CN101448526A - Seneca valley virus based compositions and methods for treating disease - Google Patents

Abstract

The present invention relates to a novel RNA picornavirus that is called Seneca Valley virus ('SVV'). The invention provides isolated SW nucleic acids and proteins encoded by these nucleic acids. Further, the invention provides antibodies that are raised against the SVV proteins. Because SVV has the ability to selectively kill some types of rumors, the invention provides methods of using SVV and SVV polypeptides to treat cancer. Because SVV specifically targets certain tumors, the invention provides methods of using SW nucleic acids and proteins to detect cancer. Additionally, due to the information provided by the tumor-specific mechanisms of SVV, the invention provides methods of making new oncolytic virus derivatives and of altering viruses to have tumor-specific tropisms.

Description

Based on the compositions of Xi Nijia paddy virus and the method for treatment disease

This application requires following priority: United States Patent (USP) 60/664,442, (being filed on March 23rd, 2005); United States Patent (USP) 60/726,313 (being filed on October 13rd, 2005); United States Patent (USP) 11/335,891 (being filed on January 19th, 2006).These applications are introduced this and are sentenced it all as reference.

The copyright protection of holding in contained in the disclosure.When the disclosure as United States Patent and Trademark Office file or when record, the copyrighter does not oppose that anyone carries out duplicate copy to this patent document or this patent disclosure; Otherwise will keep all copyright power.

All patent applications of quoting in this manual, publish patent application, patent, text and the article with authorizing examined all introduce this and sentence it all as reference, so that state of the art of the present invention is described more fully.

Background technology

Virus therapy has prospect very much aspect treatment of cancer.Tumor cell cracking performance virus is intended to infect specifically and killing tumor cell.Compare with other treatment scheme (as chemotherapy or radiotherapy), natural and or the tumor cell cracking performance virus of through engineering approaches may be more effective and toxicity is littler.In addition, tumor cell cracking performance virus therapy is employed to be the virus with replication capacity, so it is the at present unique therapy that can amplify curative effect in the site of expectation.

The key of treatment of cancer is to realize the High Fragmentation rate of the cancerous cell of relative normal cell.A lot of reasons causes and is difficult to reach above-mentioned target, and described reason comprises that related cell type is extensive and various, make cancerous cell distribution whole body owing to shifting, and difference biology between cancerous cell and normal cell is very little.Although obtained some progress, needed all the time existing treatment of cancer is improved.

In the past, the surgeon attempts basically not pass through to patient harm surgery operating removing tumor.But because transfer in early days health the unknown the site and be retained in the there and fail to detect, so can not guarantee survival even tumor in situ excised fully.Some research proposal is because because the tumor resection cell produces angiogenesis inhibitor, surgery is got involved may promote remote transfer.At last, under a lot of situations, tumor is grown again and is got back to original site behind the exenterate.The purpose of radiotherapy is optionally to destroy great majority proliferating cells rapidly to damage on the basis that other cell is a cost.Yet tumor cell can or have resistance or escapes radiotherapy by be in non-splitting status during treating by becoming.In addition, radiotherapy is not always optionally to enlivening splitted a lot of normal cell, and these normal cells (for example the cell in the bone marrow, gastrointestinal tract cell and hair follicle cell etc.) also can be killed and wounded in treatment.

Therefore the same with radiotherapy, the selectivity of chemotherapy is also incomplete, also can destroy a lot of normal cells, and because the splitting status of the drug resistance of cell and/or cell and can not kill and wound all tumor cells.Therefore since the chemotherapy and radiation utilization be the trickle sensitivity differences that exists between normal cell and the cancerous cell, make that their therapeutic index is very narrow.For any treatment of cancer pattern, little therapeutic index clearly is not desired characteristic.Therefore expectation overcomes the new cancer treatment method of these limitations.

Tumor cell cracking performance viral therapy a kind of new Therapeutic Method that comes to this.At first, attempt to utilize and to carry the genetically modified replication defective virus of cytotoxicity and carry out treatment of cancer.Yet their find the to transduce efficient of tumor cell is low, the insufficient and target tumor optionally fully not at the tumor mass internal diffusion.In order to overcome these limitations, perhaps virus is improved so that it can optionally duplicate in tumor cell, perhaps seek natural virus with tumour-specific.Therefore these tumor cracking performances virus have sends these viruses by local injection or system and optionally duplicates, spreads by tumor mass and the characteristic (Fig. 1) of killing tumor cell.

Although this class anti-cancer therapies of redetermination has certain advantage in early days in treatment, owing to exist a lot of limitations may limit them as cancer treatment drugs.Therefore need the new tumor cell cracking performance virus that can be used in treatment of cancer badly.

Summary of the invention

It is found that a kind of novel picornavirus (hereinafter being called Xi Nijia paddy virus (SVV)), their natural characteristic comprises: the ability of the tumor cell of some type of selective killing.As illustrated in following Example, SVV is the killing tumor cells frenulum optionally, has neural tropism (neurotropic) characteristic.In most of the cases, kill and wound 50% tumor cell and the required virus quantity of 50% normal cell (that is EC, ₅₀Value) differs 10000 times.Also obtain same result in the experiment in vivo, wherein, the tumour transplatation thing in the mice has optionally been removed.And experimental result shows that SVV does not have toxicity to normal cell in the body, in the complete mice of immunodeficiency or immunologic function, even systemic administration is up to 1 * 10 ¹⁴Vp/kg (carrier or virion number/kilogram) can not cause death yet, and not observe clinical symptoms.

SVV is 1 * 10 ⁸Can bring into play curative effect under the low like this dosage of vp/kg, and can reach 100000 very high therapeutic index.Effect is very strong, shows 100% ground a large amount of tumor of setting up in advance in mice thoroughly to be eradicated (embodiment 11).Under the situation without any auxiliary therapy, only just can reach this effect by simple systematicness injection.In addition, SVV injection mice at least 200 days, any clinical symptoms both do not occurred and had not occurred tumor recurrence yet after accepting injection.Can be with the paramount titre of SVV purification, and in allowing cell line (permissive cell line) can with 200000 virion number/cells produce.And then, for the cancer types of selecting, have considerable prospect aspect safety, the effective and new treatment line based on the viral therapy of SVV.In addition, SVV has the genome of little and easy operating, simply and fast biocycle and duplicating biology of being well known, therefore can stand modification.Allow to adopt the method that generates the SVV that modifies to make it to have new cell or tissue specificity tropism to these characteristics of small part, and then point to because original SVV separated strain and infecting the new tumor type of resistance based on the treatment of SVV.

Correspondingly, the invention provides isolating virus, it comprises and has and SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21,168, perhaps the length of any in these sequences continuous part that is at least 50 nucleotide has the nucleotide sequence of at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity, perhaps with these sequences in any length continuous part of being at least 10,15 or 20 nucleotide have 95% conforming nucleotide sequence.Isolating nucleic acid of the present invention can be RNA or DNA.

Various aspects of the present invention provide isolating nucleic acid, its comprise with literary composition in SVV nucleic acid SEQIDNO sequence in any continuous part have at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% conforming nucleotide sequence, wherein, the length of this continuous part for example is at least about 20,25,50,75,100,150,200,250,300,350,400,450,500,750,1000,1250,1500,2000 or 2500 nucleotide.This SVV nucleic acid SEQ ID NO sequence comprises for example SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21 and 168.

Various aspects of the present invention provide isolating protein or polypeptide, its comprise with literary composition in the aminoacid SEQ ID NO sequence of SVV in any continuous part have at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% conforming aminoacid sequence.Wherein, the length of this continuous part at least for example is about 5,6,7,8,9,10,15,20,30,40,50,60,70,80,90,100,125,150,175,200,225,250,275,300 or 350 aminoacid.These SVV aminoacid SEQ IDNO sequence comprises for example SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22 and 169.

On the other hand, the invention provides isolating nucleic acid, its length that comprises with SEQ ID NO:168 or SEQ ID NO:168 is at least 20,50, the continuous part of 100 or 200 nucleotide has at least 95%, 96%, 97%, 98% or 99% conforming nucleotide sequence, this isolating nucleic acid can comprise the specific part of SEQ ID NO:168, including, but not limited to: the untranslated region (UTR) of SVV5 ' end that is positioned at the 1-666 position nucleotide of SEQ ID NO:168, be positioned at the coded sequence of SVV polyprotein of the 667-7209 position nucleotide of SEQ ID NO:168, the SVV that is positioned at the 667th to 903 nucleotide of SEQ ID NO:168 leads the coded sequence of peptide, be positioned at the proteic coded sequence of SVV VP4 of the 904-1116 position nucleotide of SEQ ID NO:168, be positioned at the proteic coded sequence of SVVVP2 of the 1117-1968 position nucleotide of SEQ ID NO:168, be positioned at the proteic coded sequence of SVV VP3 of the 1969-2685 position nucleotide of SEQ ID NO:168, be positioned at the proteic coded sequence of SVV VP1 of the 2686-3477 position nucleotide of SEQ ID NO:168, be positioned at the proteic coded sequence of SVV 2A of the 3478-3504 position nucleotide of SEQ ID NO:168, be positioned at the proteic coded sequence of SVV 2B of the 3505-3888 position nucleotide of SEQ ID NO:168, be positioned at the proteic coded sequence of SVV 2C of the 3889-4854 position nucleotide of SEQ ID NO:168, be positioned at the proteic coded sequence of SVV 3A of the 4855-5124 position nucleotide of SEQ IDNO:168, be positioned at the proteic coded sequence of SVV 3B of the 5125-5190 position nucleotide of SEQID NO:168, be positioned at the proteic coded sequence of 5191-5823 position nucleotide SVV 3C of SEQ ID NO:168, be positioned at the proteic coded sequence of SVV 3D of 5824-7209 position nucleotide of SEQ ID NO:168 and the 3 ' UTR of SVV that is positioned at the 7210-7280 position nucleotide of SEQ ID NO:168.

On the one hand, the invention provides and use SVV 2A albumen, SVV to lead the method that peptide or other SVV albumen or its polypeptide portion are closed the protein translation of (shut off) host cell.On the one hand, such SVV albumen can be used for by close the translation of host protein in host cell internal interference or inhibition " cap binding protein complex ".

On the other hand, the invention provides the method for using SVV 2A albumen or other SVV albumen or its polypeptide portion to come scinderin or polypeptide.

On the one hand, the invention provides isolating nucleic acid, described isolating nucleic acid can be under high, medium strictness or low stringency condition with SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21,168 or with any length at least in these sequences be the continuous part hybridization of 50 nucleotide.

On the other hand, the present invention also provides carrier, described carrier contains and SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21,168, and perhaps the length of any in these sequences continuous part that is at least 50 nucleotide has at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% conforming nucleotide sequence.Carrier compositions can also comprise the proteic SEQ ID of coding SVV NO:168 nucleotide sequence zone.

The present invention also provides the isolating polypeptide by following nucleic acid coding, described nucleic acid and SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21,168, perhaps the length of any in these sequences continuous part that is at least 50 nucleotide has at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% concordance.The present invention also provides the isolating polypeptide by following nucleic acid coding, and described nucleic acid has at least 95%, 96%, 97%, 98% or 99% concordance with the proteic SEQID NO:168 of coding SVV nucleotide sequence zone.

On the one hand, the invention provides isolating polypeptide, described polypeptide comprise with SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,169 or these sequences in any length be at least 10 amino acid whose continuous parts and have at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% conforming aminoacid sequence.

On the other hand, the invention provides isolating polypeptide, described polypeptide comprises length with SEQ ID NO:169 and is at least 9,10,15,20 or 50 amino acid whose continuous parts and has at least 95%, 96%, 97%, 98% or 99% conforming aminoacid sequence.The continuous part of exemplary SEQ IDNO:169 comprises but not only is confined to: comprise the proteinic zone of SVV, for example be positioned at the peptide of leading of 1-79 position residue), be positioned at the VP4 of 80-150 position residue, be positioned at the VP2 of 151-434 position residue, be positioned at the VP3 of 435-673 position residue, be positioned at the VP1 of 674-937 position residue, be positioned at the 2A of 938-946 position residue, be positioned at the 2B of 947-1074 position residue, be positioned at the 2C of 1075-1396 position residue, be positioned at the 3A of 1397-1486 position residue, be positioned at the 3B of 1487-1508 position residue, be positioned at the 3C of 1509-1719 position residue, be positioned at the 3D of 1720-218 position residue.

On the other hand, the invention provides can specificity in conjunction with the isolated antibody of following polypeptide, described polypeptide comprises and SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,169, and perhaps the length of any in these sequences is at least 9,10,15 or 20 amino acid whose continuous parts and has at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% conforming aminoacid sequence.The antibody that generates can combine with any protein epitope or antigen of SEQ ID NO:2 or 169.And this antibody can be polyclonal antibody, monoclonal antibody or chimeric antibody.

On the one hand, the invention provides isolating SVV or and derived virus or its correlated virus.These viral genome sequences comprise with SEQ ID NO:1 or SEQ ID NO:168 and have at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% conforming sequence.

On the other hand, the invention provides isolating virus, it is whole identification marks and the nucleotide sequence of PTA-5343 that described virus has Unite States Standard culture collection center (ATCC) patent preserving number.Part virus of the present invention is pointed to the separated strain of PTA-5343 and the mutant of variant, homology strain, relevant strain, derived virus and PTA-5343; Or the sequence of the responsible SVV virus determined (wild strain and mutant the two) cancerous cell cracking performance characteristic modified and other the viral variant, homology strain, derived virus and the mutant that obtain.

The present invention also provides isolating SVV, and it has following characteristics: the single stranded RNA genome of about 7.5kb or about 7.3kb (positive-sense strand (+)); Diameter is about 27nm; Capsid contains at least three kinds of proximate protein that are respectively about 31kDa, 36kDa and 27kDa of molecular weight.Buoyant density in cesium chloride (CsCl) density gradient is about 1.34g/mL; With in tumor cell, have replication capacity.On the one hand, the capsid protein of 31kDa (VP1) can contain the sequence with SEQ IDNO:8, and perhaps the 674-937 position residue of SEQ ID NO:169 has at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% conforming aminoacid sequence; The capsid protein of 36kDa (VP2) can contain the NO:4 with SEQ ID, and perhaps the 151-434 position residue of SEQ ID NO:169 has at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% conforming aminoacid sequence; The capsid protein of 27kDa (VP3) can contain the IDNO:6 with SEQ, and perhaps the 435-673 position residue of SEQ ID NO:169 has at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% conforming aminoacid sequence.

On the other hand, the invention provides SVV derived virus or correlated virus, it has following characteristics: the ability of duplicating in tumor cell, tumor cell tropism and do not have lysis in normal cell.The SVV correlated virus comprises SVV sample picornavirus, comprises the separated strain that derives from following USDA: MN 88-36695, NC 88-23626, IA 89-47552, NJ90-10324, IL 92-48963, CA 131395, LA 1278, IL 66289, IL 94-9356, MN/GA 99-29256, MN 99197 and SC 363649.Do not have in following characteristic if SVV sample picornavirus is natural: the ability of duplicating in the tumor cell, tumor tropism and do not have lysis in normal cell can make it to obtain these characteristics by sudden change SVV sample picornavirus.Can be by relatively this class sudden change of sequential design of SVV sample picornavirus and SVV, and make and suddenly change in this SVV sample picornavirus, make its aminoacid sequence and SVV is in full accord or basically identical (in specific zone).On the other hand, virus can be duplicated in the tumor cell type with neuroendocrine characteristic.

On the other hand, the invention provides: contain the virus of the present invention of effective dose and pharmaceutically acceptable carrier pharmaceutical composition, contain virus of the present invention cell, contain the virolysis thing of virus antigen of the present invention and the virus antigen of the isolating and purification that obtains from virus of the present invention.

On the other hand, the invention provides the method for purification virus of the present invention, comprising: use virus infected cell; Reclaim cell lysate; Make cell lysate accept an at least density gradient centrifugation; With from gradient, separate this virus.

On the one hand, the invention provides the treatment method for cancer, comprise giving effective dose virus or its derived virus.In order to treat cancer, wherein, virus has following genome sequence, described sequence and SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21,168, or the part of SEQ ID NO:1 or SEQ ID NO:168 has at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% a conforming sequence.On the one hand, the invention provides the treatment method for cancer, it comprises: virus or its derived virus of giving effective dose.In order to treat cancer, wherein, this virus has following genome sequence, and described sequence and SEQ ID NO:1 have at least 95%, 96%, 97%, 98% or 99% concordance.This virus for example can be, SVV mutant, SVV sample picornavirus or Cardioviruses.SVV sample piconavirus for example can be, a kind of in the following separated strain: MN 88-36695, NC 88-23626, IA89-47752, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197 and SC 363649.SVV sample piconavirus can be wild type or mutant.

On the other hand, the invention provides the treatment method for cancer, comprise the virus with capsid protein coding region that gives effective dose, described capsid protein coding region is contained and is at least 75,100,200 or 500 nucleotide sequences with the 904-3477 position nucleotide of SEQ ID NO:3,5,7, SEQ ID NO:169 or their length and has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% conforming sequence.The present invention also provides the treatment method for cancer, comprise the virus that gives effective dose with capsid protein, described capsid protein comprises and SEQ ID NO:4,6,8, the 80-937 amino acids residue of SEQ ID NO:169, perhaps the length of above-mentioned sequence is at least 25,50 or 100 amino acid whose continuous parts and has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% conforming sequence.

On the one hand, the invention provides the method that suppresses cancer progression, comprise cancer cell is contacted with virus or its derived virus, wherein, virus or its derived virus are specifically in conjunction with cancerous cells (cancerous cell), wherein, this virus has the genome that contains following sequence, and described sequence and SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21 or 168 have at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% concordance.

On the other hand, the invention provides the method that suppresses cancer progression, comprise cancer cell is contacted with virus or its derived virus, wherein, virus or its derived virus infect cancerous cells specifically, wherein, this virus has the genome sequence that contains following sequence, and the continuous part that the length of described sequence and SEQ ID NO:168 or SEQ ID NO:168 is at least 50,100,200 or 500 nucleotide has at least 95%, 96%, 97%, 98% or 99% concordance.

On the other hand, the invention provides the method for killing and wounding cancer cell, comprise cancer cell is contacted with virus or its derived virus of effective dose, wherein, this virus has the genome sequence that contains following sequence, and described sequence and SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21 or 168 have at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% concordance.

On the other hand, the invention provides the method for killing and wounding cancer cell, comprise cancer cell is contacted with virus or its derived virus of effective dose, wherein, this virus has the genome sequence that contains following sequence, and the continuous part that the length of described sequence and SEQ ID NO:168 or SEQ ID NO:168 is at least 50,100,200 or 500 nucleotide has at least 95%, 96%, 97%, 98% or 99% concordance.

In these methods at cancer, this virus can be picornavirus.This picornavirus can be Cardioviruses (cardiovirus), equine rhinoviruses (erbovirus), aphtho virus (aphthovirus), ridge virus (kobuvirus), hepatovirus (heaptovirus), the lonely virus of secondary intestinal (parechovirus), prompt Shen virus (teschovirus), enterovirus (enterovirus), rhinovirus (rhinoviruss), SVV and SVV sample picornavirus.Cardioviruses is selected from vilyuisk people's encephalomyelitis virus, match Le Shi murine encephalomyelitis virus and encephalomyocarditis virus.SVV can be that the ATCC preserving number is the virus of PTA-5343, or contain the virus of following nucleotide sequence, described sequence and SEQ ID NO:1, or the continuous part that the length of SEQ ID NO:168 or these sequences is at least 50,100,200 or 500 nucleotide has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% concordance.This SVV sample picornavirus can be following virus, described virus contains the NO:168 with SEQ ID, or the length of this sequence continuous part sequence that is at least 50,100,200 or 500 nucleotide has and is at least 95%, 96%, 97%, 98% or 99% concordance.SVV sample picornavirus can be, for example, and from the separated strain of one of following virus: MN88-36695, NC 88-23626, IA 89-47752, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN99197 and SC 363649.SVV sample picornavirus can be wild type or saltant.

The present invention also provides the method for the cell that kills and wounds abnormality proliferation, comprises making virus of the present invention and this cells contacting.On the one hand, the cell of abnormality proliferation is a tumor cell; This method on the other hand, tumor cell is selected from: human small cell lung carcinoma, human retina blastoma, human neuroblastoma, people's medulloblastoma, mice neuroblastoma, Wei Ermushi tumor and people's nonsmall-cell lung cancer etc.

The present invention also provides the method for treatment experimenter's the tumor patient's condition (neoplastic condition), comprises the virus of the present invention that gives effective dose to mammal.On the one hand, tumor neogenetic thing situation is a neuroendocrine carcinoma; On the other hand, the experimenter is a mammal; On the other hand, the experimenter behaves.

The present invention also provides the method for producing virus of the present invention, comprising: this virus is reclaimed in cultivation with the cell of this viral infection with from cell or culture supernatant under the condition that allows virus replication.An aspect of this method, cell is the PER.C6 cell.An aspect of this method, cell is the H446 cell.Another aspect of this method, each cell can produce and surpass 200000 virions.

On the other hand, the invention provides the method that detects virus of the present invention, comprising: contain isolation of RNA the test substance of virus of the present invention from suspection; RNA corresponding at least 15 continuous nucleotides of SEQ ID NO:1 or SEQ ID NO:168 is carried out labelling; RNA and test substance that labelling is crossed are detected (probing); Detection is through the RNA and the combination that separates from the RNA of test substance of labelling, wherein in conjunction with the existence of indicator virus.On the other hand, the invention provides nucleic probe, it is corresponding at least 15 continuous nucleotides of SEQ ID NO:1 or SEQ ID NO:168 or its complementary series.

(a) the present invention also provides the method for making tumor cell cracking performance virus, and this method comprises: (a) SVV genome sequence and virus genome sequence to be measured are compared; (b) identify amino acid whose difference between the genome sequence encoded polypeptides of first SVV genome sequence encoded polypeptides and virus to be measured at least; (c) genome sequence of sudden change virus to be measured makes and to reduce 1 at least by the polypeptide of the group group sequential coding of virus to be measured and the amino acid whose difference between the SVV genome sequence encoded polypeptides; (d) the genome sequence transfection of the virus to be measured after will suddenling change is advanced in the tumor cell; Whether (e) measure tumor cell is infected by the genome sequence cracking performance ground of the virus to be measured after the sudden change.On the one hand, the aminoacid that virus to be measured is suddenlyd change is positioned at structural region, for example in the capsid coding region.On the other hand, the aminoacid that suddenlyd change of virus to be measured is arranged in non-structural region.

One side in the method for making tumor cell cracking performance virus, the SVV genome sequence is the SVV virus isolated strain of PTA-5343 available from the ATCC preserving number, or obtain the virus that contains following sequence, described sequence and SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21,168 or the concordance of its continuous part be at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.On the one hand, the SVV genome sequence is the SVV virus isolated strain of PTA-5343 available from the ATCC preserving number, or containing the virus of following sequence, the concordance that described sequence and SEQ ID NO:168 or its length are at least the continuous part of 50,100,200 or 500 nucleotide is at least 95%, 96%, 97%, 98% or 99%.This method on the other hand, the step of the virus genome sequence to be measured of suddenling change comprises that the cDNA to the genome sequence with virus to be measured suddenlys change.This method on the other hand, the step of the virus genome sequence to be measured of transfection sudden change comprises transfection RNA, wherein, this RNA produces from the cDNA with virus genome sequence to be measured of sudden change.

In the method for making tumor cell cracking performance virus on the other hand, this virus to be measured is picornavirus.This virus to be measured comprises prompt Shen virus, enterovirus, rhinovirus, Cardioviruses, equine rhinoviruses, aphtho virus, ridge virus, hepatovirus, the lonely virus of secondary intestinal or prompt Shen virus.On the other hand, this virus to be measured is Cardioviruses.On the other hand, this viral SVV sample picornavirus to be measured.SVV sample picornavirus can be for example from the virus of following separated strain: MN88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197 and SC 363649.On the other hand, the aminoacid difference of identifying in the method for making tumor cell cracking performance virus is the difference of the sequence of SVV capsid protein and viral capsid proteins to be measured.In the method for making tumor cell cracking performance virus on the other hand, the genome sequence of virus to be measured is selected from vilyuisk people's encephalomyelitis virus, plug Le Shi murine encephalomyelitis virus and encephalomyocarditis virus.On the other hand, virus genome sequence to be measured is selected from encephalomyocarditis virus.On the other hand, encephalomyocarditis virus, SVV sample picornavirus or other virus to be measured are selected from the separated strain that contains following nucleotide sequence, it is the SVV virus of PTA-5343 that described nucleotide sequence contains with the ATCC preserving number, the continuous part that perhaps SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21,168, or its length is at least 50,100,200 or 500 nucleotide has at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% conforming sequence.

In the method for making tumor cell cracking performance Cardioviruses on the other hand, virus to be measured with the aminoacid difference of SVV in the capsid protein zone of SVV, wherein, at SEQ ID NO:4,6,8, the 80-937 position residue of SEQ ID NO:169, the 80-150 position residue of SEQ ID NO:169, the comparison amino acid difference is different in the 151-434 position residue of SEQ ID NO:169, the 435-673 position residue of SEQ ID NO:169 or the 674-937 position residue of SEQ ID NO:169.

The present invention also provides the method for making the mutant virus of the cell type tropism with change, and this method comprises: (a) create the virus mutant library of containing multiple nucleotide sequence; (b) with this virus mutant library transfection to allowing in the cell, make it produce the various mutations precursor virus; (c) separate the various mutations precursor virus; (d) isolating various mutations precursor virus and nonpermissive cell are hatched; (e) reclaim the mutant virus that nonpermissive cell produces, preparation has the mutant virus of the tropism of change thus.On the other hand, this method can also comprise step: (f) hatch the mutant virus of recovery and (g) reclaim the mutant virus of nonpermissive cell generation in nonpermissive cell.On the other hand, this method also comprises repeatedly repeating step (f) and (g).On the other hand, this virus mutant library create from following parental array, described parental array comprise and SEQ IDNO:1,3,5,7,9,11,13,15,17,19,21,168 or the sequence of its continuous part;

Have in manufacturing aspect of method of cell type tropism of change, hatch in porous high flux platform, wherein this platform contains different nonpermissive cell types in each hole.Aspect this, this method can comprise also that this platform of screening contains the hole of the mutant virus of cell killing with evaluation.On the other hand, screen by the absorbance of analyzing each hole.

Have in manufacturing change the cell type tropism mutant virus method on the other hand, nonpermissive cell is a tumor cell.

