivity of the recombinant virus can be effectively reduced, the recombinant virus can be infected, replicated and proliferated in tumor cells, and the sensitivity of the recombinant virus to the tumor cells is improved.

Description

Constructs, oncolytic viruses with improved sensitivity and uses thereof

Technical Field

The invention relates to the field of biotechnology and gene therapy, in particular to a construct, oncolytic virus with improved sensitivity and pharmaceutical application.

Background

The oncolytic virus has good anti-tumor effect, can selectively replicate on tumor cells to directly crack and kill the tumor cells, and the released tumor-associated antigen activates the anti-tumor immunity of an organism through antigen presentation, thereby achieving the effect of inhibiting tumor growth and even completely eliminating tumors. The anti-tumor immune response caused by infected tumor cell debris can be amplified by the addition of immunostimulatory factors. In order to further improve the therapeutic effect of oncolytic virus, some immunoregulation or immune activation genes can be inserted into a virus genome, so that the virus can express corresponding genes while replicating on tumor cells and releasing around tumors, thereby improving the tumor immune microenvironment and enhancing the anti-tumor immunity.

IL12, interleukin 12, a cytokine with immunomodulatory activity.

The oncolytic virus is used for expressing IL12, so that the IL12 level in the tumor is improved, the systemic toxic and side effects caused by the traditional intravenous administration can be avoided, and the safety is improved. Although the prior art reports that the oncolytic virus expresses IL12, such as patent CN202010145491.4, the specific construction strategy of the oncolytic virus and the insertion site of a foreign gene (such as IL12) are different, and the influence on the biological activity of the constructed oncolytic virus is unknown. There have been no reports of sensitivity to oncolytic viruses expressing IL 12.

IL12 is applied to the anti-tumor field, and the constructed novel oncolytic virus HSV1-IL12(+) has the advantages of high efficiency, strong specificity and the like, can also cause specific anti-tumor immune response, and improves the sensitivity on tumor cells.

Disclosure of Invention

The present application is directed to solving, at least to some extent, one of the technical problems in the background art set forth above.

To this end, in a first aspect of the invention, the invention proposes a construct. According to an embodiment of the invention, the vector of the construct is derived from HSV, and the construct carries an immunostimulant gene coding box which is arranged at the position of the UL23 gene. The inventor unexpectedly and surprisingly found in experiments that the construct can continuously and highly express the immunostimulant when the gene coding frame of the immunostimulant is arranged at the position of UL23 gene.

According to an embodiment of the present invention, the above-mentioned construct may further comprise at least one of the following additional technical features:

according to embodiments of the invention, ICP47 and the double copy ICP34.5 of the constructs are knocked out,

knocking out a replication nonessential gene ICP47 in HSV genome, and improving the expression of MHC-I on the surface of a tumor cell infected by virus and the capability of presenting cell antigen; by knocking out double copies of ICP34.5, the replication of Herpes Simplex Virus (HSV) in normal cells is limited, and the herpes simplex virus can selectively replicate in tumor cells, thereby improving the safety of the HSV virus in administration.

According to the embodiment of the present invention, the immune stimulator gene coding frame is located between the 387-521 nucleotides of the UL23 gene sequence. It should be noted that the coding sequence of the UL23 gene described herein is encoded by using the first nucleotide of the initiation codon of the UL23 gene as the 1 st position, and the sequence of the UL23 gene can be referred tohttps:// www.ncbi.nlm.nih.gov/nuccore/NC_001806.2report＝genbank&from＝46609&to＝ 47803&strand＝true. According to the embodiment of the present invention, the location of the immunostimulatory factor gene coding frame between the 387-521 nucleotides of the UL23 gene sequence is obtained by knocking out the 388-520 nucleotides of the UL23 gene sequence, and then inserting the immunostimulatory factor gene coding frame between the 387-521 nucleotides of the UL23 gene sequence.

According to an embodiment of the invention, the IL-12 gene coding cassette is operably linked to a promoter comprising at least one selected from CMV, CAG, EF1 a, Rous sarcoma virus long terminal repeat (RSV LTR), metallothionein I (MTI), and the immunostimulant is selected from at least one of GM-CSF, IL2, IL5, IL12, IL15, IL24 and IL 27.

According to an embodiment of the invention, the promoter is CMV.

According to an embodiment of the invention, the immunostimulatory factor is IL 12.

