CN1259106C - A kind of preparation method of anti-cancer targeting gene virus drug - Google Patents

Abstract

The invention belongs to the field of biotechnology. The invention provides a preparation method of an anti-cancer targeting gene virus drug. The invention constructs an adenovirus lacking a 55Kda gene and carries an exogenous anti-cancer gene, which is named Ad5-ZD55-gene, overcomes the shortcoming that the ONYX-015 virus cannot carry an exogenous anti-cancer gene, and improves the anti-cancer effect. The adenovirus mutant constructed by the invention can be used to develop an effective new anticancer drug for treating tumors.

Description

A kind of preparation method of anti-cancer targeting gene virus drug

Technical field :

The invention belongs to the field of biotechnology. In particular, it relates to a preparation method of an anti-cancer targeting gene virus drug.

Background technology :

Malignant tumors seriously endanger human life and health. The number of new cancer cases in my country exceeds 1.6 million each year (the total number of cancer patients exceeds 10 million). Cancer may overtake cardiovascular and cerebrovascular diseases as the number one cause of death for humans.

Gene therapy is a biological high-tech program that has emerged in the past 10 years. Tumor gene therapy programs account for more than 60% of the entire gene therapy program, and it may be one of the most promising programs for treating malignant tumors. Currently, there are two types of vectors for gene therapy: viral and non-viral. Non-viral vectors include: naked DNA liposomes and DNA wrapped in other substances; viral vectors include: retroviruses, adenoviruses, adeno-associated viruses, and herpes viruses. Virus-mediated DNA transfer method has been developed rapidly in recent years, among which adenovirus vector is more commonly used in tumor gene therapy.

Adenovirus is a double-stranded DNA virus with a genome length of 36kb, divided into early and late transcription regions. The E1 region is deleted (or both E1 and E3 are deleted) to obtain a traditional adenovirus vector, which can insert and express foreign genes within 8kb in length. Adenovirus vectors can infect many types of cells, including resting cells, and can be cultured on a large scale to obtain high-titer virus pure products. These characteristics make adenoviruses a common carrier for tumor gene therapy. Traditional adenoviral vectors cannot replicate themselves to produce progeny viruses. Although traditional adenoviral vectors have achieved some good results in animal experiments for tumor gene therapy, there is no clear clinical efficacy so far. The inability of adenoviral vectors to transfect tumor cells targetedly is considered to be the main reason for its limited clinical efficacy.

As early as a hundred years ago, someone discovered that a very small number of tumor patients who were infected with viruses showed temporary regression of tumors, and thus began to try tumor virus therapy. Genetic engineering technology is used to change the genome of the virus so that it can specifically replicate and spread in tumor cells, and then kill the tumor cells, and the released progeny virus spreads to adjacent tumor cells, causing a panic attack in the tumor tissue. This kind of chain reaction eventually spreads to the whole tumor and completely eliminates it, but it does not damage normal tissues. The application of specific multiplication virus in tumor has brought new dawn to tumor treatment.

The gene therapy of tumor has no significant clinical progress, but the virus therapy of tumor has made significant progress. The mutant adenovirus (d11520, also called ONYX-015) developed by ONXY Pharmaceutical Company of the United States is to delete the E1b55K protein gene of the adenovirus genome, so that the mutant adenovirus can replicate in large quantities in p53-deficient tumor cells. Cannot replicate in normal cells. Using ONYX-015 combined with conventional chemotherapy in the treatment of 30 patients with head and neck cancer who had relapsed after chemotherapy, 8 of them were in complete remission, 1 of which had a tumor with a diameter of more than 10 cm also completely regressed, and 11 of the tumors shrunk by more than half. up to 63% (Nature Medicine 2000). At present, it has entered Phase III clinical trials, and more than 200 patients have been treated. This is currently the most effective tumor biological treatment method. ONYX-015 combined with chemotherapy has achieved 63% curative effect in clinical treatment of head and neck cancer, but ONYX-015 alone (not combined with chemotherapy) has a very poor curative effect, only 15-20%. In order to overcome the unsatisfactory curative effect of using ONYX-015 alone, academician Liu Xinyuan proposed in 2000 (Chinese Journal of Tumor Biotherapy, 8(1), 2001, P1-), a new strategy: gene virus therapy strategy for tumors. Anticancer genes are added to the tumor-specific proliferating virus, and the gene therapy of tumors is combined with virus therapy. On the one hand, this new type of tumor treatment strategy retains the effective specificity of virus therapy, and on the other hand, it uses a large number of virus replication to make the anti-cancer genes it carries a large number of amplification and high-efficiency expression. Improve anticancer efficacy.

