CN114703186A - Tumor specific promoter and application thereof - Google Patents

Abstract

The invention belongs to the field of tumor immunotherapy, and particularly relates to a tumor-specific promoter and its application. The technical problem to be solved by the present invention is that the tumor-specific proliferation ability of the oncolytic adenovirus is limited. The solution of the present invention to solve the above problems is to provide a core promoter element, which not only has an independent function of initiating gene expression, but also can be used alone as a promoter, and can also be used as an element to construct a promoter. Its nucleotide sequence is shown in SEQ ID No.1; or there are 1 or several base insertion, deletion and/or substitution mutations in the nucleotide sequence shown in SEQ ID No.1, and still have Nucleic acid molecules that function as promoters. The modified tumor-specific promoter of the present invention has better tumor-specific promoter function, and the oncolytic adenovirus constructed with this promoter has the characteristics of being able to specifically proliferate in tumor cells but not in normal cells, and is a An excellent immunotherapy drug against a variety of tumors.

Description

Tumor specific promoter and its application

technical field

The invention belongs to the field of tumor immunotherapy, and particularly relates to a tumor-specific promoter and its application.

Background technique

Surgery, radiotherapy and chemotherapy are the conventional methods for the treatment of tumors. In recent years, tumor immunotherapy has developed rapidly and has become a hot spot in clinical tumor treatment research. Oncolytic adenovirus is a recombinant replicating adenovirus. By inserting a tumor-specific promoter or deleting the relevant genes required for the virus to proliferate in normal cells, the virus that only proliferates in tumor cells can specifically kill tumor cells and kill normal cells. Cells have no cytotoxic activity. Oncolytic adenovirus has excellent characteristics such as easy production, high efficiency and clinical safety. In recent years, oncolytic adenovirus has become a hot spot in the field of tumor therapy because of its innovation and efficacy.

Although oncolytic adenovirus has made some progress in tumor treatment, it has not shown complete anti-tumor effect in most clinical experiments. It mainly faces several challenges, including the limited tumor-specific proliferation ability of oncolytic adenovirus. , Intratumoral infection spread is limited, monotherapy cannot cope with tumor complexity, tumor suppressor microenvironment inhibits the effect of oncolytic adenovirus, etc. Aiming at the challenges of oncolytic adenovirus applications, the main solutions include optimizing tumor-specific promoters to enhance intratumoral proliferation, expressing pro-apoptotic proteins to sensitize tumor cells, optimizing transport modes, and mediating immune regulation.

The tumor-specific and efficient proliferation ability is the source of innovation of oncolytic adenovirus, and it is also one of the biggest challenges in clinical application. In order to ensure the safety of oncolytic adenovirus in normal cells and ensure that it only proliferates in tumor cells, there are mainly two ways: one is to use tumor-specific promoters to control the genes necessary for virus proliferation, thereby controlling the virus's proliferation. Proliferation; the second is to delete genes that are necessary for virus proliferation in normal cells but not required in tumor cells, so that the virus can only proliferate in tumor cells. The application of the former has become an important strategy in targeted gene virus therapy. .

Human telomerase reverse transcriptase (hTERT) has been confirmed to be highly expressed in a variety of malignant tumor tissues compared with normal tissues, such as lung cancer, esophageal cancer, breast cancer, thyroid cancer, melanoma, cervical cancer, Rectal cancer, kidney cancer and leukemia cells and other malignant tumors. Therefore, the telomerase promoter can be used as a tumor-specific promoter for oncolytic adenovirus therapy. Existing studies have shown that the hTERT promoter has limited activity as a tumor-specific promoter, and it is difficult to achieve good results in practical applications.

There is an urgent need in the field to optimize and restructure tumor-specific promoters to improve the tumor-specific and efficient proliferation ability of oncolytic adenoviruses, thereby realizing the efficient and specific killing ability of oncolytic adenoviruses, which provides the research and development of tumor immunotherapy in the field. New valid option.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is that the tumor-specific proliferation ability of the oncolytic adenovirus is limited. The technical solution of the present invention to solve the above problem is to provide a core activation element. The core promoter element has an independent function of initiating gene expression, and can be used as a promoter alone or as an element to construct a promoter.

The core startup element:

1), the nucleotide sequence is shown in SEQ ID No.1;

or:

2) A nucleic acid molecule that has one or several base insertion, deletion and/or substitution mutations in the nucleotide sequence shown in SEQ ID No. 1 and still has the function of a promoter.

Preferably, the above-mentioned 1 or several base insertion, deletion and/or substitution mutations are 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base insertion, deletion and/or Replacement mutation.

Further, the nucleotide sequence of the above-mentioned core promoter element is shown in any one of SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5.

The present invention also provides a promoter containing the above-mentioned core promoter element.

Further, the nucleotide sequence of the above-mentioned promoter is shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No.8.

Wherein, at least one E2F binding site is also inserted into the above promoter.

Wherein, the nucleotide sequence of the above-mentioned E2F binding site is shown in SEQ ID No.15. Or it is a nucleic acid molecule with one or several base insertion, deletion and/or substitution mutations in the nucleotide sequence shown in SEQ ID No. 15, and still has the function of E2F binding site.

Preferably, the above-mentioned 1 or several base insertion, deletion and/or substitution mutations are 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base insertion, deletion and/or Replacement mutation.

Further, the E2F binding site in the above-mentioned promoter is inserted at the 5' end and/or 3' end of the above-mentioned core promoter element.

Further, the nucleotide sequence of the above promoter is shown in SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13 or SEQ ID No.14.

The present invention also provides the application of the above-mentioned core promoter element or the above-mentioned promoter in the preparation of virus or plasmid. Further, the virus is adenovirus. Further, the adenovirus is an oncolytic adenovirus.

The present invention also provides a recombinant vector containing the above-mentioned core promoter element or the above-mentioned promoter.

Wherein, the above-mentioned recombinant vector is a plasmid vector or a viral vector.

Wherein, the above-mentioned viral vector is an adenovirus vector, an adenovirus-associated virus or a retrovirus vector.

Among them, the above-mentioned viral vector is an oncolytic virus.

Further, the above-mentioned oncolytic virus is oncolytic adenovirus, oncolytic parvovirus, oncolytic herpes virus, oncolytic pox virus, oncolytic vesicular stomatitis virus, oncolytic measles virus, oncolytic myxoma virus, oncolytic reverse At least one of the oncolytic virus, the oncolytic reovirus, and the oncolytic vaccinia virus.

Wherein, the above-mentioned plasmid vectors are pDC316, pDC311, pDC312, pDC315, pDC511, pDC512, pDC515, pDC516, pShuttle, pShuttle-CMV, pCTAP-Shuttle series plasmids, pNTAP-Shuttle series plasmids, pAdTrack, pAdTrack-CMV, pacAd5 series plasmids , at least one of pHBAd series plasmids or pXC1 plasmids.

Wherein, the oncolytic adenovirus described in the above recombinant vector is an adenovirus whose serotype belongs to subgenus A, subgenus B, subgenus C, subgenus D, subgenus E, subgenus F or subgenus G.

Wherein, the oncolytic adenovirus described in the above recombinant vector can be selected from serotypes 12, 18, 31 and 61 belonging to subgenus A, and serotypes 3, 7, 11, 14, 16, Types 21, 34, 35, 55, 66, 68, 76 to 79 belong to the C subgenus 1, 2, 5, 6, 57 and 89; belong to the D subgenus 8, 9, 13, 15, 17, 19, 20, 22 to 30, 32, 33, 36 to 39, 46, 48, 49, 53, 54, 56, 58 to 60, 62 to 65, 67, 69 to 75, 80 to 88, 90 to Type 103; type 4 belonging to subgenus E, type 40 and type 41 belonging to subgenus F; type 52 and other adenovirus belonging to subgenus G.

Wherein, in the oncolytic adenovirus described in the above-mentioned recombinant vector, the expression of E1A and/or E1B-19K gene is driven by the above-mentioned core promoter element or the above-mentioned promoter.

Wherein, the E1A described in the above recombinant oncolytic adenovirus is E1A (Delta24) with 24 bp in the middle deleted, and the nucleotide sequence is shown in SEQ ID No.16. Wherein, the nucleotide sequence of E1B 19K is shown in SEQ ID No.17.

The present invention also provides a host cell containing the above-mentioned recombinant vector. Further, the above-mentioned host cells are eukaryotic cells.

On this basis, the present invention provides an antitumor drug. The antitumor drug is prepared by adding the above-mentioned recombinant carrier with pharmaceutically acceptable auxiliary components.