Experiments prove that when the IL-12 gene coding frame is arranged at the UL23 gene position of the HSV vector, the construct can continuously and highly express IL-12, not only can propagate and infect in tumor cells in a highly selective manner, but also can have the function of immunoregulation, and ensure that the tumor cells are thoroughly eliminated by the immune response of an organism.

According to an embodiment of the invention, the HSV is HSV 1. The inventor finds that HSV2 can cause genital infection, and further selects HSV1, so that the safety is further improved.

According to an embodiment of the invention, the coding frame of the IL12 gene has the nucleotide sequence shown as 1) SEQ ID NO 1; 2) a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity compared to 1).

atggagccgctggtgacctgggtggtccccctcctcttcctcttcctgctgtccaggcagggcgctgcctgcagaaccagtgagtgctgttttcaggacccgccatatccggatgcagactcaggctcggcctcgggccctagggacctgagatgctatcggatatccagtgatcgttacgagtgctcctggcagtatgagggtcccacagctggggtcagccacttcctgcggtgttgccttagctccgggcgctgctgctacttcgccgccggctcagccaccaggctgcagttctccgaccaggctggggtgtctgtgctgtacactgtcacactctgggtggaatcctgggccaggaaccagacagagaagtctcctgaggtgaccctgcagctctacaactcagttaaatatgagcctcctctgggagacatcaaggtgtccaagttggccgggcagctgcgtatggagtgggagaccccggataaccaggttggtgctgaggtgcagttccggcaccggacacccagcagcccatggaagttgggcgactgcggacctcaggatgatgatactgagtcctgcctctgccccctggagatgaatgtggcccaggaattccagctccgacgacggcagctggggagccaaggaagttcctggagcaagtggagcagccccgtgtgcgttccccctgaaaaccccccacagcctcaggtgagattctcggtggagcagctgggccaggatgggaggaggcggctgaccctgaaagagcagccaacccagctggagcttccagaaggctgtcaagggctggcgcctggcacggaggtcacttaccgactacagctccacatgctgtcctgcccgtgtaaggccaaggccaccaggaccctgcacctggggaagatgccctatctctcgggtgctgcctacaacgtggctgtcatctcctcgaaccaatttggtcctggcctgaaccagacgtggcacattcctgccgacacccacacagaaccagtggctctgaatatcagcgtcggaaccaacgggaccaccatgtattggccagcccgggctcagagcatgacgtattgcattgaatggcagcctgtgggccaggacgggggccttgccacctgcagcctgactgcgccgcaagacccggatccggctggaatggcaacctacagctggagtcgagagtctggggcaatggggcaggaaaagtgttactacattaccatctttgcctctgcgcaccccgagaagctcaccttgtggtctacggtcctgtccacctaccactttgggggcaatgcctcagcagctgggacaccgcaccacgtctcggtgaagaatcatagcttggactctgtgtctgtggactgggcaccatccctgctgagcacctgtcccggcgtcctaaaggagtatgttgtccgctgccgagatgaagacagcaaacaggtgtcagagcatcccgtgcagcccacagagacccaagttaccctcagtggcctgcgggctggtgtagcctacacggtgcaggtgcgagcagacacagcgtggctgaggggtgtctggagccagccccagcgcttcagcatcgaagtgcaggtttctgattggctcatcttcttcgcctccctggggagcttcctgagcatccttctcgtgggcgtccttggctaccttggcctgaacagggccgcacggcacctgtgcccgccgctgcccacaccctgtgccagctccgccattgagttccctggagggaaggagacttggcagtggatcaacccagtggacttccaggaagaggcatccctgcaggaggccctggtggtagagatgtcctgggacaaaggcgagaggactgagcctctcgagaagacagagctacctgagggtgcccctgagctggccctggatacagagttgtccttggaggatggagacaggtgcaaggccaagatgtga(SEQ ID NO:1)。

According to an embodiment of the invention, the construct has the nucleotide sequence shown as a) SEQ ID NO 2; b) a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity compared to a).

