CN100489091C - Recombinant herpes simplex virus capable of excreting angiostatin and endostatin protein and its application in treating lung cancer - Google Patents
Recombinant herpes simplex virus capable of excreting angiostatin and endostatin protein and its application in treating lung cancer Download PDFInfo
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- CN100489091C CN100489091C CNB200510053471XA CN200510053471A CN100489091C CN 100489091 C CN100489091 C CN 100489091C CN B200510053471X A CNB200510053471X A CN B200510053471XA CN 200510053471 A CN200510053471 A CN 200510053471A CN 100489091 C CN100489091 C CN 100489091C
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Abstract
The present invention relates to a recombinant herpes simplex virus and a method of gene treat of human tumour by using the recombinant virus, particularly relates to a recombinant herpes simplex virus with blood vessel chalone and endodermis chalone albumen, and its application in treating lung cancer. The invention also relates to medicine compound containing the recombinant virus. The recombinant herpes simplex virus of the invention can represent blood vessel chalone and endodermis chalone amalgamation albumen having effect of restraining the generation of blood vessel in the tumour cell, and the virus disintegratively increases in the tumour, such that the purpose of treating lung cancer by restraining the generation of blood vessel and disintegratively decomposing tumour cell is achieved.
Description
Technical field
The present invention relates to a kind of recombinant herpes simplex virus and adopt this recombinant virus to carry out the gene therapy method of human tumor.Particularly relate to the recombinant herpes simplex virus that has angiostatin and endostatin protein, and the application in treatment or prevention lung cancer.Also relate to the pharmaceutical composition that contains this recombinant virus.
Background technology
Lung cancer is one of human modal malignant tumour, and it takes place, develops and shifts is an extremely complicated unusual process of polygene regulation and control.When making a definite diagnosis, this disease mostly is non-early stage greatly, so often result of treatment is relatively poor.Recent statistics shows that the lung cancer mortality ascensional range occupies first of all kinds of tumours, has reached 17.54/10 ten thousand people to the nineties in 20th century, and this shows that it is serious day by day to the harm of human health.In recent years, along with development, especially the DNA recombinant technology and the gene transfer technique of Medical Molecular Biology technology and theory are progressively ripe, utilize gene transfer technique in body, to import goal gene, intervene in order to molecule link, for new way has been opened up in the treatment of lung cancer lung cancer morbidity.
Studies show that, lung cancer be because the change of the inactivation of the activation of some proto-oncogene, cancer suppressor gene and apoptosis-related genes causes cell proliferation and dead unusual result (Mistudomi T et al., Cancer and Chemotherapy, 1996,23:990-996).The process that it is the multistage, multistep is rapid, polygene participates in has heterogeneic change in succession or simultaneously in different steps.The gene therapy of lung cancer be exactly at lung cancer in generation, development and evolution process, the activation of cellular proto-oncogene and these nodes of the inactivation of cancer suppressor gene adopt technology such as known gene fragment importing, transfer and reorganization to eliminate the function of cancer suppressor gene in activated oncogene and the propagation tumour.Put it briefly, gene therapy comprises two portions: the one, and the somatic cell gene function of recovery unconventionality expression or disappearance; The 2nd, introduce the gene in other sources that therapeutic value is arranged.In the lung cancer gene therapy, except considering to break the growth of tumor rule, correct outside its pernicious performance, also need to consider the functional rehabilitation and the reinforcement of tumor by local microenvironment and the antitumor system of whole body thereof.
At present, the gene therapy method of studying more lung cancer has: the treatment of (1) cancer suppressor gene: the cancer suppressor gene treatment is a kind of gene replacement therapy, promptly substitutes the cancer suppressor gene of inactivation with the cancer suppressor gene of wild-type, thereby reaches the purpose of treatment tumour.What research was more at present is with exogenous wild type p53 gene therapy lung cancer; (2) Antisense gene therapy: i.e. opposite with the oncogene sequence one section Nucleotide of synthetic or oncogene oppositely is connected on the plasmid, through carrier they are delivered to tumour cell, so as transcribe or translation skill on the overexpression of blocking-up oncogene; (3) suicide gene therapy: the suicide gene therapy poison mediation activation prodrug gene therapy of pretending illness again.Its mechanism is after some genes are imported into tumour cell, makes prodrug activation by expressing special enzyme.The nucleic acid metabolism of these medicines tumour cell capable of blocking causes the death of tumour cell.Douglas etc. are with several enzymes and prodrug combined utilization, and as Isocytosine deaminase and 5-flurocytosine, Cytochrome P450 2B1 and endoxan and xanthine-guanine ribose transferring enzyme and 6-Thioxanthine combined utilization have obtained good effect; (4) immunogene treatment: the immunogene treatment is to adopt certain method or carrier that goal gene is imported tumour cell or effector cell---tumor infiltrating lymphocyte (TIL), to express then in the recipient cell input patient body of goal gene, by improve the human immune system to the identification of tumour cell, suppress and kill and wound, thereby a kind of method that tumour is treated; (5) ribozyme gene treatment; And (6) multidirectional drug resistant gene treatment etc. (Albelda S M, Chest, 1997, III Suppl:145-149).
Though carried out utilizing the antitumor rate of the gene therapy for cancer that variety of way carries out and the preclinical experimentation on animals of security aspect, up to now, its clinical effectiveness is all unsatisfactory but generally speaking.In general, not antitumor inefficiency, be exactly not have (BuenoR., Chest1999,116 (3 Suppl): 444S-45S; Arriola EL et al.Oncogene 1999,18 (4): 1081-91).The reason that this situation occurs undoubtedly is diversified, but thereby the genetic modification of cancer therapy mainly is subjected to and can't enough obtaining the restriction that enough high-caliber genetic expression obtains clinical effectiveness in the target cell of quantity greatly.
At present transgenosis is the most effective and to use maximum methods be to carry out gene therapy methods with virus as carrier in the gene therapy, wherein studies maximum retroviral vectors that is.U.S.'s nineteen ninety-five approval enters 106 of clinical gene therapies, and virus-mediated has 92, accounts for 85%, and 76 of retrovirus account for sum more than 50%.With the retrovirus is that carrier can duplicate sustainable expression alien gene with exogenous origin gene integrator to nucleus with cell fission.But in security, have problems,, may cause the viral integrase position unusual, thereby patient becomes the dangerous person that cancer is carried because retrovirus is inserted into host DNA at random.In addition, virus has and recovers the retroviral possibility of wild-type again behind the packing transfered cell, may cause the potential virus infection to the host.