Have in manufacturing change the cell type tropism mutant virus method on the other hand, the step of creating the virus mutant library comprises: the polynucleotide that have with the corresponding to sequence of part of parental array (i) are provided; (ii) these polynucleotide that suddenly change in order to generate multiple different mutant polynucleotide sequence; (iii) the polynucleotide of various mutations are connected in the carrier with the virus genome sequence except the contained virus genome sequence part of the polynucleotide of step (i), create the virus mutant library in view of the above.On the one hand, Bing Du genome sequence is from picornavirus.On the other hand, Bing Du genomic sequence contain with SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21,168 or the concordance of its continuous part be at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence.On the other hand, the genomic sequence of virus contains following sequence, and the concordance that the length of described sequence and SEQ ID NO:168 or this sequence is at least the continuous part of 50,100,200 or 500 nucleotide is at least 95%, 96%, 97%, 98% or 99%.On the one hand, contain the virus examination SVV sample picornavirus that concordance that length with SEQ ID NO:168 sequence or this sequence is at least the continuous part of 50,100,200 or 500 nucleotide is at least 95%, 96%, 97%, 98% or 99% sequence.On the other hand, create in the step in virus mutant library, carry out step sudden change (ii) in the polynucleotide by nucleotide is inserted at random.On the one hand, carry out the insertion at random of nucleotide by trinucleotide mutagenesis (TRIM).On the other hand, insert at least a portion coding epi-position label of the nucleotide in the polynucleotide.On the other hand, create in the step in virus mutant library, in the capsid coding region of polynucleotide, carry out step sudden change (ii).

The present invention also provides the method for making the mutant virus of the cell type tropism with change, this method comprises: the mutant nucleotide library of (a) creating virus, wherein this structure comprises: provide the polynucleotide sequence in coding viral capsid zone, these polynucleotide that suddenly change in order to generate multiple different mutant capsid coded polynucleotide sequence; Be connected in the carrier with the virus genome sequence except the capsid coding region with capsid coded polynucleotide, create this viral mutant polynucleotide sequence library in view of the above various mutations; (b) with the library transfection of mutant polynucleotide sequence to allowing in the cell, thereby produce the various mutations precursor virus; (c) separate the various mutations precursor virus; (d) isolating various mutations precursor virus and nonpermissive cell are hatched; (e) reclaim the mutant virus that nonpermissive cell produces, make the mutant virus of tropism in view of the above with change.On the other hand, this method can also comprise step: (f) hatch the mutant virus of recovery and (g) reclaim the mutant virus of nonpermissive cell generation in nonpermissive cell.On the other hand, this method also comprises repeatedly repeating step (f) and (g).On the other hand, create from following parental array in this virus mutant library, described parental array comprise and SEQ IDNO:1,3,5,7,9,11,13,15,17,19,21,168 or the sequence of its continuous part; On the other hand, by being inserted in the capsid coded polynucleotide at random, nucleotide suddenlys change.Insert at least a portion coding epi-position label of the nucleotide of capsid coded polynucleotide at random.On the other hand, carry out the insertion at random of nucleotide by TRIM.On the other hand, a plurality of different mutant capsid coded polynucleotide sequences contain 10 ⁸Or 10 ⁹Above different capsid coded polynucleotide sequence.This a plurality of mutant polynucleotide sequences library can be from for example Cardioviruses or SVV sample picornavirus.

On the one hand, the mutant SVV that makes the cell type tropism with change comprises: the cDNA library of (a) creating the SVV mutant; (b) generate corresponding SVV RNA by this SVV mutants cDNA library; (c) with SVV RNA transfection to allowing in the cell, to generate multiple SVV virus mutant; (d) separate various mutations body SVV; (e) isolating various mutations body SVV is hatched with non-permission tumor cell; (f) reclaim the mutant SVV that cracking ground has infected non-permission tumor cell, preparation has the mutant SVV of the tropism of change thus.On the other hand, this method also comprises step: (g) the mutant SVV and the nonpermissive cell that reclaim are hatched; (h) the mutant SVV of the non-permission tumor cell of recovery lytic infection.On the other hand, this method further comprises: repeating step (g) and (h) repeatedly.On the one hand, hatch in porous high flux platform, wherein this platform contains different nonpermissive cell types in each hole.On the other hand, this method comprises that also this platform of screening contains the hole of the mutant virus of cell killing with evaluation.On the other hand, screen by the absorbance of analyzing each hole.On the one hand, various mutations body SVV capsid polynucleotide sequence is contained in the cDNA library of this SVV mutant.On the other hand, this various mutations body SVV capsid polynucleotide sequence produces the random mutation from nucleotide.On the other hand, at least a portion of the nucleotide sequence that inserts at random coding epi-position label.On the other hand, carry out the insertion at random of nucleotide by TRIM.On the other hand, the generation of the cDNA library of SVV mutant is the SVV of PTA-5343 from the ATCC preserving number.On the other hand, the cDNA library of SVV mutant produces from the SVV that contains following sequence, described sequence and SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21,168, or the concordance that the length of above-mentioned sequence is at least the continuous part of 50,100 acid, 200 or 500 nucleotide is at least 99%, 95%, 90%, 85%, 80%, 75%, 70% or 65%.On the one hand, the cDNA library of SVV mutant produces from the SVV that contains following sequence, and the concordance that described sequence and SEQ ID NO:168 or its length are at least the continuous part of 50,100 acid, 200 or 500 nucleotide is at least 95%, 96%, 97%, 98% or 99%.On the other hand, non-permission tumor cell is a tumor cell line or from the isolating tumor cell type of patient (tumor cell-type).

The present invention also provides the method for making the mutant virus with tumor cell type tropism in vivo, and this method comprises: (a) create the virus mutant library of containing multiple nucleotide sequence; (b) with this virus mutant library transfection to allowing in the cell, thereby produce the various mutations precursor virus; (c) separate the various mutations precursor virus; (d) isolating multiple virus mutant is administered to the mammal that suffers from tumor, wherein, this mammal is not the natural host of the mutant virus of sudden change not; (e) be recovered in the virus of duplicating in the tumor, thereby make mutant virus in vivo with tumor cell type tropism.On the one hand, the step in establishment virus mutant library comprises: the polynucleotide that coding viral capsid proteins zone is provided; In order to generate a plurality of different mutant capsid coded polynucleotide sequences, these polynucleotide suddenly change; Be connected to contain with capsid coded polynucleotide and encode in the carrier of the virus genome sequence many thuja acids, create the virus mutant library in view of the above except capsid with this various mutations.On the other hand, the virolysis ground infected tumor cell that step (e) is reclaimed.Make in vivo have tumor cell type tropism mutant virus method on the other hand, tumor is allograft thing, syngeneic tumor, homologue tumor or transgenic tumor.On the other hand, mammal is a mice.

In all methods, virus can be picornavirus among the present invention.This picornavirus can be the virus of the genus under Cardioviruses, equine rhinoviruses, aphtho virus, ridge virus, hepatovirus, the lonely virus of secondary intestinal, prompt Shen virus, enterovirus, rhinovirus or the SVV.This virus can be Cardioviruses.This virus can be SVV sample picornavirus.This virus can be SVV.This SVV virus can be that the ATCC preserving number is the SVV of PTA-5343, the SVV that perhaps contains following sequence, described sequence and SEQ ID NO:1,3,6,7,9,11,13,15,17,19,21,168 or the concordance of the continuous part of these sequences be at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.In addition, Cardioviruses can be selected from vilyuisk people's encephalomyelitis virus, plug Le Shi murine encephalomyelitis virus and encephalomyocarditis virus.On the one hand, SVV sample picornavirus is selected from: MN 88-36695, and NC 88-23626, IA 89-47552, NJ90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197 and SC 363649.On the other hand, the arbitrary virus that is comprised among the present invention is selected from the separated strain with following sequence identity: with the ATCC preserving number be the SVV of PTA-5343, or SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21,168 and the continuous part of above-mentioned sequence, or other are considered to be at least 99%, 95%, 90%, 85%, 80%, 75%, 70% or 65% because of the concordance of the sequence homology separated strain relevant with SVV.

On the other hand, arbitrary virus that is comprised among the present invention and ATCC preserving number are the SVV of PTA-5343; SEQ ID NO:168; Or SEQ ID NO:1, the sequence identity that 168 length is at least the continuous part of 100,200,300,400,500,750,1000,1500 or 2000 nucleotide is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.

The present invention also provides the tumor cell cracking performance virus for preparing by any method of making virus mutant disclosed herein.On the one hand, the invention provides method with tumor cell cracking performance viral therapy tumor patient, this method comprises: (a) will be by the prepared tumor cell cracking performance inactivation of virus of any manufacture method disclosed herein, and it is noninfective viral that tropism was lost efficacy with this tumor cell cracking performance to make tumor cell cracking performance virus become; (b) patient that tormented by tumor.On the other hand, this treatment patient's method also comprises toxin is connected on the virus of deactivation.

On the other hand, the invention provides the method with SVV treatment tumor patient, this method comprises: (a) with the SVV inactivation of virus, it is noninfective viral that tropism was lost efficacy with this tumor cell cracking performance to make SVV become; (b) inactivation is got the patient that SVV is tormented by tumor.On the other hand, the method for this treatment tumor patient also comprises toxin is connected on the SVV of deactivation.

On the other hand, provided by the present invention contain deactivation SVV or the SVV attenuation gets the SVV compositions.On the other hand, the invention provides to contain and be inserted into the SVV that the capsid zone must comprise epitope peptide.

The present invention also provides the method with SVV treatment tumor patient, and this method comprises: (a) create mutant SVV, it contains the epi-position label of coding at capsid; (b) toxin is connected on this epi-position label; (c) the mutant SVV that this is connected with toxin be subjected to tumor torment the patient.On the one hand, this establishment comprises: the polynucleotide of the epi-position of will encoding label are inserted in the capsid coding region Polynucleotide sequence of SVV.On the one hand, the cell type tropism enzyme of mutant SVV is not changed.On the other hand, this method also comprises deactivation mutant SVV, makes it not have infectivity or reproducible not.

The present invention also puies forward the method that has been total to the tumor cell in the test sample, comprising: (a) separate tumor sample from the patient; (b) with tumor sample and epi-position label SVV hatch; (c) by detecting the tumor cell of epi-position label filtration in conjunction with SVV.

On the one hand, the present invention also provides the method that detects tumor cell in vivo, comprising: (a) give the SVV of the epi-position label of patient's deactivation, wherein, put together labelling on the epi-position label; (b) detection patient's labelling.In the method kind of detection tumor cell of the present invention, this SVV can be the mutant SVV that generates by method disclosed herein.

On the one hand, the invention provides the method for the tumor cell in the test sample, comprising: (a) from experimenter's isolated cell sample; (b) cell sample and SVV (or SVV sample picornavirus) are hatched; (c) cell and the SVV specific antibody (or SVV sample picornavirus specific antibody) that are recovered in the step (b) are hatched; (d) cell sample of screening binding antibody, wherein, binding antibody indicates this sample to contain tumor cell.

On the one hand, the invention provides and measure the method whether experimenter is suitable for accepting the SVV treatment, comprising: (a) from experimenter's isolated cell sample; (b) cell and SVV are hatched; (c) cell of step (b) and the antibody incubation of anti-SVV; (d) detect on the cell or the existence of the antibody of anti-SVV in the cell, wherein, positive detection indication experimenter is suitable for accepting the SVV treatment.

The cell sample of screening binding antibody or the existence that detects anti-SVV antibody can be carried out in conjunction with the constant region of anti-SVV antibody or the second antibody of non-epi-position calmodulin binding domain CaM by adding.Wherein, second antibody is puted together or labelling with detectable label.This detectable label can be for example fluorescent chromophore, for example fluorescein.After second antibody was with detectable label labelling, this detectable label can be detected, for example detected by fluorescence microscope.Can be from experimenter's cell from the biopsy of experimenter's tissue.The biopsy of tissue can be taken from experimenter's tumor or the experimenter's that suspection contains tumor cell zone.Directly can also be used for the evaluation of tumor cell with the SVV of fluorescent chromophore labelling.

In addition, the method with the producing SVV treatment tumor patient's condition, that detect the tumor patient's condition is applied to wild type SVV Strain, mutant (comprise modification or mutant) SVV, SVV correlated virus, SVV sample picornavirus and other tumour-specific virus of the present invention.

Virus of the present invention and its compositions can be used to treat the medicine of the disease that reaches mentioned herein.In addition, virus of the present invention and its compositions can be used to treat the disease that reaches mentioned herein.Therefore, an aspect of of the present present invention provides SVV (or mutant virus, derived virus, correlated virus or its compositions) purposes in oncotherapy, perhaps is used to prepare the purposes of the medicine that is used for treating cancer in preparation.

The SVV and the SVV sample virus that are used for gene therapy: the replication defective SVV that expresses genes of interest can be used for delivery of gene to correct genopathy.SVV or SVV sample virus equally also can be used as the delivery vector of siRNA to stop the expression of any specific gene.Replication defective virus can be in complementary cell system and/or is grown in the presence of the helper virus of the function that its disappearance is provided for recombinant virus.

The IRES of known picornavirus plays important effect in the gene expression in the tissue specificity mode.The IRES element of SVV and SVV sample virus can be used to replace the IRES element of other picornaviruses.This strategy can be used to generate the virus of the tumor tropism with change.On the one hand, the invention provides SVV or, its objective is in tissue-specific mode and express two genes under the promoter from the IRES element of SVV correlated virus.

The characteristic of oneself's cutting of the 2A protease of SVV can be used to use a promoter and transcription stop signals sequence to express more than one gene equally.On the one hand, the invention provides the SVV with self-cutting function or the 2A peptide of SVV correlated virus, its objective is under the regulation and control of a promoter and a poly (A) signal and can express two or more albumen equally.On the other hand, the present invention also provides and has utilized SVV or SVV correlated virus 3C protease to cut polypeptide to carry out eukaryotic proteinic production.On the other hand, the present invention also provides and has utilized the peptide of leading of SVV or SVV sample virus to close protein synthesis in tumor cell or other purpose cell.

The virus-like particle of SVV can be produced or is used as vaccine and identified specific cell type in blended cell colony.

Preservation information

Following material all is stored in Unite States Standard and cultivates preservation center (ATCC), 10801University Blvd., and Manassas, Virginia, 20110-2209, U.S.A., the microorganism preservation budapest treaty that is used for proprietary program according to international endorsement carries out.Be given the ratification in this patent application, all cancelled unconditional all about the restrictive clause that obtains this preservation material.Associated materials comprises: Xi Nijia paddy virus (SVV, ATCC preserving number PTA-5343).Preservation date: on July 25th, 2003.

The accompanying drawing summary

Fig. 1 is the sketch map that carries out viral therapy with tumor cell cracking performance virus.Send viral mode by local injection virus or system, virus is delivered in the tumor mass, virus has optionally duplicates, sends out the also characteristic of killing tumor cell in tumor tissues.

Fig. 2 shows is that the SVV that obtains behind the purification dyes with uranium acetate (uranyl acetate) and uses transmissioning electric mirror checking.The diameter of spherical virus particle is about 27nm.

Fig. 3 shows is Electronic Speculum picture after SVV infects the PER.C6 cell.What show among the figure is crystalline inclusion and big vesicular body.

Fig. 4 A is the analysis to SVV RNA.With guanidinium isothiocyanate and use Trizol (available from Invitrogen company, Carlsbad, phenol extraction method CA) is extracted the geneome RNA of SVV.In 1.25% degeneration agarose gel electrophoresis, RNA is analyzed, with seeing and imaging after EB (EtBr) dyeing.Can observe the remarkable band of the geneome RNA of SVV in No. 2 swimming lane, the genomic size of indication total length SVV is about about 7.5kb.

Fig. 4 B shows is to comprise the genome structure of picornavirus of SVV and the sketch map of the protein product that generates by polyprotein processing.

What Fig. 5 A-5E showed is nucleotide sequence (SEQ ID NO:1) and the amino acid sequence coded (SEQ ID NO:2) thereof of SVV.The termination codon of 5671-3 position is represented with " * ".Generally speaking, if the nucleotide of a certain exact position still can not be determined then be expressed as " n " in the sequence of announcing.If therefore contain " n " in the codon, then corresponding aminoacid then is expressed as " x ".

What Fig. 6 A-6D showed is the nucleotide sequence (SEQ IDNO:1) of most of SVV genome total lengths.Nucleotide sequence is derived from TCC preserving number: PTA-5343, date saved: the SVV separated strain on July 25th, 2003.

Fig. 7 A-7B show by SEQ ID NO:1 amino acid sequence coded (SEQ ID NO:2).

What Fig. 8 showed is the nucleotide sequence (SEQ ID NO:3) of SVV part 1B or VP2 coding region.This sequence is consistent with the 429th nucleotide of 4-in the SEQ ID NO:1 sequence.

Fig. 9 shows is the proteic aminoacid sequence of VP2 (SEQ ID NO:4) by the part SVV of SEQ ID NO:3 coding.The aminoacid of the 143rd of 2-is consistent in listed SEQ ID NO:4 sequence and the SEQ ID NO:2 sequence.

What Figure 10 showed is the 1C of SVV or the nucleotide sequence of VP3 protein-coding region (SEQ IDNO:5).This sequence is consistent with the 430th-1146 nucleic acid among the SEQ ID NO:1.

What Figure 11 showed is the proteic aminoacid sequence of SVV VP3 (SEQ ID NO:6) of being encoded by SEQ ID NO:5.This sequence is consistent with the sequence of the 144th the-the 382nd amino acids in the SEQ ID NO:2 sequence.

What Figure 12 showed is 1D or the proteic nucleotide sequence of VP1 (SEQ IDNO:7) of coding SVV.The 1147th the-the 1923rd consensus nucleic acid sequence among this sequence and the SEQ ID NO:1.

What Figure 13 showed is the proteic aminoacid sequence of SVV VP1 (SEQ ID NO:8) of being encoded by SEQ ID NO:7.The sequence of listed SEQ ID NO:8 is consistent with the sequence of the 383rd the-the 641st amino acids among the SEQ ID NO:2.

What Figure 14 showed is the nucleotide sequence (SEQ ID NO:9) of SVV2A protein-coding region.The 1924th the-the 1965th consensus nucleic acid sequence among this sequence and the SEQ ID NO:1.

What Figure 15 showed is the proteic aminoacid sequence of SVV2A (SEQ ID NO:10) of being encoded by SEQ ID NO:9.The sequence of listed SEQ ID NO:10 is consistent with the sequence of the 642nd the-the 655th amino acids among the SEQ ID NO:2.

What Figure 16 showed is the nucleotide sequence (SEQ ID NO:11) of SVV 2B protein-coding region.The 1966th the-the 2349th consensus nucleic acid sequence among this sequence and the SEQ ID NO:1.

What Figure 17 showed is the proteic aminoacid sequence of SVV2B (SEQ ID NO:12) of being encoded by SEQ ID NO:11.The sequence of listed SEQ ID NO:12 is consistent with the sequence of the 656th the-the 783rd amino acids among the SEQ ID NO:2.

What Figure 18 showed is the nucleotide sequence (SEQ ID NO:13) of SVV2C protein-coding region.The 2350th the-the 3315th consensus nucleic acid sequence among this sequence and the SEQ ID NO:1.

What Figure 19 showed is the proteic aminoacid sequence of SVV 2C (SEQ ID NO:14) of being encoded by SEQ ID NO:13.The sequence of listed SEQ ID NO:14 is consistent with the sequence of the 784th the-the 1105th amino acids among the SEQ ID NO:2.

What Figure 20 showed is the nucleotide sequence (SEQ ID NO:15) of SVV 3A protein-coding region.The 3316th the-the 3585th consensus nucleic acid sequence among this sequence and the SEQ ID NO:1.

What Figure 21 showed is the proteic aminoacid sequence of SVV 3A (SEQ ID NO:16) of being encoded by SEQ ID NO:15.The sequence of listed SEQ ID NO:16 is consistent with the sequence of the 1106th the-the 1195th amino acids among the SEQ ID NO:2.

What Figure 22 showed is the nucleotide sequence (SEQ ID NO:17) of SVV3B protein-coding region.The 3586th the-the 3651st consensus nucleic acid sequence among this sequence and the SEQ ID NO:1.

What Figure 23 showed is the proteic aminoacid sequence of SVV3B (SEQ ID NO:18) of being encoded by SEQ ID NO:17.The sequence of listed SEQ ID NO:18 is consistent with the sequence of the 1196th the-the 1217th amino acids among the SEQ ID NO:2.

What Figure 24 showed is the nucleotide sequence (SEQ ID NO:19) of SVV 3C protein-coding region.The 3652nd the-the 4284th consensus nucleic acid sequence among this sequence and the SEQ ID NO:1.

What Figure 25 showed is the proteic aminoacid sequence of SVV 3C (SEQ ID NO:20) of being encoded by SEQ ID NO:19.The sequence of listed SEQ ID NO:20 is consistent with the sequence of the 1218th the-the 1428th amino acids among the SEQ ID NO:2.

What Figure 26 showed is the coding proteic nucleotide sequence of SVV 3D (SEQ ID NO:21).The 4285th the-the 5673rd consensus nucleic acid sequence among this sequence and the SEQ ID NO:1.

What Figure 27 showed is the proteic aminoacid sequence of SV V3D (SEQ ID NO:22) of being encoded by SEQ ID NO:21.The sequence of listed SEQ ID NO:22 is consistent with the sequence of the 1429th the-the 1890th amino acids among the SEQ ID NO:2.

That Figure 28 A-28H shows is the result that SVV SEQ ID NO:2 and different cardiovirus members are carried out the aminoacid sequence comparison.Described Cardioviruses member is encephalomyocarditis virus (EMCV for example; The encephalomyocarditis virus class), plug Le Shi murine encephalomyocarditis virus (TMEV; The Theilovirus kind), rat TMEV sample virus (TLV; The Theilovirus kind) and Vilyuisk people's encephalomyelitis virus (VHEV; The Theilovirus kind).The characteristic sequence of different Cardioviruseses is as follows: SEQ ID NO:23 (EMCV-R), 24 (EMCV-PV21), 25 (EMCV-B), 26 (EMCV-Da), 27 (EMCV-Db), 28 (EMCV-PV2), 29 (EMCV-Mengo), 30 (TMEV/DA), 31 (TMEV/GDVII), 32 (TMEV/BeAn8386), 33 (TLV-NGS910) and 34 (VHEV/Siberia-55).

Location number is not corresponding to the numbering of listed sequence among Figure 28.What "/" represented is cleavage site, and generation final functional product in cutting back takes place at this place polyprotein, for example compares the 1st and the 157th in 1A (VP4) zone; In 1B (VP2) zone the 159th and the 428th; The 430th and the 668th of 1C (VP3); In 1D (VP1) zone the 670th and the 967th; In the 2A zone the 969th and the 1111st; In the 2B region the 1112nd and 1276; In the 2C zone the 1278th and 1609; In the 3A zone the 1611st and 1700; In the 3B zone the 1702nd and 1723; In the 3C zone the 1725th and 1946; In the 3D zone the 1948th and 2410.Comparison has also shown can be by potential conservative or the similarity between the virus sequence of standard sequence analysis programme mensuration.Use BioEdit 5.0.9 and Clustal W to generate this comparison.

Figure 29 has listed the final polypeptide product corresponding to the SVV of SEQ ID NO:2.The ATP that underlines some conservative motif: the 2A/2B " cutting " (NPGP, SEQ IDNO:111), the 2C that are marked with runic in conjunction with (GxxGxGKS/T (SEQ ID NO:112) and hyhyhyxxD), 3B (VPg)/RNA assembling residue (Y), 3C (pro) activate site residue (H, C, H); 3D (pol) motif (KDEL/IR (SEQ ID NO:113), PSG, YGDD (SEQ ID NO:114), FLKR (SEQ ID NO:115)).

What Figure 30 listed is the picornavirus kind, and it is used for carrying out sequence analysis to determine the race relation (phylogeneticrelationship) (referring to the embodiment 4 of part i) of SVV and these picornaviruses with SEQ ID NO:1 and 2.

Analysis SVV (SEQ ID NO:4) that Figure 31 shows and race (phylogenetic relationship) relation of other picornavirus aspect the VP2 sequence.Figure has shown the crosslinked tree in ortho position (Bootstraped-joining tree) (referring to the embodiment 4 of part i) of guiding expansion.

Figure 32 has shown the crosslinked tree in ortho position (referring to the embodiment 4 of part i) of the proteic guiding expansion of the VP3 between SVV (SEQ ID NO:6) and other picornaviruses.

Figure 33 has shown the crosslinked tree in ortho position (referring to the embodiment 4 of part i) of the proteic guiding expansion of the VP1 between SVV (SEQ ID NO:8) and other picornaviruses.。

Figure 34 has shown the crosslinked tree in ortho position (referring to the embodiment 4 of part i) of the guiding expansion of the P1 albumen (for example 1A, 1B, 1C and 1D) between SVV (for example among part P1 albumen-SEQ ID NO:2 the 2nd to the 641st amino acids) and other picornaviruses.

Figure 35 has shown the crosslinked tree in ortho position (referring to the embodiment 4 of part i) of the proteic guiding expansion of the 2C between SVV (SEQ ID NO:14) and other picornaviruses.

Figure 36 has shown the crosslinked tree in ortho position (referring to the embodiment 4 of part i) of the proteic guiding expansion of the 3C (pro) between SVV (SEQ ID NO:20) and other picornaviruses.

Figure 37 has shown the crosslinked tree in ortho position (referring to the embodiment 4 of part i) of the proteic guiding expansion of the 3D (po l) between SVV (SEQ ID NO:22) and other picornaviruses.

Figure 38 shows is the amino acid whose actual percentage of the proteic concordance of VP2 (referring to the embodiment 4 of part i) between SVV (SEQ ID NO:4) and other picornaviruses.

Figure 39 shows is the amino acid whose actual percentage of concordance (referring to the embodiment 4 of part i) in the VP3 albumen between SVV (SEQ ID NO:6) and other picornaviruses.

Figure 40 shows is the amino acid whose actual percentage of the proteic concordance of VP1 (referring to the embodiment 4 of part i) between SVV (SEQ ID NO:8) and other picornaviruses.

Figure 41 shows is SVV (part capsid or part P1 (among the SEQ ID NO:2 the 2nd to the 641st amino acids)) and other picornaviruses between the amino acid whose actual percentage of the proteic concordance of P1 (referring to the embodiment 4 of part i).