TCGATGTGTCTGTCCTCCGGAAGGGCCCCCAACACGATGTTTGTGCCGGGCAAGGTCGGCGGGATGAGGGCCACGAACGCCAGCACGGCCTGGGGGGTCATGCTGCCCATAAGGTATCGCGCGTCttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttggtaccgagctcggatccactagtccagtgtggatggagccgctggtgacctgggtggtccccctcctcttcctcttcctgctgtccaggcagggcgctgcctgcagaaccagtgagtgctgttttcaggacccgccatatccggatgcagactcaggctcggcctcgggccctagggacctgagatgctatcggatatccagtgatcgttacgagtgctcctggcagtatgagggtcccacagctggggtcagccacttcctgcggtgttgccttagctccgggcgctgctgctacttcgccgccggctcagccaccaggctgcagttctccgaccaggctggggtgtctgtgctgtacactgtcacactctgggtggaatcctgggccaggaaccagacagagaagtctcctgaggtgaccctgcagctctacaactcagttaaatatgagcctcctctgggagacatcaaggtgtccaagttggccgggcagctgcgtatggagtgggagaccccggataaccaggttggtgctgaggtgcagttccggcaccggacacccagcagcccatggaagttgggcgactgcggacctcaggatgatgatactgagtcctgcctctgccccctggagatgaatgtggcccaggaattccagctccgacgacggcagctggggagccaaggaagttcctggagcaagtggagcagccccgtgtgcgttccccctgaaaaccccccacagcctcaggtgagattctcggtggagcagctgggccaggatgggaggaggcggctgaccctgaaagagcagccaacccagctggagcttccagaaggctgtcaagggctggcgcctggcacggaggtcacttaccgactacagctccacatgctgtcctgcccgtgtaaggccaaggccaccaggaccctgcacctggggaagatgccctatctctcgggtgctgcctacaacgtggctgtcatctcctcgaaccaatttggtcctggcctgaaccagacgtggcacattcctgccgacacccacacagaaccagtggctctgaatatcagcgtcggaaccaacgggaccaccatgtattggccagcccgggctcagagcatgacgtattgcattgaatggcagcctgtgggccaggacgggggccttgccacctgcagcctgactgcgccgcaagacccggatccggctggaatggcaacctacagctggagtcgagagtctggggcaatggggcaggaaaagtgttactacattaccatctttgcctctgcgcaccccgagaagctcaccttgtggtctacggtcctgtccacctaccactttgggggcaatgcctcagcagctgggacaccgcaccacgtctcggtgaagaatcatagcttggactctgtgtctgtggactgggcaccatccctgctgagcacctgtcccggcgtcctaaaggagtatgttgtccgctgccgagatgaagacagcaaacaggtgtcagagcatcccgtgcagcccacagagacccaagttaccctcagtggcctgcgggctggtgtagcctacacggtgcaggtgcgagcagacacagcgtggctgaggggtgtctggagccagccccagcgcttcagcatcgaagtgcaggtttctgattggctcatcttcttcgcctccctggggagcttcctgagcatccttctcgtgggcgtccttggctaccttggcctgaacagggccgcacggcacctgtgcccgccgctgcccacaccctgtgccagctccgccattgagttccctggagggaaggagacttggcagtggatcaacccagtggacttccaggaagaggcatccctgcaggaggccctggtggtagagatgtcctgggacaaaggcgagaggactgagcctctcgagaagacagagctacctgagggtgcccctgagctggccctggatacagagttgtccttggaggatggagacaggtgcaaggccaagatgtgacagatatccagcacagtggcggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtGGATCCCCGGGTACCGAGCTCCCCATTGTTATCTGGGCGCTTGTCATTACCACCGCCGCGTCCCCGGCCGATATCTCACCCTGGTCGAGGCGGTGTTGTGTGGTGTAGATGTTCGCGATTGTCTCGG(SEQ ID NO:2)。

In a second aspect of the invention, the invention features an oncolytic virus with increased sensitivity.

According to an embodiment of the invention, the oncolytic virus carries the construct described above. According to the oncolytic virus provided by the embodiment of the invention, HSV1 carries IL12, so that the targeting of a virus strain to tumor cells is enhanced, and the anti-tumor immune response of an organism is activated by expressing IL12 protein through exogenous IL12 genes. The oncolytic virus expressing IL12 according to the embodiment of the invention can make the virus replicate in a tumor and continuously express IL12 at high level locally by means of intratumoral injection, and avoids systemic toxic and side effects caused by intravenous administration.

According to embodiments of the invention, the oncolytic virus may be continuously and highly expressing IL 12.

In a third aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises the construct as described above or the oncolytic virus as described above. The pharmaceutical composition according to the embodiment of the invention can take effect at low dose, and has good killing effect on tumor cells.