Invention content :

The technical problem to be solved by the present invention is to overcome the shortcoming that viral vectors in tumor gene therapy cannot effectively amplify specifically in tumors, improve the current situation that ONYX-015 cannot carry exogenous genes and lack lethality, and combine gene therapy of tumors with Combining the advantages of the two in virus therapy provides a kind of recombinant adenovirus that can replicate and proliferate in tumor cells, but not proliferate in normal cells, and can efficiently express anti-cancer genes in tumor cells.

The invention provides a preparation method of an anti-cancer targeting gene virus drug. The method is a method for constructing a tumor-specific proliferating adenovirus expressing an anti-cancer gene, and is characterized in that: (1) 2269-3327 bp of the adenovirus is deleted by a site-directed PCR method. A SV40 polyA tail was added after the E1B 19Kda gene. The single enzyme cutting site Bgl II added at the SV40 polyA tail; (2) the anti-cancer gene is cloned into the multiple cloning site of the plasmid pCA13, and the expression frame of the therapeutic gene is cut out with BglII enzyme (it contains the HCMV promoter, anti-cancer gene, SV40poly A tail), inserted into the modified adenovirus vector; (3) transfect HEK-293 cells with the modified adenovirus vector containing the anti-cancer gene and the large adenovirus plasmid pBHGE ₃ or pBHG ₁₀ to establish a therapeutic Recombinant adenovirus; (4) isolation of recombinant adenovirus for treatment.

The present invention names this type of virus Ad5-ZD55-gene according to the difference of the inserted gene, such as Ad5-ZD55-sFLT-1, Ad5-ZD55-IL-12 and the like.

The invention constructs an adenovirus lacking a 55Kda gene and carrying an exogenous anticancer gene ZD55-gene, which overcomes the shortcoming that the ONYX-015 adenovirus cannot carry an exogenous anticancer gene, and improves the anticancer effect. The present invention applies site-directed mutagenesis PCR technology, deletes the 55Kda protein of the wild-type adenovirus, and adds an enzyme cutting site at the same time, which can be conveniently inserted into the expression frame of the anti-cancer gene, followed by the termination signal of the SV40 PolyA tail. The bp2269-3327 of the adenovirus deleted in the present invention is the one with the largest deletion of 55Kda protein gene bases in the mutant adenovirus, which provides more space for pZD55 to insert foreign anti-cancer genes, which is another advantage of our vector , and ONYX-015 does not contain exogenous gene insertion sites at all, so it cannot insert exogenous anti-cancer genes.

The recombinant virus constructed by the present invention can not only selectively reproduce and proliferate specifically in tumor cells, but also has an anti-cancer gene that kills tumor cells. The large number of copies of the specific virus in cancer cells makes the copy number of the anti-cancer gene large Increase. The close synergy between gene therapy and viral therapy of tumors has been realized.

The adenovirus mutant constructed in the present invention can be used to develop an effective new anticancer drug for treating tumors, and is used for treating tumors with p53 gene or p53 pathway defects. A pharmaceutical composition can be developed, which contains any recombinant adenovirus described in the present invention, such as the mutual combination of carrying A anticancer gene and carrying B anticancer gene, and can also be compounded with one of the following compounds Treatment: Chemotherapeutic drugs; biotoxins; immunosuppressive compounds, monoclonal antibodies, etc.

