CN110628728A

CN110628728A - Recombinant oncolytic gene-adenovirus of targeted cancer and construction method and application thereof

Info

Publication number: CN110628728A
Application number: CN201810651914.2A
Authority: CN
Inventors: 章康健; 方先龙; 刘新垣; 顾锦法; 倪爱明
Original assignee: Shanghai Yuan Song Biotechnology Co Ltd
Current assignee: Shanghai Yuan Song Biotechnology Co Ltd
Priority date: 2018-06-22
Filing date: 2018-06-22
Publication date: 2019-12-31

Abstract

The invention discloses a recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 for targeting cancer, wherein the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 drives a virus E1A gene by a survivin promoter, and simultaneously, an E1B gene region is knocked out from a virus genome, and a VGLL4 expression frame segment is inserted on the basis. The invention also discloses a construction method of the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 and application thereof in preparing a medicament for treating cancer. The recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 has good treatment effect in anticancer tests by means of specific replication of adenovirus vectors in cancer cells.

Description

Recombinant oncolytic gene-adenovirus of targeted cancer and construction method and application thereof

Technical Field

The invention relates to the technical field of recombinant oncolytic gene-adenovirus, in particular to a recombinant oncolytic gene-adenovirus targeting cancer and a construction method and application thereof.

Background

Cancer severely affects human health and development, is affected by medical and environmental conditions, and has a higher than global average cancer mortality rate in china. The traditional therapy of tumor has the defects of poor curative effect, high mortality rate, high recurrence rate after prognosis and the like, so people begin to put great hope on the emerging tumor treatment method. Emerging strategies for treating tumors are also under constant development, and currently, relatively approved strategies include immunotherapy, gene therapy, oncolytic virus therapy, and the like.

Currently, cancer is treated only with gene therapy or tumor-specific targeting viruses, which have very limited efficacy. However, the anti-cancer gene is inserted into the tumor-specific virus, so that the virus therapy and the gene therapy are cooperated, namely, the multiple treatment effects of the biological treatment strategy are exerted at the same time, and the safety of the virus is greatly improved and the curative effect of the gene therapy is enhanced. The virus vector in the targeted gene-virus therapy can remarkably play a role in killing tumors, moreover, the copy number of the gene in the targeted gene-virus therapy can be increased in a chain manner along with the replication and proliferation of the virus, and meanwhile, the expression quantity of the gene can be greatly improved. In conclusion, the targeting gene-virus treatment strategy has the unique advantages of high targeting property, safety, gene expression and the like, greatly promotes the long-term development of the targeting gene-virus treatment, and plays an important role in finally overcoming cancers for human beings. However, the current gene vectors are not able to specifically select tumor cells in human body, nor express anticancer genes in tumor cells with high efficiency, resulting in weak gene therapy effect and possibility of accidental injury to normal cells of human body.

In addition, for early cancer, the existing method mostly adopts a surgical operation method to remove tumors, the effect is ideal, but some tumors have long hiding period, are not easy to be discovered as soon as possible, and miss the optimal time for treatment. While the operation methods in the middle and late stages are not feasible, chemotherapy or radiotherapy is generally carried out, and the traditional chemoradiotherapy means has the defects of poor curative effect and high side effect. The current medical level, whether surgery or drug therapy is very harmful to patients, can not effectively prolong the survival time of patients except relieving the symptoms of the patients to a certain extent.

The Hippo pathway is a key pathway for controlling cell morphology and size, and is a kinase chain consisting of a series of protein kinases and transcription factors, which is very active in tumor cells. The end effector protein of the Hippo pathway is YAP, which is expressed in various tissues of the human body, and most of the co-transcribed downstream genes are genes promoting cell proliferation and migration. According to research reports, the expression level of YAP in various tumor cells of gastric cancer, colon cancer, lung cancer and the like is remarkably improved and is far higher than that of normal cells, so that YAP in the tumor cells becomes a new target in the hands of researchers, and is even recognized as an important treatment target with prospect.

Members of the degenerative protein family (VGLL) do not have a DNA binding domain and regulate gene transcription through a DNA binding domain. There are 4 VGLL proteins in humans, named VGLL1-4, which have no sequence similarity. VGLL4, also known as degenerate protein family member 4, is a new member of the degenerate protein family, which differs from other members in that: 1) the VGLL1-3 protein has only 1 conserved TDU domain at the N-terminus, while VGLL4 has two independent conserved TDU domains at the C-terminus; 2) VGLL1 and VGLL3 are expressed primarily in the placenta, VGLL2 is expressed in skeletal muscle, and VGLL4 is the only gene family member expressed in the heart. VGLL4 is a co-transcription factor that functions by competing with YAP for TEAD transcription factor binding through the TDU domain. Therefore, the VGLL4 protein is said to be a promising therapeutic gene against YAP-driven human cancers.