In a fourth aspect, the present invention proposes the use of a construct as described above, an oncolytic virus as described above or a pharmaceutical composition as described above for the manufacture of a medicament for the treatment or prevention of a tumour.

According to an embodiment of the invention, the medicament is for selectively killing tumor cells.

According to an embodiment of the invention, the tumor is pancreatic cancer, ovarian cancer, pharyngeal squamous carcinoma, liver cancer, melanoma.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a plasmid map of pMD18-HOM-hIL12 according to an embodiment of the present invention;

FIG. 2 is a PCR gel electrophoresis assay of a recombinant virus constructed according to an embodiment of the present invention;

FIG. 3 is a diagram of a sequencing structure of a recombinant virus constructed according to an embodiment of the present invention;

FIG. 4 is a graph showing the results of expression of hIL12 in Vero cells according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

In the present invention, when the oncolytic virus of the present invention is used for treating a tumor, it is particularly preferable that the immunostimulatory factor is an immunostimulatory factor that can be used for tumor treatment. Examples of "immunostimulatory factors" include, but are not limited to, cytokines (e.g., IL2, IL12, IL15, GM-CSF, IFN γ, etc.), chemokines (e.g., IL8), growth factors (e.g., IL 5).

The "IL 12" is also called IL-12 or interleukin 12, and is a cytokine with immunoregulation function. Early studies of the effects of various cancer types revealed their broad antitumor potential. Wherein "hIL 12" is human interleukin 12.

The "IL 12 gene coding box" as described in the present application refers to a nucleic acid sequence capable of expressing functional IL12, i.e., the IL12 segment encoded by the nucleic acid sequence is a segment capable of realizing the function of IL12, or the IL12 segment encoded by the nucleic acid sequence is an essential functional region of IL 12.

The 'ICP 47 and ICP34.5 knocked-out' refers to that ICP47 and ICP34.5 genes are silenced, and the expression amount of the two genes is obviously reduced compared with HSV-1 without the ICP47 and ICP34.5 knocked-out.

The term "UL 23 gene inactivation" as used herein refers to insertion inactivation, i.e., the original function of UL23 gene is lost after insertion of foreign gene at the position of UL23 gene.

As used herein, "operably linked" refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as promoter sequences and the like, are capable of performing their intended function of regulating the transcription and translation of the foreign gene.

The following embodiments utilize the CRISPR/Cas9 system to construct recombinant HSV-1 virus, which specifically includes plasmid construction, recombinant screening, virus identification and the like. The biological activity of the recombinant HSV-1 virus specifically comprises the expression condition of hIL12, the infection and killing capacity of the virus on tumor cells and the like.

Example 1 construction of recombinant herpes simplex virus

Inserting a coding hIL12 gene sequence between homologous arm sequences at the left side and the right side of a UL23 gene to obtain a pMD18T-HOM-hIL12 plasmid, replacing a LacZ locus on the UL23 gene of an HSV-1/ICP47-/ICP 34.5-double knockout KOS virus strain by a therapeutic gene hIL12 (human IL12) through a Crisper-Cas9 system as shown in figure 1, constructing recombinant virus, and screening the successfully constructed virus by an X-gal blue-white spot screening method.

It should be noted that, since the virus has variations at some base positions during storage and use of the virus, the KOS virus strain used in this example has 99% nucleotide sequence similarity to the wild-type KOS virus strain.

The following examples 2-4 were performed to verify HSV-1-hIL12 of a single viral plaque by PCR and ELISA after multiple rounds of purification, and to evaluate the cytotoxicity of HSV1-hIL12 in different tumor cells.

Example 2 PCR validation

(1) Experimental materials:

after multiple rounds of screening, the virus is monoclonal 2K-hIL12-lacZ 11-3-1.

(2) And (3) PCR verification:

the primer is B45577UL22F/B48431UL24R, and the size of the PCR product is 4051 bp.

The primer sequence is as follows:

B45577UL22F	GAGGCGGTTGGATTGTGCAAGAGAC
		B48431UL24R	ACGAGAACTGCGGTCGTTGTCCTAA

and (3) PCR system:

PCR procedure:

heating at 98 deg.C for 3 min; (98 ℃, 10 s; 60 ℃, 15 s; 68 ℃, 3min) x 35 cycles; 72 ℃ for 5 min; 12 ℃ and forever.