The pZD55 vector constructed by the present invention is homologously recombined with the large plasmid pBHGE3 or pBHG10 containing most of the genes of the adenovirus to obtain the ZD55 adenovirus, which can specifically proliferate and amplify in p53-deficient tumors, but not in normal cells . At the same time, pZD55 can conveniently insert (install) the expression frame of the anti-cancer gene in and out, and package a proliferative adenovirus (Ad5-ZD55-gene) capable of expressing the anti-cancer gene. The adenovirus expressing the anti-cancer gene can specifically lyse tumor cells and express the anti-cancer gene efficiently. Compared with non-proliferative adenovirus vectors, it can specifically and greatly increase the expression of anti-cancer genes in tumor cells. The present invention is illustrated by taking Ad5-ZD55-sFLT-1 as an example.

The present invention has following beneficial effects:

1. The present invention provides a mutant adenovirus with deletion of the E1b 55Kda gene. Cell experiments prove that it can selectively proliferate in tumor cells without killing normal cells. Through animal experiments, it can be used to treat various tumors .

2. The present invention provides a method for constructing an adenovirus with E1b 55Kda gene deletion. This method can be used to construct a new type of mutant adenovirus, which is easy to master, and a cloning site for inserting an exogenous anticancer gene is added.

3. The mutant adenovirus vector constructed by the present invention can be loaded with exogenous anti-cancer genes very conveniently, and can proliferate in large quantities in tumor cells. We call it gene-virus. This vector can construct a variety of gene-viruses expressing different anti-cancer genes, providing a good basis for gene-virus therapy of tumors.

4. The gene-virus constructed in the present invention is a tumor-specific proliferating adenovirus, which can selectively reproduce and proliferate in large quantities in tumor cells. Simultaneously, the anti-cancer gene carried by the recombinant virus is amplified in large quantities along with the large-scale replication of the virus in tumor cells, so the gene-virus has a high anti-cancer effect. The close synergy between gene therapy and virus therapy for tumors has been realized.

5. The various genetic viruses constructed by the present invention have been proved by animal experiments to selectively kill tumor cells without affecting normal cells. Genes—Anti-cancer genes expressed by the virus can enhance the anti-tumor effect of the virus. This new gene virus has laid a good foundation for future use in human tumor therapy.

6. The gene virus constructed in the present invention can form a complex with one of the following compounds: other gene-viruses, chemotherapeutic drugs; biological toxins; immunosuppressive compounds, monoclonal antibodies, etc.

Specific implementation methods :

The construction of the plasmid vector pZD55 of embodiment 1.55Kda gene deletion

Unless otherwise stated, the techniques used in the present invention are conventional techniques in the art, such as molecular cloning techniques, microbiology techniques, and cell biology techniques. These techniques are fully explained in the literature, such as Sambrook's "Molecular Cloning Experiment Guideline" "wait.

The PXC1 vector was purchased from Microbix Biosystem Inc. (Toronto), Canada, and the PXC1 plasmid contained the sequence bp22-5790 of type 5 adenovirus.

A new type of adenovirus vector with E1b 55Kda gene deletion was constructed by using positional mutagenesis double PCR technique. Primers for PCR reactions:

Primer 1: 5'gcc cga cat cac ctg tgt cta gag aat g 3' (including Xba I site, paired with bp1321-1348 in pXC1)

Primer 2: 5'tca gat ggg ttt ctt cac tcc att tat cct 3' (paired with bp 2040-2011 in pXC1)

Primer 3: 5'ata aag gat aaa tgg agt gaa gaa acc cat ctg ag 3' (paired with bp 2007-2041 in pXC1, the third codon of 55Kda gene was mutated into a stop codon, mutation C2024T)

Primer 4: 5'gaa gat cta tac agt taa gcc acc tat aca aca 3' (including Bgl II site, and bp 2269-2245 in pXC1, E1B 55Kda gene reading frame mutation, C2252T, G2261T added two stop codons son)