Cancer targeted Gene-virus Therapy (Cancer Targeting Gene-Viro-Therapy, CTGVT) is constructed by adding an anti-Cancer Gene to an oncolytic virus. CTGVT, as our guiding strategy, shows considerable anticancer effects in cancer therapy. The therapy can simultaneously exert the tumor killing ability of the gene and the cell replication and lysis ability of the virus. The idea of this strategy is to utilize the ability of oncolytic virus to specifically target tumor cells, introduce foreign genes (tumor killing genes) into tumor cells, and express a large amount of killing proteins along with the amplification of the virus, thereby achieving the purpose of killing tumor cells.

In summary, finding more accurate target drugs for cancer is the key point of cancer diagnosis and treatment.

Disclosure of Invention

The invention tries to take VGLL4 as a target spot to develop a new targeted drug so as to obtain more efficient and stable curative effect and find a new direction and suggestion for treating cancer. Therefore, the E1B region is deleted on the basis of the wild type adenovirus, and the wild type promoter of E1A is replaced, so that the safety and the targeting property of the adenovirus are enhanced, and the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B with double targeting on tumor cells is constructed, and the virus targets the tumor cells in two ways: (1) the E1A is started by using a tumor-specific survivin promoter, (2) the E1B region of the adenovirus early gene is deleted, and then VGLL4 which is efficiently started by a human CMV promoter is inserted into the E1A region in the adenovirus Ad-sp-E1A-delta E1B on the basis, so that the VGLL4 and the dual-target recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B are hopefully combined to improve the curative effect of the adenovirus.

Based on the above purposes, the first aspect of the invention provides a cancer-targeting recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4, wherein the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 drives a virus E1A gene by a survivin promoter, and simultaneously the virus genome is knocked out of an E1B gene region and is inserted with a VGLL4 expression cassette fragment.

The second aspect of the invention provides a construction method of the cancer-targeted recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4, which comprises the following steps:

step one, constructing a plasmid pCA13-VGLL 4: designing primers K-VGLL4-HIND3-F and K-K-VGLL4-R by taking plasmid pcDNA-flag-VGLL4 as a template, carrying out PCR to obtain a VGLL4 gene fragment, carrying out HIND III and BamH I double enzyme digestion on the VGLL4 gene fragment and pCA13 plasmid, and connecting the VGLL4 gene enzyme digestion fragment with pCA13 enzyme digestion fragment to obtain plasmid pCA13-VGLL 4;

step two, construction of plasmid pShuttle-sp-E1A-delta E1B-VGLL4

Designing a primer one-step cloning primer-F and a primer one-step cloning R3 by taking the plasmid pCA13-VGLL4 of the first step as a template, and carrying out PCR amplification to obtain a VGLL4 expression frame fragment; then, the plasmid pShuttle-sp-E1A-delta E1B is cut by BglII; then, constructing the expression frame segments of the linearized pShuttle-sp-E1A-delta E1B and VGLL4 by a one-step cloning method to obtain a plasmid pShuttle-sp-E1A-delta E1B-VGLL 4;

step three, preparation of BJ5183 competent cell of pXYA16 plasmid

The pBHGE3 plasmid is cut by HindIII, the pAdEasy-1 plasmid is cut by SpeI, and then the two plasmids after enzyme cutting are transferred into escherichia coli BJ5183 for homologous recombination to obtain a recombinant large plasmid carrying an E3 region; then, the recombinant large plasmid is transferred into escherichia coli DH5 alpha for amplification to obtain plasmid pXYA 16; then, the plasmid pXYA16 is transferred into escherichia coli BJ5183 competence to obtain escherichia coli BJ5183 carrying pXYA16 plasmid, and the escherichia coli is prepared into competence;

step four, constructing pAd-sp-E1A-delta E1B-VGLL4 through homologous recombination

The successful pShuttle-sp-E1A-delta E1B-VGLL4 is identified in the PmeI enzyme digestion step II, FastAp dephosphorylation is carried out to obtain dephosphorylated linearized pShuttle-sp-E1A-delta E1B-VGLL4, and then the linearized pShuttle-sp-E1A-delta E1-VGLL 4 is transformed into an escherichia coli BJ5183 competent cell carrying the pXYA16 plasmid in the step III to obtain pAd-sp-E1A-delta E1B-VGLL 4;

step five, packaging of recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4

PacI digestion linearization step four plasmid pAd-sp-E1A-delta E1B-VGLL4, use the efficiency transduction Reagent Transfection of the linear fragment into HEK-293 cells, generate recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL 4.

The third aspect of the invention provides an application of the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 in preparing a medicament for treating cancer.

In a fourth aspect of the invention, a pharmaceutical composition for treating cancer is provided, wherein the pharmaceutical composition comprises the recombinant oncolytic gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL 4.

The invention has the beneficial effects that: the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 has good treatment effect in anticancer tests; meanwhile, the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 has low toxicity and good anticancer effect.