(3) Sequencing results and analysis: the PCR products were subjected to gel electrophoresis detection, as shown in FIG. 2, and the products were recovered and then sequenced, as shown in FIG. 3, to verify that UL23HOM1 has a sequence of 222bp left to UL23HOM2 right 500bp and is not mutated.

Example 3 protein ELISA identification

(1) Experimental materials:

the experimental group HSV1-hIL12-3-1 refers to 2K-hIL12-lacZ11-3-1, and the negative control is HSV-1/2K.

(2) And (3) ELISA verification: vero (African green monkey kidney cell) is taken as a host cell, and the conditions are as follows: HSV1-hIL12 and HSV-1/2K are infected with MOI (ratio of virus to cell number) of 0.01, and culture virus culture supernatants are collected at 24, 48 and 72 hours after Vero cells are infected.

(3) hIL12 expression results:

the expression results of hIL12 in Vero cells are shown in Table 1.

Table 1:

the expression result of hIL12 in Vero cells is shown in FIG. 4.

Example 4 hIL12 Virus cytotoxicity assay

(1) Experimental materials: 2K-hIL12 refers to 2K-hIL12-lacZ11-3-1, HSV-1/2K, the cell is ASPC-1 (pancreatic cancer cell); SKOV3 (human ovarian carcinoma cells); fadu (human pharyngeal squamous carcinoma cells); Hep3B2.1-7 (hepatoma cells); skmel-28 (melanoma cells).

(2) Cell killing verification: the cells were seeded in 96-well culture plates at an appropriate cell density, and after overnight culture, two viruses were added at 7 gradient concentrations (MOI ═ 10, 5, 1, 0.5, 0.1, 0.05, 0.01), cultured for 24, 48, or 72 hours, respectively, and cell viability was measured according to the CCK8 kit instructions.

(3) Cell killing results:

the toxicity of 2K-hIL12 and 2K on different tumor cells is shown in Table 2.

The 2K-hIL12 virus has good killing effect on Hep3B2.1-7 tumor cells, Skmel-28 tumor cells, ASPC-1 tumor cells, Fadu tumor cells and Skov3 tumor cells at a lower dose, and the killing power is obviously stronger than that of the 2K virus at 48 h. The sensitivity of the recombinant oncolytic virus 2k-hIL12 constructed by the invention on tumor cells is improved.

Table 1:

in the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> Dongyang photo-biological medicine research & development Co., Ltd, Dongyuan City

Guangdong Dongyangguang Pharmaceutical Co., Ltd.

<120> construct, oncolytic virus with improved sensitivity and application thereof