Figure 42 shows is the amino acid whose actual percentage of the proteic concordance of 2C (referring to the embodiment 4 of part i) between SVV (SEQ ID NO:14) and other picornaviruses.

What Figure 43 showed is the amino acid whose actual percentage of the proteic concordance of 3C (referring to the embodiment 4 of part i) of SVV (SEQ ID NO:20) and other picornaviruses.

What Figure 44 showed is the amino acid whose actual percentage of the proteic concordance of 3D (pol) (referring to the embodiment 4 of part i) of SVV (SEQ ID NO:22) and other picornaviruses.

Figure 45 analyzes VP2 albumen (about 36kDa), VP1 albumen (about 31kDa) and the VP3 albumen (about 27kDa) of SVV with the SDS-PAGE electrophoresis.The SVV of purification is carried out SDS-PAGE, and dye with silver and to make albumen as seen." MWt " swimming lane is a molecular weight marker; The swimming lane of " SVV " contains the structural protein of SVV.Give the size of three kinds of molecular weight markers and the proteic title of virus.

Figure 46 A-46B shows is the SVV amount (embodiment 7) in back blood and the tumor tissues of being administered systemically.Handle the nude mice that lotus is had the H446 tumor with SVV, dosage is 1 * 10 ¹²Vp/kg passes through tail vein injection.After injection, got blood to mice in 0,1,3,6,24,48,72 hour and the 7th day.Isolate blood plasma immediately the blood after collecting and be diluted to and infect in the culture medium, be used to infect the PER.C6 cell subsequently.After injection, gathered tumor in the 6th, 24,48,72 hour and the 7th day, tumor is cut into small pieces and is resuspended in the 1ml culture medium make CVL.

What Figure 46 C-46D showed is the related data that SVV removes in vivo.Show among the figure, compare that SVV shows the residence time in the longer in fact blood (embodiment 7), so SVV has than clearance rate in the lower body of adenovirus with the adenovirus of the similar dosage of intravenous injection.Single dose carries out intravenous injection (i.v.), and SVV can keep up to 6 hours (as Figure 46 C, Figure 46 C is the copy of 46A, and purpose is in order to compare with 46D) in blood; And adenovirus just is eliminated in one hour in blood or approach exhaustion (Figure 46 D).

That Figure 47 shows is H446 allograft thing section carrying out SABC and hematoxylin-eosin (H﹠amp; E) dyeing (embodiment 7).(dosage is 1 * 10 to lotus nude mice tail vein injection Hank ' the s balanced salt solution (HBSS) of H446 or SVV ¹²Vp/kg), the injection back was put to death mice on the 3rd day and was collected tumor tissues.By adopting the method for SABC, with the virus protein (last figure) in the specific mouse antibodies tumor cells showed of SVV.In the mice body of HBSS or SVV processing, collect tumor cell H446, use H﹠amp; The E observation of cell form (figure below) that dyes.

What Figure 48 showed is the cytotoxicity (embodiment 9) of SVV to human primary hepatocyte.Human primary hepatocyte is inoculated in 12 orifice plates of glue primordial covering, infects with SVV, dosage is 1,10,100 and 1000 granule/hole (ppc).Infect the back and detected the lactic acid dehydrogenase (LDH) of cell and the LDH content in the culture supernatant on the 3rd day respectively.Cytotoxicity is with the statement of the form of ratio, with LDH unit quantity in the culture supernatant as molecule, and in the cell maximum sum of the LDH in LDH and the supernatant as denominator.

What Figure 49 showed is the viral yield of SVV in the cell line of selecting.In order to estimate the replication capacity of SVV, infect selected normal cell and tumor cell (embodiment 9) according to 1 virion/hole (ppc) with SVV.After 72 hours, collecting cell and analysis CVL are to the titre of PER.C6 cell.For every kind of cell line, the efficient that SVV duplicates is explained with " every milliliter colony forming unit (pfu/ml) ".

Figure 50 shows is toxic action (embodiment 10) according to the nude mice and the CD1 mice of body weight.After giving virus, do not measure the average weight of each processed group mice on the same day the time.Mice was at first day SVV or PBS by the tail vein injection single dose.

Figure 51 shows is effect to H446 allograft object model.In nude mice, set up among the H446 and flow, and with mice group (n=10), and the SVV (embodiment 11) by tail vein injection HBSS or 6 various dose.The 20th day of research, HBSS group have 5 lotuses that bigger tumor is arranged (mean tumour volume is 2000mm ³) mice with 1 * 10 ¹¹Vp/kg injects (shown in the arrow).Data are expressed as mean tumour volume+standard deviation (SD).

Figure 52 shows is to accept the SVV treatment or the photo (embodiment 11) of the nude mice with the swollen thing of H446 allograft of not receiving treatment.SVV is very potent, shows thorough 100% tumor of setting up in advance of having eradicated.The mice that SVV handles does not show incumbent what clinical symptoms in after injection at least 200 days, does not have tumor recurrence yet.

What Figure 53 showed is that SVV is at external tumour-specific and the related data of curative effect (embodiment 11).Pictorialization is H446 human small cell lung carcinoma (SCLC) tumor cell (chart top) and the cell survival rate of H460 human normal cell line (chart below) after hatching with SVV.SVV is the kill tumor cell specifically, its EC ₅₀Be approximately 10 ^-3Individual virion/hole.Corresponding, SVV can not kill and wound normal cell under any concentration.

What Figure 54 described is the plasmid (embodiment 15) that contains SVV virus complete genome group sequence.Thereby the existence in ground, SVV sequence upstream T7 promoter allows the SVV sequence to generate SVV RNA molecule in vitro transcription.

What Figure 55 described is the product sketch map (embodiment 16) that makes up SVV total length or functioning gene group SVV plasmid and SVV virus subsequently.In order to obtain functioning gene group SVV clone, the complete genome group of SVV can be cloned in the carrier that has the T7 promoter.Realize cDNA clone that this point can be by preparing virus and it checked order, subsequently with the seriality fragment cloning in a plasmid, the plasmid called after " pSVV " that obtains.Have the plasmid of SVV full-length gene group can be subsequently by reverse transcription to generate SVV RNA.Then in the transfected mammalian cell that advance to allow of this SVV RNA, and can reclaim the SVV virion subsequently and carry out purification.

What Figure 56 described is the sketch map (embodiment 16) that makes up the carrier (pSVV-capsid) that contains SVV capsid protein coded sequence (the 1A-1D zone of for example encoding).PSVV-capsid carrier can be used to generate SVV capsid mutant library subsequently.

Thereby what Figure 57 showed is the method (embodiment 16) that sudden change SVV capsid generates SVV capsid mutant library.This chart is illustrated the site at random that oligonucleotide inserts plasmid.Oligonucleotide can be oligonucleotide at random, the oligonucleotide of known array, or the oligonucleotide of coding epi-position label.In the drawings, Restriction Enzyme CviJI cuts pSVV capsid DNA at random.From gel separation and the linearizing pSVV capsid DNA that is purified in the generation cutting of single site, it is connected with oligonucleotide.

What Figure 58 showed is the flow process (embodiment 16) that generates the SVV mutant library that contains the sudden change of capsid protein coding region sequence.For example from pSVV mutant library (for example according to the flow process described in Figure 57), separate the coding region that obtains capsid protein with gel-purified by restriction enzyme digestion.Digestion cuts out the capsid coding region thereby the carrier that contains the SVV full length sequence equally also carries out enzyme action.To be connected with the pSVV carrier of the wild type capsid sequence that has lacked it from the capsid coding region of pSVV mutant library then, contain total length SVV mutant (called after " pSVVFL " carrier) library thereby create, various mutations is contained in the capsid coding region in this library.

Figure 59 has introduced the conventional method (embodiment 16) of manufacturing the SVV virion that contains the capsid mutant.Thereby the pSVVFL carrier is carried out reverse transcription generate SVV RNA.SVV RNA transfection is advanced to allow in the cell, wherein produce SVV mutant virus granule.This virion cell lysis separates the SVV virion colony of containing multiple capsid variant (SVV capsid library) then.

Figure 60 shows is that screening can the specific infection tumor cell and the conventional method that do not infect the SVV capsid mutant of non-tumor cell.SVV capsid library and tumor cell or interested tissue are hatched.After hatching for the first time, clean cell to remove the SVV virion that those fail to enter cell.Subsequently cell is continued to cultivate up to observing viral cracking performance.Collect the SVV capsid mutant of culture supernatant then with separation energy cracking performance ground infected tumor cell.(counter-screen) is preceding oppositely screening, and the known permission cell line of these viral infection is made its growth.Oppositely the process of screening is as follows: the SVV capsid mutant virus and the normal cell of energy infected tumor cell are hatched.Reclaim residual uncombined virus in the culture supernatant, recovery has the mutant SVV virus of tumour-specific thus.Can repeat this process to be further purified the SVV of isolating tumour-specific.

Figure 61 introduced detect virus mutant whether in conjunction with and/or the traditional method of infection cell system.Traditional detection method is the strain efficient deficiency that seems for auxocyte, and for example culture bottle is cultivated.This makes a large amount of screenings in the virus mutant library relate to multiple different cell strain become loaded down with trivial details.

Figure 62 has shown the high throughput method of the present invention (embodiment 16) of the virus mutant that is used to screen the ability with the different cell lines of specific infection.In the figure, a large amount of different tumor cell lines are grown in 384 orifice plates.In every hole, add Virus Sample (for example sample in SVV capsid library).From those demonstrate the hole of cytopathic effect, collect culture medium, allow cell line (for example H446 for SVV or PER.C6) any virus in described culture medium that can increase by in culture bottle or big tissue culture's ware, infecting.Viral growth, thus can isolation of RNA and carry out sequencing analysis, to determine inserting the encoded polypeptides sequence that mutagenesis inserts by the oligonucleotide of capsid.Subsequently can to this peptide before itself detecting specified it whether combine specifically with tumor cell type.

What Figure 63 showed is the sketch map (embodiment 16) of another high-throughput screening method.Tumor cell or normal cell are grown in porous plate.Virus joined detect cell in each hole and whether can be killed and wounded by the cracking of virus-mediated property.By read in each hole absorbance value can analyze rapidly by the pair cell pathologic effect.From the viral growth in the hole that demonstrates cytopathic effect, and further in external (tumor cell and Normocellular detection again) and body inner model (detecting the viral graft that whether can kill and wound in the mice), detect.

Figure 64 has shown can analyze the SVV capsid mutant (order according to occurring is respectively SEQ ID NO:45-48) with new tumour-specific tropism, to generate the tumor-selective peptide.These SVV capsid mutants of the ability of infected tumor's cell line check order to having specifically, thereby determine the peptide of the polynucleotide encoding of insertion.Can determine conforming aminoacid sequence from this successful capsid mutant.Subsequently the peptide with consensus sequence is detected, whether can be to determine them specifically in conjunction with paid close attention to ground tumor cell type.Can subsequently this tumor-selective peptide section be connected on toxin or the medicine, to be used as the tumour-specific targeting vector.

Figure 65 introduces at first detecting SVV capsid library in vivo.Transplant specific tumor for mice (comprising normal mouse, athymic mouse, nude mice, CD-1 transgenic mouse etc.), give these injected in mice SVV derived virus library, for example SVV capsid library subsequently.At point sometime, reclaim tumor cell from mice, in these mices, show as tumor and removed fully.From the initial tumor sample, separate obtaining virion, and in allowing cell strain, cultivate.

Figure 66 has shown the Clinical detection flow process to SVV derived virus of the present invention.

Figure 67 is to the various application of the SVV virus derived virus (having new tumor tropism) of those epi-position labels of encoding in capsid.They can be used as screening reagent, by the existence of detection epi-position label, thereby check whether there is specific tumor cell in tissue sample.Perhaps, toxin or other treatment medicine are linked to each other with this epi-position, giving the patient subsequently should virus.Further, SVV wild type or its derived virus are opened by irradiation or deactivation, thereby this virion itself is as therapy equipment (therapeutic device).Owing to contain apoptosis-induced peptide, virion so cell death inducing; Or this granule can connect toxin or some other medicine, thus should virus as specificity to delivery apparatus.

What Figure 68 showed is the basic biocycle of picornavirus.

Figure 69 compares the polypeptide length of SVV and other picornavirus.

The aminoacid that Figure 70 has listed picornavirus 2A sample NPG/P albumen (order according to occurring is respectively SEQ ID NO:49-110) compares.This sequence is the 635th the-the 656th residue among the SEQID NO:2 of listed SVV.

Figure 71 list for the aminoacid sequence of EMCV-R (SEQ ID NO:23).

Figure 72 list for the aminoacid sequence of EMCV-PV21 (SEQ ID NO:24, registration number CAA52361).

Figure 73 list for the aminoacid sequence of EMCV-B (SEQ ID NO:25, registration number P17593).

Figure 74 list for the aminoacid sequence of EMCV-Da (SEQ ID NO:24, registration number P17594).

Figure 75 list for the aminoacid sequence of EMCV-Db (SEQ ID NO:27).

Figure 76 list for the aminoacid sequence of EMCV-PV2 (SEQ ID NO:28, registration number CAA60776).

Figure 77 list for the aminoacid sequence of EMCV-mengo (SEQ ID NO:29, registration number AAA46547).

Figure 78 list for the aminoacid sequence of TMEV/DA (SEQ ID NO:30, registration number AAA47928).

Figure 79 list for the aminoacid sequence of TMEV/GDVII (SEQ ID NO:31, registration number AAA47929).

Figure 80 list for the aminoacid sequence of TMEV/BeAn8386 (SEQ ID NO:32, registration number AAA47930).

Figure 81 list for the aminoacid sequence of TLV-NGS910 (SEQ ID NO:33, registration number BAC58035).

Figure 82 list for the aminoacid sequence of VHEV/Siberia-55 (SEQ ID NO:34, registration number AAA47931).

What Figure 83 A-83H showed is SVV full-length gene group sequence (SEQ ID NO:168) and encoded polypeptides aminoacid sequence (SEQ ID NO:169) thereof.SVV full-length gene group sequence source is from the SVV of ATCC separated strain (the ATCC preserving number is PTA-5343).The special characteristic of SVV genome sequence has for example been described the proteinic specific coding zone of cutting from polyprotein matter sequence herein.

That Figure 84 A-84D shows is SVV full-length gene group sequence (SEQ ID NO:168).This sequence source is from the SVV of ATCC separated strain (the ATCC preserving number is PTA-5343).

What Figure 85 A-85B showed is the aminoacid sequence (SEQ IDNO:169) of SVV total length polyprotein matter, and it is by the 667th to 7209 nucleotide coding among the SEQ ID NO:168.

Figure 86 carries out race relation or epidemiological analysis to the SVV full-length gene group and the polyprotein matter sequence that are derived from SEQ ID NO:168 and 169.SVV is a kind of exclusive virus, the close Cardioviruses that is similar to of race, but on the tree that separates.Because the high-caliber sequence identity between SVV and SVV sample Microrna, and because the antibody that is produced by SVV sample picornavirus has and SVV virus bonded ability (vice versa), therefore SVV sample picornavirus most possibly is positioned at identical tree with SVV or belongs to identical genus (referring to Figure 87-89).

Figure 87 A-87D has shown that SVV virus and the nucleotide sequence of some SVV sample picornavirus in the zone of coding P1 structural region and 2A compare.Particularly, compare at VP2 (part)-VP3-VP1-2A (part).Listed SVV sequence is SEQ ID NO:170; The sequence of listed isolating IA 89-47752 is SEQ ID NO:171; The sequence of listed isolating CA 131395 is SEQ ID NO:172; The sequence of listed isolating NC 88-23626 is SEQ ID NO:173; The sequence of listed isolated M N 88-36695 is SEQ IDNO:174; The sequence of listed isolating NJ90-10324 is SEQ ID NO:175; The sequence of listed isolating IL 92-48963 is SEQ ID NO:176; The sequence of listed isolating LA1278 (97-1278) is SEQ ID NO:177, and the consensus sequence of listing is SEQID NO:178.

Figure 88 carries out nucleotide sequence relatively with isolating IA 89-47752 and CA 131395 in 2C coding region (part) with SVV.Listed SVV sequence is SEQ ID NO:179; The sequence of listed isolating IA 89-47752 is SEQ ID NO:180; The sequence of listed isolating CA131395 is SEQ ID NO:181; And the consensus sequence of listing is SEQ IDNO:182.

Figure 89 A-89B carries out nucleotide sequence relatively with isolating NC 88-23626, MN 88-36695, IA89-47752, NJ 90-10324, IL 92-48963, LA 97-1278 and CA 131395 in 3D polymerase coding region (part) and 3 ' UTR zone with SVV.Listed sequence is: SVV sequence (SEQ ID NO:183), NC 88-23626 (SEQ ID NO:184), MN 88-36695 (SEQ ID NO:185), IA 89-47752 (SEQ ID NO:186), NJ90-10324 (SEQ ID NO:187), IL 92-48963 (SEQ ID NO:188), LA97-1278 (SEQ ID NO:189), CA 131395 (SEQ ID NO:190) and consensus sequence (SEQ ID NO:191).

The SVV of Figure 90 A-90E single dose can be reduced in the size of the tumor of transplanting in the mice body effectively and suppress its growth.Figure 90 A shows is the size of the SVV H446 people SCLC tumor that can reduce transplanting and suppresses its growth (ED ₅₀=0.0007).What Figure 90 B showed is that SVV can reduce the blastomatous size of Y79 human retina of transplanting and suppress its growth (ED ₅₀=0.0007).Figure 90 C shows is the size of SVV H69AR people SCLC-MDR (multi-drug resistance) tumor that can reduce transplanting and suppresses its growth (ED ₅₀=0.05).Figure 90 D shows is the size of the SVV H1299 people HSCLC tumor that can reduce transplanting and suppresses its growth (ED ₅₀=4.8).What Figure 90 E showed is that SVV can reduce the size of the NIE-115 mice neuroblastoma of transplanting and suppress its growth (ED in A/J mice (normal mouse that immunologic function is perfect) body ₅₀=0.001).

Figure 91 shows is the EMCV that compares of the sequence with SVV and the molecular model of TMEV capsid structure.The molecular model combination algorithm is used for the antigenicity prediction, and it is used to select to be suitable for producing the peptide sequence of polyclonal antibody.Brown part is represented the β sheet among the figure, and green portion is represented the α spiral, yellow representative be that the length that is used to generate polyclonal antibody is 12 amino acid whose peptides.The particular sequence of selecting (in the VP2 zone) is because it shows the exposure of good surface according to model.

What Figure 92 A-92D showed is the specificity of anti-SVV polyclonal antibody.The negative contrast of 92A is with the immunofluorescence picture that shows with nonspecific anti-mice serum and second antibody dyeing back behind the SVV infection cell.What 92B and 92C showed is with behind the SVV infection cell, the immunofluorescence imaging after dyeing with mouse anti SVV serum (1:50 dilution) and second antibody (fluorescein-labeled anti-mice Ig).Figure 92 D shows can be used to carry out the polyclone anti-SVV antibody of virus in conjunction with test.As shown in the figure, because before cell is placed on ice to stop SVV virus internalization, so immunofluorescence shows that SVV accumulates in the cell outline periphery.

The GP102 serum that Figure 93 shows is among the SVV and experimental result (seeing embodiment 18).In and titre (the maximum dilution meter of neutralization virus can be done 100%) be 1:100.

What Figure 94 showed is the neutralization test (see embodiment 18) of anti-SVV antiserum to MN88-36695.In and titre be 1:560.

What Figure 95 A and Figure 95 B were described is the ortho position cladogram.These trees are to make up with PHYLIP (being used to infer that the race of race relation infers the compressed package computer program, Phylogeny InferencePackage Computer Programs for Inferring Phylogenies) to form.And shown the mutual relation that SVV and 7 kinds of SVV sample picornaviruses obtain when carrying out the sequence comparison at P1 and part 2A district (Figure 95 A) and genomic 3 ' terminal (Figure 95 B).

Detailed Description Of The Invention

Term " virus ", " virion ", " virion ", " virion " can Alternate.

Term " virion " and " vector particles " can Alternates, and can be understood widely, for example refer to that it has formed and has infective virion. For example: viral vectors among the present invention transforms or is transfected into suitable cell or clone to produce infectious particles.

Term " derived virus ", " mutant strain ", " variant " and " modification " (modified) can Alternate. Derived virus, mutant strain, variant or modification can have nucleotide sequence or the amino acid sequence that there are differences with masterplate nucleic acid sequence or amino acid sequence. For example the SVV of SVV derived virus, mutant strain, variant or modified has and the nucleotide sequence or the amino acid sequence that there are differences with wild type SVV (the ATCC preserving number is PTA-5343).

" SVV sample picornavirus " described herein refers to that those and SVV are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% in the uniformity of nucleic acid level (referring to SEQ ID NO:168, SVV full-length gene group sequence among Figure 84 and Figure 83). Sequence relatively is not limited only to full genome analysis, also can concentrate on genomic specific region, for example 5 ' UTR, structural proteins coding region, non-structural protein coding region, 3 ' UTR or their part. For a person skilled in the art, the genome sequence that is used for sequence length-specific relatively enough is determined and correlation/possibility of SVV. And this enough length is also closely related with the uniformity percentage that exists. For example, the sequence length during the sequence comparison can be for example to be 20,50,100,200,300,400,500,750,1000,1500,2000 or 2500 nucleotides. Shorter such as infructescence, those skilled in the art can know, for example the uniformity between two sequences is very high, therefore can think that the two has correlation. Yet when considering sequence conservation at least, this instruction can be quantitative, because more conservative between relevant kind than other in genomic specific region. In addition, if produce from the antiserum of virus can in and SVV SVV is allowed the infection of cell, and this antiserum can also in conjunction with other viruses (for example this antiserum is used for indirect immunofluorescence and detects virus), can be SVV sample picornavirus by other viruses of this antiserum combination artificially then. According to purpose of the present invention, SVV virus can comprise Cardioviruses. The permission cell of SVV or clone are including, but not limited to Y79, NCI-H446, N1E-115, NCI-H1770, NCI-H82, PER.

NCI-H69AR, SK-NEP-1, IMR-32, NCI-H187, NCI-H209, HCC33, NCI-H1184, D283Med, SK-N-AS, BEK PCB3E1, ST, NCI-H1299, DMS153, NCI-H378, NCI-H295R, BEK, PPASMC, PCASMC, PAoSMC, NCI-H526, OVCAR-3, NCI-H207, ESK-4, SW-13,293, Hs 578T, HS 1.Tes and LOX IMVI.

When being used for herein, term " cancer ", " cancer cell ", " oncocyte (neoplastic cells) ", " knurl (neoplasmia) ", " tumour ", " tumour cell " can Alternates, refer to the cell of showing relative oneself's growth property, they have showed excrescent phenotype, it is characterized in that significantly losing the control of on cell proliferation. Tumour cell and can for pernicious also can be optimum. According to the present invention, preferred tumor cell type is the cell with characteristic of neural taxis (neurotropic).

" uniformity " or " uniformity " percentage of in the literary composition two or more nucleotide sequences or protein sequence, refer to when adopting for example Protein-Protein BLAST (the protein-protein BLAST comparison program (Altschul in the GenBank database of sequence comparison algorithm, S.F., Gish, W., Miller, W., Myers, E.W.﹠ Lipman, D.J. (1990) ＂ Basic local alignmenet search tool. ＂J.Mol.Biol.215:403-410))) or same visual inspection. Relatively or during the maximum uniformity of comparison, identical or identical amino acid residue or the nucleotides with particular percentile of two or more sequences (comprising subsequence), or use the range estimation detection technique to analyze. The BLAST logical algorithm in the article that Altschul etc. delivers, have detailed introduction (referring to Altschul et al,J.Mol. Biol..215:403-410 (1990)), BLAST software on the common web page of American National biotechnology information centre (NCBI), can obtain (http://www.ncbi.nlm.nih.gov/)。

For example, the term " uniformity is at least 90% " that is used for herein refers to compare with reference polypeptide sequence (or polynucleotides), and in fact uniformity refers to more than 90% between 90% to 100%. Lift a case in point, if with a length be that 100 amino acid whose polypeptide compare, if inconsistent amino acid number is no more than 10% (that is, 100 the insides have 10) in sequence to be measured and the reference sequences, then be expressed as more than 90%. Similar more also can carry out in sequence more to be measured with between with reference to polynucleotides. These difference amino acid both can be randomly dispersed in for the form of point mutation full length amino acid sequence among, also can for example have one or more positions of the variable-length of 10 differences (90% uniformity) to cluster in 100 amino acid up to maximum tolerance level. Difference can be defined as nucleic acid or amino acid whose replacement, insertion or disappearance. If consistency level is more than 85-90%, the result is independent of program and gap parameter group, and this type of high-caliber uniformity can be estimated by reading, and does not usually rely on software.

Concept " high stringency ", " medium stringency " and " low stringency " refer to the hybridization conditions of nucleic acid. High stringency refers to need to have very high conforming condition between target nucleic acid sequence and the probe nucleic acid sequence in order to make target and probe that annealing or hybridization occur; Low stringency refers to need to have low conforming condition between target nucleic acid sequence and the probe nucleic acid sequence in order to make target and probe that annealing or hybridization occur. The strict degree of hybridization can be controlled by the salinity in the buffer solution or the temperature of hybridizing. High salt concentration can reduce the strict degree of hybridization, and high temperature then can improve the strict degree of hybridization. Although the variation of stringency is depended on length and nucleic acid thereof that the regional inner nucleotide sequence of hybridization occurs and is formed, described below the representative of conditions of height, moderate or low stringency in the exemplary condition. Hybridization buffer commonly used is SSC (sodium chloride-trisodium citrate), usually be mixed with 20 * storage liquid (0.3M trisodium citrate, 3M sodium chloride). Under high stringency hybridization conditions, the working solution concentration of SSC can be 0.1 *-0.5 * (trisodium citrate of 1.5-7.5mM, the NaCl of 15-75mM), and hybridization temperature is set as 65 ℃; Under the moderate hybridization conditions, the working solution concentration of SSC is 0.5 *-2 * (trisodium citrate of 7.5-30mM, the sodium chloride of 75-300mM), and hybridization temperature is set as 55-62 ℃; The working concentration of SSC under the low hybridization conditions is 2 *-5 * (trisodium citrate of 30-75mM, the sodium chloride of 300-750mM), and hybridization temperature is set as 50-55 ℃. Note that these conditions that provide only are exemplary, do not think limitation.