Drawings

FIG. 1 is a diagram showing the results of PCR with the plasmid pcDNA-flag-VGLL 4.

FIG. 2 is a diagram showing the results of double digestion with Hind III and BamH I of linearized plasmid pCA 13.

FIG. 3 is a diagram showing the results of HindIII and BamH I double digestion of VGLL 4.

FIG. 4 is a diagram showing the results of double digestion with Hind III and BamH I of plasmid pCA13-VGLL 4.

FIG. 5 is a diagram showing the results of BglII cleavage of plasmid pShuttle-sp-E1A-. DELTA.E 1B.

FIG. 6 is a diagram showing the results of BglII cleavage of plasmid pShuttle-sp-E1A-. DELTA.E 1B-VGLL 4.

FIG. 7 is a diagram showing the results of MluI cleavage of plasmid pAD-sp-E1A- Δ E1B-VGLL 4.

FIG. 8 is a schematic diagram of the construction of recombinant oncolytic gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL 4.

FIG. 9 is an identification of VGLL4 gene of recombinant oncolytic gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL 4.

FIG. 10 shows the wild virus identification of the recombinant type of E1A wild-type promoter of recombinant oncolytic gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL 4.

FIG. 11 shows the wild virus identification of recombinant type of E1B region of recombinant oncolytic gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL 4.

FIG. 12 cytotoxicity assay of recombinant oncolytic gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL 4.

FIG. 13 is a safety experiment of recombinant oncolytic gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL 4.

FIG. 14 shows that q-PCR and Western blot detect the expression level of VGLL4 of Ad-sp-dE1B-VGLL4 in hepatoma cells.

FIG. 15 shows the effect of q-PCR on the transcription level of the gene downstream of VGLL4 after hepatoma cells are infected with Ad-sp-dE1B-VGLL 4.

FIGS. 16-17 show the transcription and expression level of E1A detected by q-PCR and Western blot.

FIGS. 18-20 are flow assays for detecting apoptotic changes.

FIG. 21 shows apoptosis observed by Hoechst33342 staining.

FIG. 22 shows the observation of apoptosis in western blot assays.

FIG. 23 shows the q-PCR and Western blot detection of survivin expression levels in hepatoma cells and normal liver cells.

FIG. 24 shows the expression level of E1A, which is an early essential protein, in liver cancer cells (HuH-7 and Hep3B) and normal liver cells (HLF) of Ad-sp-E1A- Δ E1B-VGLL 4.

Detailed Description

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. Unless otherwise indicated, the techniques employed in the present invention are conventional in the art, such as molecular cloning techniques, microbiological techniques, cell biology techniques, and the like.

The cell lines, main equipment and instruments, primers and plasmids of this example are as follows.

1. The cell lines of this example are shown in Table 1

TABLE 1 cell lines

2. Main equipment and instruments are shown in Table 2

TABLE 2 Main Equipment and instrumentation

3. PCR primer sequences, see in particular Table 3

The sequences shown in Table 3 were synthesized by Huajin or Piracetam Biotechnology Ltd.

TABLE 3PCR primer sequences

4. Plasmids, see in particular Table 4

TABLE 4 plasmids

Example 1

The construction method of the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 for targeting cancer is as follows: step one, construction of plasmid pCA13-VGLL4

(1) VGLL4 gene fragment obtained by PCR method

The VGLL4 gene fragment is obtained by PCR with the plasmid pcDNA-flag-VGLL4 as a template.

The PCR reaction system is as follows:

the reaction procedure for PCR was:

PCR products were run on a 1% agarose gel. The PCR results are shown in FIG. 1. Wherein, lane 1 is DNA Marker (Trans2K Plus DNA Marker); lane 2 is H₂O (negative control); lane 3 is the pcDNA-flag-VGLL4PCR product.

The results show that: the target band is between 909bp and 750bp and 1000bp, which indicates that the VGLL4 gene fragment is successfully amplified.

(2) Purification of PCR products

And (2) purifying the PCR product by using an agarose gel DNA recovery kit for the VGLL4 gene fragment obtained by the PCR in the step one (1), and storing the recovered product at-20 ℃ for later use.

(3) HindIII and BamH I double digestion pCA13 plasmid

An enzyme digestion reaction system:

the reaction system is subjected to water bath at 37 ℃ for more than 30min, and then gel electrophoresis and tapping recovery are carried out, thus obtaining the purified linearized plasmid pCA 13. The cleavage results are shown in FIG. 2. Among them, lanes 1-3 are the HindIII and BamH I double digestion products of pCA 13; lane 4 is DNA Marker (DL 15000).

The results show that: the target band is 6916bp, between 5000bp and 7500bp, which indicates that pCA13 is successfully linearized.