<130> 2020-07-15

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1989

<212> DNA

<213> Artificial Sequence

<220>

<223> IL12 Gene coding frame sequence

<400> 1

atggagccgc tggtgacctg ggtggtcccc ctcctcttcc tcttcctgct gtccaggcag 60

ggcgctgcct gcagaaccag tgagtgctgt tttcaggacc cgccatatcc ggatgcagac 120

tcaggctcgg cctcgggccc tagggacctg agatgctatc ggatatccag tgatcgttac 180

gagtgctcct ggcagtatga gggtcccaca gctggggtca gccacttcct gcggtgttgc 240

cttagctccg ggcgctgctg ctacttcgcc gccggctcag ccaccaggct gcagttctcc 300

gaccaggctg gggtgtctgt gctgtacact gtcacactct gggtggaatc ctgggccagg 360

aaccagacag agaagtctcc tgaggtgacc ctgcagctct acaactcagt taaatatgag 420

cctcctctgg gagacatcaa ggtgtccaag ttggccgggc agctgcgtat ggagtgggag 480

accccggata accaggttgg tgctgaggtg cagttccggc accggacacc cagcagccca 540

tggaagttgg gcgactgcgg acctcaggat gatgatactg agtcctgcct ctgccccctg 600

gagatgaatg tggcccagga attccagctc cgacgacggc agctggggag ccaaggaagt 660

tcctggagca agtggagcag ccccgtgtgc gttccccctg aaaacccccc acagcctcag 720

gtgagattct cggtggagca gctgggccag gatgggagga ggcggctgac cctgaaagag 780

cagccaaccc agctggagct tccagaaggc tgtcaagggc tggcgcctgg cacggaggtc 840

acttaccgac tacagctcca catgctgtcc tgcccgtgta aggccaaggc caccaggacc 900

ctgcacctgg ggaagatgcc ctatctctcg ggtgctgcct acaacgtggc tgtcatctcc 960

tcgaaccaat ttggtcctgg cctgaaccag acgtggcaca ttcctgccga cacccacaca 1020

gaaccagtgg ctctgaatat cagcgtcgga accaacggga ccaccatgta ttggccagcc 1080

cgggctcaga gcatgacgta ttgcattgaa tggcagcctg tgggccagga cgggggcctt 1140

gccacctgca gcctgactgc gccgcaagac ccggatccgg ctggaatggc aacctacagc 1200

tggagtcgag agtctggggc aatggggcag gaaaagtgtt actacattac catctttgcc 1260

tctgcgcacc ccgagaagct caccttgtgg tctacggtcc tgtccaccta ccactttggg 1320

ggcaatgcct cagcagctgg gacaccgcac cacgtctcgg tgaagaatca tagcttggac 1380

tctgtgtctg tggactgggc accatccctg ctgagcacct gtcccggcgt cctaaaggag 1440

tatgttgtcc gctgccgaga tgaagacagc aaacaggtgt cagagcatcc cgtgcagccc 1500

acagagaccc aagttaccct cagtggcctg cgggctggtg tagcctacac ggtgcaggtg 1560

cgagcagaca cagcgtggct gaggggtgtc tggagccagc cccagcgctt cagcatcgaa 1620

gtgcaggttt ctgattggct catcttcttc gcctccctgg ggagcttcct gagcatcctt 1680

ctcgtgggcg tccttggcta ccttggcctg aacagggccg cacggcacct gtgcccgccg 1740

ctgcccacac cctgtgccag ctccgccatt gagttccctg gagggaagga gacttggcag 1800

tggatcaacc cagtggactt ccaggaagag gcatccctgc aggaggccct ggtggtagag 1860

atgtcctggg acaaaggcga gaggactgag cctctcgaga agacagagct acctgagggt 1920

gcccctgagc tggccctgga tacagagttg tccttggagg atggagacag gtgcaaggcc 1980

aagatgtga 1989

<210> 2

<211> 3252

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of construct

<400> 2

tcgatgtgtc tgtcctccgg aagggccccc aacacgatgt ttgtgccggg caaggtcggc 60

gggatgaggg ccacgaacgc cagcacggcc tggggggtca tgctgcccat aaggtatcgc 120

gcgtcttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 180

atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 240

cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 300

tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 360

agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 420

tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 480

aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta 540

gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag 600

ctggctagcg tttaaactta agcttggtac cgagctcgga tccactagtc cagtgtggat 660

ggagccgctg gtgacctggg tggtccccct cctcttcctc ttcctgctgt ccaggcaggg 720

cgctgcctgc agaaccagtg agtgctgttt tcaggacccg ccatatccgg atgcagactc 780

aggctcggcc tcgggcccta gggacctgag atgctatcgg atatccagtg atcgttacga 840

gtgctcctgg cagtatgagg gtcccacagc tggggtcagc cacttcctgc ggtgttgcct 900

tagctccggg cgctgctgct acttcgccgc cggctcagcc accaggctgc agttctccga 960

ccaggctggg gtgtctgtgc tgtacactgt cacactctgg gtggaatcct gggccaggaa 1020

ccagacagag aagtctcctg aggtgaccct gcagctctac aactcagtta aatatgagcc 1080

tcctctggga gacatcaagg tgtccaagtt ggccgggcag ctgcgtatgg agtgggagac 1140

cccggataac caggttggtg ctgaggtgca gttccggcac cggacaccca gcagcccatg 1200

gaagttgggc gactgcggac ctcaggatga tgatactgag tcctgcctct gccccctgga 1260

gatgaatgtg gcccaggaat tccagctccg acgacggcag ctggggagcc aaggaagttc 1320

ctggagcaag tggagcagcc ccgtgtgcgt tccccctgaa aaccccccac agcctcaggt 1380