Though the virus vector that research is used for lung cancer therapy comparatively fully is retrovirus (retrovius) and adenovirus (AV).But its shortcoming is also fairly obvious: 1) as indicated above, reversal of viral has and recovers the retroviral possibility of wild-type again, thereby produce the virus with replication behind the packing transfered cell, and there is the risk of virus infection in the host; 2) since retrovirus be random integration to host genome, thereby may cause " insertion mutagenesis "---can cause the activation that some insert near the proto-oncogene sites, bring out cancer; 3) retrovirus packing foreign DNA ability is very limited, and it generally can only pack the foreign DNA less than 8kb; 4) retrovirus also requires the target cell surface will have the corresponding acceptor of retrovirus.For adenovirus, its shortcoming also is fairly obvious: 1) adenovirus almost can infect all cells, therefore lacks specificity; 2) adenovirus be because can not be incorporated on the host chromosome, thus can not the required product of continuous expression, and expression time is of short duration, need repeat to give the carrier formulation treatment; 3) virion of adenovirus can cause strong immunne response, and possible immune response stimulating can reduce the effect of treatment repeatedly gradually.
In addition, the virus vector that is usually used in oncotherapy, as retrovirus and adenovirus, be nonreplicative virus vector, limited the diffusion of virus in tumour owing to not realizing duplicating of tumour-specific, can only rely on the effect of its gene pairs tumour of carrying fully, thereby its clinical effectiveness is very limited.
Summary of the invention
Lung cancer is the same with other tumours, and its growth, transfer and prognosis all depend on vasculogenesis.Based on this principle, the applicant attempts to utilize this principle of generation at gene molecule level blocking-up lung cancer nutrient vessel, effectively treats lung cancer.
The most promising technical scheme of utilizing the angiogenesis inhibitor mode to treat lung cancer is at present: with one or more anti-angiogenic medicaments/albumen long duration of action, thereby reach an effect stable, that continue.But there are problems in present this imagination of technical enforcement: how to select effective especially anti-angiogenic medicaments/albumen of lung cancer therapy or gene, how to improve the efficient of the importing body of gene, the security that how to improve gene therapy reduces immunogenicity and the toxic action of carrier to body, how to improve gene in vivo with external conversion capability or the like.Technical scheme of the present invention has overcome a series of problems that exist in the prior art, has realized the target of effective treatment lung cancer.
In order to overcome available technology adopting retrovirus commonly used and adenovirus carrier as the existing many-sided defective of Vectors in Gene Therapy, the present invention has selected for use the I herpes simplex virus type (HSV-1) that has lacked portion gene as carrier.
HSV-1 is a kind of common human virus that can infect neurone and non-neuronal cell, and it has the icosahedral capsid 162 capsid protein subunits be made of of surface coverage by the glycoprotein adventitia.It is double-stranded linear dna virus, about 153kb, about 84 albumen of encoding.Rose since 1980, HSV-1 just is used for the treatment of cancer and other diseases as carrier in gene therapy.
Compare with other carriers commonly used, HSV-1 has many advantages: 1) have maximum volume, can insert the foreign gene of 30kb at least and do not influence the assembling of virus; 2) it has good genetic stability, and most reorganization HSV-1 virus is at infection animal repeatedly, and after external a large amount of breedings, still keeping its original heredity is the presentation feature again, thereby it is safe; 3) infection ability is strong, can infect polytype host cell, and except hiding in neurone, it also can infect multiple tissue, comprises muscle, tumour, lung, liver, pancreas islet.The infection ability of HSV-1 can be suitable with adenovirus, and is almost stronger about 1000 times than retroviral infection ability; 4) in addition, to the existing deep research of the metainfective human pathology of HSV-1, molecular biology and clinicing aspect, and set up corresponding animal model, this compares with the virus that other new suggested is used for carrying out gene therapy, understanding to HSV-1 is many completely, thereby clinical use is as safe as a house; 5) HSV-1 infects in the mode of nonconformity host cell chromosome, thereby does not have the possibility that causes the host cell genetic mutation, thereby compares with retrovirus, and what security will be high is many; 6) clinically, developed the medicine that multiple anti-HSV-1 infects, put into practice clinically as Acyclovir (ACV) or Ganciclovir (GCV) etc. and to be used for the treatment of herpesvirus infection for many years, in case thereby clinical gene therapy is out of control, said medicine can effectively suppress viral growth, thereby interrupt infecting, for the individuality that carries out gene therapy provides further safety precaution; 7) the HSV-1 immunostimulation a little less than, different with adenovirus, HSV-1 virus has shelters infected host cell, the ability that makes it not to be subjected to body immune system to be attacked; 8) thus HSV-1 can carry out genetically engineered with good conditionsi in the tumour cell of target specific duplicating.
Its last item characteristic makes HSV-1 become first tumour cracking performance virus vector (the Martuza RLet al.Science 1991 that is used for cancer therapy; 252 (5007): 854-6).Known, early gene when virus, for example thymus gland kinases (tk) or ribonucleotide reductase gene are (just, ICP6 gene among the HSV) after quilt is lacked, HSV-1 has can duplicate and cause cytolysis in the cell that increases sharply, but can not or breed the characteristic of duplicating in the cell slowly in mitosis anaphase.G207 is the HSV-1 muton that is used for cancer therapy at present clinically: this virus is derived from the HSV-1F strain of wild-type, wherein cause the two copy of the virus ICP34.5 gene of encephalitis to carry out the deletion mutantion of 1kb in pairs, and in the ICP6 of coding nucleotide reductase enzyme gene, insert the lacZ gene and make its inactivation, and kept the TK gene, make HSV can be in tumour massive duplication, but can not in normal cell, duplicate.This genetic modification has weakened the neurotoxicity of HSV-1, and G207 is preferably duplicated in the tumour cell of fast breeding.Experiment confirm, in vivo with external, G207 can effectively kill the human tumor of various cell types, and it confirms equal no cytotoxicity in human body and animal body by coming I phase clinical experiment; Simultaneously, owing to kept the TK gene, can use some conventional antiviral such as the Acyclovir (ACV) or the Ganciclovir (GCV) of anti-acycloguanosine etc., prevent the excessive toxic reaction that causes that duplicates of virus.
The inventor is on the basis of G207 carrier, the angiostatin by selecting angiogenesis inhibitor and the fusion gene of Endostatin replace the lacZ gene among the G207, obtained except effect the recombinant virus AE618 that also has the function of angiogenesis inhibitor with the effective cracking tumour of energy thereby make up.