Using the PXC1 plasmid as the template of the PCR reaction, the first PCR reaction was carried out with primer 1 and primer 2, and a 719bp fragment was recovered by electrophoresis. Using the PXC1 plasmid as the template of the PCR reaction, the second PCR reaction was carried out with primers 3 and 4, and a 270bp fragment was recovered by electrophoresis. The products of the two PCR reactions have a paired sequence of 34 bp. The two PCR products are mixed as a template, and the third PCR reaction is performed with primer 1 and primer 4, and a 955 bp fragment is recovered by electrophoresis. The 955bp PCR product was digested with Xba I+Bgl II, and cloned into the pXC1 plasmid digested with Xba I+BglII, named pXC1-D55. Compared with PXC1, PXC1-D55 lacks the bp2269-bp3327 of the PXC1 vector, and mutates the third codon of the E1b 55Kda gene into a stop codon, and behind the stop codon of the E1b 19Kda protein, it is the E1b 55Kda gene Two stop codons were added to the reading frame.

The pCA13 plasmid was purchased from Microbix Biosystem Inc (Toronto) in Canada. PCA13 contains the adenovirus type 5 sequence bp22-5790, and the 342 to 3523 bp fragment of the E1 region is deleted, and the human cytomegalovirus (HCMV) IE promoter (- 299-+72) and SV40 polyA tailed signal. The PCA13 vector was digested with BamH I+Bgl II, and the 160bp fragment was recovered by electrophoresis, that is, the SV40 polyA tail was recovered, and the fragment was cloned into pXC1-D55 digested with Bgl II, identified by enzyme digestion, and the forward clone was named pZD55. A SV40 polyA tail was added behind the E1b19Kda protein gene. There is only one BglII site in pZD55. In front of the BglII site is the SV40 polyA tail. The BglII site is not in the reading frame of the adenovirus gene, nor in the adenovirus promoter. It can be used to insert the gene expression frame without affecting the expression of other genes in the adenovirus. Adenovirus replication. But pZD55 contains the left arm sequence for homologous recombination with adenovirus.

The pZD55 plasmid was sequenced, and the sequencing results were consistent with the expected results, indicating that the vector was constructed successfully.

...GATTTTCTGGCCATGCATCTGTGGAGAGCGGTTGTGAGACA

CAAGAATCGCCTGCTACTGTTGTCTTCCGTCCGCCCGGCGATAA

TACCGACGGAGGAGCAGCAGCAGCAGCAGGAGGAAGCCAGGC

GGCGGCGGCAGGAGCAGAGCCCATGGAACCCGAGAGCCGGCC

TGGACCCTCGGGAATGAATGTTGTA TAG GTGGCT TAA CTGTA

T

AGATCTGGAAGGTGCTGAGGTACGAT

GAGACCCGCACCAGGTGCAGACCCTGCGAGTGTGGCG...

The SV4O poly A tail sequence is in the box.

AGATCT is a Bgl II site.

TAG and TAA are the stop codons of the reading frame of the E1b 55K Da gene.

Embodiment II. The expression frame of anticancer gene is inserted into pZD55, constructs gene virus plasmid

pZD55-gene (the genes here can be the products of suicide genes CD and TK; the products of genes Trail, Blys and Smae with strong anticancer effects; cytokines 1L-12, 1L-2, IFN and GM-CSF; apoptosis genes ICF, Caspase-3, Caspase-8, Caspase-9, Bax products; Angiogenesis inhibitory genes, sflt-1, Angicstatin, Endcstatin, TIMP-4, PAI products. Any gene, now only sFLT-1 gene as an example).