(4) HindIII and BamH I double enzyme digestion VGLL4 gene fragment

The VGLL4 gene fragment obtained by PCR in step one (1) was digested with Hind III and BamH I, and the digestion results are shown in FIG. 3. Wherein lanes 1-3 are HindIII and BamH I double digestion products of VGLL4 gene fragment; lane 4 is DNASMarker (DL 15000).

The result shows that the target band of enzyme digestion is 891bp, which indicates that VGLL4 is successfully linearized.

(5) DNA ligase ligation of linearized pCA13 and VGLL4 fragment

An enzyme digestion reaction system:

the reaction system is incubated in a heat preservation bucket at the temperature of 16 ℃ for more than 1h to obtain plasmids pCA13-VGLL4, and the plasmids pCA13-VGLL4 are placed at the temperature of-20 ℃ for preservation. The identification scheme of plasmid pCA13-VGLL4 is shown in FIG. 4. Wherein, Lane 1 is DNA Marker (DL 15000); lanes 2-3 are the HindIII and BamH I double digests of pCA 13.

The results show that the cleavage product has two bands: 6916bp and 819bp, which shows that the plasmid pCA13-VGLL4 is successfully constructed.

Step two, construction of plasmid pShuttle-sp-E1A-delta E1B-VGLL4

(1) Obtaining VGLL4 expression cassette fragment by PCR

And (3) amplifying a specific fragment by using the designed primer by using the plasmid pCA13-VGLL4 of the step one as a template.

The PCR reaction system is as follows:

the reaction procedure for PCR was:

the PCR product was run on a 1% agarose gel and purified.

(2) BglII single-restriction enzyme plasmid pShuttle-sp-E1A-delta E1B

An enzyme digestion reaction system:

and (3) carrying out water bath on the reaction system at 37 ℃ for 30min, carrying out gel electrophoresis, tapping, recovering and purifying to finally obtain the purified linearized plasmid pShuttle-sp-E1A-delta E1B. The cleavage results are shown in FIG. 5. Wherein, lane 1 is DNA Marker (1kb DNAsder); lane 2 shows the BglII cleavage product of pShuttle-sp-E1A-E1B.

The result shows that the target band of the enzyme digestion is 8163bp, which is between 8000bp and 10000bp, and the pShuttle-Sp-E1A-delta E1B is proved to be successfully linearized.

(3) Construction of plasmid pShuttle-sp-E1A-delta E1B-VGLL4 by one-step cloning method

The kit used in this step is 2 × one step seamless cloning kit from Ethicon Biotech, Inc., Beijing.

The reaction system is as follows:

and (3) placing the reaction system at 50 ℃ for 30min, and then placing the one-step cloning product at-20 ℃ for storage for later use.

(4) Screening for successfully recombined plasmids

And (4) transforming the one-step cloning product obtained in the step two (3) into DH5 alpha competent cells, coating a Kana resistance plate, picking a plurality of monoclonal colonies every other day, shaking the bacteria, extracting plasmids to identify whether the recombination is successful, and selecting the plasmids with successful recombination for the next test.

(5) Identification of plasmid pShuttle-sp-E1A-delta E1B-VGLL4

And (3) carrying out single enzyme digestion identification on the successfully recombined recombinant plasmid pShuttle-sp-E1A-delta E1B-VGLL4 in the step two (4).

The enzyme digestion identification is as follows:

after 30min at 37 ℃, electrophoresing the product of digestion, and observing the map to screen the correct plasmid. The cleavage results are shown in FIG. 6. Wherein, Lane 1 is DNA Marker (1kbDNA ladder); lane 2 shows the BglII cleavage product of pShuttle-sp-E1A-. DELTA.E 1B-VGLL 4.

The result shows that two bands appear after single enzyme digestion of 1 and BglII, which are 6183bp and 1456bp respectively, which indicates that the plasmid is successfully constructed; 2. the band for enzyme digestion is 6183bp, which is between 6000bp and 8000bp, and the pShuttle-Sp-delta E1B-VGLL4 is a correct plasmid.

Step three, constructing escherichia coli BJ5183 competent cells carrying pXYA16 plasmid

The pBHGE3 plasmid was digested with HindIII, the pAdEasy-1 plasmid was digested with SpeI, and the two digested plasmids were co-transferred to Escherichia coli BJ5183 (purchased from Beijing all-type gold Biotechnology Co., Ltd.) for homologous recombination to obtain a recombinant plasmid carrying the E3 region. The recombinant large plasmid is transferred into escherichia coli DH5 alpha (purchased from Beijing all-style gold biotechnology, Inc.) for amplification, and after plate coating, a single clone is selected to obtain plasmid pXYA 16. The plasmid pXYA16 is transferred into Escherichia coli BJ5183 competence to obtain Escherichia coli BJ5183 carrying pXYA16 plasmid, and the Escherichia coli is prepared into competence.