Because primary tumor and metastatic tumor thereof need new vasculogenesis to support its growth and existence, and known nonsmall-cell lung cancer needs higher vasculogenesis.For these reasons, the applicant has selected angiostatin and Endostatin fusion gene to be used for treating targetedly lung cancer as therapeutic gene.
Angiostatin (angiostatin) is the Angiostatin that O ' Reilly equals discovery in 1994, it derives from tumour cell, be the crack fragment of proplasmin 1-4kringles, can strong specificity suppress vascular endothelial cell proliferation, stop new vessel to generate.Ambs etc. studies show that, the cDNA of angiostatin is implemented in transfection melanoma cell B16F10 behind the plasmid PMB75.6, the vitro culture tumor proliferation is suppressed, be inoculated in the C57BL/6 mouse respectively and will screen the clone that obtains variable expression (high expression level in low), the result shows that tumor growth suppresses degree and is directly proportional with the angiostatin expression amount, but effect is ofer short duration.With the clone intravenous injection inoculation of high expression level, after 26 days, lung tumor metastatic carcinoma tubercle number obviously reduces (＜3/side), and control group has half mouse more than 150/side, prompting angiostatin high expression level can effectively suppress primary or metastatic tumo(u)r, is dose-dependently, and needs to use repeatedly.
Endostatin (endostatin) is another Angiostatin that O ' Reilly etc. found in 1997, it is the C-terminal fragment of the collagen VIII factor, it is suitable with angiostatin that it suppresses the vascular endothelial cell proliferation effect, and both are better than other Angiostatins by blood vessel formation against function.Yoon etc. with transfection the tumour cell intravenous injection of endostatin gene in the mouse body, the lung metastatic tumour seldom appearred or does not occur after 4 weeks.And subcutaneous injection fails to suppress the formation of lung metastatic tumour, may be different relevant with different tissues Endostatin expression amount; The tumour cell combined inoculation of they have also the used 25% transfection tumour cell of endostatin gene and 75% untransfected is subcutaneous in mouse, finds that its tumour cell that suppresses effect that primary tumo(u)r forms and 100% transfection endothelium suppressor gene is suitable.Prompting is as long as a small amount of endothelium suppressor gene transfection tumor cell just can suppress growth of tumour cell.Research also shows, Endostatin mainly suppresses in the different organs formation of former or metastatic carcinoma, and is relatively poor for established tumor effect.
In the present invention, we have adopted the virus of similar G207 molecule framework, as the primary virus vector, have made up the recombinant viral vector of expressing anti-angiogenic proteins.The fusion gene that carries is subjected to the regulation and control of people's elongation factor gene (hEF-1) promotor, and has the signal peptide of IL12 at its 5 ' end.We adopt at the coding region of ICP6 insertion angiostatin and Endostatin fusion gene and make the ICP6 inactivation, thereby obtain new recombinant virus AE618.
Owing in fact be subjected to the influence of various factors at the medicine of human body effect, for example degradation speed in dissimilar tumours of the perviousness of tumor by local, medicine, tumour are to susceptibility of vascularity or the like, whether can effectively realize the therapeutic purpose of its expection based on the recombinant virus of The Theory Construction, must be by experiment, particularly the checking of animal model could be set up.Especially, because different tumor tissues origin is different, often difference is bigger.In addition, the generation of tumour is based on transgenation, and this gene that indicates tumour cell has unstable, this make gene therapy medicament effect often with greatly differing from each other of theory vision in advance, make that the validity of medicine is unpredictable.Therefore, we are by the concrete experiment among the embodiment hereinafter, but have verified that further we make up the result of treatment of the recombinant herpes simplex virus of the excreting angiostatin of acquisition and endostatin protein.Our experimental data confirms that the AE618 recombinant virus can not only kill lung carcinoma cell by the mode cracking of virus replication, can also suppress growth of tumor by its excretory anti-angiogenic proteins.
On the one hand, but the present invention relates to the recombinant herpes simplex virus of a kind of excreting angiostatin and endostatin protein, two ICP34.5 genes that it is characterized in that described herpes simplex virus HSV-1 are all by excalation, and non-ly duplicate that necessary gene is inner to insert angiostatin and the Endostatin fusion gene makes this gene inactivation at it.
Recombinant herpes simplex virus as indicated above, wherein said non-to duplicate necessary gene be the ICP6 gene.
Recombinant herpes simplex virus as indicated above, it can optionally duplicate in the lung cancer tumour cell.
Recombinant herpes simplex virus as indicated above, its mode that can pass through virus replication can infect to target cell tumour cell in addition optionally at lung cancer tumour internal diffusion.
Recombinant herpes simplex virus as indicated above, it can divide vigorous lung cancer tumour cell by the killing of mode cracking performance of virus replication.
Recombinant herpes simplex virus as indicated above, its excretory angiostatin and Endostatin fusion rotein can effectively suppress vasculogenesis, thereby suppress the lung cancer growth of tumor.
Recombinant herpes simplex virus as indicated above, it is a temperature sensitive mutant, is being higher than described recombinant virus propagation inactivation under 39.5 ℃ the condition.
Recombinant herpes simplex virus as indicated above, it is to conventional antiviral AVC, GVC sensitivity, and conventional antiviral AVC, GVC can suppress the growth of described recombinant herpes simplex virus.
On the one hand, but the present invention relates to the method for the recombinant herpes simplex virus of a kind of structure excreting angiostatin and endostatin protein, the steps include:
1) two of excalation herpes simplex virus HSV-1 ICP34.5 genes;
2) make the ICP6 gene inactivation at the inside of the ICP6 of HSV-1 gene insertion angiostatin and Endostatin fusion gene.
On the other hand, the present invention relates to a kind of pharmaceutical composition that contains above-mentioned recombinant herpes simplex virus.
Pharmaceutical composition as indicated above, wherein said pharmaceutical composition can be injection, propellant and other conventional pharmaceutical dosage form forms.
The pharmaceutical composition that comprises medicine of the present invention can be by known method preparation, for example by mixing with pharmaceutically acceptable carrier.The example of the method for this carrier and preparation can be found from Remington ' s pharmaceutical science.In order to form a pharmaceutically acceptable composition that is suitable for effective administration, said composition will contain the described medicine of significant quantity.Effective amount of drug will be according to the condition of the various factors such as individuality, weight, sex and age and change.Other the factor comprises mode of administration.Pharmaceutical composition can be by all means to individual administration, for example partial (comprising in the tumour) and whole body.
Medicine of the present invention can come administration with the wide range of therapeutic medicine type that tradition is used for the vehicle of administration.For example, described medicine can be with the form of various dosage, as tablet, and capsule (each sheet comprise regularly discharge and sustained release formulation), pill, pulvis, granula, solution, suspension, liposome and other possible pharmaceutical dosage form modes, or by injection system, directly administration in vein or tumour.