The pCA13 vector was purchased from Microbix Biosystem Inc. (Toronto), Canada. PCA13 contains the sequence bp22-5790 of type 5 adenovirus, and the 342 to 3523 bp fragment of the E1 region is deleted (but it contains the adenovirus left arm recombination sequence), and the E1 deletion region is inserted Human cytomegalovirus (HCMV) IE promoter (-299-+72) and SV40 polyA tailing signal, containing multiple multiple cloning sites between IE and tailing signal, Sal I, Hind III, EcoRI, EcoR V , Xba I, Xho I, BamH I. Any one of the above-mentioned genes is inserted forward into the multiple cloning site of pCA13 by genetic manipulation to construct the plasmid pCA13-gene. The plasmid pCA13-gene was digested with Bgl II to cut out the expression frame of the gene. This expression cassette contains hCMV promoter, anti-cancer gene and SV40 polyA tail. Then this expression frame was cloned into the pZD55 plasmid which was digested by Bgl II and dephosphorylated, and constructed into plasmid pZD55-gene.

Now only take the sFLT-1 gene as an example to illustrate. FLT-1 is the receptor of human vascular endothelial cell growth factor VEGF, sFLT-1 is the extracellular region of the receptor, it can bind with VEGF, thereby inhibiting the activity of VEGF, so the high expression of sFLT-1 can inhibit the promotion of vascularity by VEGF growth, thereby inhibiting the nutrient supply of tumors and achieving the purpose of killing tumors.

Using polymerase chain reaction (PCR) technology, amplify the 1014bp extracellular region cDNA (sFLT-1) of the FLT-1 gene from the human cardiomyocyte cDNA library, and introduce EcoR I and BamH I two restriction enzymes at the end of the gene site

Primer 5: 5'tag aat tca tgg tca gct act ggg ac 3'

(contains EcoRI site, paired with FLT-1 bp1-18)

Primer 6: 5'tga gga tcc tta atg ttt cac agt gat gaa tgc 3'

(contains BamHI site, paired with FLT-1 bp1014-994)

After the PCR reaction, the 1029bp fragment was recovered and cut with EcoR I+BamH I, and the resulting gene fragment was inserted into the pCA13 plasmid, named pCA13-sFLT-1. The sFLT-1 sequence was determined, and the results were as follows (consistent with theoretical data):

Gaattcatggtcagctactgggacaccggggtcctgctgtgcgcgctgctcagctgtctgcttctcacag

gatctagttcaggttcaaaattaaaagatcctgaactgagtttaaaaggcacccagcacatcatgcaagca

ggccagacactgcatctccaatgcagggggggaagccagcccataaatggtctttgcctgaaatggtgagt

aaggaaagcgaaaggctgagcataactaaatctgcctgtggaagaaatggcaaacaattctgcagtact

ttaaccttgaacacagctcaagcaaaccacactggcttctacagctgcaaatatctagctgtacctacttca

aagaagaaggaaacagaatctgcaatctatatatttattagtgatacaggtagacctttcgtagagatgtac

agtgaaatccccgaaattatacacatgactgaaggaagggagctcgtcattccctgccgggttacgtcac

ctaacatcactgttactttaaaaaagtttccacttgacactttgatccctgatggaaaacgcataatctggga

cagtagaaagggcttcatcatatcaaatgcaacgtacaaagaaatagggcttctgacctgtgaagcaaca

gtcaatgggcatttgtataagacaaactatctcacacatcgacaaaccaatacaatcatagatgtccaaata

agcacaccacgcccagtcaaattacttagaggccatactcttgtcctcaattgtactgctaccactcccttg

aacacgagagttcaaatgacctggagttaccctgatgaaaaaaataagagagcttccgtaaggcgacga

attgaccaaagcaattcccatgccaacatattctacagtgttcttactattgacaaaatgcagaacaaagac

aaaggactttatacttgtcgtgtaaggagtggaccatcattcaaatctgttaacacctcagtgcatatatatg

ataaagcattcatcactgtgaaacattaaggattc

The plasmid pCA13-sFLT-1 was digested with Bgl II, and a 1583bp fragment was recovered, which contained hCMV promoter, human sFLT-1 gene and SV40 polyA tail. Then this expression frame was cloned into the pZD55 plasmid which was digested by Bgl II and dephosphorylated, and constructed into plasmid pZD55-sFLT-1.