Meanwhile, the present invention also provides a method for preparing the above-mentioned recombinant vector. The method includes the following steps:

a), the above-mentioned core promoter element or the above-mentioned promoter is operably connected to the gene required for adenovirus propagation, and constructed into the shuttle plasmid;

b), transferring the shuttle plasmid and adenovirus backbone plasmid constructed in step a) into packaging cells, and packaging to obtain an oncolytic adenovirus.

Wherein, the gene required for adenovirus propagation described in the above method is E1A and/or E1B-19K gene.

Wherein, the shuttle plasmid described in the above method is pDC316.

Wherein, at least one of the adenovirus backbone plasmids pBHGloxdelE13cre, pBHGfrtdelE13FLP, pAdEasy-1, pAdEasy-2, pBHGE3i or pBHGE10i described in the above method;

Wherein, the packaging cells described in the above method are HEK293 cells.

Wherein, the above-mentioned shuttle vector and adenovirus backbone plasmid were transferred into HEK293 cells using Lipofectamine 3000 for oncolytic adenovirus packaging.

The beneficial effects of the present invention are as follows: the present invention creatively obtains a modified tumor-specific core promoter element and a tumor-specific promoter, and has better tumor-specific promoter functions. The oncolytic adenovirus was constructed with this promoter, which has the advantage of specific targeting that can specifically proliferate in tumor cells but not in normal cells, and is an excellent immunotherapy drug for a variety of tumors. Experiments have proved that the tumor-specific core promoter element and tumor-specific promoter of the present invention are only active in tumor cells and inactive in normal cells, and are safe and specific. The oncolytic adenovirus of the present invention can more effectively kill various tumor cells in vitro, but does not kill normal cells. More importantly, in vivo experiments, the oncolytic adenovirus of the present invention can effectively inhibit tumor growth, which provides a new option for the development and application of immunotherapy drugs in the art.

Description of drawings

Figure 1 RT-PCR detection of human telomerase reverse transcriptase mRNA expression in different human cells.

Figure 2 Detection of hTERT promoter mutations in different human tumor cells.

Figure 3 Schematic diagram of the hTERT WT promoter.

Figure 4. Detection of hTERT point mutation promoter activity by double fluorescein assay. * means p<0.05; ** means p<0.01.

Figure 5. Detection of hTERT point mutation core promoter element activity by dual fluorescein assay. * is p<0.05; ** is p<0.01; *** is p<0.001.

Figure 6. Double fluorescein assay to detect the activity of hTERT point mutant promoter after modification of E2F binding site. * is p<0.05; ** is p<0.01; *** is p<0.001.

Figure 7 RT-PCR detection of E1A mRNA expression in cells infected with oncolytic adenovirus OAd-null.

Figure 8 Western blot detection of E1A protein expression in cells infected with oncolytic adenovirus OAd-null.

Fig. 9 The in vitro killing activity of oncolytic adenovirus OAd-null detected by CCK8 assay. * is p<0.05; ** is p<0.01; *** is p<0.001.

Figure 10 Tumor-killing activity of OAd-null oncolytic adenovirus in different tumor models. A. Tumor volume and tumor size after OAd-null oncolytic adenovirus treated A375 tumor, symbol × indicates tumor regression; B. Tumor volume and tumor size after OAd-null oncolytic adenovirus treated U87MG tumor; C. OAd- Tumor volume and tumor size after null oncolytic adenovirus treatment of SKOV3 tumors. * means P<0.05; ** means P<0.05.

Detailed ways

The tumor therapy of oncolytic viruses, especially oncolytic adenoviruses, still faces some challenges, one of which is the limited tumor-specific proliferation ability of oncolytic adenoviruses.

The present invention has carried out a lot of creative work in order to enhance the intratumoral proliferation ability of the oncolytic virus, especially the tumor-specific proliferation ability. Surprisingly, the present invention finds that a high-efficiency tumor-specific promoter can be obtained by specific modification of the hTERT promoter. Of course, this promoter can also be used in other fields besides the preparation of oncolytic adenoviruses, such as for the preparation of cis-transformed expression plasmids, for the preparation of other oncolytic viruses, such as oncolytic parvovirus, oncolytic herpes virus, Oncolytic vesicular stomatitis virus, oncolytic measles virus, oncolytic myxoma virus, oncolytic retrovirus, oncolytic reovirus, oncolytic vaccinia virus, etc.

Previous work has sequenced the hTERT promoter region of various tumor cells, and found two mutations in the promoter, C mutation to T, resulting in high expression of hTERT gene. We expect that these two mutations may be related to the specific activation ability of the hTERT promoter, and may be used on the promoter of oncolytic adenovirus to improve its therapeutic effect. The wild-type hTERT promoter (hTERT WT, SEQ ID No. 2), C69T mutant promoter (SEQ ID No. 6), C47T mutant promoter (SEQ ID No. 7) and DT double mutant promoter (SEQ ID No. .8) The fragment was ligated into a plasmid and transfected into a variety of cells. The experimental results show that the reporter gene controlled by the above promoter is basically not expressed in normal cells, but is strongly expressed in various tumor cells, and the expression ability of the mutant promoter is stronger than that of the wild type, and the tumor-specific DT double mutant promoter is tumor-specific. Sexual expression is the strongest.

Further, the present invention used a truncated 181bp core promoter segment (SEQ ID No. 1), which was mutated with C69T and/or C47T corresponding to the wild type. First, it was found that this segment has a promoter function, which can initiate the expression of luciferase in various tumor cells. The 181bp core promoter segment of C69T mutation (SEQ ID No. 3), C47T mutation (SEQ ID No. 4) and DT double mutation (SEQ ID No. 5) significantly improved the promoter activity, especially The 181bp core promoter segment of DT double mutation has the strongest promoter activity. That is, the truncated 181bp core promoter segment is a core promoter element. The core promoter element has an independent function of initiating gene expression and can be used as a promoter; it can also be used as an element to construct other promoters.

Subsequently, in order to further improve the tumor-specific expression ability of the above promoter, we used the method of inserting the E2F binding site on the E2F-1 promoter into the above promoter for further modification. The results showed that the insertion of the E2F binding site on the E2F-1 promoter could effectively enhance the tumor-specific expression ability of the above promoters. One of skill in the art can insert one or more (the plurality can be 2, 3, 4, 5, 6 or more) and select an appropriate insertion site. In one example of the present invention, an E2F binding site is inserted upstream and/or downstream of the hTERT 181 bp core fragment of the promoter. Constructed to insert an E2F binding site between the promoters -181bp and -182bp, insert an E2F binding site between the promoters +4bp and +5bp, and insert an E2F binding site into each of the above two positions at the same time. The modified promoters can improve their tumor-specific expression ability.

It can be seen that the above-mentioned core promoter elements and promoters are particularly suitable for preparing recombinant vectors that need to be specifically expressed in tumor cells. Such as plasmid vector or viral vector.

On this basis, the present invention also provides an oncolytic virus preparation and tumor treatment plan involving the above-mentioned core promoter element and promoter. The above-mentioned promoters can be used to construct various oncolytic viruses in which the expression of genes mainly used for replication or propagation is activated, so that new oncolytic viruses can be prepared. Such as oncolytic adenovirus, oncolytic parvovirus, oncolytic herpes virus, oncolytic pox virus, oncolytic vesicular stomatitis virus, oncolytic measles virus, oncolytic myxoma virus, oncolytic retrovirus, oncolytic reovirus Orphan virus, oncolytic vaccinia virus, etc.

Certainly, the promoter provided by the present invention is suitable for participating in the construction of new oncolytic adenovirus. For example, in adenoviruses of different serotypes, the original endogenous promoter is replaced, and the expression of E1 and other essential genes for proliferation is activated, thereby controlling the replication and proliferation of adenovirus in tumor cells, and obtaining an oncolytic with improved tumor-specific proliferation ability. type adenovirus. It can be used for the preparation of oncolytic adenoviruses of various serotypes, such as serotypes 12, 18, 31 and 61 belonging to subgenus A, and 3, 7, 11, 14, 16, Types 21, 34, 35, 55, 66, 68, 76 to 79 belong to the C subgenus 1, 2, 5, 6, 57 and 89; belong to the D subgenus 8, 9, 13, 15, 17, 19, 20, 22 to 30, 32, 33, 36 to 39, 46, 48, 49, 53, 54, 56, 58 to 60, 62 to 65, 67, 69 to 75, 80 to 88, 90 to Type 103; Type 4 belonging to the E subgenus, Type 40 and 41 belonging to the F subgenus; Type 52 belonging to the G subgenus.