(1) And a PmeI single enzyme digestion step II identifies successful pShuttle-sp-E1A-delta E1B-VGLL4, and the reaction system is as follows:

and (3) placing the reaction system in a water bath kettle at 37 ℃ for 1 hour to obtain linearized pAd-sp-E1A-delta E1B-VGLL4, and continuing the next dephosphorylation experiment.

(2) FastAp dephosphorylation of linearized pShuttle-sp-E1A-. DELTA.E 1B-VGLL4, the reaction system is as follows:

the reaction system is placed in a water bath kettle at 37 ℃ for 1 hour, and then the next conversion experiment is continued.

(3) And (2) recombining pShuttle-sp-E1A-delta E1B-VGLL4 in BJ5183 competent cells carrying the plasmid pXYA16 of the third step to obtain pAd-sp-E1A-delta E1B-VGLL4, wherein the concrete steps are as follows:

A) taking dephosphorylated linearized pShuttle-sp-E1A-delta E1B-VGLL4 in the step four (3), transforming the dephosphorylated linearized pShuttle-sp-E1-delta E1-VGLL 4 into the BJ5183 competent cells, coating plates, selecting bacteria, and shaking the bacteria overnight;

B) and carrying out plasmid extraction on the cultured bacterial liquid by using a small plasmid extraction kit to obtain pAd-sp-E1A-delta E1B-VGLL 4.

(4) Enzyme cutting identification, screening and recombination successful pAd-sp-E1A-delta E1B-VGLL4 plasmid, and converting into DH5 alpha for large-scale amplification.

The enzyme digestion system is as follows:

and (3) placing the reaction system in a constant-temperature water bath kettle at 37 ℃ for reaction for 30min, and then carrying out electrophoresis identification. Converting the correctly identified pAd-sp-E1A-delta E1B-VGLL4 plasmid into DH5 alpha, plating, picking out a monoclonal colony, shaking bacteria, extracting plasmid, and performing enzyme digestion identification. The enzyme digestion reaction system is as follows:

and (3) placing the reaction system in a constant-temperature water bath kettle at 37 ℃ for reaction for 30min, and then carrying out electrophoresis identification. FIG. 7 shows the restriction result of MluI. Wherein, lane 1 is DNA Marker (1kb DNA ladder); lane 2 is the MluI cleavage product of pAD-sp-E1A- Δ E1B-VGLL 4.

The results showed that there were 5 bands in the product of Mlu I digestion of plasmid pAd-Sp-. DELTA.E 1B-VGLL4, indicating that pAd-Sp-. DELTA.E 1B-VGLL4 is the correct plasmid.

(1) Cell plating

Spreading a 6-well plate of HEK-293 cells to make the cell density reach 60% -80% on day 2.

(2) And carrying out enzyme digestion on the plasmid pAd-sp-E1A-delta E1B-VGLL4 in the third step by PacI to obtain a linearized plasmid pAd-sp-E1A-delta E1B-VGLL 4. The enzyme digestion reaction system is as follows:

(3) transfection of plasmids and packaging of viruses

The linearized plasmid pAd-sp-E1A-delta E1B-VGLL4 obtained in step five (2) was transfected into HEK-293 cells (6-well plate) according to the instructions of the Effectene Transfection Reagent kit to generate recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4, and the gene-virus structure diagram is shown in FIG. 8. Collecting virus liquid after virus is completely diseased for about 7-10 days, and storing at-80 ℃ for later use.

Example 2 identification of recombinant oncolytic Gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL4

(1) Viral genomic DNA was extracted according to the instructions of the blood genome extraction kit

(2) PCR identification of the VGLL4 gene carried by Ad-sp-E1A-delta E1B-VGLL4 of step four

The PCR reaction system is as follows:

the reaction procedure for PCR was:

after PCR was completed, electrophoresis was performed. The results are shown in FIG. 9. Wherein, lane 1 is DNA Marker (Trans2K Plus DNA Marker); lane 2 is the PCR product of Ad-sp-E1A- Δ E1B-VGLL 4.

The result shows that the target band is 891bp, and slightly below 1000bp, which proves that VGLL4 is successfully amplified.

(3) And C, identifying whether wild viruses of the E1A wild type promoter recombination type exist in the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 constructed in the step four by PCR.

The PCR reaction system is as follows:

the reaction procedure for PCR was:

after the PCR was completed, the PCR product was identified by electrophoresis using 1% agarose gel. The results are shown in FIG. 10. Wherein lanes 1-4 are Ad-sp-E1A- Δ E1B-VGLL 4; lane 5 is H₂O; lane 6 is HEK 293 genome; lane 7 is Wt-Ad; lane 8 is DNA Marker (Trans2K Plus DNA Marker).

The result shows that no target band exists in all the lanes of the samples to be tested, which indicates that no E1A promoter exists in all the samples to be tested, i.e. no wild toxic pollution exists.