Xi Nijia paddy virus (SVV):

SVV is a kind of in undiscovered novel RNA virus before. Compare with the characteristic picornavirus of previous discovery, the Cardioviruses in SVV and the picornaviridae is more closely related, (referring to international patent application: PCT/US2004/031594). The result of the sequence analysis between SVV and other Cardioviruses has been discussed in PCT/US2004/031594. In this article with its whole introducing in literary composition as a reference. Since in PCT/US2004/031594 SVV being carried out rising in the sequence analysis, Microrna research group is just discussed whether should listing SVV in picornaviridae as the newcomer. Figure 86 has shown the tree of the genetic affinity between the member in the picornaviridae.

To known picornavirus (referring to international patent application no: PCT/US2004/031504) carry out initial sequence relatively after, can obtain two kinds of races (phylogenetic classification) classification: (1) is included in cardiovirus with SVV as new genus; Or (2) appointment SVV is a new genus. During the application this patent, SVV has been designated as the newcomer of cardiovirus. But after further analyzing, find that SVV has several features different from Cardioviruses. For example: some Cardioviruses genome contains inside poly (C) tail (tract) that prolongs at 5 ' UTR, and SVV does not then contain poly (C). The two is except variant on 5 ' section sequence information, also the endogenous RES (IRES) of SVV has been carried out being figure and compares with other picornavirus. Find that from result relatively SVV IRES belongs to the IV type, and the IRES of Cardioviruses is the II type. Cardioviruses has long 2A protease (150 amino acid), and SVV has short 2A protease (9 amino acid). On the size of 2A albumen and other albumen (leading peptide, 3A albumen etc.), also there is very big-difference between SVV and the Cardioviruses. From to learning the research of other picornaviruses, these albumen have participated in and the interactional process of host cell, comprise taxis and virulence. Last owing to have some zones so different from Cardioviruses in the whole genome sequence of SVV, therefore think that SVV is the new picornavirus of a class. In addition, found the picornavirus of many strains uniqueness at USDA, compared with respect to other picornavirus with SVV that it is more similar to SVV. Therefore, ICTV (ICVT) thinks that according to the result of study of picornavirus seminar SVV is a new class picornavirus, and called after Xi Nijia paddy virus. But by up to the present, the viral generic that the picornavirus of the uniqueness of still finding at USDA for SVV or those picornavirus class of called after SVV sample (in the text we) is well defined.

Many SVV sample picornaviruses of finding at USDA and SVV have 95-98% in nucleic acid level homology (illustrating, referring to Figure 87-89). For in the antiserum energy of a kind of virus (MN 88-36695) and SVV, and should virus also to other can in and the serum of SVV react. SVV sample picornavirus derives from pig, so pig is likely the permission host of SVV and other SVV sample picornavirus. Isolated SVV sample virus comprises following several Strain (but being not limited to this) of separating from USDA: MN88-36695 from pig, NC 88-23626, and LA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; With SC 363649. SVV sample picornavirus also comprises the Cardioviruses very near with SVV (the cross reaction degree of the antibody of the result by sequence analysis and virus and anti-SVV antigen is determined). Therefore think that in the present invention SVV has following characteristics: (1) is very near or belong to the member of Cardioviruses with the cardiovirus of picornavirus family; (2) be newcomer in the picornavirus family, the SVV sample Microrna virus that comprises SVV among this newcomer and be not classified into the member of other genus.

The same with Cardioviruses, according to virus genomic compositing characteristic, common cause of disease attribute and separablely in the 0.1M NaCl solution of pH value between 5-7 go out the characteristic such as virion (referring to by " Cardioviruses (picornavirus) " these chapters and sections (Scraba in R.G.Webseter and A.Granoff and " virology encyclopedia " (second edition) write in 1999, D. wait and write)), SVV and other picornavirus can be made a distinction. The genome of SVV is comprised of strand justice RNA, and size is 7310 nucleotides, and contain one long be poly (A) tail (referring to Figure 83 A-83H, Figure 84 A-84D, SEQ ID NO:168) of 30 nucleotides. Because SVV belongs to picornavirus, it has the most conservative feature that all picornaviruses have: (i) geneome RNA has infectivity, can directly be used to transfection and advance in the cell and skip " virus receptor in conjunction with-enter the step of viral life cycle "; (ii) hold the non-translational region (UTR) that long (600-1200bp) arranged (for SVV genomic 5 ', be positioned at the 1-666 position of SEQID NO:168), 3 ' short non-translational region (is about 50-100bp, for SVV, be positioned at the 7210-7280 position of SEQ ID NO:168); (iii) 5 ' UTR contains the secondary structure of clover sample, and known its is internal ribosome entry site (IRES) (for example, being positioned at the 300-360 position of SEQ ID NO:168). Cardioviruses has II type IRES, and SVV has IV type IRES; (iv) the remaining genome sequence single polyprotein (for SVV, the 667-7209 position nucleotide coding polyprotein of SEQ ID NO:168 (SEQ ID NO:169)) of encoding; (v) the genome two ends are all modified, and 5 ' end is connected with a little basic protein " Vpg ", and 3 ' end is by polyadenylation (for SVV, being positioned at the 7281-7310 position of SEQ ID NO:168).

The invention provides SVV Strain (the ATCC preserving number is PTA-5343) and the SVV complete genome group information of separation. At first the large fragment in the SVV nucleic acid that separates is checked order, the result is shown in Fig. 5 A-5E and Fig. 6 A-6D. This SVV nucleic acid is to derive from PTA-5343 separated strain (containing most SVV genome sequences), is referred to herein as SEQ ID NO:1. The result of this nucleotide sequence translation shows that the major part of single polyprotein of SVV is coded by SEQ ID NO:1. All be listed among Fig. 5 A-E and Fig. 7 A-7B by the 1st polypeptid acid sequence to the 5673rd nucleotide coding among the SEQ ID NO:1, at this called after SEQ ID NO:2. Obtained since then SVV full-length gene group and it seems it almost is the sequence of full-length gene group, and it is listed among Figure 83 A-83H and the SEQ ID NO:168, the total length polyprotein of nucleic acid 667-7209 coding SVV, and the amino acid sequence of this polyprotein is listed among Figure 83 A-83H and the SEQ ID NO:169.

(or purifying) of the invention provides separation comprises SEQ ID NO:3, the part of 5,7,9,11,13,15,17,19 and 21 SEQ ID NO:1; And the part of the SEQ ID NO:168 that separates, comprising: the coding region (5125-5190) of the coding region (3889-4854) of the coding region (3478-3504) of the coding region (1969-2685) of the coding region (904-1116) of 5 ' UTR zone (1-666), the coding region (667-903) of leading peptide, VP4 albumen, the coding region (1117-1968) of VP2 albumen, VP3 albumen, the coding region (2686-3474) of VP1 albumen, 2A albumen, the zone (3505-3888) of 2B albumen, 2C albumen, the coding region (4855-5124) of coding 3A albumen, 3B albumen, the zone (5191-5823) of coding 3C albumen, the coding region (5824-7209) of 3D albumen and the zone (7210-7310) that coding comprises 3 ' UTR of poly (A) tail. The present invention also provides the nucleic acid of the separation of above-mentioned specificity part partly. The present invention also provides mutated viruses or the derived virus of the part of such separation. SEQ ID NO:1 can be subcloned in the expression vector with 168 the part of separating, thereby separates the polyprotein of these parts codings. In addition, the present invention also provide under height, moderate and low hybridization stringency condition can with SEQ ID NO:1 or 168 with and any part nucleotide sequence of hybridizing mutually. Following table has been listed the nucleic acid of the coding SEQ ID NO:168 of coding SVV albumen. The invention provides SVV albumen or its part of separation (or purifying). Also listed the amino acid sequence corresponding to the SVV albumen of the polyprotein sequence of in SEQ ID NO:169, listing in the table.

Table A: SVV genome and protein characteristic

 

The SVV feature	Location among the SEQ ID NO:168	Location among the SEQ ID NO:169
			5′UTR	1-666	N/A (nothing)
Lead peptide	667-903 (leading peptide-coding sequence)	1-79
			VP4	904-1116 (VP4 coded sequence)	80-150
VP2	1117-1968 (VP2 coded sequence)	151-434
			VP3	1969-2685 (VP3 coded sequence)	435-672
VP1	2686-3474 or 3477 (VP1 coded sequence)	674-936 or 937
			2A	3478-3504 (2A coded sequence)	938-946
2B	3505-3888 (2B coded sequence)	947-1074
			2C	3889-4854 (2C coded sequence)	1075-1396
3A	4855-5124 (3A coded sequence)	1397-1486
			3B	5125-5190 (3B coded sequence)	1487-1508

 

3C	5191-5823 (3C coded sequence)	1509-1719
			3D	5824-7209 (3D coded sequence)	1720-2181
3′UTR	7210-7310	N/A

The SVV that the invention provides separation leads peptide sequence peptide (it has the amino acid sequence of the 79th residue of 1-among the SEQ ID NO:169), and it is by the 667-903 position nucleotide coding of SEQ ID NO:168.

The invention provides the SVV VP4 albumen (1A) (it has the amino acid of the 150th residue of 80-among the SEQ ID NO:169) of separation, this section sequence is by 904-1116 position nucleotide coding among the SEQ ID NO:168.

The invention provides the SVV VP2 albumen (1B) (it has the amino acid of the 434th residue of 151-among the SEQ ID NO:169) of separation, this section sequence is by 1117-1968 position nucleotide coding among the SEQ ID NO:168. The present invention also provides the SVV VP2 albumen (1B) (it has the amino acid sequence of SEQ ID NO:4) that separates, and is listed in (corresponding with the 2nd the-the 143rd amino acids residue among the SEQ ID NO:2) among Fig. 9. The amino acid sequence of part SVV VP2 albumen is listed in (corresponding with 4-429 position nucleotides among the SEQ ID NO:1) among Fig. 8 by SEQ ID NO:3 coding.

The invention provides the SVV VP3 albumen (1C) (it has the amino acid sequence of the 673rd residue of 435-among the SEQ ID NO:169) of separation, this section sequence is by 1969-2685 position nucleotide coding among the SEQ ID NO:168. The present invention also provides SVV VP3 (1C) albumen (amino acid sequence with SEQ ID NO:6) that separates, and is listed in (corresponding with the 144th the-the 382nd amino acids residue among the SEQ ID NO:2) among Figure 11. The amino acid sequence of SVV VP3 albumen is listed in (corresponding with 430-1146 position nucleotides among the SEQ ID NO:1) among Figure 10 by SEQ ID NO:5 coding.

The invention provides the SVV VP1 albumen (1D) (it has the amino acid sequence of the 937th residue of 674-among the SEQ ID NO:169) of separation, this section sequence is by 2686-3477 position nucleotide coding among the SEQ ID NO:168. The present invention also provides the SVV VP1 albumen (1D) of the amino acid sequence with SEQ ID NO:8 that separates, and is listed in (corresponding with the 383rd the-the 641st amino acids residue among the SEQ ID NO:2) among Figure 13. The amino acid sequence of SVV2A albumen is listed in (corresponding with 1147-1923 position nucleotides among the SEQ ID NO:1) among Figure 12 by SEQ ID NO:9 coding.

The invention provides the SVV2A albumen (it has the amino acid sequence of the 946th residue of 938-among the SEQ ID NO:169) of separation, this section sequence is by 3478-3504 position nucleotide coding among the SEQ ID NO:168. The present invention also provides the SVV 2A albumen (it has the amino acid sequence of SEQ ID NO:10 position residue) that separates, and is listed in (corresponding with the 642nd the-the 655th amino acids residue among the SEQ ID NO:2) among Figure 15. The amino acid sequence of SVV VP1 albumen is listed in (corresponding with 1924-1965 position nucleotides among the SEQ ID NO:1) among Figure 14 by SEQ ID NO:7 coding.

The invention provides the SVV 2B albumen (it has the amino acid sequence of the 1074th residue of 947-among the SEQ ID NO:169) of separation, this section sequence is by 3505-3888 position nucleotide coding among the SEQ ID NO:168. The present invention also provides the SVV 2B albumen (with corresponding among the SEQ ID NO:12) that separates, and is listed in (corresponding with the 656th the-the 783rd amino acids residue among the SEQ ID NO:2) among Figure 17. The amino acid sequence of SVV 2B albumen is listed in (corresponding with 1966-2349 position nucleotides among the SEQ ID NO:1) among Figure 16 by SEQ ID NO:11 coding.

The invention provides the SVV 2C albumen (it has the amino acid sequence of the 1396th residue of 1075-among the SEQ ID NO:169) of separation, this section sequence is by 3889-4854 position nucleotide coding among the SEQ ID NO:168. The present invention also provides the SVV2C albumen (it has the amino acid sequence of SEQ ID NO:14 position residue) that separates, and is listed in (corresponding with the 784th the-the 1105th amino acids residue among the SEQ ID NO:2) among Figure 19. The amino acid sequence of SVV 2B albumen is listed in (corresponding with 2350-3315 position nucleotides among the SEQ ID NO:1) among Figure 18 by SEQ ID NO:13 coding.

The invention provides the SVV 3A albumen (it has the amino acid sequence of the 1486th residue of 1397-among the SEQ ID NO:169) of separation, this section sequence is by 4855-5124 position nucleotide coding among the SEQ ID NO:168. The present invention also provides the SVV3A albumen (it has the amino acid sequence of SEQ ID NO:16 position residue) that separates, and is listed in (corresponding with the 1106th the-the 1195th amino acids residue among the SEQ ID NO:2) among Figure 21. The amino acid sequence of SVV 3A albumen is listed in (corresponding with 3316-3585 position nucleotides among the SEQ ID NO:1) among Figure 20 by SEQ ID NO:15 coding.

The invention provides SVV 3B (VPg) albumen (it has the amino acid sequence of the 1508th residue of 1487-among the SEQ ID NO:169) of separation, this section sequence is by 5125-5190 position nucleotide coding among the SEQ ID NO:168. The present invention also provides the SVV 3B albumen (it has the amino acid sequence of SEQ ID NO:18 position residue) that separates, and is listed in (corresponding with the 1196th the-the 1217th amino acids among the SEQ ID NO:2) among Figure 23. The amino acid sequence of SVV3B albumen is listed in (corresponding with 3586-3651 position nucleotides among the SEQ ID NO:1) among Figure 22 by SEQ ID NO:17 coding.

The invention provides SVV3C (pro or the protease) albumen (corresponding with 1509-the 1719th amino acids residue among the SEQ ID NO:169) of separation, this section sequence is by 5191-5823 position nucleotide coding among the SEQ ID NO:168. The present invention also provides the SVV3C albumen (with corresponding among the SEQ ID NO:20) that separates, and is listed in (corresponding with the 1218th the-the 1428th amino acids residue among the SEQ ID NO:2) among Figure 25. The amino acid sequence of SVV 3C albumen is listed in (corresponding with 3652-4284 position nucleotides among the SEQ ID NO:1) among Figure 24 by SEQ ID NO:19 coding.

The invention provides SVV 3D (pol or the polymerase) albumen (it has 1720-the 2181st amino acids residue of SEQ ID NO:169) of separation, this section sequence is by 5824-7209 position nucleotide coding among the SEQ ID NO:168. The present invention also provides the SVV 3D albumen (it has the amino acid sequence of SEQ ID NO:22) that separates, and is listed in (corresponding with the 1429th the-the 1890th amino acids residue among the SEQ ID NO:2) among Figure 27. The amino acid sequence of SVV 3C albumen is encoded by SEQ ID NO:19, be listed among Figure 24 (corresponding with 4285-5673 position nucleotides among the SEQ ID NO:1, nucleotides 5671-5673 is terminator codon " tga ", is expressed as asterisk " * " in the amino acid sequence tabulation).

Nucleic acid provided by the present invention comprises RNA and these two kinds of forms of DNA, and the complementary series of the sequence in the tabulation that provides in the tabulation of acquiescence.

Therefore, the SVV nucleotides of separation is numbered SEQ ID NO:168 (length is 7310 nucleotides), and the polyprotein of its coding has the amino acid sequence shown in the SEQ ID NO:169. The SVV nucleotide sequence that separates is that the SEQ ID NO:1 of 5752 nucleotides is represented by length, and its coding has the polyprotein of the amino acid sequence shown in the SEQ ID NO:2. The SVV genome sequence is translated into single polyprotein matter, and it is sheared into various downstreams " transcription product ". The present invention contains the nucleotide sequence of all fragments of SEQ ID NO:168 and SEQ ID NO:1, and all are by the polypeptide of these fragment codings.

The full length amino acid sequence of SVV polyprotein is represented by SEQ ID NO:169, and the 667-7209 position nucleotides of its coding SEQ ID NO:168. The major part of this section total length SVV polyprotein amino acid sequence is by 1-5673 position nucleotide coding among the SEQ ID NO:1. Polyprotein is sheared into three amyloid protein precursors, is respectively P1, P2 and P3 (referring to Fig. 4 B). P1, P2 and P3 further are cut into less protein product, and the shearing product of the structural region of P1 (1ABCD or capsid zone) is 1ABC, VP0, VP4, VP2, VP3 and VP1. The shearing product of non-structural protein P2 (2ABC) is 2A, 2BC, 2B and 2C. The shearing product of non-structural region P3 albumen (3ABCD) is 3AB, 3CD, 3A, 3C, 3D, 3C ' and 3D '.

In certain embodiments, provide the nucleic acid that separates among the present invention, it comprises: (i) coded sequence in 1ABCD or capsid zone (the 904th the-the 3477th nucleotides among the SEQ ID NO:168); (ii) coded sequence of 1ABC (the 2685th nucleotides of 904-among the SEQ ID NO:168); (iii) coded sequence of VP0 (904-1968 position nucleotides among the SEQ ID NO:168); (iv) coded sequence of 2ABC (3478-4854 position nucleotides among the SEQ ID NO:168; 1924-3315 position nucleotides among the SEQ ID NO:1); (v) coded sequence of 2BC (3505-4854 position nucleotides or SEQ ID NO:1 kind 1966-3315 position nucleotides among the SEQ ID NO:168); (iii) coded sequence of 3ABCD (the 5673rd nucleotides of 3316-of 4855-7209 position nucleotides or SEQ ID NO:1 among the SEQ ID NO:168); (iv) coded sequence of 3AB (among the SEQ ID NO:168 among 4855-5190 position nucleotides or the SEQ ID NO:1 3316-3651 position nucleotides); (v) coded sequence of 3CD (among the SEQ ID NO:168 among 5191-7209 position nucleotides or the SEQ ID NO:1 3652-5673 position nucleotides). The present invention also provides by the albumen of above-mentioned sequential coding or polypeptide, and corresponding fragment.

The basic capsid structure of picornavirus forms 20 bodies by the mutual close-packed arrays of 60 substances (protomer), and each substance is comprised of 4 peptide species VP1, VP2, VP3 and VP4. This four peptide species is sheared by initial substance VP0. The diameter of SVV virion is about 27nm (referring to Fig. 2), on the size consistent with other picornavirus (latter's diameter is about 27-30nm).

The duplicating dynamics of picornavirus is very fast, finishes the about 5-10 of one-period hour (typically being about 8 hours) (referring to replicative cycle schematic diagram of Figure 68 picornavirus). When with receptors bind after, geneome RNA just discharges in the endochylema from particle. Geneome RNA is directly translated by polymer subsequently, but will be in metainfective 30 minutes, and intracellular protein is synthetic to be reduced suddenly, almost reduces to 0. This phenomenon is called as " closing ", also is the main cause of cell pathology effect (cpe). The translation appearance of closing is to be caused by the cutting in conjunction with compound (CBC) of the cap of the 220kDa of host cell. CBC relates to the combination to the m7G cap of 5 ' end in translation initiation stage of all eukaryotic mrnas. It seems that the cutting of CBC be caused by 2A protease.

5 ' UTR of virus contains IRES. Usually, when the eucaryon translation initiation, ribosomes is incorporated into the cap of 5 ' terminal methyl, then scans to find first AUG initiation codon along mRNA. And IRES has broken through this process, and it can allow the RNA of picornavirus to continue to transcribe after CBC degrades. In one embodiment, the invention provides the nucleic acid of the separation that contains SVV IRES, wherein, IRES is included in the 5 ' UTR. In one embodiment, SVV IRES also can be from the 300-366 position nucleotides of SEQ ID NO:168. 5 ' the UTR of SVV is positioned at the 666th nucleotides place of 1-of SEQ ID NO:168.

The polyprotein of virus is slit into polyprotein P1, P2 and P3 (referring to Fig. 4 B) by the 2A proteolytic cleavage at first. Further shear event is undertaken by 3C (being the major protein enzyme of picornavirus) albumen. The RNA polymerase (3D) that one of shearing product of 3C albumen relies on for viral RNA, its copy geneome RNA is to produce antisense strand. Antisense strand is the synthetic masterplate of positive-sense strand (genome) RNA. The part positive-sense strand is translated out extra virus protein product, and it is packed some positive-sense strand in capsid and forms new virion.

Although the molecular mechanism of action between genome and the capsid protein it be unclear that, think that positive-sense strand rna gene group is wrapped in the capsid protein of preprocessing. In all Microrna Viral infections, the situation of hollow capsid is very common. P1 polyprotein precursor is cut into the substance that is comprised of VP0, VP3 and VP1, be assembled into capsid by these substances, they connect the packaging gene group that combines. Make virion ripe and have an infectivity shearing that to depend on viral inner autocatalysis VP0 be VP2 and VP4. When lysis, the new virion that forms just is released.

The present invention provides also that to have with ATCC patent preserving number be the viral on all four feature of PTA-5343 and the separated strain of nucleotide sequence. Virus of the present invention can be pointed to PTA-5343 separated strain, variant, homology strain, derive strain or mutant strain, and other picornaviruses, described other picornaviruses are that the tumour cell cracking performance spy's who is defined as responsible SVV (wild strain or mutant strain described herein) SVV sequence has been carried out the virus of modifying.

The present invention also provides specific antibody, and described antibody is anti-: the epitope of the following SVV protein of SVV (ATCC patent preserving number is PTA-5343) and separation. The SV protein of described separation has SEQ ID NO:2,4,6,8,10,12,14,16,18, the amino acid sequence of 20,22 and 169 (comprise complete polyprotein: VP4, VP2, VP3, VP1,2A, 2B, 2C, 3A, 3B, 3C, 3D and part thereof, consist of the amino acid sequence of the SEQ ID NO:169 of these protein referring to aforementioned Table A). The present invention also provide specificity anti-by SEQ ID NO:1 or 168 fragment or the epi-position of the coded protein of part.

The comparative analysis of the RNA sequence of various Cardioviruses separated strains shows the nucleotides uniformity that has greater than 45% between genome. Cardioviruses can subclassification be that (EMCV-is Mengo for example, B, R for EMC sample virus again; And MM, ME, Columbia-SK), match Le Shi sample virus (" TMEV ", BeAn for example, DA and GD VII strain) and Vilyuisk are viral.

The SVV sequence is analyzed with other viruses, found that SVV is a kind of Cardioviruses (figure of embodiment 4 and reference herein). If with EMCV and the TMEV Cardioviruses as standard, SVV obviously is not typical Cardioviruses so. However, also have some differences between EMCV and the TMEV, especially with the difference of 5 ' UTR the most obviously (Pevear et al., 1987,J.Virol.，61:1507-1516). To SVV and EMCV and TMEV cluster and the polyprotein (P1,2C, the 3C that carry out so many they^proAnd 3D^polThe district; Referring to Figure 31-37) ethnicity when analyzing, find SVV and may be Cardioviruses very.

SVV is very similar to Cardioviruses on the race, but be determined it on the tree that separates (referring to Figure 86). SVV may be in the genus that separates, because: the IRES of (1) SVV is IV type (and the IRES of Cardioviruses is the II type); (2) multiple unique virus (SVV sample picornavirus) is compared more similar to SVV (referring to embodiment 18 and Figure 87-89) with other picornavirus. In those energy and SVV can be in conjunction with the antibody of the SVV of SVV sample picornavirus to the antibody of the antibody of the infection that allows cell line or anti-SVV. Therefore SVV sample picornavirus can be used for any method of the present invention, comprise treatment of cancer, wherein, determined SVV sample picornavirus be natural tumour cell cracking performance or manufacture and have the tumour cell cracking performance (for example according to the SVV sequence by design in the genomic sudden change of SVV sample picornavirus). In one embodiment, MN 88-36696 is used for method of the present invention and is used for treating cancer.

The method for the treatment of cancer

The present invention adopts the virus of modifying to carry out treatment of cancer with reference to the cancer cell cracking performance characteristic of SVV. These viruses comprise picornavirus (comprising SVV sample picornavirus), derived virus, variant viral, mutated viruses or homology virus. The present invention shows that wild type SVV (the ATCC preserving number is PTA-5343) has the ability of selective killing class tumour cell. For example SVV can optionally kill and wound those neurogenics (neurotropic) or have the tumour cell of neuroendocrine function (neuroendocrine), comprises small cell lung cancer cell (SCLC) or neuroblastoma. The tumour cell that other available viruses of the present invention are treated comprises following several (but being not limited to this): Adrenal Pheochromocytoma, gastrinoma (causing Zollinger-Ellison syndrome), glucagonoma of pancreas, insulinoma, cephaloma (comprising medullary carcinoma of thyroid gland), multiple endocrine knurl syndrome, endocrine tumor of pancreas, Chromaffionoma, VIPomas (VIPoma), Islet Cell Tumors and pheochromocytoma.

In the embodiment, the invention provides method from SVV sample picornavirus to object that treat or reduce NET by give SVV or. And one or more neuroendocrine tumor signs of neuroendocrine tumor or expression (or cross and express), including, but not limited to: NTR (neurotensin receptor), ATOH (GL11, Myc, GRP acceptor, GRP, neuronspecific enolase (NSE), carcinomebryonic antigen (CEA), Chromogranin A, NCAM, IgF2, BCL-2, Sonic hedgehog (sonic hedgehog) signal path and chemokine receptors.

The neuroendocrine lung neoplasm that also comprises Four types among the present invention. A most serious type is ED-SCLC (SCLC), and the speed of growth and diffusion velocity are the fastest in all cancer cells. Large cellular neural endocrine knurl is very rare, except the size of the cell that forms tumour from SCLC is different, other side (such as prognosis and methods for the treatment of etc.) is all similar with SCLC. Carcinoid (Carcinoid tumors) comprises other two types nervus pulmonalis incretion cancer as carcinoid is known, and these two types is typical carcinoid and atypia carcinoid.