gagattctcg gtggagcagc tgggccagga tgggaggagg cggctgaccc tgaaagagca 1440

gccaacccag ctggagcttc cagaaggctg tcaagggctg gcgcctggca cggaggtcac 1500

ttaccgacta cagctccaca tgctgtcctg cccgtgtaag gccaaggcca ccaggaccct 1560

gcacctgggg aagatgccct atctctcggg tgctgcctac aacgtggctg tcatctcctc 1620

gaaccaattt ggtcctggcc tgaaccagac gtggcacatt cctgccgaca cccacacaga 1680

accagtggct ctgaatatca gcgtcggaac caacgggacc accatgtatt ggccagcccg 1740

ggctcagagc atgacgtatt gcattgaatg gcagcctgtg ggccaggacg ggggccttgc 1800

cacctgcagc ctgactgcgc cgcaagaccc ggatccggct ggaatggcaa cctacagctg 1860

gagtcgagag tctggggcaa tggggcagga aaagtgttac tacattacca tctttgcctc 1920

tgcgcacccc gagaagctca ccttgtggtc tacggtcctg tccacctacc actttggggg 1980

caatgcctca gcagctggga caccgcacca cgtctcggtg aagaatcata gcttggactc 2040

tgtgtctgtg gactgggcac catccctgct gagcacctgt cccggcgtcc taaaggagta 2100

tgttgtccgc tgccgagatg aagacagcaa acaggtgtca gagcatcccg tgcagcccac 2160

agagacccaa gttaccctca gtggcctgcg ggctggtgta gcctacacgg tgcaggtgcg 2220

agcagacaca gcgtggctga ggggtgtctg gagccagccc cagcgcttca gcatcgaagt 2280

gcaggtttct gattggctca tcttcttcgc ctccctgggg agcttcctga gcatccttct 2340

cgtgggcgtc cttggctacc ttggcctgaa cagggccgca cggcacctgt gcccgccgct 2400

gcccacaccc tgtgccagct ccgccattga gttccctgga gggaaggaga cttggcagtg 2460

gatcaaccca gtggacttcc aggaagaggc atccctgcag gaggccctgg tggtagagat 2520

gtcctgggac aaaggcgaga ggactgagcc tctcgagaag acagagctac ctgagggtgc 2580

ccctgagctg gccctggata cagagttgtc cttggaggat ggagacaggt gcaaggccaa 2640

gatgtgacag atatccagca cagtggcggc cgctcgagtc tagagggccc gtttaaaccc 2700

gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg 2760

tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa 2820

ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca 2880

gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg 2940

cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg 3000

gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 3060

ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 3120

cccgtggatc cccgggtacc gagctcccca ttgttatctg ggcgcttgtc attaccaccg 3180

ccgcgtcccc ggccgatatc tcaccctggt cgaggcggtg ttgtgtggtg tagatgttcg 3240

cgattgtctc gg 3252

Claims

1. A construct, wherein the vector of the construct is derived from HSV, and wherein the construct carries an immunostimulatory factor gene coding box and locates the immunostimulatory factor gene coding box at the UL23 gene position.

2. The construct of claim 1, wherein the ICP47 and double copy ICP34.5 genes of the construct are knocked out.

3. The construct of claim 1, wherein the immunostimulatory factor gene coding cassette is located between nucleotides 387-521 of the UL23 gene sequence.

4. The construct of claim 1, wherein the immunostimulatory factor gene coding cassette is operably linked to a promoter comprising at least one member selected from the group consisting of CMV, CAG, EF1 a, RSV LTR, MTI, and wherein the immunostimulatory factor is selected from at least one member selected from the group consisting of GM-CSF, IL2, IL5, IL12, IL15, IL24, and IL 27; optionally, the immunostimulatory factor is IL 12.

5. The construct according to claim 4, wherein the coding cassette of the IL12 gene has the nucleotide sequence as shown below,

1) 1, SEQ ID NO;

2) a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% homology to 1).

6. The construct of claim 4, wherein the construct has a nucleotide sequence as shown below,

1) a nucleotide sequence shown as SEQ ID NO. 2;

7. An oncolytic virus having increased sensitivity comprising the construct of any one of claims 1-6.

8. The oncolytic virus of claim 7, wherein the virus expresses IL 12.

9. A pharmaceutical composition comprising the construct of any one of claims 1 to 6 or the oncolytic virus with increased sensitivity of any one of claims 7 to 8.

10. Use of the construct of any one of claims 1 to 6, the oncolytic virus of increased sensitivity of any one of claims 7 to 8 or the pharmaceutical composition of claim 9 for the manufacture of a medicament for the treatment or prevention of a tumor, optionally a tumor cell, wherein the tumor is pancreatic cancer, ovarian cancer, pharyngeal squamous carcinoma, liver cancer, melanoma.