Those skilled in the art know that the preparation of oncolytic viruses, especially oncolytic adenoviruses, requires the use of some commonly used vectors. Generally, shuttle vectors and backbone vectors are used. The shuttle vector can be selected from pDC316, pDC311, pDC312, pDC315, pDC511, pDC512, pDC515, pDC516, pShuttle, pShuttle-CMV, pCTAP-Shuttle series of vectors, pNTAP-Shuttle series of vectors, pAdTrack, pAdTrack-CMV, pacAd5 series of vectors, pHBAd Serial vectors, pXC1, etc. The backbone vector can be selected from pBHGloxdelE13cre, pBHGfrtdelE13FLP, pAdEasy-1, pAdEasy-2, pBHGE3i, pBHGE10i and the like. By co-transfecting the constructed shuttle vector and the backbone vector into cells, the packaging and further proliferation of the virus can be realized.

In one example of the present invention, plasmid pDC316 was used as a shuttle plasmid, and the modified hTERT promoter and the E1A/E1B-19K fragment necessary for adenovirus propagation were ligated into the plasmid. pBHGlox(delta)E1,3Cre was also used as a backbone plasmid. The constructed shuttle vector and backbone vector were co-transfected into HEK293 cells and packaged to obtain an oncolytic adenovirus. The obtained oncolytic adenovirus specifically and efficiently expresses the E1A gene, and at the same time shows better anti-tumor effect in in vitro and in vivo anti-tumor tests.

The promoter of the invention has tumor specificity and can be used to construct various tumor-targeted drugs such as oncolytic adenovirus and tumor targeting plasmid. The constructed oncolytic adenovirus can specifically proliferate in tumor cells but not in normal cells, can effectively kill tumor cells, inhibit tumor growth, and has efficacy, safety and specificity.

"Gene" or "coding sequence" refers to a nucleotide sequence or region of DNA or RNA that "encodes" a particular protein. When placed under the control of an appropriate regulatory region, such as a promoter, the coding DNA sequence is transcribed into RNA and translated into a polypeptide. A gene may also contain several operably linked segments, such as a promoter, 5' leader sequence, intron, coding sequence and 3' untranslated sequence, and may also contain a polyadenylation site or signal sequence. A chimeric or recombinant gene is a gene that is not normally found in nature, eg, a gene in which, eg, a promoter, is not associated in nature with part or all of the transcribed DNA region. "Gene expression" refers to the process by which a gene is transcribed into RNA and/or translated into an active protein.

The method of the present invention will be further described below in conjunction with the examples.

The main reagents used in the examples are:

Anti-E1A antibody was purchased from Santa Cruz Biotechnology.

Balb/c nude mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

The reverse transcription kit and SYBR dye detection kit used for RT-PCR were purchased from Nanjing Novizan Biotechnology Co., Ltd.

CCK8 detection reagents were purchased from MCE Company.

Dual-luciferase detection kit was purchased from Promega Corporation.

All other reagents are imported or domestic analytically pure products.

Example 1 Detection of mRNA expression of human telomerase reverse transcriptase in different human cells

The cells used in this example are human-derived normal cells, human embryonic lung fibroblasts MRC-5 (ATCC: CCL-171) and human primary skin fibroblasts PCS-201-010 (ATCC: PCS-201-010 ^TM ), human Source tumor cells: malignant glioblastoma cells U87MG (ATCC: HTB-14), malignant melanoma cells A375 (ATCC: CRL-1619), lung cancer cells A549 (ATCC: CRM-CCL-185), breast cancer cells MCF- 7 (ATCC: HTB-22), cervical cancer cells Hela (ATCC: CRM-CCL-2) and ovarian cancer cells SKOV3 (ATCC: HTB-77). Cells were harvested, and total cellular RNA was extracted and reverse transcribed into cDNA. The expression of human telomerase reverse transcriptase was detected by RT-PCR, and GAPDH was used as an internal reference.

As shown in the results (Fig. 1), in normal cells MRC-5 and PCS-201-010, human telomerase reverse transcriptase was hardly expressed, while in tumor cells of 6 different tumor origins, human telomerase reverse transcriptase was reversed. transcriptases are highly expressed.

Example 2 Detection of hTERT promoter mutation in different human tumor cells

The tumor cells U87MG, A375, A549, MCF-7, Hela and SKOV3 were collected, the cell genome was extracted, and the hTERT promoter nucleic acid sequence was sequenced.

The experimental results showed (Figure 2): in U87MG cells, there are partial C69T and C47T double mutations in the hTERT promoter; in A375 cells, there is a C69T mutation; in other cells, there is no mutation in the hTERT promoter. Therefore, it was considered whether these mutations could be applied to the promoter optimization of oncolytic adenovirus and the improvement of oncolytic effect.

Example 3 Preliminary optimization of hTERT promoter

1. Construction of hTERT promoter point mutation

In this example, the -378-+77 region sequence of the hTERT promoter (from the TERT 5' regulatory region of human chromosome 5, sequence ID: NG_055467.1), a total of 455 bp, is used, which is marked as hTERT WT (marked as L-WT in Figure 3 ). ) sequence (SEQ ID No. 2) (see Figure 3). By studying its sequence, the segment containing 181 bp from -181 to +77 was selected as the core region with strong promoter activity, which was marked as 181-WT sequence (SEQ ID No. 1) (see Figure 3). In Figure 3, +1 indicates the first nucleotide in the mRNA sequence, and -1 indicates the first 5' nucleotide to the transcription start site. Both C69T (nucleotide C at -69) and C47T (nucleotide C at -47) indicate that the hTERT promoter sequence has a single cytosine C to thymine T mutation. DT indicated that the sequence had the above-mentioned double mutation of C→T at positions -47 and -69. The relevant nucleic acid sequences are shown in Table 1:

Table 1. Various hTERT promoter nucleic acid sequences

The pGL3-basic plasmid (purchased from Microbix Biosystems Inc) can be used for the detection of promoter activity, that is, the multiple cloning site region is located upstream of the firefly luciferase gene. After the pGL3-basic plasmid was digested with restriction enzymes XhoI and HindIII, the above-mentioned hTERT WT, 181-WT, 181-C69T, 181-C47T, 181-DT, L-C69T, L-C47T and L -DT fragments were ligated with the digested pGL3-basic, respectively, and constructed as pGL3-L-WT, pGL3-181-WT, pGL3-181-C69T, pGL3-181-C47T, pGL3-181-DT, pGL3-L- C69T, pGL3-L-C47T and pGL3-L-DT were sequenced correctly.

2. Detection of hTERT point mutation promoter activity

The pGL3-basic plasmid carries the firefly luciferase gene, which can be used to detect the activity of the promoter. 96-well plates were plated at 1.5×10 ⁴ cells per well for U87MG, 1.5×10 ⁴ cells per well for A375, and 1× each for MRC-5, PCS-201-010, A549, MCF-7, Hela, and SKOV3 10 ⁴ cells were incubated overnight at 37°C.

Grouping: (1) Control

(2) pGL3-basic (100ng)+pRL-TK (internal reference plasmid 10ng)

(3) pGL3-L-WT (100ng)+pRL-TK (internal reference plasmid 10ng)

(4) pGL3-L-C69T (100ng)+pRL-TK (internal reference plasmid 10ng)

(5) pGL3-L-C47T (100ng)+pRL-TK (internal reference plasmid 10ng)

(6) pGL3-L-DT (100ng)+pRL-TK (internal reference plasmid 10ng)

The plasmids in the above groups were transfected into each cell with Lipofectamine 3000 for 24 hours and the double fluorescence was detected. Note: pRL-TK is the HSV TK promoter to promote the expression of Renilla luciferase, and pCMV-TK is the CMV promoter to promote the expression of Renilla luciferase. The internal reference plasmid used for MRC-5 and PCS-201-010 was pCMV-TK.

The results showed (Fig. 4) that in normal cells MRC-5 and PCS-201-010 cells, almost no firefly luciferase was detected. In tumor cells, the L-DT double-mutated promoter activity was significantly higher than that of the L-WT sequence, indicating that the hTERT WT promoter (-378～+77) double-mutated the activity was significantly enhanced.

3. Detection of hTERT point mutation core promoter element promoter activity

To further test the promoter activity of the core region of the truncated 181bp core promoter element, 96-well plates were plated with 1.5×10 ⁴ cells per well for U87MG, 1.5×10 ⁴ cells per well for A375, MRC-5, PCS-201- 010, A549, MCF-7, Hela and SKOV3 were incubated at 1×10 ⁴ cells per well overnight at 37°C.