Do not accept the limitation of opinion, the ability of SVV specific killing tumour cell including, but not limited to: copy optionally, close that cell protein is synthetic, apoptosis, enter by the tumor-selective cell and to cause cracking, tumor-selective translation, tumor-selective protein cleavage, tumor-selective rna replicon and their combination.

Compare with other tumour cell cracking performance viruses (adenovirus that comprises modification), SVV has following good feature: (i) SVV has high selectivity for the cancer with nerve characteristic, and described cancer comprises: the SVV of SCLC, Wei Ermushi tumour, retinoblastoma and neuroblastoma-for example to neuroendocrine tumor selectively will be up to 10000 times; (ii) compare with the chemotherapy data, the cell killing specificity of SVV will be above 1000 times; (iii) (dosage is 10 the mouse systematicness being given SVV¹⁴Virion/kilogram) when treating, do not find any toxic and side effect; (iv) effect of SVV is very powerful, show can 100% ground to eradicate the large tumour of on mouse, setting up in advance, and without the recurrence of tumor growth; (v) SVV can pass through the paramount titre of purifying, and in allowing cell line, can reach the granule number of each cell generation above 200000; (vi) SVV very little (diameter of SVV virion is no more than 30nm) therefore compares with other tumour cell cracking performance virus, can permeate better and propagate; (vii) SVV copies rapidly (being less than 12 hours); (viii) SVV without any modification is suitable for as the specificity antineoplastic medicine.

In addition, initial research (referring to embodiment 6) shows some additive factor of existence, and it is so that SVV becomes the good tool for tumour cell cracking performance viral therapy: the neutralizing antibody that (i) does not contain anti-SVV in the human serum; (ii) SVV is not suppressed by complement; (iii) SVV can not cause that haemagglutination occurs the HRBC. All these factors that act on SVV make it than its tumour cell cracking performance virus, show the longer body-internal-circulation time (embodiment 7).

The invention provides the method for selective killing neonatal cell in cell mass (neoplastic cell). The method comprises: SVV and the described cell mass of effective dose are in contact with one another under certain condition, and virus can be transduceed and be entered into the neonatal cell of cell colony under this condition, copies and kill and wound neonatal cell. Except SVV in vivo the method for killing tumor cell,, method of the present invention comprises such embodiment, tumour cell wherein can be cultivated external after infecting with SVV for (1); (2) in the presence of non-tumor cell, cultivate; (3) cell (tumour cell and non-tumor cell) is the mammiferous cell comprising people's cell. External cell is cultivated and is infected with SVV multiple use can be arranged. For example Infection in Vitro can be used as producing the method for a large amount of SVV viruses, as measuring or detect the method that in cell mass, whether has tumour cell, perhaps as screening mutant SVV whether can specific target with kill and wound different tumour cells or types of organization.

The present invention also provides the method for external treatment cancer, and wherein, the patient isolates cancer cell from human cancer, in vitro culture, uses the SVV of selective killing tumour cell to infect, and non-tumor cell is fed back in the patient body. In addition, as the method that gives SVV to the patient, will infect with SVV from isolated tumour cell in the patient body, subsequently at once in the patient body of feedback. In the embodiment, tumour cell originates from hematopoietic cell. Perhaps, receive treatment before the normal cell of in vitro culture is fed back (such as radiotherapy or chemotherapy) tumour cell in the patient body is all destroyed. In one embodiment, this methods for the treatment of also can be used for destroying patient's bone marrow cell.

The coated SVV of polymer can be used to SVV is targeted to the cell of any particular type. This coated strategy also can be used for overcoming the antibody for SVV.

SVV has potential antitumor activity, its anti-tumor cell type with nerve feature. SVV does not show that to the normal cell that detects lysis is active. SVV does not still have lysis to people's liver cell of former generation. Below table 1 summed up preliminary research, described research be carried out in order to determine that external SVV is to the lysis effect of the tumor cell type selected.

Table 1:SVV is to the lysis effect of the tumor cell type of selection

 

Clone	Cell type	EC 50(virion/cell)
			H446	People SCLC	0.0012
PER.C6	People embryo retinoblastoma	0.02
			H69AR	The SCLC-multi-drug resistance	0.035
293	People through the AD5DNA conversion	0.036
			Y79	People's retinoblastoma	0.00035
IMR32	People's cerebral nerve blastoma	0.035
			D283	The human brain cerebellum medulloblastoma	0.25
SK-N-AS	People's cerebral nerve blastoma	0.474
			N1E-115	The mouse neuroblastoma	0.0028
BEKPCB3E1	BEK cell through the Ad5E1 conversion	0.99
			H1299	The non-SCLC of people	7.66
ST	The pig testis cell	5.9
			DMS153	People SCLC	9.2
BEK	The BEK cell	17.55
			M059K	The human brain glioblastoma	1,061
PK15	Porcine kidney cell	1,144
			FBRC	Peptide bovine retina cell	10,170

 

HCN-1A	People's brain cell	23,708
			H460	People LCLC	30000 (deactivations)
Neuro 2A	The mouse neuroblastoma	30000 (deactivations)
			DMS79	People SCLC	30000 (deactivations)
H69	People SCLC	30000 (deactivations)
			C8D30	The mouse brain cell	30000 (deactivations)
MRC-5	The HFL fibroblast	30000 (deactivations)
			HMVEC	The new vessels endothelial cell	30000 (deactivations)
HMVEC	Adult vascular endothelial cell	30000 (deactivations)
			A375-S2	People's malignant melanoma cell	30000 (deactivations)
SK-MEL-28	Malignant melanoma cell	30000 (deactivations)
			PC3	The human prostate cell	30000 (deactivations)
PC3M2AC6	The human prostate cell	30000 (deactivations)
			LnCap	The human prostate cell	30000 (deactivations)
DU145	The human prostate cell	30000 (deactivations)

What the table 1-A of below listed is permission clone and the nonpermissive cell system that SVV infects. Table shows is the lysis sexuality of SVV and selective.

Table 1-A SVV is in external cancer cell cracking performance ability and selective

^*EC50:, all after administration, detected EC50 on the 3rd day as not dated especially

Table 1-A lists is the admissibility result of experiment of carrying out in 165 kinds of primary cells representing 22 kinds of tissues and cell line from the SVV of 8 different kinds.The result shows that nearly all adult normal cell all is the nonpermissive cell of SVV.13 kinds of former generation adult cell cultures through check are non-permissions.12 kinds only have 3 kinds of cell cultures in the cattle, sheep, pig of check and former generation normal cell culture be admissibility, and be the smooth muscle cell of pig.This result and the natural host of SVV are that the imagination of pig is consistent.Except the smooth muscle cell of pig, it is admissibility that neuroendocrine cancer cell line or most of embryo cell line are only arranged.

Muroid experiment (referring to embodiment) shows that SVV can be with efficient and high specific killing tumor cell specifically in vivo.Studies show that in these bodies with other tumor cell cracking performance virus and compare that SVV has lot of advantages.For example, influencing the key factor that tumor cell cracking performance oncovirus goes to eradicate the ability of the tumor of having set up is the penetrance of virus.In research, find based on the not effect of SCLC tumor of the carrier of Ad5 to athymic mouse to adenovirus vector.On immunohistochemical experiment result's basis, find that adenovirus can not see through the tumor cell of setting up in advance.On the contrary, SVV can remove H446SCLC tumor in the nude mouse body after the single system administration.The size of SCC very little (diameter is less than 30nm), therefore in tumor tissues than the easier infiltration of other virus with send out.Therefore the smaller size smaller of SVV can successfully permeate and remove the tumor of having set up to it and played effect.

In the test, set up tumor model in vivo, the therapeutic effect behind the analysis single dose intravenous injection SVV with nude mouse and the complete mice of immunologic function.These tumor models comprise: (1) H446 (people SCLC); (2) Y79 (human retina blastoma); (3) H69AR (people's multi-drug resistance SCLC); (4) H1299 (people NSCLC); And (5) N1E-115 (mice neuroblastoma).The result of the test of these detections is referring to Figure 90 A-E and embodiment 11.Detection shows the therapeutic effect of SVV in above-mentioned all models, and the ED of curative effect and in vitro tests ₅₀Be consistent with the effect of the swollen thing of interior therapeutic people allograft.In the complete mice neuroblastoma model of treatment N1E-115 immunologic function, SVV has shown the anti-tumor activity that under normal immune system.

When the cancer patient accepted chemotherapy, the situation of chemotherapy resistance might appear producing.The patient this situation can occur usually, and this moment, patient's prognosis was very poor, and did not probably have other alternative treatment method.Generally, the reason of generation chemotherapy resistance is that an a kind of albuminoid family that is called as multi-drug resistance albumen (MRPs) expression has taken place, crossed and express or active increasing.The patent applicant equally also finds: the tumor cell of certain kind is relevant to the sensitivity of the SVV also expression with the chemotherapy resistance of cancerous cell or MRP.H69 is the strain cell strain responsive to chemotherapy (amycin), but external SVV is had resistance.And H69AR is a strain chemotherapy resisting cell strain, crosses expression MRPs in its cell, but in vitro tests SVV is treated responsive (referring to table 1).These evidences show that crossing between expression and the sensitivity of cell to the SVV lethal effect of MRPs (comprising MDP) is closely related.In a word, the invention provides with SVV and kill and wound the method that those cross the cell of expressing MRP.

The present invention also provides and has treated those with SVV and because of cell the unusual (method of the disease that causes of cellular abnormality propagation for example takes place.Method is that SVV is contacted with abnormal cell, thereby makes part or all of abnormal cell destroyed.SVV can be used to treat various because of the unusual disease that causes takes place cell, comprises following disease (but being not limited to this): the tumor cell that shows as neuroendocrine tumour or neurofibroma.

Neuroendocrine tumor can be identified by a lot of methods.For example some neuroendocrine tumour can produce and secrete multiple polypeptides hormone and amine substance.Specific some clinical symptoms that causes of some meeting comprises: carcinoid, Zollinger-Ellison syndrome, hyperglycemia, glucagonoma of pancreas and WDHA syndrome etc. in these materials.The specific molecular sign of these symptoms comprises the baseline and/or the post-stimulatory level of materials such as gastrin, insulin, glucagon, vasoactive intestinal peptide in 5-HIAA in the urine, serum or the blood plasma.Part carcinoid and about 1/3 endocrine pancreas cancer do not show any Clinical symptoms, so claim that they are " no function tumor ".Therefore, to those tumor related symptoms and unconspicuous patient, then detect those common tumor-marker molecules usually as Chromogranin A, pancreatic polypeptide, serum neuron specific enolase and glycoprotein hormones subunit etc.In these tumor-marker molecules, Chromogranin A (although function it be unclear that) is a kind of very responsive and special serum mark molecules for many neuroendocrine tumors.Because at the commitment of those PD neuroendocrine tumour, when not having other hormone secretion, Chromogranin A significantly improves.Therefore for neuroendocrine tumor widely, Chromogranin A is considered to the optimum tumor markers of using with therapeutic evaluation that is used to diagnose, its 50-100% that can raise usually in various neuroendocrine tumor patient bodies.The serum of Chromogranin A and the compound size of blood plasma level reflection tumor, and the independent tag of visible peristalsis visible intestinal peristalsis carcinoid patient prognosis in can be used as.

The present invention also provides by SVV and the pharmaceutical composition pharmaceutically can received carrier formed.This type of pharmaceutical composition contain the SVV of effective dose in pharmaceutically can received carrier, be applicable to that therefore dosage according to a unit carries out the part or is administered systemically.The preparation type of medicine comprises the outer injection of aseptic intestinal or suspension, the outer injection of aseptic non-intestinal or oral administration solution or suspension, water-in-oil emulsion, oil-in-water emulsion and other related preparations.Intestinal prescription outer and that the outer medicine of non-intestinal is sent is that those skilled in the art is known.The lyophilizing and/or the reconstituted form that comprise SVV in the pharmaceutical composition.Can received pharmaceutical carriers comprise saline solution, protamine sulfate (Elkins-Sinn, Inc., Cherry Hill, NJ), water, water solublity buffer (for example phosphate buffer and Tris buffer or viral supernatant (Polybrene, Sigma chemical reagents corporation, St.Louis, MO)), phosphate buffered saline(PBS) (PBS) and sucrose solution.Selecting suitable pharmaceutical carriers is a very dark knowledge in fact.These solution all need to carry out sterilization treatment and do not contain other particulate matters except that SVV.Also containing in the medicine on the materia medica can received complementary material, from but the condition of solution near physiological conditions.These complementary materials comprise pH value regulator, toxicity regulator and related reagent (for example sodium acetate etc.).Auxiliary substance also can strengthen the ability of SVV infection cell.

Give the SVV of host or experimenter's sufficient dosage, thereby in tumor cell, duplicate inhibition tumor growth or destroyed tumor cell by virus.The method of utilizing SVV to carry out treatment of cancer comprises systematicness, regionality or local these several modes of virus of carrying.Dosage comprises safe dose, can develop dosage and section's tolerance dose.Select suitable dosage to guarantee to obtain curative effect and destroyed tumor cell.Even extremely after the system type administration, the therapeutic index of SVV also is at least 10, ideally may be at least 100 or more than 1000.Generally speaking, the dosage of SVV is at 1 x 10 ⁸To 1 x 10 ¹⁴Between the vp/kg.The exact dose of administration depends on multiple factor, comprises age, body weight, patient's the size of sex tumor and order of severity etc.Virus can give according to one or many dosage, and concrete number of times depends on host's immunoreation ability.By doctor in charge's decision is that single dose or multiple dose are treated, and decision dosage level and administering mode.If necessary, reduce patient's immunoreation by using various immunosuppressant, thereby help the replication capacity of repetitively administered and/or enhanced virus.Antitumor virus therapy among the present invention can be united use with other antineoplastic agent therapy.Administering mode can have a variety of, for example utilizes liposome-mediated, direct injection, conduit infusion, local topical, suction or the like.In addition, the DNA copy of SVV geneome RNA or its part also can be directly as administering modes, and DNA will transcribe and produce virion or specificity SVV polypeptide in cell subsequently.

Viral dosage with therapeutic effect can significantly improve patient's symptom or prolong patient's life-span.Thereby carry out toxicity and the treatment effect that related experiment is determined virus by pair cell culture or laboratory animal.For example measure LD ₅₀(cause experimental animal or cell the viral dosage of half death to occur, for virus, dosage unit is vp/kg) and ED ₅₀(half experimental animal or cell are had the viral dosage (vp/kg) for the treatment of effect) or EC ₅₀(half experimental animal or cell being had the virus concentration (virion/cell) for the treatment of effect) referring to table 1.Dosage ratio between poisonous effect and the therapeutic dose is a therapeutic index, and it can be LD ₅₀With ED ₅₀Or EC ₅₀Between ratio.Should have the virus of efficient therapeutical effect by first-selected those.The data that obtain from cell culture test and zooscopy can be used as the reference of setting at human body using dosage scope.In the circulation composition of certain limit, viral dosage (comprises ED ₅₀And EC ₅₀) rare or do not have toxicity.Dosage can change in this scope accordingly, and this depends on dosage form and the administering mode that is adopted.

On the other hand, also provide another treatment cancer patient's method here, comprised the dosage of the virus drugs for the treatment of at host's body.The neoplasm is here organized the normally tissue of abnormality proliferation, and these tissues are likely malignant tumor tissue.Accordingly, whether virus can distribute is through to whole tumor tissues or lump, depends on the ability of virus copy choice in tumor tissues.Be applicable to that accepting tumor that method provided by the present invention treats should have and have a liking for neural attribute.

Produce and make the method for virus among the present invention:

The method of producing and make this high titre and high yield virus is an another aspect of the present invention.Just as previously mentioned, can be purified into the SVV virion of high titre, and the output in allowing cell strain also can surpass 200,000 virion/cells.Have the cell strain of producing the high yield virion comprise several (but being not limited to this): PER.C6 (Fallaux et al, Human Gene Therapy.9:1909-1917,1998), H446 (ATCC# HTB-171) and table a kind of EC ₅₀Be lower than other cell strain of 10.

For example can take following method to cultivate picornavirus: the interested virus of purification is had a liking for bacterial plaque, and picking is single has a liking for bacterial plaque and amplification in allowing cell line (for example PER.C6).From allow cell, obtain thick pure virolysis thing (CVL) by multigelation, be used to infect a large amount of permission cells subsequently.Allow cell can be cultured in the various tissue cultures ware, for example contain 50 x 150cm of various culture medium ²Culture dish.These culture medium comprise contain 10% hyclone (Biowhitaker, Walkersvile, MD) and 10mM MgCl ₂(Sigma, St Louis, Dulbecco ' s modified Eagle culture medium MO) (DMEM, Invitrogen, Carlsbad, CA).Reclaim the cell that infects between 48 hours or when complete cytopathology effect (CPE) occurring back 12 hours of infection, centrifugal 10 minutes of 4 ℃ of 1500rpm.With cells and supernatant re-suspended cell precipitation and carry out multigelation and handle.By 4 ℃ of 1500rpm final CVL clarification that becomes in the time of centrifugal 10 minutes.With density gradient centrifugation purified virus granule.The effective purification SVV virion of two circulation CsCl density gradient centrifugation for example: first step density gradient centrifugation (CsCl density is respectively CsCl 1.24g/ml and 1.4g/ml) and back to back second is taken turns density gradient centrifugation (CsCl density is 1.33g/ml).Detect the concentration of the virion behind the purification: A260 etc. with absorption spectrometry to be estimated as 9.5 x 10 at 1 o'clock ¹²Individual virion (referring to by " Cardioviruses (picornavirus) " these chapters and sections (Scraba, D. etc. write) in R.G.Webseter and A.Granoff and " virusology encyclopedia " (second edition) write in 1999).Have a liking for bacterial plaque and/or median tissue culture infective dose (TCID by bioassay standard ₅₀) determine the infection titer of purification methamphetamine hydrochloride.And this method is applicable to PER.C6 or other any cell type when.The SVV viral yield that obtains from the PER.C6 cell surpasses 200,000 virions/cell usually, and the ratio of virion and PFU is about 100.The SVV viral yield that obtains from other permission cell strain (H446-ATCC# HTB-171) is at least with top the same even higher.Also can carry out the SVV purification by the mode of affinity column.

In addition, also there is the correlation step in the extensive good manufacture practice flow process (GMP) that many commercializations order about to be used to purification SVV virus.The present invention equally also on the basis of reference purification of adenoviral, has designed the method for purification SVV.These methods comprise utilizes the density of SVV self to come purification, because the density of SVV is very close with adenovirus, therefore can carry out copurification with adenovirus.

Detect and study the method for tumor

The present invention also provides and has utilized in the invention related virus to come the patient is carried out the method that tumor cell detects.Cell sample can be from the patient, by with cell sample with contain the epi-position label SVV (or other the invention in mentioned virus with tumour-specific, tumour-specific Cardioviruses mutant for example) hatches, the sample that those combine SVV is screened by detecting the epi-position label.Another method, thus whether can cause that by detecting SVV lysis finishes screening process.If SVV can cause lysis really, perhaps SVV can combine by cell specific and in the sample, this may point out may contain in the sample can by SVV in conjunction with and/or the tumor that infects

In addition, SVV also can be used to detect tumor cell in vivo.In the method, at first will contain the SVV deactivation of epi-position label, but the SVV after the deactivation still can with the combining of tumor cell specific, the just replication capacity of forfeiture.By detecting the epi-position label, whether combine with SVV thereby detect tumor cell.Can specificity finish the detection of epi-position label in conjunction with the antibody of epi-position label by adopting, same, antibody itself also can be with corresponding certain labelling (for example fluorescent labeling), perhaps uses to have two of labelling and resist and detect.

Conventional detection comprises uses any virus of mentioning in the invention to come specific targeting to detect the tumor cell of any type.Specific tumor type comprises neuroendocrine type tumor, for example retinoblastoma, SCLC, neuroblastoma, glioma and medulloblastoma.

The present invention equally also carries with utilizing SVV to study the method for tumor cell.The destroyed tumor cell of SVV specific selectivity, then toxicity is very little even do not have toxicity to non-tumor cell.Just because of have these characteristics, SVV can be used to study tumor or provide find the new tumour-specific gene or the possibility of signal path.In other words, can duplicate breeding by specific clothes within it just because of tumor cell has the SVV that some characteristic is, and normal cell is not showed any toxic action.Be tested and appraised out a certain new tumour-specific gene and/or signal path, just might design corresponding treatment antibody or small-molecule drug, and determine by screening whether these medicines have antitumor action.

The present invention also provide identify all to SVV respond the method for tumor cell.The cell of gathering that obtains is hatched with corresponding SVV,, thereby identify those the cell of SVV by reaction by the situation of duplicating of detection cell killing or virus.Available several different methods detects the cell killing situation, and equally also there is corresponding skill (for example MTS analytic process (referring to the corresponding chapters and sections of introducing high flux screening herein)) in this.The method that detects the virus replication situation also has a lot, and a lot of skills (for example CPE observational method, have a liking for bacterial plaque analytic process, DNA quantitative analysis method, FACS detect methods such as virus quantity in the tumor cell, RT-PCR detection viral RNA) are equally also arranged.Here the cell of being mentioned all is a cancerous cell.Cancerous cell among the embodiment comprises the tumor cell line of foundation or isolating tumor cell (but being not limited to this) in the mammalian body.The mammal here is meant the people, and cell also derives from the cancer patient.

The method of the cancerous cell that evaluation responds to SVV can be used for finding the admissibility tumor cell line or the tumor tissues that are applicable to that SVV duplicates.In addition, by detecting the feature of permission type tumor cell, thereby identify the feature of the tumor cell that those can be infected by SVV and kill.Find that these special types help to instruct the new target spot of searching tumour medicine.Equally, identify that the tumor cell to responding property of SVV also can be used to the cancer patient is screened, be suitable for accepting the SVV treatment to determine those patients.

The antibody of anti-SVV or SVV sample picornavirus (monoclonal antibody or polyclonal antibody) can be used to virus in conjunction with test, thereby the patient who is fit to accept the treatment of SVV or SVV sample picornavirus is carried out pre-examination.Can carry out pre-examination according to following process: (1) is separated in patient's body and is obtained cell sample, can obtain by aspiration biopsy; (2) cell sample is hatched dyeing with the antibody of anti-SVV or anti-SVV sample picornavirus; (3) with two anti-detections that are connected with corresponding molecular marker (dyestuff that fluorescein or other can be used for detecting or fluorescent dye).Two anti-can specific identification and anti-in conjunction with anti-SVV or anti-SVV sample Microrna.If one anti-be the rabbit source, so two anti-should be able to specific identification and in conjunction with the immunoglobulin of rabbit; (4) detection molecules labelling.If molecular marker is a fluorescein, then can detect with fluorescence microscope.(the 3rd step also can select to resist one of SVV or anti-SVV sample picornavirus anti-ly to carry out direct labelling (being applicable to monoclonal antibody), if one anti-ly be polyclonal antibody, then recommended to use the method for indirect labelling--utilize tape label two anti---detects).Infect if patient's tumor cell is suitable for SVV or SVV sample picornavirus, then this patient is suitable for accepting SVV or SVV sample picornavirus is treated.In conjunction with in testing, if the antibody staining result is positive, the tumor cell that then proves the patient is the permission cell of SVV in virus.Referring to the fluorescent image of 92B-92C, being the permission cell of SVV and being infected of demonstration by SVV.

When in conjunction with test the patient being carried out pre-examination with virus, cell sample can come from the patient, also can be the section of suspecting the tissue that contains tumor cell.Tissue slice is cut into plurality of sections and hatches with SVV, the antibody with anti-SVV or anti-SVV sample picornavirus carries out immunohistochemical analysis subsequently.

The present invention also provides the method that detects SVV.This method is to be based upon on the antibody basis of the anti-SVV polypeptide of specificity mark.In addition, also can be to be based upon on the basis of nucleic acid hybridization.From SVV, isolate RNA, and carry out respective markers (for example radioactive label, chemical method signal, fluorescent labeling or the like) and be prepared into probe.Isolation of RNA from sample to be tested is combined in (analog material that perhaps has identical function) on the cellulose acetate film, detects with the SVVRNA probe rna of tape label, thereby detects amount in conjunction with last probe.Obtaining on the basis of corresponding sequence, can RNA carried out direct or indirect order-checking by the method for PCR.Used PCR is a real-time quantitative PCR.

Change hiv chemokine tropism's method:

The invention provides the method that makes up SVV mutant (or variant or derived virus), and change has taken place in the cell type tropism of these mutants.Make it obtain special cell type tropism thereby also SVV sample picornavirus can be suddenlyd change.Since SVV and SVV sample picornavirus mutant have specificity in conjunction with and the ability of killing tumor cell, therefore can be used for the treatment of those and originally wild type SVV or SVV sample picornavirus be had tumor cell in conjunction with resistance.

Simple genome and structure primary or wild type SVV can be used to transform, thus can targeting in conjunction with other tumor.Therefore the pattern of infection of these derived virus is extended, can be used for the treatment of the tumor of non-neuropathic and still keep very high therapeutic index (being consistent with primary SVV).A kind of possible mode of carrying out targeted therapy is that tumour-specific peptide section or part are connected on the virus.

In order to select can be used to carry out the virus of treatment of cancer, the decorum of the present invention make up and the method for screening tumor cell cracking performance virus base.With one section of coding at random the gene of peptide section sequence be inserted at random in the original SVV genome in the nucleotide sequence of coding capsid protein.The diversity in peptide section library is 10 at random ⁸, therefore have enough sample sizes, should be able to obtain can specificity in conjunction with the peptide section of tumor tissues.

Studies show that with normal cell in giving and compare that tumor cell has many differences: the membrane permeability of (1) tumor cell is higher; (2) tumor has specific stromal cell characteristic, for example the receptor that the blood vessel endothelium cellulation can the express cell type specific; (3) certain receptor of tumor cell differential expression, antigen and extracellular matrix protein (Arap, W.et al, Nat.Med., 2002,8 (2): 121-127; Kolonin, M.et al, Curr.Opin.Chem.Biol., 2001,5 (3): 308-313; St.Croix, B.et al, Science.2000,289 (5482): 1997-1202).These studies show that it is discrepant between tumor tissues and the normal structure on molecular level, this targeted medicine carries out anticancer therapy provides possibility.What deserves to be mentioned is, by in mouse model, utilizing the method for the blood vessel of going back to the nest, the peptide section of existing a lot of acquisitions through screening be used to carry out the targeted cytotoxic drug (Arap, W.et al, Science.1998,279 (5349): 377-380), short apoptosis polypeptide (Ellerby, H.M.et al, Nat.Med..1999,17 (8): 768-774), inhibitors of metalloproteinase (Koivunen, E.et al, Nat.Biotechnol.1999,17 (8): 768-774), cytokine (Curnis, F.Etal, Nat.Biotechnol..2000,18 (11): 1185-1190), fluorescent material (Hong.F.D.and Clayman, G.L., Cancer Res..2000,60 (23): 6551-6556) and gene (Trepel, M.et al, Hum.Gene Ther., 2000,11 (14): 1971-1981).These cancer target polypeptide are proved to be to occupy to be increased curative effect of medication and reduces toxic effect.