(4) PCR identification of whether wild virus of E1B region recombination type exists in Ad-sp-E1A-delta E1B-VGLL4

The PCR reaction system is as follows:

PCR conditions were as above, and run electrophoresis was used after PCR was completed. The results are shown in FIG. 11. Wherein, lane 1 is DNA Marker (Trans2K Plus DNA Marker); lanes 2-5 are Ad-sp-E1A- Δ E1B-VGLL 4; lane 6 is H₂O; lane 7 is HEK 293 genome; lane 8 is Wt-Ad.

The result shows that no target band exists in all the lanes of the samples to be tested, which indicates that no E1B area exists in all the samples to be tested, i.e. no wild virus pollution exists.

Example 3 determination of the Titers of the recombinant oncolytic Gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL4

The principle of virus titer determination is to count the number of cells positive for hexon staining by immunocytochemistry to determine the number of viruses with infectious activity, and then determine the titer of the amplified and purified adenovirus according to the instruction on the adenovirus titer kit.

Example 4 evaluation of anticancer Effect of recombinant oncolytic Gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL4

(1) MTT test for toxicity of recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 to cells.

In order to further discuss the killing effect of the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 on tumor cells, an MTT method is selected to detect the killing capability of the adenovirus on the tumor cells. The same tumor cells were infected with Ad-sp-E1A- Δ E1B-VGLL4 (hereinafter referred to as Ad-sp-VGLL4) and the corresponding unloaded virus Ad-sp-E1A- Δ E1B (hereinafter referred to as Ad-sp) at the same MOI concentration, and the survival rate of the tumor cells was measured by MTT method every 24 hours.

Spreading normal cells or tumor cells in a 96-well plate, adding 20ul of thiazole blue solution (4mg/ml) into each well after a certain time, discarding the liquid in each well after four hours, adding 150 microliter of dimethyl sulfoxide, oscillating for about 15min, and detecting the light absorption value of each well by using an enzyme-labeling instrument. Substituting into formula to calculate cell survival rate and drawing cell survival curve. The relevant statistical analysis is shown in FIG. 12.

As can be seen from FIG. 12, the killing ability of the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 to Hep3B and HuH-7 hepatoma cell lines is obviously greater than that of the unloaded virus Ad-sp-E1A-delta E1B along with the increase of days.

And (4) conclusion: the capability of the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 in killing tumor cells is stronger than that of the no-load control virus, and the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 has stronger killing property on liver cancer cells.

(2) Safety experiment of recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 on cells

In order to further detect the safety of the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 to normal cells, an MTT method is selected to detect the killing of the normal cells after the adenovirus is infected. We tested the above recombinant oncolytic gene-adenovirus killing of 3 normal cells as follows: human normal liver cell HLF, human normal fibroblast HPF, and human normal stomach cell Ges-1. The relevant statistical analysis is shown in FIG. 13.

As can be seen from FIG. 13, the cell survival rate of the three normal cells did not change much with the increase of multiplicity of infection (MOI), and Ad-sp-VGLL4 and Ad-sp were hardly toxic to the three normal cells.

The results show that the recombinant oncolytic gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL4 still exhibits safety to three normal cell lines even at high MOI treatment.

Example 5 testing whether Virus Ad-sp-E1A- Δ E1B-VGLL4 transcribes and expresses VGLL4 protein normally

In order to identify whether the virus Ad-sp-E1A-delta E1B-VGLL4 constructed by us can normally transcribe and express VGLL4 protein, the transcription and expression levels of VGLL4 are detected by respectively applying a q-PCR technology and a Western blot technology, and the downstream gene expression of VGLL4 is detected in cells.

One, real-time q-PCR technology detection

(one) extraction of RNA

Total RNA was extracted from each group of cells according to TRIzon Reagent instructions and stored at-80 ℃ until use for detection of mRNA expression levels.

(II) reverse transcription of RNA

(1) Determination of RNA concentration

(2) Configuring a reverse transcription system:

the reverse transcription reaction procedure is as follows:

37℃ 15min

98℃ 5min

4℃ forever

(III) q-PCR

(1) Preparing a q-PCR reaction system:

(2) adding the reaction system into a 96-well plate, carefully covering the 96-well plate with the Q-PCR special membrane, and pressing the membrane along the same direction by using a special card without directly contacting the surface of the membrane by hands.

(3) Centrifuging a 96-well plate at 3000rpm for 1 min;

(4) the real-time fluorescent quantitative PCR reaction is carried out on a Bio-Rad fluorescent quantitative PCR instrument, and the reaction program is as follows:

second, Western Blot technical detection

Western blot for detecting the expression level of E1A protein, VGLL4 protein and apoptosis-related protein in tumor cells comprises the following specific steps:

before starting the experiment, SDS polyacrylamide gel with proper concentration is prepared according to the molecular weight of the target protein, and the concentration of the polyacrylamide separation gel is determined according to the molecular weight of the target protein detected by separation, wherein the formulas of 10% separation gel and 4% concentrated gel are shown in Table 5.