Grouping: (1) Control

(2) pGL3-basic (100ng)+pRL-CMV (internal reference plasmid 5ng)

(3) pGL3-L-WT (100ng)+pRL-CMV (internal reference plasmid 5ng)

(4) pGL3-181-WT (100ng)+pRL-CMV (internal reference plasmid 5ng)

(5) pGL3-181-C69T (100ng)+pRL-CMV (internal reference plasmid 5ng)

(6) pGL3-181-C47T (100ng)+pRL-CMV (internal reference plasmid 5ng)

(7) pGL3-181-DT (100ng)+pRL-CMV (internal reference plasmid 5ng)

The plasmids in the above groups were transfected into each cell with Lipofectamine 3000 for 24 hours and the double fluorescence was detected. Note: pCMV-TK is the CMV promoter to promote the expression of Renilla luciferase.

The results showed (Fig. 5) that in normal cells MRC-5 and PCS-201-010 cells, almost no firefly luciferase was detected. In tumor cells, compared with the 181-WT sequence, the promoter activity of the 181-DT double mutation was significantly increased, indicating that the promoter activity was significantly enhanced after the double mutation of the 181bp core promoter element.

4. Further optimization of hTERT double mutant promoter

The E2F-1 promoter has also been reported in the art for oncolytic adenovirus therapy and can selectively proliferate in Rb-deficient cells. The E2F binding site on the E2F-1 promoter is a key site involved in the binding of the E2F-RB complex, and the nucleic acid sequence of the E2F binding site is TCGGCGGCTCGTGGCTCTTTCGCGGCAAAAAGGATTTGGCGCG TAAAAGTGG (SEQ ID No. 15). The present invention considers that it may further enhance its activity by cooperating with the hTERT promoter. The present invention inserts the E2F binding site between -181bp and -182bp on the basis of L-WT and L-DT to form E2F up, and inserts +4bp to + E2Fdown is formed between 5 bp, and E2F up+down is formed by insertion between the two sites (see Table 1 and Figure 3). After the pGL3-basic plasmid was digested with restriction enzymes XhoI and HindIII, L-WT-E2F up, L-WT-E2F down, L-WT-E2F up+down, L-DT shown in Figure 3 -E2F up, L-DT-E2F down and L-DT-E2F up+down fragments were ligated with the digested pGL3-basic to construct pGL3-L-WT-E2F up, pGL3-L-WT-E2F down, pGL3-L-WT-E2F up+down, pGL3-L-DT-E2Fup, pGL3-L-DT-E2F down and pGL3-L-DT-E2F up+down were sequenced correctly.

96-well plates were plated at 1.5×10 ⁴ cells per well for U87MG, 1.5×10 ⁴ cells per well for A375, and 1× each for MRC-5, PCS-201-010, A549, MCF-7, Hela, and SKOV3 10 ⁴ cells were incubated overnight at 37°C.

Grouping:

(1)Control

(2) pGL3-basic (100ng)+pRL-TK (internal reference plasmid 10ng)

(3) pGL3-L-WT (100ng)+pRL-TK (internal reference plasmid 10ng)

(4) pGL3-L-WT-E2F down (100ng)+pRL-TK (internal reference plasmid 10ng)

(5) pGL3-L-WT-E2F up (100ng)+pRL-TK (internal reference plasmid 10ng)

(6) pGL3-L-WT-E2F up+down (100ng)+pRL-TK (internal reference plasmid 10ng)

(7) pGL3-L-DT (100ng)+pRL-TK (internal reference plasmid 10ng)

(8) pGL3-L-DT-E2F down (100ng)+pRL-TK (internal reference plasmid 10ng)

(9) pGL3-L-DT-E2F up (100ng)+pRL-TK (internal reference plasmid 10ng)

(10) pGL3-L-DT-E2F up+down (100ng)+pRL-TK (internal reference plasmid 10ng)

The plasmids in the above groups were transfected into each cell with Lipofectamine 3000 for 24 hours and the double fluorescence was detected. The internal reference plasmid used for MRC-5 and PCS-201-010 was pCMV-TK 50ng, and the internal reference plasmid used for other tumor cells was pRL-TK10ng.

The results showed (Fig. 6) that in normal cells MRC-5 and PCS-201-010 cells, almost no firefly luciferase was detected, but Renilla luciferase could be detected. In tumor cells, the promoter activity of pGL3-L-DT E2F down was significantly increased compared with that of pGL3-L-DT sequence, but not in pGL3-L-DT-E2F down and pGL3-L-DT-E2F up+down. There is a significant difference, so the L-DT-E2F down sequence was selected as the optimal promoter sequence in the subsequent experiments, marked as mhTRET, with a total of 504bp (SEQ ID No. 12).

Example 4 Packaging and functional verification of oncolytic adenovirus OAd-hTERT (hTERT as promoter) and OAd-null (mhTERT as promoter)

After the pDC316 plasmid was digested with restriction enzymes XbaI and HindIII, hTERT WT and mhTERT (the optimal promoter L-DT-E2F in Example 3 (SEQ ID No. 12)) were mixed with adenovirus, respectively. The E1A/E1B-19K (nucleotide sequence shown in SEQ ID No. 18) necessary for proliferation was linked into the digested pDC316 plasmid to construct pDC316-hTERT and pDC316-mhTERT, and an EcoRI was added between hTERT and E1A Restriction site for easy verification.

Lipofectamine 3000 was used to transfer pDC316-hTERT and adenovirus backbone plasmid pBHGlox(delta)E1,3Cre into HEK293 cells for oncolytic adenovirus packaging, labeled as oncolytic adenovirus OAd-hTERT; mhTERT and the adenovirus backbone plasmid pBHGlox(delta)E1,3Cre were transferred into HEK293 cells for oncolytic adenovirus packaging, labeled as oncolytic adenovirus OAd-null.

MRC-5, PCS-201-010, U87MG, A375, A549 and SKOV3 were plated in 6-well plates with 3×10 ⁵ cells per well and cultured at 37°C overnight. The next day, A549 cells were infected with Ad-GFP (adenovirus expressing GFP protein), H101, OAd-hTERT and OAd-null according to MOI(pfu)=64 and the remaining cells according to MOI(pfu)=32. After 24 hours of infection, the cell pellets were collected, and the mRNA and protein expression levels of E1A were detected by RT-PCR and Western blot, respectively. H101 was a positive control oncolytic adenovirus (purchased from Shanghai Sunway Biotechnology Co., Ltd.).

As shown in the results (Figure 7 RT-PCR detection and Figure 8 Western blot detection), in normal cells MRC-5 and PCS-201-010 cells, the mRNA and protein expressions of E1A were hardly detected. In tumor cells, the mRNA and protein expression of E1A could be detected, and compared with H101 and OAd-hTERT, E1A was significantly up-regulated after OAd-null infection, indicating that OAd-null proliferates efficiently in tumor cells and does not affect normal cells.

Example 5 Detection of tumor-killing activity of oncolytic adenovirus OAV-hTERT and OAd-null in vitro

MRC-5, PCS-201-010, U87MG, A375, A549 and SKOV3 were plated in 96-well plates with 3×10 ³ cells per well and cultured at 37°C overnight. The next day, MRC-5, PCS-201-010, U87MG, A375 and A549 were infected with MOI(pfu)=128, 256, 512, 1024, and SKOV3 cells were infected with MOI(pfu)=31.25, 62.5, 250, 1000, respectively OAd-GFP (GFP protein after hTERTWT promoter), H101, OAd-hTERT and OAd-null prepared in Example 4. After 3-6 days of infection, the survival of cells was detected by CCK8 assay.

The results showed ( FIG. 9 ) that in the normal cells MRC-5 and PCS-201-010 cells, the cells survived better, and there was no statistical difference in the survival rate between the groups. In tumor cells, compared with OAd-GFP, H101 and OAd-hTERT could effectively kill tumor cells, while OAd-null had the strongest killing activity, with a statistical difference.

Example 6 Detection of tumor-killing activity of oncolytic adenovirus OAd-null in vivo

(1) A375 tumor model: 4-week-old Balb/c nude mice were subcutaneously inoculated with 1×10 ⁷ A375 cells, and when the tumor volume grew to about 50-100 mm ³ , administration was performed, and the groups were grouped as follows:

①Vehicle: 50μL sterile PBS;

②H101: 1×10 ⁷ pfu each, volume 50 μL;

③OAd-null: 1×10 ⁷ pfu each, volume 50 μL;

Mode of administration: once every other day, 5 times in total, intratumoral administration. Mice tumor size was measured periodically.