On the other hand, but the present invention relates to the application of recombinant herpes simplex virus in preparation treatment lung cancer drugs of a kind of excreting angiostatin and endostatin protein, two ICP34.5 genes that it is characterized in that described herpes simplex virus HSV-1 are all by excalation, and insert angiostatin in the inside of its ICP6 gene and the Endostatin fusion gene makes the ICP6 gene inactivation.
Recombinant herpes simplex virus as indicated above application in the medicine of preparation treatment and the recurrence of prevention lung cancer, described recombinant herpes simplex virus can optionally duplicate in the lung cancer tumour cell.
Recombinant herpes simplex virus as indicated above application in preparation treatment lung cancer drugs, the mode that described recombinant herpes simplex virus can pass through virus replication can infect to target cell tumour cell in addition optionally at lung cancer tumour internal diffusion.
Recombinant herpes simplex virus as indicated above application in preparation treatment lung cancer drugs, described recombinant herpes simplex virus can divide vigorous lung cancer tumour cell by the killing of mode cracking performance of virus replication.
Recombinant herpes simplex virus as indicated above application in preparation treatment lung cancer drugs, described recombinant herpes simplex virus excretory angiostatin and Endostatin fusion rotein can effectively suppress vasculogenesis, thereby suppress the lung cancer growth of tumor.
Recombinant herpes simplex virus as indicated above application in preparation treatment lung cancer drugs, described recombinant herpes simplex virus is a temperature sensitive mutant, be higher than described recombinant virus propagation inactivation under 39.5 ℃ the condition, thereby improving the security of gene therapy.
Recombinant herpes simplex virus as indicated above application in preparation treatment lung cancer drugs, described recombinant herpes simplex virus is to conventional antiviral AVC, GVC sensitivity, can suppress the growth of described recombinant herpes simplex virus by conventional antiviral AVC, GVC, thereby improve the security of gene therapy.
Recombinant herpes simplex virus as indicated above application in preparation treatment lung cancer drugs, described recombinant herpes simplex virus can be used with other cancer therapy drugs of various routines or operation, radiotherapy, chemotherapy drugs in combination.
Recombinant herpes simplex virus as indicated above application in preparation treatment lung cancer drugs, described recombinant herpes simplex virus is by the mode administration of part or systemic injection.
For those skilled in the art, range gene engineering working method of the present invention, experiment condition, conventional reagent all adopt this area the whole bag of tricks, condition and conventional reagent commonly used, existing textbook (as, the molecular cloning experiment guide, detailed record is all arranged second edition), so this specification sheets is not further described to it.Classes of agents of the present invention, carrier are reagent, the carrier that commercialization can be buied.Those skilled in the art can have no enforcement the present invention of difficulty in conjunction with relevant instruction of the prior art according to the record of this specification sheets.
The various improvement of technical scheme of the present invention are conspicuous for those skilled in the art, and it does not deviate from the scope and spirit of the present invention.Though hereinafter will further illustrate the present invention in conjunction with specific embodiment, should understand like this, claim invention required for protection should unduely not be confined in the technical scheme of this specific embodiment.In fact, the present invention attempts to comprise those those of skill in the art for chemistry or biology or association area, to the conspicuous various improvement of the technical scheme put down in writing in order to realize purpose of the present invention.All publications of mentioning in the above-mentioned specification sheets are incorporated reference at this.
Description of drawings
Fig. 1 adopts contrast to infect, or the angiostatin in the lung cell A549 of employing G207 and AE618 infection processing and the expression of Endostatin.The result shows, the Endostatin that adopts AE618 to infect significantly to have increased in the A549 cell and the expression of angiostatin.And not infected group in contrast and not with the equal expressed fusion protein not of the viral G207 group of blood vessel supressor and endothelium supressor.Second row adopts the internal reference of tubulin as albumen application of sample amount.
Fig. 2 AE618 and G207 compare the lysis effect of lung carcinoma cell H460.(A) under 0.1 M.O.I, viable count is carried out to adopting the virus of A E618 that expresses angiostatin and Endostatin fusion rotein to handle and contrast the lung carcinoma cell H460 that viral G207 handles respectively in after processing 1 to 5 day, and count results does not have significant difference; (B) adopt AE618 and G207 to handle the H460 lung carcinoma cell respectively, after processing, carried out the viable cell technology on the 3rd day.In titre is between 0.001 to 10, and handling between the technical result that obtains for two kinds does not have significant difference.This result shows, recombinant virus AE618 has kept the ability that the cracking performance to lung carcinoma cell kills and wounds, and tumour cell is had the complete inhibition effect.
Fig. 3 human vascular endothelial (HUVEC) is cultivated altogether with the H460 lung carcinoma cell that AE618 or G207 with 0.1 M.O.I infects.After processing the 3rd day and the 4th day compared with the control group that G207 handles, and adopts human vascular endothelial (HUVEC) quantity in the control group that AE618 handles significantly to descend, and obvious growth occurs and suppresses.The lung carcinoma cell that this explanation is infected by AE618 can discharge angiostatin and Endostatin fusion rotein, thereby the growth of vascular cell has been produced inhibition.
Fig. 4 is in the SCID mouse model, and AE618 is to the antitumous effect of human tumor cells.From infecting the back the 6th day, the anti-tumor in vivo effect of recombinant virus AE618 is significantly higher than G207.This figure changes with the size multiple of the original tumour of metainfective every day and represents.The lung cancer tumor model of virus infection shows, its growth velocity of tumour of injection AE618 virus also is markedly inferior to the contrast virus G207 group of not expressing angiostatin and Endostatin fusion rotein far below the PBS control group of injecting virus not.
The plasmid map of Fig. 5 plasmid pICP34.5-BLU.
Fig. 6 HSV-1ICP34.5 gene location structure.
The plasmid map of Fig. 7 plasmid pICP34.5-BLU28.
The building process of Fig. 8 recombinant virus v345281.
Fig. 9 plasmid pGT65 plasmid structure collection of illustrative plates.
The plasmid structure collection of illustrative plates of Figure 10 plasmid pBLUESCRIPT-KS.
The structure collection of illustrative plates of Figure 11 plasmid pICP6-SU6/KS.
The structure collection of illustrative plates of Figure 12 plasmid pAE.
The temperature sensitivity detected result of Figure 13 AE618.
Figure 14 AE618 is to the susceptibility result of GCV.