Firstly, sucking the alcohol between the two plates by using dust-free paper, filling separation glue, flattening the interface of the glue by using anhydrous alcohol, waiting for the drying of the separation glue, and adding 5% of concentrated glue prepared according to the formula when the alcohol is completely volatilized. Inserting a comb with the size of 1.5mm of Berkele, transferring the comb into an electrophoresis tank, pouring 1 multiplied by polyacrylamide electrophoresis liquid between two plates until the height of the electrode wires are submerged, pulling out the comb special for western blot, sequentially spotting, and performing 80V electrophoresis until a protein Marker reaches a proper position to stop electrophoresis. After electrophoresis, cutting the protein gel, cutting the PVDF membrane, placing the sponge sheet, the filter paper, the protein gel and the PVDF membrane in a cold membrane transferring solution according to the positive and negative of the electrode, then performing electric transfer, placing a special ice box on one side, and placing the whole electric transfer body under ice, wherein the electric transfer time, the time length and the protein size are determined according to 90V under general conditions.

TABLE 5SDS Polyacrylamide gel formulations

(4) Rotary film

And (3) after the electricity conversion is finished, taking down the PVDF membrane, marking the PVDF membrane by shearing corners on the front surface, and sealing the PVDF membrane in the existing prepared sealing liquid at the BD entrance for 30 min.

(5) Blocking and antibody incubation

Apoptosis-related antibodies were diluted with immunohistochemical concentrations of BSA solution, typically 1: 1000, storage conditions after use were determined by antibody company's recommended storage conditions, and after 2 hours of post-blocking room temperature, washing was carried out with a large amount of TBST. The secondary antibody of HRP-marker was incubated at room temperature without refrigeration and for a short period of time at the dilution rate specified in the second-antibody protocol. After the secondary antibody incubation was completed, the cells were washed with TBST.

(6) Development

The ECL solution is dropped on the cut PVDF membrane running with the corresponding protein by developing, and exposed and developed under a chemiluminescence imaging system.

The detection steps of the q-PCR technique and the Western blot technique are shown in example 5.

1) The detection results of the expression of the foreign gene VGLL4 are shown in FIGS. 14 and 15.

Wherein the upstream primer in the q-PCR detection method is q-VGLL4-F, and the downstream primer is q-VGLL 4-R.

As can be seen from FIGS. 14 and 15, the adenovirus Ad-sp-E1A- Δ E1B-VGLL4 greatly expresses the exogenous gene VGLL4 both at the transcription level and the protein level, which indicates that the VGLL4 gene inserted into the adenovirus can be normally transcribed and translated, and as the adenovirus is continuously replicated and proliferated, the VGLL4 gene is exponentially increased along with the continuous replication and proliferation of the adenovirus, and is more powerful than the cancer suppressor gene VGLL4 is greatly expressed in tumor cells, and the expression amount is not obtained by in vitro transfection or other ways.

2) The results of the detection of E1A expression are shown in FIGS. 16-18.

Wherein, the upstream primer in the q-PCR detection method is q-E1A-F, and the downstream primer is q-E1A-R.

E1A is used as adenovirus early protein, and has important function for replication and proliferation of adenovirus. As can be seen from FIGS. 16-18, the recombinant oncolytic gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL4 greatly expressed E1A both at the transcriptional and protein level.

And (4) conclusion: 1. the recombinant oncolytic gene-adenovirus d-sp-E1A-delta E1B-VGLL4 expresses VGLL4 protein in a large amount in hepatoma cells; 2. the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 expresses VGLL4 protein in a large amount in hepatoma cells.

Example 6 apoptosis assay of recombinant oncolytic Gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL4

The influence of adenovirus on apoptosis is detected by various methods such as Hoechst33258 staining detection, flow cytometry detection, western blot detection and the like. The results are shown in FIGS. 19-22.

(1) Flow assay for cell cycle changes and apoptosis changes

To explore the anticancer mechanism of Ad-sp-E1A- Δ E1B-VGLL4, we first investigated the effect of VGLL4 alone with Ad-sp-E1A- Δ E1B on the cell cycle: taking tumor cells HuH-7 of logarithmic growth phase, 3 × 10 per well⁵The cells are paved on a 6-well plate and cultured overnight, the next day, the tumor cells are transfected with an unloaded plasmid pCA13 and a plasmid pCA13-VGLL4 carrying genes, and after the cells are cultured in an incubator for 48 hours, cell cycle and apoptosis detection kits are used for detecting cell cycle change. Meanwhile, for the influence of PBS, Ad-sp-E1A-delta E1B, Ad-sp-E1A-delta E1B-VGLL4 on the cell cycle, taking tumor cells in logarithmic growth phase, wherein the number of the tumor cells is 3 multiplied by 10 per well⁵Individual cells were plated in 6-well plates and cultured overnight. PBS, 5MOI of virus Ad-sp-E1A- Δ E1B, Ad-sp-E1A- Δ E1B-VGLL4, was added and cell cycle changes were detected after 48 hours of culture in an incubator.