(2) U87MG tumor model: 4-week-old Balb/c nude mice were subcutaneously inoculated with 2×10 ⁶ U87MG cells, and when the tumor volume grew to about 50-100 mm ³ , the administration was administered, and the groups were grouped as follows:

①Vehicle: 50μL sterile PBS;

②OAd-null: 1×10 ⁷ pfu each, volume 50 μL;

Administration: once a week, a total of 2 times, intratumoral administration. Mice tumor size was measured periodically.

(3) SKOV3 tumor model: 4-week-old Balb/c nude mice were subcutaneously inoculated with ⁵ ×10 SKOV3 cells, and when the tumor volume grew to about 50-100 mm ³ , the drugs were administered, and the groups were grouped as follows:

①Vehicle: 50μL sterile PBS;

②OAd-null: 1×10 ⁷ pfu each, volume 50 μL;

Administration: once a week, a total of 2 times, intratumoral administration. Mice tumor size was measured periodically.

From the results (Fig. 10A), in the A375 tumor model, the Vehicle group had faster tumor growth. Compared with Vehicle, H101 inhibited tumor growth to a certain extent, with a tumor inhibition rate of 33%. On the other hand, OAd-null oncolytic adenovirus could effectively inhibit tumor growth, with a tumor inhibition rate of 77%, and tumor regression occurred in 2 mice (symbol X indicates tumor regression). In the U87MG model (Fig. 10B), the tumor growth in the Vehicle group was also relatively fast. Compared with the Vehicle, the OAd-null oncolytic adenovirus could effectively inhibit the tumor growth, and the tumor inhibition rate reached 79%. In the SKOV3 model (Fig. 10C), the tumor growth in the Vehicle group was also relatively fast. Compared with Vehicle, OAd-null oncolytic adenovirus could effectively inhibit tumor growth, with a tumor inhibition rate of 75%.

Other nucleotide sequences used in the present invention:

SEQ ID NO. 16 (nucleotide sequence of E1A (DELTA24)):

ATGAGACATATTATCTGCCACGGAGGTGTTATTACCGAAGAAATGGCCGCCAGTCTTTTGGACCAGCTGATCGAAGAGGTACTGGCTGATAATCTTCCACCTCCTAGCCATTTTGAACCACCTACCCTTCACGAACTGTATGATTTAGACGTGACGGCCCCCGAAGATCCCAACGAGGAGGCGGTTTCGCAGATTTTTCCCGACTCTGTAATGTTGGCGGTGCAGGAAGGGATTGACTTACTCACTTTTCCGCCGGCGCCCGGTTCTCCGGAGCCGCCTCACCTTTCCCGGCAGCCCGAGCAGCCGGAGCAGAGAGCCTTGGGTCCGGTTTCTATGCCAAACCTTGTACCGGAGGTGATCGATCCACCCAGTGACGACGAGGATGAAGAGGGTGAGGAGTTTGTGTTAGATTATGTGGAGCACCCCGGGCACGGTTGCAGGTCTTGTCATTATCACCGGAGGAATACGGGGGACCCAGATATTATGTGTTCGCTTTGCTATATGAGGACCTGTGGCATGTTTGTCTACAGTAAGTGAAAATTATGGGCAGTGGGTGATAGAGTGGTGGGTTTGGTGTGGTAATTTTTTTTTTAATTTTTACAGTTTTGTGGTTTAAAGAATTTTGTATTGTGATTTTTTTAAAAGGTCCTGTGTCTGAACCTGAGCCTGAGCCCGAGCCAGAACCGGAGCCTGCAAGACCTACCCGCCGTCCTAAAATGGCGCCTGCTATCCTGAGACGCCCGACATCACCTGTGTCTAGAGAATGCAATAGTAGTACGGATAGCTGTGACTCCGGTCCTTCTAACACACCTCCTGAGATACACCCGGTGGTCCCGCTGTGCCCCATTAAACCAGTTGCCGTGAGAGTTGGTGGGCGTCGCCAGGCTGTGGAATGTATCGAGGACTTGCTTAACGAGCCTGGGCAACCTTTGGACTTGAGCTGTAAACGCCCCAGGCCATAAGGTGTAAACCTGTGATTGCGTGTGTGGTTAACGCCTTT GTTTGCTGAATGAGTTGATGTAAGTTTAATAAAGGGTGAGATAATGTTT

SEQ ID NO. 17 (nucleotide sequence of E1B 19K)

TATATAATGCGCCGTGGGCTAATCTTGGTTACATCTGACCTCATGGAGGCTTGGGAGTGTTTGGAAGATTTTTCTGCTGTGCGTAACTTGCTGGAACAGAGCTCTAACAGTACCTCTTGGTTTTGGAGGTTTCTGTGGGGCTCATCCCAGGCAAAGTTAGTCTGCAGAATTAAGGAGGATTACAAGTGGGAATTTGAAGAGCTTTTGAAATCCTGTGGTGAGCTGTTTGATTCTTTGAATCTGGGTCACCAGGCGCTTTTCCAAGAGAAGGTCATCAAGACTTTGGATTTTTCCACACCGGGGCGCGCTGCGGCTGCTGTTGCTTTTTTGAGTTTTATAAAGGATAAATGGAGTGAAGAAACCCATCTGAGCGGGGGGTACCTGCTGGATTTTCTGGCCATGCATCTGTGGAGAGCGGTTGTGAGACACAAGAATCGCCTGCTACTGTTGTCTTCCGTCCGCCCGGCGATAATACCGACGGAGGAGCAGCAGCAGCAGCAGGAGGAAGCCAGGCGGCGGCGGCAGGAGCAGAGCCCATGGAACCCGAGAGCCGGCCTGGACCCTCGGGAATGA

SEQ ID NO. 18 (nucleotide sequence of E1ADELTA24-E1B 19K):

ATGAGACATATTATCTGCCACGGAGGTGTTATTACCGAAGAAATGGCCGCCAGTCTTTTGGACCAGCTGATCGAAGAGGTACTGGCTGATAATCTTCCACCTCCTAGCCATTTTGAACCACCTACCCTTCACGAACTGTATGATTTAGACGTGACGGCCCCCGAAGATCCCAACGAGGAGGCGGTTTCGCAGATTTTTCCCGACTCTGTAATGTTGGCGGTGCAGGAAGGGATTGACTTACTCACTTTTCCGCCGGCGCCCGGTTCTCCGGAGCCGCCTCACCTTTCCCGGCAGCCCGAGCAGCCGGAGCAGAGAGCCTTGGGTCCGGTTTCTATGCCAAACCTTGTACCGGAGGTGATCGATCCACCCAGTGACGACGAGGATGAAGAGGGTGAGGAGTTTGTGTTAGATTATGTGGAGCACCCCGGGCACGGTTGCAGGTCTTGTCATTATCACCGGAGGAATACGGGGGACCCAGATATTATGTGTTCGCTTTGCTATATGAGGACCTGTGGCATGTTTGTCTACAGTAAGTGAAAATTATGGGCAGTGGGTGATAGAGTGGTGGGTTTGGTGTGGTAATTTTTTTTTTAATTTTTACAGTTTTGTGGTTTAAAGAATTTTGTATTGTGATTTTTTTAAAAGGTCCTGTGTCTGAACCTGAGCCTGAGCCCGAGCCAGAACCGGAGCCTGCAAGACCTACCCGCCGTCCTAAAATGGCGCCTGCTATCCTGAGACGCCCGACATCACCTGTGTCTAGAGAATGCAATAGTAGTACGGATAGCTGTGACTCCGGTCCTTCTAACACACCTCCTGAGATACACCCGGTGGTCCCGCTGTGCCCCATTAAACCAGTTGCCGTGAGAGTTGGTGGGCGTCGCCAGGCTGTGGAATGTATCGAGGACTTGCTTAACGAGCCTGGGCAACCTTTGGACTTGAGCTGTAAACGCCCCAGGCCATAAGGTGTAAACCTGTGATTGCGTGTGTGGTTAACGCCTTT GTTTGCTGAATGAGTTGATGTAAGTTTAATAAAGGGTGAGATAATGTTTAACTTGCATGGCGTGTTAAATGGGGCGGGGCTTAAAGGGTATATAATGCGCCGTGGGCTAATCTTGGTTACATCTGACCTCATGGAGGCTTGGGAGTGTTTGGAAGATTTTTCTGCTGTGCGTAACTTGCTGGAACAGAGCTCTAACAGTACCTCTTGGTTTTGGAGGTTTCTGTGGGGCTCATCCCAGGCAAAGTTAGTCTGCAGAATTAAGGAGGATTACAAGTGGGAATTTGAAGAGCTTTTGAAATCCTGTGGTGAGCTGTTTGATTCTTTGAATCTGGGTCACCAGGCGCTTTTCCAAGAGAAGGTCATCAAGACTTTGGATTTTTCCACACCGGGGCGCGCTGCGGCTGCTGTTGCTTTTTTGAGTTTTATAAAGGATAAATGGAGTGAAGAAACCCATCTGAGCGGGGGGTACCTGCTGGATTTTCTGGCCATGCATCTGTGGAGAGCGGTTGTGAGACACAAGAATCGCCTGCTACTGTTGTCTTCCGTCCGCCCGGCGATAATACCGACGGAGGAGCAGCAGCAGCAGCAGGAGGAAGCCAGGCGGCGGCGGCAGGAGCAGAGCCCATGGAACCCGAGAGCCGGCCTGGACCCTCGGGAATGA