Embodiment
Technical scheme of the present invention will adopt following non-limiting example further instruction.
Embodiment 1: the structure of recombinant virus AE618
(a) preparation method of the F strain virus DNA of HSV-1 virus:
The F strain of the HSV-1 virus that is used to recombinate is buied by U.S. ATCC company.Under the condition of MOI=1, infect the Vero cell of cultivating (U.S. ATCC company) with this virus.When treating after 24-36 hour that big portion cell presents cytopathy (CPE), collecting cell also changes the centrifugal removal cell culture fluid of 15ml centrifuge tube over to.Adding the 0.3ml cell pyrolysis liquid (contains: 100Mm NaCl, 10Mm Tris.HClpH 8,25Mm EDTA pH8 and 0.5%SDS) cracking cells infected and handled 1 hour with RNaseA (10mg/ml) 65 degree, above-mentioned lysate is used the extracting of isopyknic phenol/chloroform (1:1) mixed solution again with isopyknic phenol earlier, the aqueous phase solution that contains DNA adds the 7.5M Ammoniom-Acetate of 1/2 volume and the raw spirit of 2 times of volumes after transferring to new centrifuge tube, leave standstill the centrifugal viral DNA precipitation that obtains after 10 minutes, in 4 ℃ of preservations, prepare original HSV-1 viral DNA with 20-100ul Tris/EDTA damping fluid dissolving back.
(b) the concrete experimental procedure of missing gene ICP34.5:
Make up plasmid pICP34.5BLU28:
The HSV-1F strain virus DNA that gets 5 μ g purifying obtains including the fragment of a 14509bp of ICP34.5 gene with restriction enzyme PstI digestion and separation and purification.This fragment so with XhoI and SapI (NEWENGLAND) digestion separate obtain a 5853bp than small segment, this XhoI/SapI fragment is gone into the XhoI of plasmid pBLUESCRIPT and EcoRV and is held and obtain plasmid pICP34.5-BLU (Fig. 5) in that SapI end is mended flat rear clone with the T4DNA synthetic enzyme, plasmid pICP34.5-BLU Bsu36I (NEW ENGLAND, BIOLABS) and BstEII digestion obtain 1699bp (BstEII/Bsu36I) and two fragments of 7089bp (Bsu36I/BstEII), the former includes the big portion of ICP34.5 gene 3 ' end, after the electrophoretic separation, the 7089bp fragment is preserved and the 1699bp fragment further digests with AseI, obtain the fragment of a 705bp (AseI/Bsu36I) and a 994bp (BstEII/AseI) after the electrophoretic separation, as shown in Figure 6, the latter comprises the ICP34.5 gene from all later alkali yl coding sequences of the 84th thymus nucleic acid (being equivalent to the 28th amino acid), meanwhile synthetic double chain oligonucleotide (normal chain: 5 ' GTTACCGTTTAAACAT3 ', anti-chain: 5 ' TAATGTTTAAACG3 '), the one end is BstEII, the other end is AseI, at last, with above-mentioned 7089bp (Bsu36I/BstEII), 705bp (AseI/Bsu36I) and oligonucleotide chain mix, adding T4 ligase enzyme and corresponding damping fluid at room temperature react after 30 minutes and change intestinal bacteria over to, obtain plasmid pICP34.5-BLU28 (Fig. 7), the the 84th to 991 base of the ICP34.5 gene coding region that wherein comprises replaced by above-mentioned synthetic oligonucleotide, thereby all removed after the 28th amino acid of ICP34.5 gene coding region 3 ' end.
(c) the building process (see figure 8) of recombinant virus v345281.
Recombinant plasmid pICP34.5BLU28 (Fig. 7) obtains to have the recombinant virus (v345281) (detailed process of cotransfection is seen Fig. 8) that lacks the ICP34.5 gene with the HSV-1F strain virus DNA cotransfection Vero cell of purifying.
The method that the HSV-1 viral DNA of plasmid pICP34.5-BLU28 and 5 μ g purifying is recommended by the product description of company with LIPOFECTAMINE (GIBCOBRL) changes in the Vero cell of cultivation jointly, based on homologous recombination mechanism, original ICP34.5 gene is substituted by the dna sequence dna in the plasmid in the viral genome, and the HSV-1 virus of generation reorganization, from said process, obtain 92 viral plaques altogether, infect the Vero cell of on 96 porocyte culture plates, growing respectively, after treating that pathology appears in whole cells, collect virus and get part virus by above-mentioned method extraction viral DNA, Southern Blotting is adopted in selecting of recombinant virus, at first, (991bp BstEII/AseI) is marked with as masterplate preparation to be removed the fragment of ICP34.5 gene order with 0.3 μ g
32The probe of P (random primer labelingkit, GIBCO BRL), then, every strain virus is got 0.1 μ g point on the cellulous film, above-mentioned probe is hybridized 15 hour whether existing with identifying virus ICP34.5 gene regions under 60 degree, in above-mentioned 92 strain virus, there is 5 strains performance that the ICP34.5 disappearance is arranged, do gel electrophoresis with BamHI and NcoI after the DNA digestion to this 5 strain virus, and BamHI/AseI (1315bp) segment of further downcutting from plasmid pICP34.5-BLU28 is that masterplate obtains
32The probe of P mark is made Southern and is identified.Find that a wherein strain is that two ICP34.5 gene copies all lack called after v34528.Identify through dna sequencing, confirm that two ICP34.5 gene all is removed.
(d) insert the concrete experimental procedure of the fusion gene of people's Endostatin and Angiostatin in the position of gene ICP6:
Structure has the fusion gene expression cassette that contains Endostatin and Angiostatin and the recombinant plasmid pAE of ICP6 flanking sequence, the viral DNA cotransfection VERO cell of recombinant plasmid pAE and recombinant virus V34528, acquisition insert the recombinant virus AE618 of the fusion gene of expressing Endostatin and Angiostatin in the ICP6 gene location.
The concrete experimental procedure of construction recombination plasmid pAE:
1. at first the ICP6 gene clone is obtained plasmid pICP6-SU6/KS in cloning vector pBlueScriptKS.