Plasmids constructed in a similar way include pZD55-Trail, pZD55-TK, pZD55-CD, pZD55-IL12, pZD55-Smac, pZD55-IFNβ, pZD55-Endostatin, pZD55-Angiostatin and their derivatives, etc.

The recombinant virus construction of the adenovirus of embodiment III.E1b 55Kda gene deletion:

The 293 cell line was purchased from Microbix Biosystem Inc. (Toronto) in Canada, and was transformed from human embryonic kidney cells with sheared type 5 adenovirus DNA. It contains and expresses the E1 region of adenovirus type 5, and the adenovirus DNA has a high transfection rate to it. We co-transfect any pZD55-gene (which contains the left arm sequence of homologous recombination with adenovirus) and the plasmid pBHGE3 containing the right arm of adenovirus into 293 cell line through Effectene. For specific methods, see Qigen The company's operating instructions. The PBHG-E3 plasmid was purchased from Microbix Biosystem Inc. (Toronto), Canada. It contains the right arm of adenovirus type 5, and contains the E3 region and most of the other genes of the adenovirus. After pZD55-gene and pBHGE3 were co-transfected into 293 cells, virus plaques appeared in 10-14 days. After 2 times of purification of virus plaques, amplification was carried out, DNA of recombinant adenovirus was extracted, DNA digestion analysis, PCR analysis, The adenovirus strain with correct recombination is confirmed, that is, the adenovirus with 55Kda gene deletion and insertion into the target gene expression cassette is named Ad5-ZD55-gene (sometimes referred to as ZD55-gene).

Taking the sFLT-1 gene as an example, the plasmid pZD55-sFLT1 and pBHGE3 were co-transfected into 293 cells, and after 10-14 days, the virus plaques were picked out, and the viral plaques were purified twice, a small amount of amplification was performed, and the viral DNA was extracted. PCR identification of recombinant adenovirus strains, identification of the primers used:

Primer 7: 5'Aga gcc cat gga acc cga ga 3' (paired with Ad5 bp 2200-2219)

Primer 8: 5'Cat cgt acc tca gca cct tcca 3' (paired with Ad5 bp 3353-3332)

Primer 9: 5'Tcc gac tgt ggt tgc ttc atg 3' (paired with Ad5 bp 2999-3019)

Using viral DNA as a template, primer 7 and primer 8 perform PCR reaction to amplify a fragment of 1840bp, while using wild-type viral DNA as a template, a fragment of 1153bp can be amplified. Using viral DNA as a template, the PCR reaction of primer 9 and primer 8 cannot amplify a DNA fragment, while using wild-type viral DNA as a template, a 354bp fragment can be amplified. The PCR reaction proved that the selected adenovirus clone was correct, the E1b 55Kda gene was removed, and the expression cassette of the sFLT-1 gene was added. The adenovirus in which the E1b 55Kda gene is deleted and inserted into the sFLT-1 gene expression cassette is named Ad5-ZD55-sFLT-1. Ad5-ZD55-sFLT-1 is a type 5 adenovirus, the E1b 55Kda gene of the adenovirus has been deleted (bp2269-bp3327 deletion), and a SV40polyA tail is added behind the E1b 19Kda protein gene, and a Bgl II bit is added behind the SV40polyA tail point, and a gene expression cassette containing a CMV promoter, a human sFLT-1 gene, and an SV40 polyA tail was inserted into the restriction site. The other DNA sequences of the virus are identical to those of adenovirus type 5.

The adenovirus Ad5-ZD55-sFLT-1 multiplied in 293 cells, and the adenovirus was purified by cesium chloride gradient centrifugation. For specific operation methods, see the operating instructions of Microbix Biosystem Inc. (Toronto).

Recombinant adenovirus constructed in a similar way also includes Ad5-ZD55-Trail, Ad5-ZD55-TK, Ad5-ZD55-CD, Ad5-ZD55-IL 12, Ad5-ZD55-Smac, Ad5-ZD55-Endostatin, Ad5-ZD55 -Angiostatin and its derivatives, etc.