sequence listing

<110> Sichuan University

<120> Tumor-specific promoters and their applications

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 258

<212> DNA

<213> Artificial Sequence

<400> 1

ccaggaccgc gctccccacg tggcggaggg actggggacc cgggcacccg tcctgcccct 60

tcaccttcca gctccgcctc ctccgcgcgg accccgcccc gtcccgaccc ctcccgggtc 120

cccggcccag ccccctccgg gccctcccag cccctcccct tcctttccgc ggccccgccc 180

tctcctcgcg gcgcgagttt caggcagcgc tgcgtcctgc tgcgcacgtg ggaagccctg 240

gccccggcca cccccgcg 258

<210> 2

<211> 455

<212> DNA

<213> Artificial Sequence

<400> 2

tggcccctcc ctcgggttac cccacagcct aggccgattc gacctctctc cgctggggcc 60

ctcgctggcg tccctgcacc ctgggagcgc gagcggcgcg cgggcgggga agcgcggccc 120

agacccccgg gtccgcccgg agcagctgcg ctgtcggggc caggccgggc tcccagtgga 180

ttcgcgggca cagacgccca ggaccgcgct ccccacgtgg cggagggact ggggacccgg 240

gcacccgtcc tgccccttca ccttccagct ccgcctcctc cgcgcggacc ccgccccgtc 300

ccgacccctc ccgggtcccc ggcccagccc cctccgggcc ctcccagccc ctccccttcc 360

tttccgcggc cccgccctct cctcgcggcg cgagtttcag gcagcgctgc gtcctgctgc 420

gcacgtggga agccctggcc ccggccaccc ccgcg 455

<210> 3

<211> 251

<212> DNA

<213> Artificial Sequence

<400> 3

cgcgctcccc acgtggcgga gggactgggg acccgggcac ccgtcctgcc ccttcacctt 60

ccagctccgc ctcctccgcg cggaccccgc cccgtcccga ccccttccgg gtccccggcc 120

cagccccctc cgggccctcc cagcccctcc ccttcctttc cgcggccccg ccctctcctc 180

gcggcgcgag tttcaggcag cgctgcgtcc tgctgcgcac gtgggaagcc ctggccccgg 240

ccacccccgc g 251

<210> 4

<211> 251

<212> DNA

<213> Artificial Sequence

<400> 4

cgcgctcccc acgtggcgga gggactgggg acccgggcac ccgtcctgcc ccttcacctt 60

ccagctccgc ctcctccgcg cggaccccgc cccgtcccga cccctcccgg gtccccggcc 120

cagccccttc cgggccctcc cagcccctcc ccttcctttc cgcggccccg ccctctcctc 180

gcggcgcgag tttcaggcag cgctgcgtcc tgctgcgcac gtgggaagcc ctggccccgg 240

ccacccccgc g 251

<210> 5

<211> 251

<212> DNA

<213> Artificial Sequence

<400> 5

cgcgctcccc acgtggcgga gggactgggg acccgggcac ccgtcctgcc ccttcacctt 60

ccagctccgc ctcctccgcg cggaccccgc cccgtcccga ccccttccgg gtccccggcc 120

cagccccttc cgggccctcc cagcccctcc ccttcctttc cgcggccccg ccctctcctc 180

gcggcgcgag tttcaggcag cgctgcgtcc tgctgcgcac gtgggaagcc ctggccccgg 240

ccacccccgc g 251

<210> 6

<211> 455

<212> DNA

<213> Artificial Sequence

<400> 6

tggcccctcc ctcgggttac cccacagcct aggccgattc gacctctctc cgctggggcc 60

ctcgctggcg tccctgcacc ctgggagcgc gagcggcgcg cgggcgggga agcgcggccc 120

agacccccgg gtccgcccgg agcagctgcg ctgtcggggc caggccgggc tcccagtgga 180

ttcgcgggca cagacgccca ggaccgcgct ccccacgtgg cggagggact ggggacccgg 240

gcacccgtcc tgccccttca ccttccagct ccgcctcctc cgcgcggacc ccgccccgtc 300

ccgacccctt ccgggtcccc ggcccagccc cctccgggcc ctcccagccc ctccccttcc 360

tttccgcggc cccgccctct cctcgcggcg cgagtttcag gcagcgctgc gtcctgctgc 420

gcacgtggga agccctggcc ccggccaccc ccgcg 455

<210> 7

<211> 455

<212> DNA

<213> Artificial Sequence

<400> 7

tggcccctcc ctcgggttac cccacagcct aggccgattc gacctctctc cgctggggcc 60

ctcgctggcg tccctgcacc ctgggagcgc gagcggcgcg cgggcgggga agcgcggccc 120

agacccccgg gtccgcccgg agcagctgcg ctgtcggggc caggccgggc tcccagtgga 180

ttcgcgggca cagacgccca ggaccgcgct ccccacgtgg cggagggact ggggacccgg 240

gcacccgtcc tgccccttca ccttccagct ccgcctcctc cgcgcggacc ccgccccgtc 300

ccgacccctc ccgggtcccc ggcccagccc cttccgggcc ctcccagccc ctccccttcc 360

tttccgcggc cccgccctct cctcgcggcg cgagtttcag gcagcgctgc gtcctgctgc 420

gcacgtggga agccctggcc ccggccaccc ccgcg 455

<210> 8

<211> 455

<212> DNA

<213> Artificial Sequence

<400> 8

tggcccctcc ctcgggttac cccacagcct aggccgattc gacctctctc cgctggggcc 60

ctcgctggcg tccctgcacc ctgggagcgc gagcggcgcg cgggcgggga agcgcggccc 120

agacccccgg gtccgcccgg agcagctgcg ctgtcggggc caggccgggc tcccagtgga 180

ttcgcgggca cagacgccca ggaccgcgct ccccacgtgg cggagggact ggggacccgg 240

gcacccgtcc tgccccttca ccttccagct ccgcctcctc cgcgcggacc ccgccccgtc 300

ccgacccctt ccgggtcccc ggcccagccc cttccgggcc ctcccagccc ctccccttcc 360

tttccgcggc cccgccctct cctcgcggcg cgagtttcag gcagcgctgc gtcctgctgc 420

gcacgtggga agccctggcc ccggccaccc ccgcg 455

<210> 9

<211> 504

<212> DNA

<213> Artificial Sequence

<400> 9

tggcccctcc ctcgggttac cccacagcct aggccgattc gacctctctc cgctggggcc 60

ctcgctggcg tccctgcacc ctgggagcgc gagcggcgcg cgggcgggga agcgcggccc 120

agacccccgg gtccgcccgg agcagctgcg ctgtcggggc caggccgggc tcccagtgga 180

ttcgcgggca cagacgccca ggaccgcgct ccccacgtgg cggagggact ggggacccgg 240

gcacccgtcc tgccccttca ccttccagct ccgcctcctc cgcgcggacc ccgccccgtc 300

ccgacccctc ccgggtcccc ggcccagccc cctccgggcc ctcccagccc ctccccttcc 360

tttccgcggc cccgccctct cctcggcggc tcgtggctct ttcgcggcaa aaaggatttg 420

gcgcgtaaaa gtggcggcgc gagtttcagg cagcgctgcg tcctgctgcg cacgtgggaa 480

gccctggccc cggccacccc cgcg 504

<210> 10

<211> 507

<212> DNA

<213> Artificial Sequence

<400> 10

tggcccctcc ctcgggttac cccacagcct aggccgattc gacctctctc cgctggggcc 60

ctcgctggcg tccctgcacc ctgggagcgc gagcggcgcg cgggcgggga agcgcggccc 120

agacccccgg gtccgcccgg agcagctgcg ctgtcggggc caggccgggc tcccagtgga 180

ttcgcgggca cagacgctcg gcggctcgtg gctctttcgc ggcaaaaagg atttggcgcg 240

taaaagtggc caggaccgcg ctccccacgt ggcggaggga ctggggaccc gggcacccgt 300

cctgcccctt caccttccag ctccgcctcc tccgcgcgga ccccgccccg tcccgacccc 360

tcccgggtcc ccggcccagc cccctccggg ccctcccagc ccctcccctt cctttccgcg 420

gccccgccct ctcctcgcgg cgcgagtttc aggcagcgct gcgtcctgct gcgcacgtgg 480

gaagccctgg ccccggccac ccccgcg 507

<210> 11

<211> 556

<212> DNA

<213> Artificial Sequence

<400> 11

tggcccctcc ctcgggttac cccacagcct aggccgattc gacctctctc cgctggggcc 60

ctcgctggcg tccctgcacc ctgggagcgc gagcggcgcg cgggcgggga agcgcggccc 120

agacccccgg gtccgcccgg agcagctgcg ctgtcggggc caggccgggc tcccagtgga 180

ttcgcgggca cagacgctcg gcggctcgtg gctctttcgc ggcaaaaagg atttggcgcg 240

taaaagtggc caggaccgcg ctccccacgt ggcggaggga ctggggaccc gggcacccgt 300

cctgcccctt caccttccag ctccgcctcc tccgcgcgga ccccgccccg tcccgacccc 360

tcccgggtcc ccggcccagc cccctccggg ccctcccagc ccctcccctt cctttccgcg 420

gccccgccct ctcctcggcg gctcgtggct ctttcgcggc aaaaaggatt tggcgcgtaa 480

aagtggcggc gcgagtttca ggcagcgctg cgtcctgctg cgcacgtggg aagccctggc 540