The plasmid pGT65 (Fig. 9) of fusion gene expression cassette that contains Endostatin:Angiostatin is available from InvivoGen Co..The preparation of plasmid pICP6-SU6/KS:
Get 2 μ g plasmid pBLUESCRIPT-KS (Figure 10) with the digestion after 2 hours under 37 degree in the corresponding damping fluid of 50ul of each 10 unit of restriction enzyme BamHI and SacI, separate and after carrying DNA purification column (QIAGIN) purifying for a short time, obtain the plasmid fragment with 1% agargel electrophoresis, get the HSV-1F strain virus DNA that the preceding method purifying obtains that passes through of 5 μ g simultaneously, adopt restriction enzyme BglII and SacI under same digestion condition, to digest, and separation and purification obtains including the fragment of a 3573bp of ICP6 gene, this BglII/SacI fragment is with after the BamHI/SacI fragment of above-mentioned pBLUESCRIPT-KS plasmid is mixed, the T4DNA ligase enzyme and the corresponding reaction buffer that add 5 units reach cumulative volume 20ul, reaction was got the DH5 Ecoli cell that 5ul changes sensitization over to after 30 minutes under the room temperature.The bacterium colony that grows after cultivating 18 hours on the substratum that contains 100 μ g/ml ampiciline with the plasmid of the QIAGEN cover liquid of purifying in a small amount, extracts and enzyme is cut evaluation and obtained plasmid pICP6-SU6/KS (Figure 11).
2. with Endostatin, the fusion gene expression cassette of Angiostatin inserts plasmid pICP6-SU6/KS and obtains recombinant plasmid pAE.
The structure of plasmid pAE:
Above-mentioned plasmid pICP6-SU6/KS has two BamHI point of contacts (as described in Figure 11) in the ICP6 gene of the HSV-1 that inserts, after this plasmid digests under these conditions with BamHI, adding mixing solutions that 5ul contains 10 unit T4 DNA synthetic enzyme and four kinds of thymus nucleic acids (concentration is 10uM separately) continues 37 degree reactions 30 minutes flat to mend, remove the fragment (764bp) between the two BamHI point behind the electrophoresis purifying and obtain plasmid fragment (5736pb), simultaneously, to contain Endostatin, the plasmid pGT65 (Fig. 9) of the fusion gene expression cassette of Angiostatin digests with XhoI and SwaI with reaction conditions similar to the above and mends flat two ends with T4 DNA synthetic enzyme under similarity condition, obtain containing the fragment (2854pb) of Endo:Angio genetic expression unit behind the electrophoresis purifying, change over to after the pICP6-SU6/KS plasmid fragment of this fragment and above-mentioned 5736pb is connected with the T4 dna ligase and obtain plasmid pAE in the intestinal bacteria.The inscribe zymogram of plasmid pAE is seen Figure 12.
3. with the viral DNA cotransfection VERO cell of recombinant plasmid pAE and recombinant virus V34528,
The recombinant virus AE618 of the fusion gene of expressing Endostatin and Angiostatin is inserted in acquisition in the ICP6 gene location.
With the method viral same with above-mentioned structure v34528, the v34528 viral DNA of plasmid pAE and purifying is changed in the Vero cell of cultivation jointly, based on above-mentioned identical homologous recombination mechanism, the sequence of ICP6 genes encoding section is replaced by the dna sequence dna of the Endo:Angio fusion gene ceneme among the pAE, in 53 strain virus that obtain, identify whether include Endo:Angio fusion gene ceneme with PCR method.The sequence of PCR upstream and downstream primer is selected from Endo:Angio fusion gene ceneme inside, and the length of its theoretical PCR product should be 330bp.The sequence of upstream and downstream primer is as follows:
Upstream: 5 ' ACGCATCTTCTCC TTTGACGG3 '
Downstream: 5 ' GTCCCTCTGTAGTTCTTTCCATT3 '
The PCR condition is: 0.05 μ g viral DNA masterplate, 0.5 TaqDNA of unit synthetic enzyme, 5%DMSO and 10mMMgCl
2, at first press 94 ℃ 45 afterwards at 94 ℃ of following inactivations 5 ' " and-52 ℃ 45 "-72 ℃ 45 " 25 circulations of do.PCR product gel electrophoresis result shows that it is recombinant virus that 9 strains are wherein arranged, identify through order-checking, confirm in the ICP6 gene in these viral genome that the 536th to the 1300th base pair is substituted by the fragment of above-mentioned Endo:Angio genetic expression unit by design requirements, while, two ICP34.5 gene copies all lacked the 84th to 991 base of its coding region, this viral nomenclature is AE618, and the growth and breeding on the Vero cell of the AE618 virus after the screening and separating is placed on subzero 80 degree Celsius and preserves.
Cultivate the clone that is adopted
Non-small cell lung cancer cell is that NCI-H460 (ATCC HTB-177) and A549 (ATCCCCL-185) derive from American type culture collection.With this cell cultures (Dulbecco ' s modified Eagle medium) in the DMEM substratum that is added with 10% foetal calf serum (FCS).
Human umbilical vein endothelial cells (HUVEC) is incubated at the MCDB 107 that is added with the fibroblast growth factor (1 μ g/ml) that 2%FCS and Medulla sus domestica extract partly be rich in, and (JRH Biosciences, Lenexa is KS) in the substratum.
Embodiment 2: detect Expression of Fusion Protein in the cancer cells that infects AE618
Before the infection, under 37 ℃, cultivate lung cancer A549 cell (1 * 10 at 10cm plate middle plateform
6) 4 hours.Then it is adopted G207, AE618 or blank to infect (each infection titer M.O.I.=1.0) respectively, and metainfective cell is continued to cultivate 24 hours.Adopt PBS flushing culturing cell sample twice, from plate, cell is scraped gently subsequently, and the employing cell lysis buffer solution (50mM Tris-HCl pH7.5,150mM NaCl, 1%NP40,0.5% Sodium desoxycholate 0.1%SDS) comes lysing cell.Whole product of cell lysis are boiled, and (Bio-Rad, Hercules CA) detect protein concn through the Bradford method.Albumen (the 100 μ g) sample of getting equivalent carries out electrophoretic separation on 15% acrylamide gel under reductive condition.With the albumen behind the electrophoresis change film to the Immobilon-P transfer film (Millipore, Bedford, MA), then film is placed 5% skim-milk to seal, and under 4 ℃, be placed among the TBS (TBS-T) that contains 0.2% Tween-20 and spend the night, at room temperature educated 1 hour subsequently with a temperature resistance.Adopt TBS-T to wash film 3 times, each 5 minutes.Anti-purchase ﹠amp at one of Endostatin and angiostatin in R; D Systems Inc. (Minneapolis, MN), at γ-tubulin (sc-7396 is the same) one anti-purchase in Santa Cruz Biotechnology Inc. (Santa Cruz, CA).(Pierce, Rockford IL) resist as two with the anti-goat-anti body of horseradish peroxidase bonded rabbit in employing.Protein develops the color by luminol chemiluminescence reagent (Santa Cruz).