Example IV. Detecting the Killing Ability of Recombinant Adenovirus to Tumor Cells in Vitro

Take Ad5-ZD55-sFLT-1 as an example. Ad5-ZD55-sFLT-1, ONYX-015 and wild type 5 adenovirus Ad5 were used to infect 293 cells, liver cancer cell line Hep3B cells, colorectal cancer cell line SW620 and normal human embryonic lung cells. Cells were inoculated into 6-well plates at 2×10 ⁵ /well, and infected with 1×10 ⁵ pfu each of Ad5-ZD55-sFLT-1, ONYX-015, and Ad5 respectively. After 48 hours, the entire cell culture medium was collected and used after 3 freeze-thaws The virus titer of 293 cell line was determined. For specific methods, refer to the operating instructions of Microbix Biosystem Inc. (Toronto). The result is: 293 Hep3B SW620 normal embryonic lung cells Ad5ONYX-015Ad5-ZD55-sFLT-1 1×10 ⁵ 1×10 ⁵ 1×10 ⁵ 1×10 ⁵ 8×10 ⁴ 8×10 ⁴ 2×10 ⁵ 6×10 ⁴ 7×10 ⁴ 5×10 ⁴ ＜1×10 ¹ ＜1×10 ¹

It was proved that Ad5-ZD55-sFLT-1 can specifically replicate in tumor cells (up to 8×10 ⁴ ), but not in normal cells (only 1×10 ¹ ). The constructed adenovirus expressing anti-cancer gene E1b 55Kda gene deletion can specifically replicate and proliferate in tumor cells and kill tumor cells.

Example V. Detecting the Ability of Recombinant Viruses to Express Anticancer Genes in Vitro

In order to identify the ability and expression level of the adenovirus with E1b55Kda gene deletion to express anti-cancer genes, a luciferase gene virus Ad5-ZD55-Luciferase was constructed using a method similar to the above, and an adenovirus with deletion of the E1 region that could not replicate in tumors was constructed. Adenovirus Ad5-CA13-Luciferase. Luciferase gene is a luciferase reporter gene, purchased from Promega. The quantitative measurement of luciferase gene refers to the product manual of Promega Company.

Ad5-ZD55-Luciferase, Ad5-CA13-Luciferase were respectively infected with liver cancer cell line Hep3B cells, colorectal cancer cell line SW620, breast cancer cell line Bcap-37 and normal human embryonic lung cells. In 24 plates, inoculate 2×10 ⁴ pfu virus per well, infect Ad5-ZD55-Luciferase, Ad5-CA13-Luciferase 1×10 ² pfu respectively, collect cells after 24 hours, 48 hours, 72 hours and 96 hours, After lysing the cells, measure the luciferase intensity. The results showed that after 24 hours, Ad5-ZD55-Luciferase was the same as Ad5-CA13-Luciferase, and after 48 hours, 72 hours, and 96 hours in Hep3B, SW620, and Bcap-37 cells, Ad5-ZD55-Luciferase was much higher than Ad5-CA13 -Luciferase, but there was no significant difference between Ad5-ZD55-Luciferase and Ad5-CA13-Luciferase in normal human embryonic lung cells. Infected cancer cells, Ad5-ZD55-Luciferase peaked at 96 hours and doubled with time. The peak of Ad5-CA13-Luciferase was at 48 hours, and the expression level began to decrease after 48 hours.

Experiments have shown that the Ad5-ZD55 vector can express foreign genes, and with the replication and proliferation of the vector, the gene expression level increases. Compared with Ad5-CA13, an adenovirus vector that cannot proliferate, the Ad5-ZD55 vector can increase gene expression in tumor cells thousands of times.