cccggccacc cccgcg 556

<210> 12

<211> 504

<212> DNA

<213> Artificial Sequence

<400> 12

tggcccctcc ctcgggttac cccacagcct aggccgattc gacctctctc cgctggggcc 60

ctcgctggcg tccctgcacc ctgggagcgc gagcggcgcg cgggcgggga agcgcggccc 120

agacccccgg gtccgcccgg agcagctgcg ctgtcggggc caggccgggc tcccagtgga 180

ttcgcgggca cagacgccca ggaccgcgct ccccacgtgg cggagggact ggggacccgg 240

gcacccgtcc tgccccttca ccttccagct ccgcctcctc cgcgcggacc ccgccccgtc 300

ccgacccctt ccgggtcccc ggcccagccc cttccgggcc ctcccagccc ctccccttcc 360

tttccgcggc cccgccctct cctcggcggc tcgtggctct ttcgcggcaa aaaggatttg 420

gcgcgtaaaa gtggcggcgc gagtttcagg cagcgctgcg tcctgctgcg cacgtgggaa 480

gccctggccc cggccacccc cgcg 504

<210> 13

<211> 507

<212> DNA

<213> Artificial Sequence

<400> 13

tggcccctcc ctcgggttac cccacagcct aggccgattc gacctctctc cgctggggcc 60

ctcgctggcg tccctgcacc ctgggagcgc gagcggcgcg cgggcgggga agcgcggccc 120

agacccccgg gtccgcccgg agcagctgcg ctgtcggggc caggccgggc tcccagtgga 180

ttcgcgggca cagacgctcg gcggctcgtg gctctttcgc ggcaaaaagg atttggcgcg 240

taaaagtggc caggaccgcg ctccccacgt ggcggaggga ctggggaccc gggcacccgt 300

cctgcccctt caccttccag ctccgcctcc tccgcgcgga ccccgccccg tcccgacccc 360

ttccgggtcc ccggcccagc cccttccggg ccctcccagc ccctcccctt cctttccgcg 420

gccccgccct ctcctcgcgg cgcgagtttc aggcagcgct gcgtcctgct gcgcacgtgg 480

gaagccctgg ccccggccac ccccgcg 507

<210> 14

<211> 556

<212> DNA

<213> Artificial Sequence

<400> 14

tggcccctcc ctcgggttac cccacagcct aggccgattc gacctctctc cgctggggcc 60

ctcgctggcg tccctgcacc ctgggagcgc gagcggcgcg cgggcgggga agcgcggccc 120

agacccccgg gtccgcccgg agcagctgcg ctgtcggggc caggccgggc tcccagtgga 180

ttcgcgggca cagacgctcg gcggctcgtg gctctttcgc ggcaaaaagg atttggcgcg 240

taaaagtggc caggaccgcg ctccccacgt ggcggaggga ctggggaccc gggcacccgt 300

cctgcccctt caccttccag ctccgcctcc tccgcgcgga ccccgccccg tcccgacccc 360

ttccgggtcc ccggcccagc cccttccggg ccctcccagc ccctcccctt cctttccgcg 420

gccccgccct ctcctcggcg gctcgtggct ctttcgcggc aaaaaggatt tggcgcgtaa 480

aagtggcggc gcgagtttca ggcagcgctg cgtcctgctg cgcacgtggg aagccctggc 540

cccggccacc cccgcg 556

<210> 15

<211> 52

<212> DNA

<213> Artificial Sequence

<400> 15

tcggcggctc gtggctcttt cgcggcaaaa aggatttggc gcgtaaaagt gg 52

<210> 16

<211> 1049

<212> DNA

<213> Artificial Sequence

<400> 16

atgagacata ttatctgcca cggaggtgtt attaccgaag aaatggccgc cagtcttttg 60

gaccagctga tcgaagaggt actggctgat aatcttccac ctcctagcca ttttgaacca 120

cctacccttc acgaactgta tgatttagac gtgacggccc ccgaagatcc caacgaggag 180

gcggtttcgc agatttttcc cgactctgta atgttggcgg tgcaggaagg gattgactta 240

ctcacttttc cgccggcgcc cggttctccg gagccgcctc acctttcccg gcagcccgag 300

cagccggagc agagagcctt gggtccggtt tctatgccaa accttgtacc ggaggtgatc 360

gatccaccca gtgacgacga ggatgaagag ggtgaggagt ttgtgttaga ttatgtggag 420

caccccgggc acggttgcag gtcttgtcat tatcaccgga ggaatacggg ggacccagat 480

attatgtgtt cgctttgcta tatgaggacc tgtggcatgt ttgtctacag taagtgaaaa 540

ttatgggcag tgggtgatag agtggtgggt ttggtgtggt aatttttttt ttaattttta 600

cagtttttgtg gtttaaagaa ttttgtattg tgatttttttt aaaaggtcct gtgtctgaac 660

ctgagcctga gcccgagcca gaaccggagc ctgcaagacc tacccgccgt cctaaaatgg 720

cgcctgctat cctgagacgc ccgacatcac ctgtgtctag agaatgcaat agtagtacgg 780

atagctgtga ctccggtcct tctaacacac ctcctgagat acacccggtg gtcccgctgt 840

gccccattaa accagttgcc gtgagagttg gtgggcgtcg ccaggctgtg gaatgtatcg 900

aggacttgct taacgagcct gggcaacctt tggacttgag ctgtaaacgc cccaggccat 960

aaggtgtaaa cctgtgattg cgtgtgtggt taacgccttt gtttgctgaa tgagttgatg 1020

taagtttaat aaagggtgag ataatgttt 1049

<210> 17

<211> 573

<212> DNA

<213> Artificial Sequence

<400> 17

tatataatgc gccgtgggct aatcttggtt acatctgacc tcatggaggc ttgggagtgt 60

ttggaagatt tttctgctgt gcgtaacttg ctggaacaga gctctaacag tacctcttgg 120

ttttggaggt ttctgtgggg ctcatcccag gcaaagttag tctgcagaat taaggaggat 180

tacaagtggg aatttgaaga gcttttgaaa tcctgtggtg agctgtttga ttctttgaat 240

ctgggtcacc aggcgctttt ccaagagaag gtcatcaaga ctttggattt ttccacaccg 300

gggcgcgctg cggctgctgt tgcttttttg agttttataa aggataaatg gagtgaagaa 360

acccatctga gcggggggta cctgctggat tttctggcca tgcatctgtg gagagcggtt 420

gtgagacaca agaatcgcct gctactgttg tcttccgtcc gcccggcgat aataccgacg 480

gaggagcagc agcagcagca ggaggaagcc aggcggcggc ggcaggagca gagcccatgg 540

aacccgagag ccggcctgga ccctcgggaa tga 573

<210> 18

<211> 1661

<212> DNA

<213> Artificial Sequence

<400> 18

atgagacata ttatctgcca cggaggtgtt attaccgaag aaatggccgc cagtcttttg 60

gaccagctga tcgaagaggt actggctgat aatcttccac ctcctagcca ttttgaacca 120

cctacccttc acgaactgta tgatttagac gtgacggccc ccgaagatcc caacgaggag 180

gcggtttcgc agatttttcc cgactctgta atgttggcgg tgcaggaagg gattgactta 240

ctcacttttc cgccggcgcc cggttctccg gagccgcctc acctttcccg gcagcccgag 300

cagccggagc agagagcctt gggtccggtt tctatgccaa accttgtacc ggaggtgatc 360

gatccaccca gtgacgacga ggatgaagag ggtgaggagt ttgtgttaga ttatgtggag 420

caccccgggc acggttgcag gtcttgtcat tatcaccgga ggaatacggg ggacccagat 480

attatgtgtt cgctttgcta tatgaggacc tgtggcatgt ttgtctacag taagtgaaaa 540

ttatgggcag tgggtgatag agtggtgggt ttggtgtggt aatttttttt ttaattttta 600

cagtttttgtg gtttaaagaa ttttgtattg tgatttttttt aaaaggtcct gtgtctgaac 660

ctgagcctga gcccgagcca gaaccggagc ctgcaagacc tacccgccgt cctaaaatgg 720

cgcctgctat cctgagacgc ccgacatcac ctgtgtctag agaatgcaat agtagtacgg 780

atagctgtga ctccggtcct tctaacacac ctcctgagat acacccggtg gtcccgctgt 840

gccccattaa accagttgcc gtgagagttg gtgggcgtcg ccaggctgtg gaatgtatcg 900

aggacttgct taacgagcct gggcaacctt tggacttgag ctgtaaacgc cccaggccat 960

aaggtgtaaa cctgtgattg cgtgtgtggt taacgccttt gtttgctgaa tgagttgatg 1020

taagtttaat aaagggtgag ataatgttta acttgcatgg cgtgttaaat ggggcggggc 1080

ttaaagggta tataatgcgc cgtgggctaa tcttggttac atctgacctc atggaggctt 1140

gggagtgttt ggaagatttt tctgctgtgc gtaacttgct ggaacagagc tctaacagta 1200

cctcttggtt ttggaggttt ctgtggggct catcccaggc aaagttagtc tgcagaatta 1260

aggaggatta caagtgggaa tttgaagagc ttttgaaatc ctgtggtgag ctgtttgatt 1320

ctttgaatct gggtcaccag gcgcttttcc aagagaaggt catcaagact ttggattttt 1380

ccacaccggg gcgcgctgcg gctgctgttg cttttttgag ttttataaag gataaatgga 1440