By above-mentioned immunoblot experiment, the result is presented at the AE618 of 1.0M.O.I. and infects in the A549 lung carcinoma cell after 24 hours, and the expression of Endostatin and angiostatin all significantly increases.Compare with blank, adopt above-mentioned proteic expression in the A549 cell that G207 infects without any increase (Fig. 1).The proof that this result is strong the present invention make up the endostatin-angiostatin antigen-4 fusion protein gene of integrating in the AE618 recombinant virus obtain and obtained expressing and having complete function.
Embodiment 3: the detection of the lysis effect of recombinant virus AE618
With A549 and H460 lung cancer cell line (1 * 10
5) all place 12 hole tissue culturing plates, cultivated 4 hours down at 37 ℃, come cells infected with the G207 of various concentration and AE618 recombinant virus and blank subsequently, and cell is continued to cultivate several days.After trypsin treatment, collecting cell also is resuspended in the PBS solution.In cell suspension, add isopyknic PBS that contains 0.1% phenol indigo plant.Utilize hematimeter to carry out viable count subsequently.All cytometer number averages are got the mean value of 3 these countings of increment.
Show for the cell counting result who infects 1 to 5 day the H460 lung carcinoma cell in back, compare, the viable cell number from the group that began to adopt virus treated in metainfective second day significantly descend (p＜0.001) with blank.More there is not significant difference (p〉0.05, Fig. 2 A) in viable count between the group that the group that adopts AE618 to handle and G207 handle.
In addition, same employing titre is that 0.001 to 10 AE618 and G207 infect H460 clone respectively.Handle the back and began to carry out viable count on the 3rd day.Shown in Fig. 2 B, the cell counting after two kinds of virus treated under all titres is significantly not different.
Above-mentioned experimental result shows, we have made up the still complete maintenance of the recombinant virus AE618 that the obtains splitting action of its parental virus to lung carcinoma cell.
Embodiment 4: by the restraining effect of the cancer cell excretory albumen that adopts AE618 to infect to the HUVEC growth
Human umbilical vein endothelial cells (HUVEC) (3 * 10
6) be incubated in the double-deck culture dish in 6 holes of 35mm.Cultivating after 24 hours, will be respectively the H460 lung carcinoma cell (5 * 10 of 0.1 blank, G207 and AE618 recombinant virus infection by titre
4) be inoculated in the bottom and have in the cell cultures pond of micropore of 0.4-μ m size that (Franklin Lakes NJ), and is placed on growth and has in the culture hole of HUVEC for Cat.#353090, BectonDickinson.Albumen in the substratum in above-mentioned micropore permission culture hole and cell cultures pond circulates mutually.(ICNBiomedicals Inc.) prevents the superinfection of the virus that H460 cell that HUVEC infected discharges to have added people's gamma globulin of 0.2% in this substratum.Thereby aforesaid operations make HUVEC with realized common cultivation by the H460 cell of virus infection.After processing 1 to 4 day, collector's huve cell and H460 lung carcinoma cell respectively, and adopt hematimeter to carry out viable count subsequently.All cytometer number averages are got the mean value of 3 these countings of increment.
The result shows, though because the effect of the general toxicity of the viral protein in the substratum, all descend with the growth velocity of the HUVEC cell of the H460 co-culture of cells that has infected any virus, but from the result, can be observed, after processing the 3rd to 4 day, adopt the viable count of the HUVEC cell of the group that AE618 handles significantly to be lower than group (p=0.004 and p＜0.001 of adopting G207 to handle; Fig. 3).This result shows that the Endostatin and the angiostatin fusion gene of cloning among the recombinant virus AE618 have obtained expression really, and the fusion rotein of its expression has the effect that suppresses vasculogenesis really.
Embodiment 5: experimentation on animals
The 1000000 H460 cells (1 * 10 that contain with 0.05mL
6) aseptic Hanks buffer salt solution through the subcutaneous both sides, back that are injected to SCID mouse (Charles River) respectively.Begin to adopt recombinant virus to treat tumour at implantation tumour after 16 to 18 days.With recombinant virus AE618, G207 and isopyknic PBS solution (every mouse inject 50ul respectively contain 10
7The solution of pfu virus) tumour on injection right side.Comprise 6 mouse in each treatment group.Before treatment, after (the 0th day) and the treatment (the 1st to 13 day), calculate the size of tumour respectively by formula V=L*W*W/2000.Data representation be to change with respect to the multiple of original tumour size, the 0th day the V of certain day V/ after the treatment for example.
Compare with the control group that PBS handles, from the treatment after the 2nd day (its P value is respectively: p=0.013 and p=0.009) to the 13rd day (its P value is respectively: p=0.008 and p=0.006), G207 and recombinant virus AE618 have all suppressed the H460 growth of tumor (Fig. 4) of xenograft significantly.After the treatment the 6th day, adopt that tumour size in the group of AE618 treatment continues significantly less than the group (p＜0.03) that adopts G207 to treat.This result shows that recombinant virus AE618 has significant unexpected good therapeutic action than its parental virus G207.
Statistical method
Do not having under the situation about specifying, all experiments all repeat 3 times.Adopt mean value ± standard error to represent experimental result.Adopt single tail t-to test and carry out statistical.There is significant difference in the P value less than 0.05 expression.
Embodiment 6: the temperature sensitivity of recombinant virus AE618
Because (Preston 1981 to have point mutation among the utmost point early gene ICP4 of the maternal HSV-1F strain of AE618; DeLuca andSchaffer 1985), the disappearance of its ICP6 gene can further increase its temperature sensitivity (Goldstein and Weller1988) simultaneously, thereby can predict that recombinant virus AE618 also should keep this feature, is a temperature sensitive mutant.Followingly will confirm the temperature sensitivity of recombinant virus AE618 by experiment.
Behind the AE618 virus infection Vero cell of employing as indicated above reorganization, respectively 37 ℃ and 39.5 ℃ of following culturing cells 48 hours.The different time harvested cell also carries out malicious spot titration then after infection.
The result as shown in figure 13, under 37 ℃ of conditions, in 48 hours, the virus titer of AE618 has increased by three Log (i.e. three orders of magnitude, 1,000 times); And under 39.5 ℃ of conditions, AE618 stops breeding in cell, and after 48 hours, its titre has reduced by 1.5 orders of magnitude (150 times) on the contrary.This presentation of results, AE618 have kept its point mutation of wild maternal plant F virus on the ICP4 gene.This characteristic is extremely important for the security of AE618.This shows, when in case patient produces the fever symptom that is caused by inflammation when treatment, because this temperature sensitivity of AE618 is specific, its in vivo growth and breeding can be suppressed automatically, thereby lowered the danger that produces complication, improved recombinant virus AE618 greatly as the security that is applied to the gene therapy recombinant virus of human body.