Can make up SVV derived virus library from purchasing by insert one section rondom polypeptide in the viral capsid proteins zone.Shown in Figure 57, incriminate by foul means and make up the carrier that contains coding SVV capsid protein and Qu Xulie, for example " pSVV capsid protein carrier ".This capsid protein carrier (for example uses restricted enzyme (for example CviJI---flush end restriction endonuclease) to cut at random on carrier DNA by the method for sudden change subsequently.Carrier has many restriction enzyme sites, obtains accordingly only by CviJI enzyme action fragment (referring to Figure 57) once but separate with the method for glue purification subsequently.Isolating a large amount of DNA contains numerous species, they all the coding capsid protein the zone in diverse location rupture.These cutaway carrier are hatched with oligonucleotide and ligase, and the oligonucleotide of some can be connected on the cutaway carrier, therefore is inserted at random on the diverse location in coding capsid protein zone.According to said method, and can construct SVV capsid protein mutant library.

The oligonucleotide that is inserted into the capsid coding region can be a random sequence, also can right and wrong sequence (for example being predetermined sequence) immediately, or half random sequence (partial sequence is predetermined, and a part then is a random sequence in addition).Nonrandom sequence can contain the zone that coding schedule is a label.The epi-position label of these expections can be following several label (but being not limited only to this): c-myc label (being made up of-EQKLISEEDL (SEQ ID NO:35) 10 aminoacid in people's proto-oncogene myc product), HA label are derived from hemoglobin (YPYDVPDYA (SEQ ID NO:36)) and the His in human influenza virus's erythrocyte agglutination fibroin ₆Label (SEQID NO:116)-6 histidine of coding.

The polynucleotide library (for example " pSVV " capsid protein library shown in Figure 57) of coding capsid protein mutant can digest by being limited property restriction endonuclease, thereby discharges the zone of coding capsid protein.The nucleotide sequence of coding capsid protein mutant is connected to (zone that only lacks the capsid protein of encoding in this carrier) (referring to Figure 58) in the carrier that contains SVV full-length gene group sequence subsequently.Connecting product just becomes the carrier with full-length gene group sequence, and the carrier of the capsid protein mutant of encoding in a large number just can constitute mutant library.In Figure 58, the SVV mutant library is made up of many different capsid proteins, and called after " pSVVFLcapsid ".The pSVVFLcapsid vector library is carried out linearization process, thereby generate the SVV RNA (referring to Figure 59) that contains sudden change external transcribing subsequently.These mutants SVV RNA transfection is advanced to allow in the cell, thereby the sequence of SVV can be translated and produces a large amount of SVV mutant granules in host cell.In Figure 59, these SVV mutant granules are labeled as " SVV capsid protein library ".

The oligonucleotide sequence encoded polypeptide that is inserted into SVV capsid coding region can be used as the localized auxiliary original paper of targeting, thereby assists virus to carry out specific infection.When the receptor of this peptide section of the tumor cell surface expression of certain particular type, virus can navigate on this tumor cell by specific targeting, and at time multiplexed cell system and killing tumor cell, sends out simultaneously in the middle of the similar cell.This method makes identifies that novel tumor targeting peptide section and part, tumour-specific receptor, therapeutic type SVV derived virus or other viral derived virus imaginations such as (comprising the picornavirus derived virus) become possibility.

Compare with other technical method (for example polypeptide magnetic bead library with phage demonstration etc.), in external or goods body to the SVV mutant library screened many a bit.Different with other method, (for example the SVV derived virus can optionally combine with cancerous cell the detected object here, and portion duplicates within it.Different in conjunction with the method (for example phage demonstration) of carrier with the cell that before traditional searching is new, this have many incomparable advantages with the library screening method that copies as the basis.At first, the SVV library screening is based upon duplicates on the basis, has only interested viral derived virus to duplicate in target tissue or certain particular cancer cell.Process by screening/selection can obtain the extraordinary Strain of specificity, and it not only can be used as the carrier of target polypeptide, and self also can be used for the treatment of cancer.Differently in this be, the phage result displayed is only represented binding events, the also target polypeptide carrier just only that obtains.Therefore the method for SVV library screening is more effective and rapider.Secondly, in vivo or external phage show in the process of screening, reclaiming the phage obtain from target cell can increase in antibacterial, the SVV derived virus then can be directly in infection cell (or cracked infection cell culture supernatant takes place) reclaim and carry out purification.Once more, the genome of SVV littler so be easier to the breeding go down to posterity.This makes that it might be by inserting hereditary information to the capsid coding region at random, thereby guarantees to obtain optimal insertion product.Therefore make up and screen the SVV library and can produce viral efficiently derived virus.Thereby these derived virus can have the attribute of the non-neurogenic tumor of specific infection by design and screening.

By inserting oligonucleotide, can produce many deficient mutants to the capsid protein coding region.The appearance of these deficient mutants may be owing to contain termination codon in the insertion sequence, causes the polypeptide of virus normally not synthesize.The appearance of deficient mutants also may be because insertion sequence causes capsid protein to change, and can not normally assemble.Occur in order to reduce this situation, adopt the method for TRIM usually and in the design of insertion sequence, note not containing termination codon or certain monoamino-acid of encoding.

Whether suitable in order to detect the oligonucleotide sequence that is inserted into the capsid coding region, can determine by the method in RGD-SVV library (embodiment 16).The nucleotide sequence of coding SVV capsid is cut at random, for example use CviJI.The nucleotide sequence of the coding capsid of randomization linearity is connected on the oligonucleotide that encoding amino acid sequence is RGD (arginine-glycine-aspartic acid acid).The nucleotide sequence of RGD-capsid protein of will encoding subsequently is connected to and contains SVV full gene group sequence but on the carrier of disappearance coding capsid protein sequence.The RGD-SVV derived virus of producing has and expresses the plain individual ability (RGD peptide section can combine with integrin receptor) of cell-specific knot of integrating.Choose those and can successfully infect the RGD-SVV derived virus of expressing the plain cell of integration, determine whether the insertion of RGD polynucleotide by sequencing analysis.This site can be used to fix a point insert at random subsequently, nonrandom or half random sequence.

In addition, the subregion of the capsid protein of SVV and other Microrna is compared (referring to Figure 28), virus contains many non-box structures in this zone, and the similarity between these sequences is minimum.These zones may play important effect on the pattern of infection difference between the virus.Therefore these zones can be used as the insertion site of oligonucleotide, thereby carry out the sudden change of SVV capsid protein (or other viral capsid proteins).

The SVV of deactivation is used for the tumour-specific treatment

Since SVV and SVV capsid derived virus all can be positioned tumor cell and/or tissue by targeting, so SVV virion itself also can be used as the delivery vehicles of treatment.Need to optimize the cancerous cell cracking performance ability of SVV in the method, make that derived virus can target killing tumor cell.

The ability that after wild type SVV is inactivated, just no longer has the infected cell of cracking.But virus still can specificly combine and enter in the cell with target cell.All have in the various works and introduce the standard method that corresponding deactivation has the virus of replication capacity.Whole virus vaccine makes virus forfeiture replication capacity with formalin or beta-propiolactone deactivation.Wild type SVV may self just contain apoptosis-induced peptide section.SVV also can pass through the irradiation deactivation in addition.However, the virus through irradiation also should at first detect to guarantee that they still keep the ability of specific bond tumor cell, because irradiation may cause the change of albumen or capsid activity or function.In addition, some SVV mutant of generation may lack packaging signal.These specific combinations of SVV virus mutation physical ability also enter target cell, but the SVV geneome RNA that duplicates generation goes and can not have infective virion by packaged composition.However, this method is proved to be and can effectively causes death of neoplastic cells, because the SVV mutant can cause after entering host cell that host protein is synthetic and close.

The SVV derived virus that contains capsid protein sudden change also can be inactivated and be used to kill and wound cancerous cell.The SVV derived virus has the oligonucleotide of coding epi-position label to insert in the zone of coding capsid protein, causes can be used as and carries the carrier targeting of toxin to navigate on the tumor cell.Just as described in the text, can carry out random mutation and filter out Strain the SVV derived virus with tumor cell type tropism.Toxin can be connected on the epi-position label, so virus just can be loved and respected and can be delivered to tumor cell by toxin.Also the therapeutic antibodies specificity can be connected with the epi-position label in addition, thereby therapeutic antibodies be delivered to tumor cell by virus.

High flux screening:

The invention provides the high throughput method that screening has the Strain of the different cell lines of specific infection.Thereby whether has the specific infection ability by detecting cytotoxic effect detection Strain.For example, in porous plate, different cell strains are inoculated in the different holes and cultivate.Porous plate provides platform for carrying out high flux screening, and porous plate can be 384 orifice plates.Certain virus sample is joined accordingly in the air, detect virus-mediated molten cytosis whether occurred.Reclaim aerial culture medium subsequently, the viral infection in the culture medium is allowed cell strain (cultivating) in culture bottle or large organization culture dish, it is fully increased.But separation and Extraction RNA carries out sequencing analysis behind the viral growth.Judge according to sequencing result whether series jump is relevant with the change of viral infection spectrum.

Various colorimetrys and fluorescence method may be used to detect rapidly cellulotoxic effect.These methods comprise with based on the analytical method of fluorescent dye, analytical method, MTS analytic process and LDH analytic process based on ATP.Use nucleic acid dye to detect the quantity of dead cell based on comprising in the analysis side of fluorescent dye, because do not have the quantity that the nucleic acid dye of cell permeability can specificly be used to detect dead cell.If think to detect simultaneously the number of dead cell and living cells, then can just can detect the number of living cells with not having cell with probe, organelle probe or other membrane permeability indicator logotype together of the nucleic acid dye of saturating property with cell lactonase substrate, certain permeability nucleic acid dye, transmembrane potential sensitivity.For example Invitrogen company (Carlsbad CA) provides various SYTOX ^TMNucleic acid dye can be specific by containing the cell of cell membrane.EB and PI then can be used to detect dead cell or be about to dead cell.Very high affinity is arranged between these dyestuffs and the nucleic acid, therefore can be detected by the instrument of any detection light absorption value.

For example can detect by the pair cell lysate, determine to have in the damaged cell endochylema active lactic acid dehydrogenase (LDH) and be discharged in the culture supernatant.In order to detect whether there is LDH in the cells and supernatant, can in supernatant, add substrate mixture, activated LDH can be converted into the first Za with a tetrazolium salts I nucleotide by a series of enzymatic reactions.Jia Za dyestuff can detect with the instrument that detects light absorption value subsequently.In addition, utilize PMS (azophenlyene sulphuric acid formicester) also can be used to detect cytotoxicity as the MTS ([3-(4,5-dimethylthiazole-2-yl)-5 (3-carboxyl methoxyphenyl)-2-(4-sulfo group phenyl)-2H-tetrazoles, inner salt]) of electron coupling reagent.(Madison WI) provides a kind of CellTiter by name in Promega company

AQ _UeousSingle solution cell proliferation detecting kit, solution reagent directly can be joined in the cell culture hole, hatch after 1-4 hour and to detect light absorption value at wavelength 490nm place.The amount of the size of 490nm reflection Jia Za product, and how much corresponding the proliferative cell number purpose is in the latter's size and the culture.

Have many detection light absorption to be worth the high flux instrument at present, for example SpectraMax Plus384 Absorbance Platereader (Molecular Devices) can detect the absorbance value (increment be 1nm) of wavelength between 190-1000nm.The detection that this device only need just can be finished 96 orifice plates in 5 seconds, and the detection of 384 orifice plates is also only needed 16 seconds, therefore have convenient, fast and high-throughout characteristics.

Equally also can detect to Hubei Province virus replication, and this detection means also can be operated in high-throughout mode by thereby the infection indication of success is analyzed to finish.For example use real-time quantitative RT-PCR to detect the situation of virus transcription in the cells and supernatant.After the viral RNA reverse transcription was cDNA, PCR can increase and (for example utilizes the double-stranded DNA combination dye cDNA

Green, Qiagen GmbH Germany) detects.Use fluorescent agent directly to measure to the amount of PCR product.

The virus that will demonstrate cytotoxic effect in the hole is cultivated, and is carrying out follow-up in vitro tests (checking once more in tumor and normal cell) and body inner model test (detect virus and whether can kill the intravital transplanted tumor of mice).

Antibody:

The present invention also provides the antibody of specificity at virus in the patent and virus protein.Antibody in the invention has plenty of and utilizes natural operation to produce, and also has plenty of (the comprising single-chain antibody, chimeric antibody, humanized antibody and Fab etc.) of utilizing the non-natural operation to produce.The antibody that these non-natural operations produce can utilize the solid phase method of peptide synthesis to make up, also produce by reorganization, or by the library that is made of various variable heavy chain and variable light chain is screened obtain (Huse et al, Science246:1275-1281,1989).In relevant works, all have to the method that makes up chimeric antibody, humanized antibody, CDR grafted antibody or single-chain antibody and bifunctional antibody have detailed introduction (Winter and Harris, Immunol.Today14:243-246,1993; Ward et al, Nature341:544-546,1989; Harlow and Lane, Antibodies:A laboratory manual. cold spring port publishing house, 1988); Hilyard et al, Protein Engineering:A Practi calapproach, IRL Press 1992; Borrabeck, Antibody Engineering.2d ed., Oxford University Press 1995).Antibody among the present invention had both comprised complete antibody molecule, also comprised some molecule fragments, Fab for example, and F (ab ') 2, and Fv, these fragments have the ability in conjunction with the peptide section of those decision epi-positions.

Being used among the present invention generates the peptide section (for example any fragment of SEQ ID NO:2 or SEQ ID NO:169) of the SVV polypeptide of antibody and the peptide section of other virus polypeptide as immunogen, and itself does not have immunogenicity in fact.But when these hapten with after carrier (for example bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH)) is connected, then have immunogenicity after perhaps being expressed as fusion rotein.Various other molecular vehicle and with the method for hapten conjugation very detailed introduction (for example Harlow and Lane show, supra, 1988) is arranged all in other works.The method of making polyclonal antibody (for example rabbit multi-resistance, sheep multi-resistance, mice multi-resistance or other mammal multi-resistance) also has introduction in corresponding works (for example Green etc. writes ＂ Production of Polyclonal Antisera, ＂ in Immunochemical Protocols, Manson, ed., Humana Press 1992, pages 1-5; Coligan etc. write, ＂ Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, ＂ in Curr.Protocols Immunol.(1992), section2.4.1).

Also can with reference to the method for widely knowing introduced in the corresponding works obtain monoclonal antibody (Kohler and Milstein, Nature256:495,1975; Coligan et al, supra, 1992, sections 2.5.1-2.6.7; Harlow and Lane, supra, 1988).From with separating spleen cell in virus or the virus polypeptide fragment mice immunized body, merge the formation hybridoma with suitable myeloma cell.SVV polypeptide with labelling is screened hybridoma, identify that the secreted antibody of clone has specificity.Those can be expressed the hybridoma separation and Culture with desirable specificity and affinity antibodies, with source as production antibody.Polyclonal antibody also can obtain by similar method separation, for example obtains from the serum of immune animal.These antibody have the application in the method for being introduced in the present invention, and other multiple use (for example preparation standard test kit) also can be arranged.The reorganization phase antibody of expressing, for example single-chain antibody also can be as the preparation test kit.Can be with various sophisticated ways separation and purification monoclonal antibody from the culture supernatant of hybridoma.These methods comprise (Barnes et al, the in such as affinity chromatograph, sieve chromatography and ion-exchange chromatography that contains protein A-agarose gel Meth.Mol.Biol.10:79-104, Humana Press 1992); Coligan et al, supra, 1992, see sections 2.7.1-2.7.12 and sections2.9.1-2.9.3).

A certain specific antibodies can be carried out Proteolytic enzyme, perhaps express one section fragment by dna encoding.Thereby prepare antibody fragment with conjugated antigen ability.Adopt the most frequently used method, complete antibody molecule is digested with pepsin or papain, thereby obtain antibody fragment.For example antibody being produced size with the pepsin enzymolysis is the antibody fragment of 5S, called after F (ab ') 2.Subsequently this fragment is spent sulfhydryl reagent and carries out cracking, thereby and mercapto groups sealed the fracture disulfide bond at random, producing size is the Fab ' unit price fragment of 3.5S.In addition, also can utilize the papain hydrolysis antibody molecule, directly generate two monovalent Fab ' fragments and Fc fragment (see, for example, Goldenberg, U.S.Pat.NOS.4,036,945and 4,331,647; Nisonhoff et al, Arch.Biochem.Biophys.89:230.1960; Porter, Biochem.J.73:119,1959; Edelman et al, Meth.Enzymol., 1:422 (Academic Press 1967); Coligan et al, supra, 1992, see sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Another Fab of antibody is the peptide section of the single complementary determining region of coding (CDR).CDR peptide section can obtain by the polynucleotide sequence that makes up coding purpose antibody CDR.Isolation of RNA from the cell that produces antibody, again by polymerase chain reaction (PCR) obtain encoding the variable region genetic fragment (for example, Larrick et al, Methods:A Companion to Methods in Enzymology2:106,1991).

Antibody among the present invention is applicable to and carries out immunoassay that they both can be liquid phases, also can be connected on the solid phase carrier.In addition, also can in all sorts of ways these antibody are carried out corresponding labelling so that detect.Utilize the antibody in the invention can carry out competitive and noncompetitive immunoassay, comprise direct or indirect two kinds of forms, can carry out radioimmunoassay (RIA) and sandwich (immunoassay) analytic process in addition.In immunoassay (comprise the physiology sample is carried out immunohistochemical analysis), forward, reverse and two-way these three kinds of patterns are arranged with antibody test corresponding antigen molecule.In mutually deserved works, these methods all there is very detailed introduction.

Can be used for label and method that antagonist carries out labelling has a lot, in relevant works introduction is arranged all.The type that can be used for traget antibody has a lot, for example enzyme, radiosiotope, fluorescent material, gold colloidal, chemiluminescent substance, phosphorus and bioluminescence material or the like.The conventional method of introducing in the relevant works also is applicable to other antibody labeling thing or antigenic label, and all needing proves with experiment.

Only otherwise deviate from the scope and spirit that the present invention defines, allow team's said method and prescription to carry out various changes.To above-mentioned all the elements, comprise text description and accompanying drawing in the literary composition, or explain and all done detailed explanation, but the unqualified meaning.

Embodiment

The embodiment that provides below is used for the present invention is made explanations, not the purpose of restriction invention.

Embodiment 1

The amplification and the purification of virus

Xi Nika paddy virus-virus is cultivated in the PER.C6 cell: Xi Nika paddy virus is once had a liking for the bacterial plaque purification, and choose and separate good plaque and amplification in PER.C6 cell people such as (, 1998) Fallaux.From the PER.C6 cell of Xi Nika paddy viral infection, prepare thick pure virolysis thing (CVL) by three freeze-thaw cycle.The PER.C6 cell is at 50 x 150cm ²T.C. cultivate in the culture bottle, use be to contain 10% hyclone (Biowhitaker, Walkersvile, MD, the U.S.) and 10mM MgCl ₂The Eagle culture medium (DMEM, Invitrogen, Carlsbad, CA, the U.S.) of the modification of the Dulbecco of (Sigma, St Louis, MO, the U.S.).After with viral infection 30 hours, results infected cells when observing the complete CPE of appearance, and by centrifugal 10 minutes collecting cells of 4 ℃ of 1500rpm.Cell precipitation is resuspended with cells and supernatant (30ml), carry out freeze-thaw cycle subsequently three times.The CVL that obtains clarified by 4 ℃ of 1500rpm in centrifugal 10 minutes.Process two-wheeled CsCl density gradient centrifugation purified virus granule: a step gradient (density of CsCl is respectively 1.24g/ml and 1.4g/ml) and a continuous gradient subsequently centrifugal (density of CsCl is 1.33g/ml).With the virus concentration of spectrophotometric determination purification, suppose IA260=9.5 x 10 ¹²Individual virion (referring to Scraba D.G. and Palmenberg, A.C., " Cardioviruses (picornavirus) " in 1999. " virusology encyclopedia " (second editions), R.G.Webster and A GranoffEds)).Use the PER.C6 cell to have a liking for the titre of the virus of bacterial plaque detection assay purification by standard.The output of the Xi Nika paddy virus that obtains from the PER.C6 cell surpasses 200,000 virions/cell, and the ratio of virion and PFU is approximately 100 approximately.Other allows the output of cell strain (H446-ATCC# HTB-171) at least equally high even higher.

Embodiment 2

Electronic Speculum

It is online to use direct application method (application method) that Xi Nika paddy virus packets is embedded in through carrying of polyvinyl formal carbon (formvar carbon) bag quilt, with uranium acetate dyeing, uses transmission electron microscope observing subsequently.Under high-amplification-factor, the take pictures representational micro-image of virion.For transmission electron microscope observing, from embedded block, cut the ultrathin section of the PER.C6 cell of Xi Nika paddy viral infection, and under transmission electron microscope, detect the gained section.

The Xi Nika paddy virion of purified mistake is a ball-type, and diameter is about 27nm, is shown as single existence on the net carrying, or minor agglomeration may.The representative picture of Xi Nika paddy virus as shown in Figure 2.Under some area of visual field, also can see the impaired virion or the empty capsid of dye penetration.The PER.C6 cell that infects is carried out analysis of Ultrastructure can see intracytoplasmic crystalline inclusion.Fig. 3 show by the presentation graphics of the PER.C6 cell of Xi Nika paddy viral infection.By visible a small amount of bigger cystozooid (empty pocket bubble) in the cell of viral infection.

Embodiment 3

The separation of Xi Nika paddy viral nucleic acid

The separation of RNA: use Trizol (Invitrogeng company, Carlsbad, CA) geneome RNA of extracting Xi Nika paddy virus with guanidine thiocyanate and phenol extraction process.Separation process is operated according to the method that supplier recommends.Briefly, viral and the TRIZOL of 3 times of volumes and the chloroform mixed together of 249 μ l with the Xi Nika paddy of 250 μ l purification.Aqueous phase contains Xi Nika paddy viral RNA, precipitates with the isopropyl alcohol of 600ul.The ethanol that RNA is precipitated with 70% cleans twice, and drying also is dissolved in the DEPC treated water.Optical density by the 260nm place detects the amount of estimating RNA.Get a RNA, carry out electrophoretic analysis in the degeneration agarose gel 1.25 (CambrexBio Sciences Rockland Inc., Rockland, the ME U.S.), observe electrophoretic band and imaging (Fig. 4) with EB dyeing.

CDNA is synthetic: by the synthetic virus genomic cDNA of Xi Nika paddy of RT-PCR.Under standard conditions, carry out the synthetic of cDNA, use oligonucleotide or the oligo-dT of 1 μ g RNA, AMV reverse transcriptase, 14mer at random.Amplification cDNA fragment is cloned in the plasmid and order-checking.

Embodiment 4

Xi Nika paddy virus sequence is analyzed and epidemiology

Part i: Xi Nika paddy virus SEQ ID NO:1

By the nucleotide sequence analysis of SEQ ID NO:1 being determined it and other viral evolutionary relationship.The translation product of this opening code-reading frame (SEQ ID NO:2) is similar with picornavirus, arrives the termination codon of 3D polymerase from the stage casing of VP2, and length is 1890 amino acid residues (Fig. 5 A-5E and 7A-7B).3 ' the untranslated region (UTR, nucleotide 5671-5734) of 64 nucleotide that are positioned at the opening code-reading frame downstream is provided, has contained termination codon, and do not comprise the poly A tail (Fig. 5 E) of 18 residues.

3 portion gene group fragments of the Xi Nika paddy virus initial stage of carrying out is compared (result does not show), find the member that Xi Nika paddy virus is and cardiovirus (picornaviridae) race is nearest.Therefore to Xi Nika paddy virus, encephalomyocarditis virus (EMCV; The encephalomyocarditis virus kind), Theiler ' s murine encephalomyelitis virus (TMEV; The Theilovirus kind), Vilyuisk people's encephalomyelitis virus (VHEV; The Theilovirus kind) and rat TMEV sample virus (TLV; The Theilovirus kind) polyprotein has carried out comparing (Figure 28).According to this comparison, the polyprotein of Xi Nika paddy virus is processed and is processed quite with the nearest member's of cardiovirus race polyprotein.Cleavage site among Figure 28 between polypeptide is represented with ＂/＂.

At picornaviridae, the shear history of most polyproteins is to finish by one or more protease of encoding viral.Although in the ancient virus of Cardioviruses, aphtho virus, equine rhinoviruses and Tie Shi, shearing between P1-2A and the 2B is to realize by trans-acting (czs-acting) mechanism relevant with 2A sequence itself that is short in understanding, and " NPG/P " sequence has play a part crucial, here (the people such as Donnelly of the disconnection between "/" expression 2A and the 2B polypeptide, 1997 J.Gen.Virol.78:13-21).The virus of a kind of Ljungan by name in the lonely virus of secondary intestinal has this sequence (NPGP) in the upstream of the lonely viral 2A sequence of typical secondary intestinal, and or the extra 2A or the C-terminal in P1 capsid zone, during all 9 picornaviruses of having discerned belong to, 3C ^ProExecution all except that trans-acting (czs-acting) are from cleavage reaction.(promptly 2A is in the shearing of its N-terminal in enterovirus and rhinovirus, and the L at C-terminal in aphtho virus and equine rhinoviruses shears).The process that is cut into VP4 and VP2 after capsid polypeptide VP0 assembling then be can't help 3C ^ProCarry out, but finish by a kind of mechanism that might have viral RNA to participate in of the unknown.The shearing of VP0 does not take place in the lonely virus of secondary intestinal and Ke's cloth virus.Normal Cardioviruses 3C ^ProShearing site at-1 glutamine (Q) or glutamic acid (E) are arranged, glycine (G), serine (S), alanine (A) or aspartic acid (N) (table 2) are arranged in+1 site.The shearing of Xi Nika paddy virus polyprotein has confirmed this pattern, except being (table 2) the VP3/VP1 site of histidine (H)/serine (S); Yet at least one strain horse A type rhinovirus (ERAV; The aphtho virus genus) H/S might become 3A and 3B in ^VPgBetween shearing site (people such as Wutz, 1996, J.Gen. Virol77:1719-1730).