gtgaagaaac ccatctgagc ggggggtacc tgctggattt tctggccatg catctgtgga 1500

gagcggttgt gagacacaag aatcgcctgc tactgttgtc ttccgtccgc ccggcgataa 1560

taccgacgga ggagcagcag cagcagcagg aggaagccag gcggcggcgg caggagcaga 1620

gcccatggaa cccgagagcc ggcctggacc ctcgggaatg a 1661

Claims (24)

1. A core activation element, characterized in that: 1), the nucleotide sequence is shown in SEQ ID No.1; or: 2) A nucleic acid molecule that has one or several base insertion, deletion and/or substitution mutations in the nucleotide sequence shown in SEQ ID No. 1 and still has the function of a promoter. 2 . The core promoter element according to claim 1 , wherein the nucleotide sequence is shown in any one of SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5. 3 . 3. A promoter comprising the core promoter element of claim 1 or 2. 4 . The promoter according to claim 3 , wherein the nucleotide sequence is shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No.8. 5 . 5. The promoter according to claim 3 or 4, wherein at least one E2F binding site is further inserted. 6. The promoter according to claim 5, wherein the nucleotide sequence of the E2F binding site is shown in SEQ ID No. 15; or the E2F binding site is shown in the sequence SEQ ID No. 15. Nucleic acid molecules with 1 or several base insertion, deletion and/or substitution mutations in the nucleotide sequence shown in No. 15, and still have the function of E2F binding site. 7. The promoter according to any one of claims 4 to 6, wherein the E2F binding site is inserted at the 5' end and/or 3' of the core promoter element according to claim 1 or 2 end. 8. The promoter according to claim 7, wherein the nucleotide sequence is such as SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13 or Shown in SEQ ID No.14. 9. Use of the core promoter element of claim 1 or 2 or the promoter of any one of claims 3 to 8 in the preparation of oncolytic viruses or plasmids. 10. The use according to claim 9, wherein the oncolytic virus is an oncolytic adenovirus. 11. A recombinant vector comprising the core promoter element of claim 1 or 2 or the promoter of any one of claims 3 to 8. 12. The recombinant vector according to claim 11, characterized in that the recombinant vector is a plasmid vector or a viral vector. 13. The recombinant vector according to claim 12, wherein the viral vector is an adenovirus vector, an adenovirus-associated virus or a retrovirus. 14. The recombinant vector of claim 12, wherein the viral vector is an oncolytic virus. 15. The recombinant vector according to claim 14, wherein the oncolytic virus is an oncolytic adenovirus, an oncolytic parvovirus, an oncolytic herpes virus, an oncolytic pox virus, an oncolytic vesicular stomatitis virus, At least one of oncolytic measles virus, oncolytic myxoma virus, oncolytic retrovirus, oncolytic reovirus, and oncolytic vaccinia virus. 16. The recombinant vector according to claim 15, wherein the oncolytic adenovirus is a serotype belonging to subgenus A, subgenus B, subgenus C, subgenus D, subgenus E, subgenus F or , Adenoviruses of subgenus G; Further, the adenovirus is: At least one selected from type 12, 18, 31 or 61 of subgenus A; or, at least one selected from the group consisting of types 3, 7, 11, 14, 16, 21, 34, 35, 55, 66, 68, 76, 77, 78 or 79 of subgenus B, or, at least one selected from Type 1, 2, 5, 6, 57 or 89 of subgenus C; Or, selected from 8, 9, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, 46, 48, 49, 53, 54, 56, 58-60, At least one of types 62-65, 67, 69-75, 80-88 or 90-103; or, selected from type 4 of subgenus E, or, selected from type 40 or 41 of subgenus F; Alternatively, adenovirus type 52 selected from subgenus G. 17. The recombinant vector according to claim 11, wherein the plasmid vector is pDC316, pDC311, pDC312, pDC315, pDC511, pDC512, pDC515, pDC516, pShuttle, pShuttle-CMV, pCTAP-Shuttle series plasmid, pNTAP - at least one of Shuttle series plasmids, pAdTrack, pAdTrack-CMV, pacAd5 series plasmids, pHBAd series plasmids or pXC1 plasmids. 18 . The recombinant vector according to claim 15 , wherein the oncolytic adenovirus is composed of the core promoter element according to claim 1 or 2 or the promoter according to any one of claims 3 to 8 . Drives expression of E1A and/or E1B-19K genes. 19. The recombinant vector according to claim 15, wherein the E1A is E1A (Delta24) with 24bp in the middle deleted, and its nucleotide sequence is shown in SEQ ID No. 16; or, the The nucleotide sequence of E1B 19K is shown in SEQ ID No.17. 20. A host cell comprising the recombinant vector of any one of claims 11-19. 21. The host cell of claim 20, which is a eukaryotic cell. 22. An antitumor drug, characterized in that: it is prepared by adding the recombinant carrier according to any one of claims 11 to 19 and adding pharmaceutically acceptable auxiliary components. 23. The method for preparing the recombinant vector of any one of claims 11 to 19: a), constructing the core promoter element according to claim 1 or 2 or the promoter according to any one of claims 3 to 8 operably linking the genes necessary for adenovirus propagation into a shuttle plasmid; b), transferring the shuttle plasmid and adenovirus backbone plasmid constructed in step a) into packaging cells, and packaging to obtain an oncolytic adenovirus. 24. The method according to claim 23, characterized in that: at least one of the following is satisfied: The genes necessary for the proliferation of the adenovirus are E1A and/or E1B-19K; Alternatively, the shuttle plasmids are pDC316, pDC311, pDC312, pDC315, pDC511, pDC512, pDC515, pDC516, pShuttle, pShuttle-CMV, pCTAP-Shuttle series plasmids, pNTAP-Shuttle series plasmids, pAdTrack, pAdTrack-CMV, pacAd5 series At least one of plasmid, pHBAd series plasmid or pXC1 plasmid; Alternatively, the adenovirus backbone plasmid is at least one of pBHGloxdelE13cre, pBHGfrtdelE13FLP, pAdEasy-1, pAdEasy-2, pBHGE3i or pBHGE10i; Alternatively, the packaging cells are HEK293 and HEK293A cells.

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