Annexin V is a peptide that binds to phosphatidylserine that is turned out of the cell membrane during apoptosis, and therefore, fluorescein-labeled phosphatidylserine can be detected in Flow cytometry assays. Another dye, PI, is a dye that specifically stains non-intact cell membranes. Just like the dead cell membrane surface in the treatment group in the late apoptosis stage and killed by virus, the cell membrane can penetrate into the cell for staining, and in principle, the cell membrane can be analyzed in a flow mode through the kit, so that the experimental result of the cell can be further obtained. The results are shown in FIGS. 19 and 20.

(2) Observation of apoptosis by Hoechst33342 staining

When cells enter an apoptosis pathway, cell nuclei shrink and stain deepening, but Hoechst33324 dye is known as nucleic acid specific dye and has strong staining capability, and various shapes of the cell nuclei can be clearly seen under an inverted fluorescence microscope, so that the task of changing the apoptotic cells by staining can be better fulfilled. The dye is also known to have different staining abilities for different states of cells, but a uniform saturation condition is always present, which is the key point of staining. Cells were seeded at an appropriate density (3X 10) in 6-well cell culture plates⁵) Depending on the cell size and culture conditions. Cells were treated with different MOI or autoclaved PBS after different viruses (negative control). After virus treatment 2days removed all the culture medium and washed twice with PBS, stained with Hoechst33342 at a concentration of 1ug/ml for 10min, washed twice with cell grade PBS and visualized under a fluorescence microscope on a microscope stage and photographed. The results are shown in FIG. 21.

(3) The result of the western blot detection is shown in FIG. 22, and the step of the western blot detection is shown in the second step of embodiment 5.

Flow and western blot and Hoechst33342 staining conclusions: the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 significantly promotes apoptosis and mainly promotes late apoptosis of cells.

Example 7 Targeted assay of recombinant oncolytic Gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL4

The promoter of the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 constructed by the inventor is the promoter of the tumor specific expression protein Survivin, and the targeting of the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 is mainly established on the basis. Therefore, the q-PCR and Western blot methods as in example 5 are used to detect the survivin expression levels in liver cancer cells and normal human liver cells to predict targeting, wherein the upstream and downstream primers in the q-PCR process are survivin-F and survivin-R, respectively. The results are shown in FIG. 23.

And (4) conclusion: the expression of survivin at mRNA and protein levels has very significant cancer specificity.

Subsequently, a Western blot method is used for detecting the expression level of the protein of E1A in each cell after the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 is infected with liver cancer cells and normal human liver cells, so that the targeting property of the adenovirus is evaluated. The result is shown in FIG. 24, which indicates that E1A cannot be expressed in a liver normal cell line HLF after the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 is infected, and can only be specifically expressed in liver cancer cell lines Huh-7 and Hep3B, and the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 is proved to have good cancer cell targeting.

Sequence listing

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Claims

1. A recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 for targeting cancer is characterized in that the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 drives a virus E1A gene by a survivin promoter, and simultaneously, the E1B gene region is knocked out from the virus genome, and an expression frame segment inserted with VGLL4 is carried on the basis.

2. The method for constructing the cancer-targeted recombinant oncolytic gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL4 of claim 1, comprising the steps of:

step one, construction of plasmid pCA13-VGLL4

Designing primers K-VGLL4-HIND3-F and K-K-VGLL4-R by taking plasmid pcDNA-flag-VGLL4 as a template, carrying out PCR to obtain a VGLL4 gene fragment, carrying out HIND III and BamH I double enzyme digestion on the VGLL4 gene fragment and pCA13 plasmid, and connecting the VGLL4 gene enzyme digestion fragment with pCA13 enzyme digestion fragment to obtain plasmid pCA13-VGLL 4;

step two, construction of plasmid pShuttle-sp-E1A-delta E1B-VGLL4

Designing a primer one-step cloning primer-F and a primer one-step cloning R3 by taking the plasmid pCA13-VGLL4 of the first step as a template, and carrying out PCR amplification to obtain a VGLL4 expression frame fragment; secondly, carrying out single digestion on the plasmid pShuttle-sp-E1A-delta E1B by using BglII, and then constructing the expression frame segments of the linearized pShuttle-sp-E1A-delta E1B and VGLL4 by adopting a one-step cloning method to obtain a plasmid pShuttle-sp-E1A-delta E1B-VGLL 4;

step three, preparation of BJ5183 competence of pXYA16 plasmid

3. An application of the recombinant oncolytic gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL4 of claim 1 in preparing a medicament for treating cancer.

4. A pharmaceutical composition for treating cancer, comprising the recombinant oncolytic gene-adenovirus Ad-sp-E1A- Δ E1B-VGLL4 of claim 1.