Example VI. Recombinant adenovirus treatment of tumor cell xenografts in nude mice

Nude mice aged 4-5 weeks were subcutaneously inoculated with the breast cancer cell line Bcap-37, and the animals were divided into groups 12 days later. The treatment group was treated with 1×10 ⁹ pfu of the gene virus Ad5-Zd55-sFLT-1, and the control group was divided into three groups: Group 1 was the PBS treatment group, Group 2 was treated with the same dose of ONYX-015 virus, and Group 3 was treated with the same dose of ONYX-015 virus. The non-proliferating virus Ad5-CA13-sFLT-1, the test results show that Ad5-ZD55-sFLT-1 can effectively kill tumor cells, the therapeutic effect is better than ONYX-015, better than Ad5-CA13-sFLT-1.

The above results fully prove that the tumor-specific proliferative adenovirus Ad5-ZD55-gene constructed by the pZD55 plasmid carrying the anti-cancer gene can realize the synergistic effect of gene therapy and virus therapy, and its therapeutic effect is better than that of gene therapy or virus therapy alone. good. Using Ad5-pZD55-Trail to treat tumors, the preliminary observation shows that the effect is more obvious.

Claims (9)

1. A method for preparing an anti-cancer targeting gene virus drug, characterized in that the method comprises the following steps: I: the construction method of the adenovirus that expresses the tumor-specific proliferation of the tumor treatment gene, it is characterized in that: (1) use the fixed-point PCR method to make the 2269-3327bp deletion of the adenovirus, and add the SV40 polyA tail after the E1B 19Kda gene , the single enzyme cutting site Bgl II added at the SV40 polyA tail; (2) the anti-cancer gene is cloned into the multiple cloning site of the plasmid pCA13, and the expression frame of the therapeutic gene is cut out with the Bgl II enzyme, which contains the HCMV promoter, Insert the anti-cancer gene and SV40 poly A tail into the modified adenovirus vector; (3) transfect HEK-293 cells with the modified adenovirus vector containing the anti-cancer gene and the large adenovirus plasmid pBHGE3 or pBHG10 to establish a therapeutic Recombinant adenovirus; (4) isolation of recombinant adenovirus for treatment; II: The recombinant adenovirus prepared in step I is used to prepare medicine according to conventional methods. 2. The preparation method of a kind of anti-cancer targeting gene virus drug according to claim 1, characterized in that the anti-cancer gene inserted into the adenovirus for tumor-specific proliferation is the product of reporter gene GFP, EGFP and Luciferase . 3. The preparation method of an anti-cancer targeting gene virus drug according to claim 1, characterized in that the anti-cancer genes inserted into the tumor-specific proliferation adenovirus are tumor suppressor genes p53, p21, RB , NF1, VHL, APC products. 4. The preparation method of an anti-cancer targeting gene virus drug according to claim 1, characterized in that the anti-cancer gene inserted into the tumor-specific proliferation adenovirus is the product of suicide genes CD and TK. 5. The preparation method of an anti-cancer targeting gene virus drug according to claim 1, characterized in that the anti-cancer gene inserted into the tumor-specifically proliferating adenovirus is the gene Trail with strong anti-cancer effect. , Blys, Smac products. 6. The preparation method of a kind of anti-cancer targeting gene virus drug according to claim 1, characterized in that the anti-cancer genes inserted into the tumor-specific proliferating adenovirus are cytokines IL-12, IL- 2. IFN, GM-CSF. 7. The preparation method of an anti-cancer targeting gene virus drug according to claim 1, characterized in that the anti-cancer genes inserted into the tumor-specific proliferating adenovirus are cell apoptosis genes ICE, caspase- 3. Caspase-8, caspase-9, Bax products. 8. The preparation method of an anti-cancer targeting gene virus drug according to claim 1, wherein the anti-cancer gene inserted into the tumor-specific proliferation adenovirus is the angiogenesis inhibitory gene sflt-1, Angiostatin, Endostatin, TIMP-4, PAI product. 9. A pharmaceutical composition comprising the recombinant adenovirus constructed according to the method of claim 1.