The shearing site of table 2 Xi Nika paddy virus and Cardioviruses

● fracture does not belong to shear event between 2A and the 2B.

The initial stage cutting (P1/P2 and P2/P3) of Xi Nika paddy virus: these initial stage cutting incidents are similar to the initial stage cutting incident of Cardioviruses, aphtho virus, equine rhinoviruses, the ancient virus of Tie Shi, comprise P1-2A by the separation of new mechanism from 2B, described new mechanism comprises NPG/P (SEQ ID NO:111) and the traditional cutting incident (table 2) of passing through 3Cpro between 2BC and P3.

P1 shears: compare by the sequence with EMCV and TMEV, (table 2) predicted in the shearing in Xi Nika paddy virus P1 capsid coding region relatively easily.

P2 shears: it is synthetic that 2C albumen has participated in RNA.The 2C polypeptide of Xi Nika paddy virus contains NTP binding motif GxxGxGKS/T (SEQ ID NO:112) (domain A) and hyhyhyxxD in the unwindase of inferring and all picornavirus 2C (wherein hy represents hydrophobic amino acid residue; Domain B) (Figure 29).

P3 shears: the prediction of P3 shearing site is also direct relatively.We understand seldom the function of 3A polypeptide.But what all picornavirus 3A albumen all contained supposition strides the film αLuo Xuanjiegou.The degree of consistency of this proteic basic sequence low (referring to Figure 28, between the 1612nd to 1701) between Xi Nika paddy virus and the Cardioviruses.

The polypeptide that is connected with genome---VPg is by the 3B regional code, very low with other Cardioviruseses concordance on aminoacid is formed, still, tertiary amino acid residue is a tyrosine, with contact of itself and viral genome 5 ' end is people such as (, 1978) Rothberg that conforms to.Referring among Figure 28 between the 1703rd to 1724.

The three dimensional structure of 4 picornavirus 3C cysteine proteinases is resolved, and the site of active amino acid residue be determined (HAV, people such as Allaire, 1994, Nature,369:72-76; People such as Bergmann, 1997, J.Virol.71:2436-2448; PV-1, people such as Mosimann, 1997, J. Mol.Biol..273:1032-1047; HRV-14, people such as Matthews, 1994, Cell, 77:761-771; AndHRV-2, people such as Matthews, 1999. Proc.Natl.Acad.Sci. U.S..96:11000-11007).The cysteine of runic labelling is a nucleophilic group among Figure 29, and the histidine of first runic labelling is a general base, and determines that the histidine of second runic labelling has specificity to glutaminic acid residue; These three amino acid residues all are (the Figure 28 that guards in (Figure 29) and all other known picornaviruses in Xi Nika paddy virus sequence; Relatively see between the 1726-1946 position for the 3C sequence).

The 3D polypeptide is the key component of the RNA polymerase of RNA dependence, Xi Nika paddy virus contains conservative motif, i.e. KDEL/IR (SEQ ID NO:113), PSG, YGDD (SEQ ID NO:114) and FLKR (SEQ IDNO:115) (Fig. 3 in the RNA polymerase that the RNA of Microrna sample virus relies on; Among Figure 28 between the 1948-2410 position).

The myristoylation of the N-terminal of P1: in most of picornaviruses, the glycine residue (when the N end methionine that occur be removed) of P1 egg precursor polypeptide by its N end, the myristic acid by a molecule of amido link covalent bond (people such as Chow, 1987, Nature.327:482-486).Therefore contain the shearing product VP0 of P1N end and VP4 egg also by myristoylation.The process of this myristoylation is finished by the myristoyl based transferase, and this transferring enzyme can be discerned 8 amino acid residue signal sequences with the glycine beginning.Now proved and in picornavirus, had a conservative motif sequence of forming by 5 amino acid residues: G-x-x-x-T/S (Palmenberg, 1989, In Molecular Aspects ofPicornavirus Infection and Detection, pp.211-241, Ed.Semler﹠amp; Ehrenfeld, Washington D.C., Amer.Soc.for Micro.).Equally do not have the lonely virus of secondary intestinal (lonely virus of the secondary intestinal of people and Ljungan virus) of the maturation cutting of VP0 not have the sign of myristoylation yet yet, but the N-terminal of the molecule sealing VOP of some types is arranged for these viruses.

The peptide sequence and the common sequence data base of indivedual Xi Nika paddy viruses are compared

Use European bio information institute (European BioinformaticsInstitute, EBI; Http:// www.ebi.ac.uk/) FASTA that provides compares analysis with every kind of Xi Nika paddy virus polypeptide (SEQ ID NO:4,6,8,10,12,14,16,18,20 and 22) with public protein sequence data base at sequence of threads.These results that relatively obtain (optimum matching) are presented in the table 3.Capsid protein (VP2, VP3 and VP1) as a whole and 2C, 3C ^ProAnd 3D ^PolTogether, nearest with the race of cardiovirus, still, the corresponding sequence race of the 2A sequence of short prediction and Ljungan virus (the lonely Tobamovirus of secondary intestinal) is nearer.The more detailed comparative result of Xi Nika paddy virus 2A nucleotide sequence and close sequence is shown in Figure 28 (more also can be referring to Figure 70 for 2A sample NPG/P albumen).

The discrete prediction polypeptide matching databases of table 3 and Xi Nijia paddy virus

^*A kind of light and, anaerobic, green sulfur bacteria t

2-Phosphoglycerate dehydratase) (2-phosphoric acid-D-glycerol liquor lyases 2)

The meaning of many bands antigen coupling of Xi Nika paddy virus 2B and Ureaplasma urealyticum or the coupling of 3A and chloracea (Chlorobium tepidum) endolase 2 is all not clear, but these dependencys might be worth further research.

The phylogeny of Xi Nika paddy virus polypeptide and other picornavirus relatively

Can and Cardioviruses calibrate (align) together Xi Nika paddy virus polypeptide (VP2, VP3, VP1,2C, 3C and 3D) and the typical member's of every kind of picornavirus species same protein compare (table 4).Service routine BioEdit v5.0.9 (Hall, 1999, Nucl. Acids.Symp.Ser.,41:95-98) and Clustal X vl.83 (people such as Thompson, 1997, Nucl.Acids Res..25:4876-4882) calibrate (alignments) and make up distance matrix (distance matrices) and unrooted in abutting connection with the tree (unrootedNeighbor-joining trees), algorithm (Satiou andNei according to Saitou and Nei, 1987, Mol.Biol.Evol..4:406-425).Method (1000 supposition repeat) by bootstrapping (bootstrap) resampling obtains ramose fiducial limit.Use TreeView1.6.6 to draw tree diagram (Page, 1996) (Figure 31-37).The distance matrix that is used to make up tree uses the value of multiple alternative corrective, and what Figure 38-44 showed is actual aminoacid concordance percentage ratio.Table 4 shows be present family's picornavirus the classification situation and these relatively in the representational virus sequence of use.

The taxonomy classification of the picornavirus that table 4 uses when comparing with Xi Nika paddy virus

* SV2 and the PEV-8 situation of classifying at present places enterovirus genus with them, yet, there is suggestion to think they can be divided into again new a genus (people such as Krumbholz, 2002; People such as 0berste, 2003).

When the data aggregate of the polypeptide in coming from all tree constructions is got up (Figure 34), the tree construction (Figure 31-33) of indivedual capsid proteins can not be represented whole tree constructions.Difficult point when this might be calibration (align) capsid polypeptide, especially when they are not full length sequence, VP2 (Figure 31) for example.However, P1,2C, 3C ^ProAnd 3D ^PolBe consistent, and the demonstration of Xi Nika paddy virus is in the same place with the TMEV cluster with EMCV.

Xi Nika paddy thigh virus is as a member of cardiovirus

The 3D of now clear and definite Xi Nika paddy virus ^Pol3D with Cardioviruses ^Pol(Figure 37 is arranged almost and same near sibship between EMCV and the TMEV; Figure 44).In other polypeptide that is considered to usually in picornavirus, guard relatively, 2C and 3C _SWAlso almost nearest with the Cardioviruses sibship, but the race of it and Cardioviruses does not have so nearly (being respectively Figure 42 and 43) between MECV and the TMEV.In extracting capsid protein (as a whole), Xi Nika paddy virus also almost with the Cardioviruses race recently, and its race is approximately and ethnic identical (～33%) between two aphtho virus species (the viral and equine rhinoviruses A type of hand-foot-mouth disease).Xi Nika paddy virus differs greatly with Cardioviruses on 2B and 3A polypeptide, and not and the detectable race of any known picornavirus.Yet this situation is not beyond example; Avian encephalomyclitis virus falls far short with hepatitis A virus (HAV) on 2A, 2B and 3A, but still temporary with HAV be divided in the hepatovirus (people such as Marvil, 1999, J.Gen.Virol., 80:653-662).

If as standard, Xi Nijia paddy virus obviously is not typical Cardioviruses with EMCV and TMEV.But, even these two kinds of viruses also there are differences, especially 5 ' UTR (people such as Pevear, 1987, J.Gen.Virol..61:1507-1516).But Xi Nika paddy virus is at its most polypeptide (P1,2C, 3C ^ProAnd 3D ^PolThe zone) last and EMCV is in the same place with the phylogenetic cluster of TMEV.Finally, the taxonomic position of Xi Nika paddy virus in picornavirus will by executive committee of ICTV (ICTV) (EC) the suggestion of Microrna research group and supportive go out plate material after decision.Two kinds of selections are arranged: i) Xi Nika paddy virus is divided into the novel species in the cardiovirus; Or ii) be decided to be new Tobamovirus for Xi Nika paddy virus.

Second portion: Xi Nika paddy virus SEQ ID NO:168

Full-length gene group (Figure 83 A-83H of Xi Nika paddy virus; SEQ ID NO:168; Embodiment 15) allowed further epidemiological study.Further the result of epidemiological study is shown in Figure 86, and Ni Kagu virus in its Chinese and Western shows with Cardioviruses to have hereditary sibship, for example EMCV and TMEV, but in different branches.

The feature of Xi Nika paddy virus full-length gene group noncoding region and coding region is as above listed in Table A.The feature of the comparison of total length Xi Nika paddy virus and EMCV and TMEV-GDVII is listed in the table of below.

Feature	Xi Nika paddy virus	Xi Nika paddy virus	EMCV	EMCV	TMEV-GDVII	TMEV-
							5′UTR	666	-	833	-	1068	-
Lead peptide	237	79	201	67	228	76
							VP4	213	71	210	70	213	71
VP2	852	284	768	256	801	267
							VP3	717	239	693	231	696	232
VP1	792	264	831	277	828	276
							2A	27	9	429	143	426	142
2B	384	128	450	150	381	127
							2C	966	322	975	325	978	326
3A	270	90	264	88	264	88
							3B	66	22	60	20	60	20
3D	1386	462	1380	460	1383	461
							3′UTR	71	-	126	-	128	-

In following table, the shearing site of Xi Nika paddy virus (full length sequence, also can referring to albumen boundary among Figure 83 A-83H with the aminoacid of runic labelling) compares with the shearing site of other Cardioviruses.

Found the virus of a plurality of uniquenesses at USDA, they have surpassed the similarity degree of Xi Nika paddy virus with other Cardioviruses with the similarity degree of Xi Nika paddy virus.These USDA virus isolated strains (member who is considered to " Xi Nika paddy virus sample picornavirus " herein) are: MN88-36695, and NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN99197 and SC 363649.These Xi Nika paddy virus sample picornaviruses and Xi Nika paddy virus have been considered to comprise a kind of new picornavirus and have belonged to.

Every kind of these Xi Nika paddy virus sample picornaviruses all is special, on the nucleic acid level and the concordance of Xi Nika paddy virus be approximately 95%-98% (for Xi Nika paddy virus with the nucleotide sequence between these USDA separated strains relatively referring to Figure 87-89).

Third part: serum research

Pig is the permission host of the USDA virus isolated strain of above-mentioned evaluation.Be inoculated into the virus isolated strain that is numbered MN88-36695 in the gnobiotic pig body and produce antiserum (GP102).These antiserums and other all USDA separated strain combination of listing above can also be with the combinations of Xi Nika paddy virus.24 kinds of common virus diseases of pigs substances of this antiserum discord react, and show that it has specificity.Equally also detected the neutralizing antibody of 1278 (nurse island, pula viruses) of porcine blood serum.Gather serum in the U.S., in 29 serum samples, have 8 positive, the titre scope from 1:57 to 1:36,500.

Whether in order to detect pig is the natural origin of Xi Nika paddy virus, gathers serum sample in multiple animal body, detects them as the ability that allows the neutralizing antibody of cell at Xi Nika paddy viral infection.In the serum and detect following carrying out: dilute multiple serum (1) according to the ratio of 1:2 and 1:4; (2) with 100 TCID ₅₀Virus mix mutually (outside the Xi Nika paddy virus; But can detect any virus and measure serum its infection that whether can neutralize); Hatched 1 hour for (3) 37 ℃; (4) join 1 x 10 ⁴In the PER.C6 cell (or other allows cell type); Hatched 3 days for (5) 37 ℃; (6) measure CPE with the MTS detection method.In and titre be defined as can 100% Zhong with the highest dilution of Xi Nika paddy virus (or other in question virus).

The neutral result of serum shows in people and the long population of Ling to have only on a small quantity or do not have neutralizing antibody.In a test, neither one contains Xi Nika paddy virucidin in 22 human serum.In another test, have only 1 to contain neutralizing antibody in 28 human serum.In the 3rd test, it is neutral not having an example in 50 serum from Amish peasant.In another test, from 4 species the animal serum neither ones of 52 primatess be neutral.

The serum neutralization results shows generally have neutralizing antibody in the farming animals animal population.In a test, be neutral from the 27/71 routine porcine blood serum on farm, in another test, be neutral from the 4/30 routine serum on anosis farm.In another test, 10/50 routine Ox blood serum is neutral, and another test, 5/35 routine field rodent serum is neutral.Because in pig, cattle and mice, found antibody Xi Nika paddy virus and/or Xi Nika paddy virus sample picornavirus there are cross reaction, may in the non-human primate animal, propagate so these data show Xi Nika paddy virus and/or Xi Nika paddy virus sample picornavirus.

Detect the thick lysate of MN 88-36695 virus, estimate it two kinds of Xi Nika paddy virus are allowed cell line (NCI-H446; HEK293) and two kinds of nonpermissive cells of Xi Nika paddy virus system (NCI-H460 and S8) cytotoxicity abilities.The cytotoxicity spectrum of MN 88-36695 is identical with Xi Nika paddy virus: the TCID of NCI-H446 ₅₀Be 1.6 x 10 ^-6The TCID of HEK293 ₅₀Be 1.3 x 10 ^-2And NCI-H460 and S8 are the nonpermissive cell strains of MN 88-36695.These data show that Xi Nika paddy virus sample picornavirus has the potentiality of the method that is applied to present sensing treatment of cancer.In one embodiment, invention provides and has utilized the use of MN 88-36695 SVV sample picornavirus in any sensing treatment of cancer, diagnosis or method for screening.

In serum neutralization test, the antiserum at MN 88-36694 and Xi Nika paddy virus is detected every kind of virus.The mice serum of anti-Xi Nika paddy virus can in and the infection of MN88-36695 and Xi Nika paddy virus (for MN 88-36695 and Xi Nika paddy virus, in the infection and titre be respectively 1:640 and 1:1000).The gnobiotic porcine blood serum of anti-MN88-36695 can in and the infection of MN 88-36695 and Xi Nika paddy virus (for MN88-36695 and Xi Nika paddy virus, in the infection and titre be respectively 1:5120 and 1:100).

These data show that Xi Nika paddy virus is associated in hereditism and serology with pig USDA virus isolated strain.

Embodiment 4

The SDS-PAGE of Xi Nika paddy viral capsid proteins and N-terminal sequencing analysis

The miniature gel electrophoresis system of Bis-Tris polyacrylamide (Novex, San Diego, Ca, the U.S.) that uses NuPAGE to record in advance carries out electrophoresis to the Xi Nika paddy virus of purification.One semigel dyes by silver to be observed, and second half is used to prepare the sample of capsid protein N terminal amino acid order-checking.Before being transferred to albumen on the film, earlier gel is immersed in the 10mM CAPS buffer, pH11,1 hour, and pvdf membrane (Amersham) soaked in methanol.Protein is transferred on the pvdf membrane.Observed in about 1 minute by the product red colouring after changeing film, cut out interested band with dissecting knife, and air drying.Use the pulsion phase sequenator,, protein is carried out automatic N-terminal order-checking by the Edman degraded.

Figure 45 has shown 3 primary structure albumen (approximately 36kDa, 31kDa and 27kDa) of purified Xi Nika paddy virus.

Embodiment 5

In the human serum sample, carry out detection at the neutralizing antibody of Xi Nika paddy virus

If in serum, be pre-existing in antibody at the specific virus carrier, might limit the application that is administered systemically of this carrier so, for example treat metastatic cancer, because the antibody that is pre-existing in can the coupling system administration carrier, and before carrier has an opportunity to transduce target tissue or organ with they neutralizations.Therefore need guarantee not carry at the neutralizing antibody that is selected as the viral vector that is administered systemically in the human body.In order to detect whether contain Xi Nika paddy virus-specific neutralizing antibody in the human serum sample, use the detection that neutralizes of the human serum sample of random acquisition.

TCID 50:, get and contain 1 x 10 in the previous day of test ⁴180 μ l PER.C6 cell suspension of individual cell, in connecing in 96 hole tissue culturing plates.With the thick virolysis thing (CVL) of Xi Nika paddy virus according to the logarithm step in DMEM (Dulbecco ' s ModifiedEagle ' s Medium) culture medium from 10 ^-0Be diluted to 10 ^-11, under each dilution factor, get 20 μ l and transfer in 3 holes in 96 orifice plates that contain the PER.C6 cell.Culture plate is placed on 37 ℃ contains 5%CO ₂Incubator in cultivate, obtain the micro-evidence of cytopathy effect (CPE) and the culture infective dose 50 (TCID of computation organization at the 3rd day reading ₅₀).

Neutralization detects: at first, add the culture medium of 40 μ l in each hole, add the heat-inactivated serum of 40 μ l subsequently in first hole, and inhale and beat mixing, carry out the 1:4 dilution and be used to screen purpose.Get 40 μ l subsequently and transfer in the next hole, carry out the twice dilution of blood serum sample.The Xi Nika paddy virus that adds 40 μ l in the hole of containing the dilute serum sample (contains 100TCID ₅₀).Culture plate was hatched under 37 ℃ 1 hour.Get 40 μ l mixed liquors and be transferred to (1 x 10 in the culture plate that contains PER.C6 ⁴Individual cell/160 μ l/ holes).Culture plate was cultivated 3 days at 37 ℃.Subsequently, obtain CPE with microscopic examination.

In detecting,, detect Xi Nika paddy virus-specific neutralizing antibody from the U.S., Europe and 22 people's serum samples of Japanese random acquisition as the above-mentioned representational neutralization of carrying out.Serum sample is carried out serial dilution, and with contain 100 TCID ₅₀The Xi Nika paddy virus of fixed amount mix.Use serum-virus mixture to infect the PER.C6 cell subsequently, and hatched 24 hours.Can block the titre of the inverse of the maximum serum dilution that CPE forms as the neutralizing antibody of measuring.In this test, do not detect the serum titer that blocking-up CPE forms, show and do not contain Zhong in the human serum sample with the neutralizing antibody of Xi Nika paddy virus.

Hatch the further Xi Nika paddy viral infection (seeing embodiment 6) that can not suppress the PER.C6 cell with human blood, show that the infection of Xi Nika paddy virus is not suppressed by complement or hemagglutinin.As a result, compare with other oncolytic virus, Xi Nika paddy virus circulation time in vivo is longer, and circulation time is to use the major issue of oncolytic adenovirus.

Embodiment 6

Xi Nika paddy virus combines with human red blood cell and hemagglutinin

Shown that multiple virus serotype is at the external erythrocytic red cell agglutination that causes separation from the blood of various animal species.Red cell agglutination or combine with erythrocyte and can cause toxicity in vivo, and influence bio distribution and viral vector effectiveness in vivo in vivo.Therefore, need be used for systemic administration to those selections treats the red cell agglutination characteristic of the viral vector of metastatic carcinoma and analyzes.

Red cell agglutination detects: in order to measure the red cell agglutination whether Xi Nika paddy virus can cause human red blood cell, carry out red cell agglutination and detect in 96 orifice plates at the bottom of the U type.The Xi Nika paddy virus of purification is carried out serial dilution in the PBS of 25 μ l (phosphate buffered saline), 1% of the adding equal volume red cell suspension in each hole.The blood sample that is used for separating red corpuscle derives from healthy individual, and heparin is as anticoagulant.By in cold PBS, erythrocyte being cleaned 3 times removing blood plasma and leukocyte, thereby prepare erythrocyte.After last the cleaning, with PBS that erythrocyte is resuspended, preparation 1% (V/V) cell suspension.With virus and the soft mixing of erythrocyte, culture plate placed hatched in the room temperature 1 hour and monitor hemagglutination.

The whole blood deactivation detects: in order to get rid of Xi Nika paddy virus by the possibility of the direct deactivation of composition in the blood, before separating plasma, at room temperature part virus was hatched 30 minutes or 1 hour with the human blood that belongs to A type, Type B, AB type and O blood group or the PBS that are added with heparin, with postoperative infection PER.C6 cell and calculate virus titer.

In according to the above-mentioned representational detection of carrying out, under the Xi Nika of any detection paddy virus-virus titre, all do not observe the erythrocytic erythrocyte aggregation reaction of different blood groups (A, B, AB and O).When Xi Nika paddy virus is mixed with the human blood sample and is at room temperature hatched 30 minutes and 1 hour, find that viral titre has increase slightly, point out virally not by the component deactivation in the blood, but infectivity is stronger under testing conditions.

Embodiment 7

Remove in the body

Blood circulation time:, the nude mice that kind is implanted with the H446 tumor is used Xi Nika paddy virus treated by tail vein injection in order to measure blood circulation time and the amount of virus in tumor.Dosage is 1 x 10 ¹²Vp/kg.Mouse blood is gathered in the 0th, 1,3,6,24,48,72 hours and the 7th day (189 hours) after injection, and after collection separated plasma from blood immediately, in infecting culture medium, dilute, and be used to infect the PER.C6 cell.After injection, put to death the mice of injection and collected tumor in 6,24,48,72 hours and the 7th day.Tumor tissues is cut into little section and is suspended from the culture medium of 1ml, by three freeze-thaw cycle virus is discharged from infection cell subsequently.Supernatant is carried out continuous log10 dilution, and in the PER.C6 cell, measure titre.The titre of Xi Nika paddy virus is expressed as pfu/ml.Tumor biopsy also carries out H﹠amp; E dyeing and immunohistochemical analysis detect the viral capsid proteins in the tumor.

Infer that 7.3% of mice body weight is a blood, measure the cyclical level of virion in blood in view of the above.In according to the above-mentioned representational detection of carrying out, giving in viral 6 hours, the cyclical level of Xi Nika paddy virus drops to zero granule, and all detects less than SVV (Figure 46 A) at later time point.In tumor, inject and still can detect Xi Nika paddy virus in back 6 hours, after this stable two the logarithm levels (Figure 46 B) that raise of Bing Du amount.Still can in tumor, detect Xi Nika paddy virus (Figure 46 B) after the injection on the 7th day.When carrying out immunohistochemical analysis, tissue slice is presented at has Xi Nika paddy virus protein (Figure 47, last lattice) in the tumor cell.When using H﹠amp; During E dyeing, tumor biopsy shows the tumor cell (Figure 47, following lattice) of some circles.

Compare with the adenovirus of the similar dosage of intravenous injection, Xi Nika paddy virus also shows significantly longer residence time in blood.After giving single intravenous injection, can there be 6 hours in Xi Nika paddy virus in blood (Figure 46 C; Figure 46 C is the copy of Figure 46 A, is used for comparing with 46D), and adenovirus was removed (Figure 46 D) from blood after about 1 hour.

Embodiment 8

The tumor cell selectivity

The cell in vitro killing activity of Xi Nika paddy virus: in order to detect the susceptibility of the mankind, cattle, pig and mouse cell, from the animal in various sources, obtain normal cell and tumor cell, and infect with Xi Nika paddy virus.The all cells type is all cultivated in culture medium by supplier's recommendation condition.Former generation human liver cell can available from In Vitro Technologies company (Baltimore, MD), and at hepatocyte culture medium (HCM ^TM, BioWhittaker/Clonetics Inc., San Diego cultivates in CA).

The cell in vitro lesion detection: for the cell that detects the sort of type to Xi Nika paddy viral infection susceptible, normal cell that monolayer is being bred and tumor cell are with the Xi Nika paddy viral infection of the purification of serial dilution.Monitor cell CPE, and compare with non-infected cells.After infection the 3rd day, carry out the MTS cell toxicant and detect, and calculate the valid density 50 (EC of each cell granulations ₅₀).Referring to following table 5 and table 6, and above-mentioned table 1A.