Embodiment 7:AE618 is to the susceptibility of conventional antiviral GCV (Ganciclovir)
Because AE618 has remained with the TK gene of HSV-1 self, thereby carries the viral the same of HSV-1TK gene with other, recombinant virus AE618 has certain susceptibility to anti-herpesvirus medicament GCV or ACV.Previous studies show that, the disappearance of ICP6 can further increase susceptibility (Yamada, the Yamamoto et al.1991 of virus to this class medicine; Mineta, Rabkin et al.1994).Followingly will verify the susceptibility of recombinant virus AE618 by concrete experiment to conventional antiviral GCV.
As indicated above, behind recombinant virus AE618 and G207 and F strain virus vero cells infection, grow not adding or be added with in the nutrient solution of GCV of different concns respectively.Also carry out malicious spot titration then at back 48 hours harvested cells of infection.
The result of Figure 14 shows, AE618 and G207 be to the susceptibility of GCV just the same (curve almost completely overlaps), and the susceptibility of its enantiopathy cytotoxic drug GCV is all than high about 10 times of HSV-1 (F) virus strain of wild-type.Data are with similarity condition but the generation titre of the virus when not containing GCV is relatively to represent (virus titer that generates when not containing GCV is 100%) among the figure.As seen from the figure, GCV concentration can suppress VAE fully in external growth when every milliliter 100 microgram.This result further shows, when adopting recombinant virus AE618 to carry out gene therapy, in case virus multiplication is out of control, can adopts conventional anti-herpesvirus therapeutical agent GCV to suppress the propagation of AE618, thereby further increase the gene therapy security of recombinant virus AE618.
Discussion of results
Angiogenesis inhibitor is a treatment human tumor approach likely.Angiostatin and Endostatin demonstrate the neovascularization that can effectively suppress in primary tumor and the metastatic tumor.In clinical application, the main limitation of this treatment approach is how to obtain to reach at tumor locus the albumen of treatment level: the dosage in experimentation on animals is calculated according to angiostatin and Endostatin, the effective dose of angiostatin should be no one every day 0.1 to more than the 1.0g, and the dosage of Endostatin should be about 20mg/kg.d; Therefore how obtaining a large amount of, high purity, highly active angiostatin and Endostatin is present urgent problem.In the present invention, we are integrated into a tumour cracking performance HSV-1 carrier G207 who effectively duplicates with angiostatin and Endostatin antigen-4 fusion protein gene, have obtained a kind of new recombinant virus thereby make up, with its called after AE618.When duplicating in the tumour cell that recombinant virus AE618 is infecting, host's tumour cell can be secreted the vessel growth that anti-angiogenic production albumen prevents to provide nutrient to solid tumor equally.Simultaneously, because AE618 is a kind of cracking performance virus, in case when the tumour cell of its infection owing to virus replication cracking takes place, thereby the virus particle of release can produce more anti-angiogenic proteins with the cell that PI more is close to.Our experimental data confirms, can secrete enough anti-angiogenic proteins by the tumour cell that AE618 infects.Can significantly suppress the growth of human endothelial cell by the albumen of the emiocytosis of having infected AE618.Simultaneously, AE618 has still kept the characteristic of the cracking tumour of its parental virus G207.The combination of these two kinds treatment approach will help it and be applied to gene therapy for cancer clinically.Because the high tumour-specific of recombinant virus AE618 is selected to duplicate and lower tissue toxicity, AE618 can become a kind of potential security medicine that is used for lung cancer therapy.
In double-deck culture systems of the present invention; the number of the HUVEC cell of the survival of cultivating altogether with the H460 lung carcinoma cell that has infected G207 significantly is lower than blank, and this surperficial people γ IgG can not protect HUVEC to make it avoid the cytotoxicity that G207 or AE618 cause completely.But count results shows equally, although adopting identical titre handles, recombinant virus AE618 still demonstrates the inhibition to the HUVEC growth that is significantly higher than G207, and this shows that the endo:angio fusion rotein by H460 emiocytosis has the effect that suppresses vasculogenesis really.Yet, since virus to HUVEC cause to cytopathy may, the effect of excretory fusion rotein may have been underestimated.Experimental result confirms in our body, compares with G207, and growth has significant inhibitory effect to AE618 for the human tumor in the SCID mouse.
In sum, the AE618 among the present invention can effectively be used for the treatment of lung cancer clinically.
Claims (6)
1. but the application of the recombinant herpes simplex virus of excreting angiostatin and endostatin protein in preparation treatment lung cancer drugs, two ICP34.5 genes that it is characterized in that described herpes simplex virus HSV-1 all are removed, and insert angiostatin and Endostatin fusion gene in the inside of its ICP6 gene and make the ICP6 gene inactivation, and described recombinant herpes simplex virus can optionally duplicate in the lung cancer tumour cell, and described recombinant herpes simplex virus can pass through the mode of virus replication optionally at lung cancer tumour internal diffusion, can infect to target cell tumour cell in addition, and can divide vigorous lung cancer tumour cell by the killing of mode cracking performance of virus replication.
2. the application of recombinant herpes simplex virus as claimed in claim 1 in preparation treatment lung cancer drugs, described recombinant herpes simplex virus excretory angiostatin and Endostatin fusion rotein can effectively suppress vasculogenesis, thereby suppress the lung cancer growth of tumor.
3. the application of recombinant herpes simplex virus as claimed in claim 1 in preparation treatment lung cancer drugs, described recombinant herpes simplex virus is a temperature sensitive mutant, be higher than described recombinant virus propagation inactivation under 39.5 ℃ the condition, thereby improving the security of gene therapy.
4. the application of recombinant herpes simplex virus as claimed in claim 1 in preparation treatment lung cancer drugs, described recombinant herpes simplex virus is to conventional antiviral ACV, GCV sensitivity, conventional antiviral ACV, GCV can suppress the growth of described recombinant herpes simplex virus, thereby improve the security of gene therapy.
5. the application of recombinant herpes simplex virus as claimed in claim 1 in preparation treatment lung cancer drugs, described recombinant herpes simplex virus can be used with other cancer therapy drugs of various routines or operation, radiotherapy, chemotherapy drugs in combination.
6. the application of recombinant herpes simplex virus as claimed in claim 1 in preparation treatment lung cancer drugs, described recombinant herpes simplex virus is by the mode administration of part or systemic injection.
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