Table 5EC ₅₀ Cell line less than 100

EC 50Cell strain less than 1	The EC50 value
		H446 (people SCLC)	0.001197
PERC6	0.01996
		H69AR (SCLC-multi-drug resistance)	0.03477
293 (human kidney cells transforms through ad5E1)	0.03615
		Y79 (human retina blastoma)	0.0003505
IMR32 (human brain, neuroblastoma)	0.03509
		D283med (human brain; The cerebellum medulloblastoma)	0.2503
SK-N-AS (human brain, neuroblastoma)	0.474
		N1E-115 (mice neuroblastoma)	0.002846
SK-NEP-1 (peritoneum oozes out for kidney, wilmsShi tumor)	0.03434
		BEKPCB3E1 (the BEK cell transforms through ad5E1)	0.99
		EC 50Cell strain less than 10 (1-10)	The EC50 value
H1299 (the non-sclc of people)	7.656
		ST (pig testis)	5.929
DMS 153 (people SCLC)	9.233

EC 50Cell strain less than 1	The EC50 value
		EC 50Cell strain less than 100 (10-100)	The EC50 value
BEK (BEK tissue)	17.55

Table 6.EC ₅₀ Surpass 1000 cell strain

M059K (human brain; Pernicious glioblast	HUVEC (people's venous endothelial cell)	CMT-64 (mice SCLC)
			KK (people's glioblastoma multiforme)	HAEC (human aorta endotheliocyte)	LLC-1 (mice LCLC))
U-118MG (people's glioblastoma multiforme)	WI38 (people's fibroblast)	RM-1 (mice prostate)
			DMS 79 (people SCLC)	MRC-5 (people's fibroblast)	RM-2 (mice prostate)
H69 (people SCLC)	IMR90 (people's fibroblast)	RM-9 (mice prostate)
			DMS 114 (people SCLC)	HMVEC (human microvascular endothelial cell (mvec)-adult)	MLTC-1 (mouse testis)
DMS 53 (people SCLC)	HMVEC (human microvascular endothelial cell (mvec)-new life)	KLN-205 (mice sqcc)
			H460 (people LCLC)	HCN-1A (human brain)	CMT-93 (mice rectum)
A375-S2 (people's malignant melanoma)	HRCE (people's renal cortex endotheliocyte)	B16F0 (mice malignant melanoma)
			SK-MEL-28 (people's malignant melanoma)		Neur0-2A (mice neuroblastoma)
PC3 (human prostate)		C8D30 (mouse brain tissue)
			PC3M2AC6 (human prostate)		PK15 (Ren sus domestica)
LNCaP (human prostate)		FBRC (tire bovine retina)
			DU145 (human prostate)		MDBK (Ren Bovis seu Bubali)
Hep3B (human hepatocellular carcinoma)		CSL 503 (the Pulmonis caprae seu ovis cell transforms through ad5E1)
			Hep2G (human hepatocellular carcinoma)		0FRC (fetal lamb retina cell)
Xi Nika paddy virus 620 (people's colon)
			Xi Nika paddy virus 839 (people's kidney)
5637 (human bladders)
			HeLaS3
S8

Finish MTS detection (CellTiter according to the operation instruction that the manufacturer provides

AQueousAssay, Promega, Madison, WI).CellTiter

AQ _UeousAssay preferably uses tetrazolium salts chemical compound (3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxyl methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salts, inner salt; MTS) and electronics coupled reagent phenazine methosulfate (phenazine methosulfate, PMS).In experiment, measured the normal cell of contact inhibition, described experiment comprises: HUVEC (Human umbilical vein endothelial cells), HAEC (human aorta endotheliocyte, Clonetics/BioWhittaker # CC-2535), Wi38 (normal person's tire lung fibroblast, ATCC # CCL-75), IMR90 (people's normal lung fibroblast, ATCC CCL-186), MRC-5 (people's normal lung fibroblast, ATCC, #C CL-171) and HRCE (people's renal cortex epithelial cell, Clonetics/BioWhittaker # CC-2554).Ni Kagu virus does not produce CPE in the normal cell Chinese and Western of any above-mentioned contact inhibition.

In following human tumor cell line, do not see the CPE:Hep3B (ATCC #HB-8064) of virus induction, HepG2 (human hepatocellular carcinoma, ATCC # HB-8065), LNCaP (human prostata cancer, ATCC # CRL-10995), PC3M-2AC6, SW620 (people's colorectum adenocarcinoma, ATCC# CCL-227), SW 839 (people's renal adenocarcinoma, ATCC # HTB-49), 5637 (human bladder cancers, ATCC # HTB-9), DMS-114 (small cell lung cancer, ATCC # CRL-2066), DMS 153 (human small cell lung carcinomas, ATCC # CRL-2064), A549 (people's pulmonary carcinoma, ATCC # CCL-185), HeLa S3 (people's adenocarcinoma of the uterine cervix, ATCC # CCL-2.2), NCI-H460 (National People's Congress's cell lung cancer, ATCC # HTB-177), KK (glioblastoma), with U-118 MG (people's glioblastoma, ATCC # HTB-15).The EC that lists among the attention-table 6 ₅₀Cell line greater than 1000 does not most possibly allow the generation of duplicating of Xi Nika paddy virus and/or virion; Although Xi Nika paddy virus still might combine and enter in the cell with these cells, but do not observe CPE, because in cell duplicating of Xi Nika paddy virus can not be taken place, can take place though perhaps duplicate really, but, some other does not observe CPE (that is the SVV genome that, duplicates can not be packaged in the virion) because entering afterwards to block to cause.But, consider in these cell line CPE can not occur, so these cell lines and their potential tumor types be good candidate that being used to detect which cell and tumor type is to allow or nonpermissive to duplicating of Xi Nika paddy virus.Although wild type Xi Nika paddy virus is tumour-specific, and display target to acting on neuroendocrine tumor, comprise small cell lung cancer and neuroblastoma, might have discrete tumor patient with some cause of disease, for example the type of their neuroendocrine tumor is that Xi Nika paddy virus is unallowed.Therefore, invention has been imagined Xi Nika paddy virus and has been derived generation, can kill and wound the tumor cell type of separation from individual patient, tumor is unallowed to wild type Xi Nika paddy virus in described patient, and separate and to comprise from these individual tumor types, for example, glioblastoma, lymphoma, small cell lung cancer, maxicell pulmonary carcinoma, melanoma, carcinoma of prostate, hepatocarcinoma, colon cancer, renal carcinoma, bladder cancer, rectal cancer and prognosis of squamous cell lung cancer.

By LDH discharge to detect (

96 Non-Radioactive CytotoxicityAssay, Promega, # G1780) the virus-mediated cytotoxicity of mensuration Xi Nika paddy to former generation human liver cell (In Vitro company).With former generation the human liver cell bed board in 12 orifice plates of glue primordial covering, infect with 1,10,100 and 1000 virion/cells of Xi Nika paddy virus (ppc).After infecting 3 hours, with infecting the growth medium that culture medium is replaced as 2ml, at CO ₂Hatched in the incubator 3 days.Detect relevant lactic acid dehydrogenase (LDH) of cell and the LDH in the culture supernatant respectively.LDH in culture supernatant amount divided by LDH amount sum in maximum cell LDH amount and the supernatant, is just obtained cytotoxicity percent.

Data presented shows among Figure 48 does not all have the virus-mediated hepatotoxicity of Xi Nika paddy under the multiple infectious condition of all detections.

Embodiment 10

Virus produces and detects

In order to detect the replication capacity of Xi Nika paddy virus, infect the contact inhibition type normal cell and the splitted tumor cell of activation of some selections with 1 virion/cell (ppc) with Xi Nika paddy virus.After infecting 72 hours, cell is carried out three multigelation circulations, centrifugal subsequently recovery supernatant together with culture medium.Supernatant is carried out continuous log10 dilution, in the PER.C6 cell, detect virus titer.For each cell line, the duplicating efficiency of Xi Nika paddy virus is represented (Figure 49) with " pfu/ml ".

Embodiment 10

Toxicity

The definition of maximum tolerated dose (MTD) is after SVV handles, and nearest dosage before the dosage of dosage limiting toxicity (DLT) occurs animal (for example mice).The definition of DLT is in the overall process of research, occurs losing weight because of giving the Xi Nika paddy animal that virus causes, the dosage when symptom and death.In baseline, the 15th day with measured the neutralizing antibody of anti-Xi Nika paddy virus on the 21st day.Detection as previously mentioned neutralizes.

Enlarge dosage (1 x 10 ⁸-1 x 10 ¹⁴Vp/kg) Xi Nika paddy virus far is that hybridization has immunocompetence mice (available from Harlan SpragueDawley, Indianapolis, IN, the U.S.) by intravenous injection to immunodeficiency nude mice and CD-1, detects MTD in 10 mices of each dosage level.Result of the test shows, in all well-tolerated that detects virus in dosage, any clinical symptoms do not occur, and lose weight (Figure 50) do not occur.Gather mouse blood at the 15th day and the 21st day, and in neutralization detects, have or not Xi Nika paddy virus-specific neutralizing antibody in the monitoring serum.The CD-1 mice of injecting Xi Nika paddy virus has produced neutralizing antibody, and the titre scope is 1/1024 to more than 1/4096.

An other toxicity research carries out in immunocompetent mice kind system (A/J) is arranged.Have been found that Xi Nika paddy virus shows have cell killing activity and replication capacity (referring to table 1) in the N1E-115 cell.Mouse cell lines N1E-115 (neuroblastoma strain, i.e. neuroendocrine tumour) is derived from A/J mice system.Therefore made up the syngeneic mouse model, wherein the N1E-115 cell is formed tumor in the subcutaneous A/J of the plantation mice body, studies its effectiveness and toxicity with Xi Nika paddy virus treated mice subsequently.

In A/J research, whether mouse mainline Xi Nika paddy virus is tolerated Xi Nika paddy virus systemic administration with definite A/J mice.Obtain hematology result and seek the toxicity indication, and can also obtain the serum chemistry result.Experimental design is shown in following table 7:

Table 7:A/J experimental design

Packet numbering #	Experimental animal (female)	Detection material	Dosage level (virion/kilogram)	Dose volume (mL/kg)	Administering mode and administration number of times	The postmortem natural law
							1	5	Blank	0	10	Carry out intravenous injection in the-sky	The 15th day
2	5	Xi Nika paddy	10 s	10	Carry out intravenous injection in the-sky	The 15th day
							3	5	Xi Nika paddy	10 11	10	Carry out intravenous injection in the-sky	The 15th day
4	5	Xi Nika paddy	10 14	10	Carry out intravenous injection in the-sky	The 15th day

The female A/J mice in age in 8-10 week available from Jackson Laboratory company (BarHarbor, Maine).Isolating Xi Nika paddy virion is kept at-80 ℃, and the time spent takes out.By melting the fresh SVV of preparation on ice, and dilute with HBSS (Hank ' s equalizing and buffering saline solution).For second group, with Xi Nika paddy viral dilution to 10 ⁷Individual virion/ml; For the 3rd group, be 10 ¹⁰Individual virion/ml; For the 4th group, be 10 ¹³Individual virion/ml.For first group, use HBSS as vehicle Control.All is placed on ice to drug solns, up to administration.

By the tail vein with the dosage of 10ml/ kg body weight to animal intravenous injection Xi Nika paddy virus.The same day animal was weighed in administration, adjusted dosage (that is the Xi Nika paddy of the injected in mice 0.200ml that 0.0200kg is heavy virus solution) according to body weight.Monitor the M ﹠ M of twice mice every day.Weigh weekly twice.The relevant information of the animal of dying animal of immediate record and any abnormal symptom of appearance.

Collect the blood preparation of all mices that when final putting to death, still survive, after death observe and detect, be used for standard hematology and serum chemistry analysis (AST, ALT, BUN, CK, LDH).When putting to death, gather following organ: brain, heart, lung, kidney, liver and gonad.Half puts every kind of organ sample rapidly to freezing in dry ice, and second half then is immersed in the formalin solution.

Obtain initial hematology result (CBC, difference) in injection Xi Nika paddy after two weeks of virus, the result is summarised in the following table 8.Each test set detects 5 mices (referring to table 7):

Table 8:A/J toxicity result-analysis of Hematology Changes

These results show, compare with hematology's feature of untreated mice, and hematology's feature of mice of Xi Nika paddy virus treated of accepting low dosage, median dose and high dose is not unusual.Can reach a conclusion from the research, systematicness gives that Xi Nika paddy virus do not find can detected toxicity indication, show the A/J mice after intravenous injection Xi Nika paddy virus to its well-tolerated.

Embodiment 11

Effect

(Indianapolis IN), is used for effect research to the female nude mice of athymism (nu/nu) in age in 6-7 week available from Harlan Sprague Dawley company.Make by hand limiter at mouse web portion right side subcutaneous vaccination 5 x 10 ⁶Individual H446 cell.Periodic measurement tumor size is calculated tumor size: π/6 x W x L according to following formula ², L=length wherein, W=tumor width.When tumor reaches 100-150mm nearly ³The time, mice (n=10) random packet.Give the Xi Nika paddy virus of mouse tail vein injection dosage increase according to 10ml/kg dosage.The HBSS of control group mice injection equivalent.Dosage increases scope from 1 x 10 ⁷To 1 x 10 ¹³Virion/kg body weight.Measure twice gross tumor volume weekly to determine antitumous effect after the injection Xi Nika paddy virus.Response is defined as the complete obiteration of xenograft fully; Partial response is defined as gross tumor volume and reduces more than 50% or 50%; Be not defined as and resemble that tumor continues growth the matched group and there is response.

The intravital tumor growth of handling with HBSS of mice is rapid, surpasses 2000mm at the 20th day gross tumor volume of testing ³(Figure 51 sees the line that has the sky diamond indicia).On the contrary, the mice (except lowest dose level is handled) of system injection that gives the Xi Nika paddy virus of all dosage all disappears in the 20th day tumor of research.In the lowest dose level group, at the 31st day of experiment, the tumor of 8 mices disappeared, and 1 mice has very big tumor, and all the other mices have little palp tumor (25mm ³).In order in the large scale tumor, to measure the anti-tumor activity of Xi Nika paddy virus,, 5 HBSS are organized tumor-bearing mice (〉 2000mm at the 20th day of research ³) one time 1 x 10 of systematicness injection ¹¹The Xi Nika paddy virus of vp/kg dosage.In the violent reduction (Figure 51) that can observe gross tumor volume with (behind the Xi Nika paddy virus injection 11 days) during examining.

The extra experiment that detects the effect of single intravenous injection Xi Nika paddy virus is to finish in the mouse tumor model of expressing the neuroendocrine mark.The tumor model that is used to detect comprise H446 (people SCLC) ( Referring to figure90A), Y79 (human retina blastoma) (referring to figure90B), H69AR (people's multi-drug resistance SCLC) ( Referring to figure90C), H1299 (people NSCLC) (referring to Figure 90 D) and N1E-115 (mice neuroblastoma) (referring to Figure 90 E).

The result shows that in all mice neuroendocrine tumor models, single intravenous injection Xi Nika paddy virus all produces effect.The result shows that also SVV is also effective in the mice neuroendocrine tumor model that the N1E-115 immunologic function perfects.

Figure 52 shows is without Xi Nika paddy virus treated (that is, handling with HBSS) or with the mice of Xi Nika paddy virus treated.As shown in the figure, untreated mice has very large tumor, and the mice of handling does not have the visible sign of tumor.In addition, for the not execution mice with Xi Nika paddy virus treated, it is long not observe tumor regrowth in (200 days) during studying.

What show among table 1, the 1A and 5 is the in vitro effects data of Xi Nika paddy virus to specific tumor cell line.Data show, the specific tumor cell type of infection of Xi Nika paddy virus-specific, and do not infect normal adult cell (except the normal cell of pig), obviously be better than other any known oncolytic virus.The cell killing specificity of Xi Nika paddy virus will be higher than 1000 times of embolic chemotherapies (the cell killing specificity values of Xi Nika paddy virus has shown above 10,000, and the cell killing specificity values of chemotherapy is approximately 10).

Ni Kagu virus shows specific cytotoxic activity in H446 people SCLC cell Chinese and Western.The Xi Nika paddy virus that cell and concentration raise was gradually hatched two days, detect cell viability subsequently, the result is shown in Figure 53.Figure 53 shows be Xi Nika paddy virus and H446 SCLC tumor cell (last figure) or hatch with normal person H460 cell (figure below) after the cells survival rate.Xi Nika paddy virus-specific killing tumor cell, EC ₅₀Be approximately 10 ^-3Individual virion/cell.On the contrary, the Xi Nika paddy virus of any concentration all can not be killed and wounded normal cell.In addition, sum up as table 1,1A-3, Xi Nika paddy virus also has cell toxicant type to some other tumor cell line, comprises SCLC multi-drug resistance tumor cell and some embryonic cells and cell line.Xi Nika paddy virus is for the EC of other tumor cell line ₅₀Scope 10 ^-3To 20,000 more than virion/cell.And Xi Nika paddy virus does not all have cytotoxicity for other multiple non-neural tumor and health adult tissue.In addition Xi Nika paddy virus to former generation human liver cell do not have cytotoxicity yet, under up to 1000 virion/cells, discharge (the seeing Figure 48) that detects by LDH.

Embodiment 12

Bio distribution of in rodent, carrying out and pharmacokinetics research

There is the immunocompromised nude mouse of H446 SCLC tumor to carry out the bio distribution and the pharmacokinetic experiment of Xi Nika paddy virus at normal mouse and lotus.This experimentation to behind the normal and immunocompromised tumor-bearing mice single intravenous injection, biodistribution, removing and the continued case of Xi Nika paddy virus.Xi Nika paddy virus (10 to each group mice single intravenous injection contrast buffer or 3 kinds of dosage ⁸, 10 ¹⁰Or 10 ¹²Vp/kg), and the monitoring clinical indication.After administration the 1st, 6,24 and 48 hours and the 1st, 2,4 and 12 weeks from the group of 5 mices, obtained blood preparation.Dosage level comprises a known poor efficiency dosage and two higher dose levels, measures the linearity of virus sweep.After administration the 24th hour and put to death in the 2nd, 4 and 12 weeks and respectively organize mice.The tissue of selecting comprises: aseptic collection liver, heart, lung, spleen, kidney, lymph node, bone marrow, brain and myeloid tissue, and use the RT-PCR that verifies to detect the existence of Xi Nika paddy viral RNA.

24 hours, the 2nd week, the 4th week and the 12nd week gathered urine sample and feces when putting to death, after administration, and detect the existence of perceptual sexually transmitted disease (STD) poison.Experimental design among this embodiment is shown in following table 9:

Table 9: Xi Nika paddy virus has the CD-1 mice and the nude mouse of SCLC tumor lotus In biodistribution

Also in having the immunocompromised nude mouse of H446 SCLC tumor, normal mouse and lotus carried out acute intravenous injection toxicity research.Have the preliminary vein experiment of carrying out in the mice of SCLC tumor to show normal mouse and lotus, Xi Nika paddy virus is up to 10 ¹⁴Be safe under the vp/kg dosage.At single intravenous injection 10 ¹⁴The Xi Nika paddy virus of vp/kg is not found clinical side effects or the sign that loses weight after two weeks.

Embodiment 13

Virus disseminating experiment in normal adult and pregnant mouse

The purpose of this EXPERIMENTAL EXAMPLE is to detect, and normal mouse has the mice of the Xi Nika paddy virus of high concentration to lean backward together with injection, and whether virus can propagated.Therefore because Xi Nika paddy virus is not duplicated in normal and non-tumor-bearing mice body, to the Xi Nika paddy virus of tumor-bearing mice injection high concentration, it is exposed in the animal of normal health and simulates clinical setting with relief.Second purpose is to detect Xi Nika paddy virus to propagate into not infected conceived DAM and propagate the potentiality of giving developmental fetus subsequently from an infected female Mus.

Three groups every group 5 other infection of homogeny of male and female CD-1 mice of experiment are first had 10 ⁸, 10 ¹⁰Or 10 ¹²The mice of vp/kg Xi Nika paddy virus is exposed to together, and monitors the existence of Xi Nika paddy virus by sample of blood, drawn.

Similar, the female Mus of Xi Nika paddy virus exposure and female Mus that some are regularly conceived mix, and detection Xi Nika paddy virus propagates into not infected pregnant female Mus from infected female Mus, and propagates the ability of giving developmental fetus subsequently.

Embodiment 14

The non-human primate experiment

Also safety, toxicity and the poisonous substance kinetics to Xi Nika paddy virus is studied in the non-human primate animal.In the dosage range discovery period, be 10 to individual monkey single intravenous injection dosage ⁸The Xi Nika paddy virus of vp/kg, and closely monitoring is infected and the toxicity clinical indication.If this dosage toleration is good, then handle other laboratory animal, up to reaching 10 with escalated dose ¹²Vp/kg.Main subsequently experiment comprises many treated animals that 3 male monkeys and female monkey are formed, and continuous 6 weeks give once the Xi Nika paddy virus of carrier or three kinds of various dose separately to every monkey weekly, and monitoring toxicity indication.The monkey of two different sexes in addition gives the Xi Nika paddy virus of independent carrier and maximum dose level level, continuous 6 weeks, and before execution, allow recovery time in extra 4 weeks.

Sample of blood, drawn during the 1st week and the 6th week after the administration.Before giving predose and putting to death, gather clinical pathology sample and hematology's sample.Obtain other blood sample after each administration, be used to detect the existence of the neutralizing antibody of SVV.

The monkey of survival is implemented euthanasia carry out comprehensive postmortem subsequently, the institute from main experiment and recovery monkey in the collection inventory in a organized way.The tissue that comes from matched group and high dose group is carried out histopathological analysis.First week and the 6th week are gathered urine sample and fecal sample after administration, to detect existing of infectious Xi Nika paddy virus.All designs of embodiment are shown in following table 10.

The multiple dose toxicological study of table 10 Xi Nika paddy virus in primate

* dosage can be adjusted according to the concrete condition of determining dosage range.

* dosage can change according to the result of dosage range discovery period

Embodiment 15

Structure has infective Xi Nika paddy virus full-length gene group and functional genome's plasmid

Hold the sequence that is about 1.5-2kb still by the order-checking except Xi Nika paddy viral genome 5 ', include 5 ' UTR, 1A albumen (VP4) and the proteic part of 1B (VP2) in the sequence of SEQ ID NO:1.In order to clone 5 ' terminal sequence of SEQ ID NO:1 disappearance, we use at high temperature still has the archaeal dna polymerase of function and can assist archaeal dna polymerase to pass through the chemical reagent of DNA secondary structure smoothly.Be the cDNA of preparation Xi Nika paddy virus the Xi Nika paddy virus-virus separated strain of PTA-5343 simultaneously from the ATCC preserving number.Xi Nika paddy viral infection is allowed cell, and for example PER.C6 isolates virion subsequently.From isolated virion, extract viral RNA and be prepared into cDNA subsequently.Independent cDNA clone is checked order, subsequently the cDNA copy of selecting is connected in the carrier that contains full-length clone.This carrier contains the T7 promoter at the upstream of Xi Nika paddy virus sequence 5 ' end.Xi Nika paddy virus full-length gene group sequence has been listed among Figure 83 A-83H and the SEQ ID NO:168.The plasmid that will contain Xi Nika paddy virus full length sequence by T7 polymerase and in vitro transcription system carries out reverse transcription, obtains full-length RNA (referring to Figure 55).The full-length RNA transfection is advanced to allow whether have infectivity (referring to Figure 55) to detect this full-length clone in the cell.

Concrete method is as follows: the separation of RNA: carry out extracting with guanidine thiocyanate-phenol extraction process (Trizol is available from Invitrogen company) and obtain Xi Nika paddy virus genome RNA.Separation process is operated according to the explanation that supplier recommends.Simple procedure is as follows: with the Xi Nika paddy virus (～3 * 10 of 250 μ l purification ¹²Individual virion) with the TRIZOL of 3 times of volumes and the chloroform mixed together of 240 μ l.Aqueous phase contains Xi Nika paddy viral RNA, precipitates with the isopropyl alcohol of 600 μ l.Be dissolved in the DEPC treated water after RNA precipitation cleaned twice drying with 70% ethanol.Estimate the amount of RNA by the optical density value of measuring the 260nm place.Get a RNA, carry out electrophoretic analysis in the degeneration agarose gel 1.25% (Cambrex Bio Sciences Rockland Inc., Rockland, the ME U.S.), observe electrophoretic band and imaging with EB dyeing.

CDNA is synthetic: by the synthetic virus genomic cDNA of Xi Nika paddy of RT-PCR.Under standard conditions, carry out the synthetic of cDNA: 1 μ gRNA, AMV reverse transcriptase, oligo-dT primer.Simultaneously also can consider to use 14 nucleotide at random.The cDNA that amplification obtains is cloned in pGEM-3Z (Promega) plasmid, subsequently the clone is carried out sequencing analysis.The sequence of viral genome 5 ' end is cloned by RACE and is checked order.Thereby reclaim all order-checkings of arrangement and cross and generate complete Xi Nika paddy virus genome sequence.

The clone of full-length gene group: design 6 pairs of SVV primers, amplify three representative cDNA fragments in the Xi Nika paddy virus full-length gene group sequence by 3 PCR reactions.In the PCR reaction, use Turbo pfu polymerase (Stratagene).At first use primer 5 ' Xi Nika paddy virus-A (SEQ ID NO:219) and Xi Nika paddy virus 1029RT-RI (SEQ IDNO:220) to amplify the virus genomic 5 ' end of Xi Nika paddy.The fragment that obtains is carried out double digestion and reclaimed purification with gel with ApaI and EcoRI.The fragment that reclaims purification is linked to each other with Nde-ApaT7 Xi Nika paddy virus (SEQ ID NO:221).The latter is contained pre-designed NdeI restriction enzyme site at 5 ' end, contains the T7 core promoter in the middle of sequence, and contains the Apal restriction enzyme site in 17 nucleotide of Xi Nika paddy virus sequence 3 ' end beginning.To connect product and connect directed cloning on the Nde I and Eco RI site of pGEM-3Z (Promega) carrier, generate p nucleotide X-03 by 3 steps.Secondly amplify virus genomic 3 ' end with primer Xi Nika paddy virus 6056 (SEQ ID NO:222) and Xi Nika paddy virus 7309NsiB (SEQ ID NO:223).Reverse primer Xi Nika paddy virus 7309NsiB is used to introduce the polyA tail that is about 30 nucleotide.Nsi I can discern the sequence of 3 ' end and be cloned in the PstI site of pGEM-3Z carrier.The PCR product that obtains is carried out enzyme action with BamHI and reclaim with gel.Amplify the fragment of covering Xi Nika paddy viral genome internal sequence with primer Xi Nika paddy virus 91 1L (SEQ ID NO:224) and Xi Nika paddy virus 6157R (SEQ ID NO:225), subsequently the PCR product is carried out enzyme action with EcoRI and BamHI and reclaim with gel.Above-mentioned two glue reclaim the sequence that product has been represented Xi Nika paddy viral genome middle part and 3 ' end.By 3 the step coupled reactions with these two fragment clonings on the EcoRI and SmaI site of pGEM-4Z carrier.Generate p nucleotide X-02.In order to generate Xi Nika paddy virus full length cDNA sequence, p nucleotide X-02 carried out double digestion with EcoRI and NsiI, obtain the fragment of 6.3kb, glue reclaims the purification rear clone on the EcoRI and PstI restriction enzyme site of p nucleotide X-03.The Xi Nika paddy virus total length plasmid that obtains at last is named as p nucleotide X-04.

Can transform 5 of p nucleotide X-04 of Xi Nika paddy virus total length plasmid ' and 3 ' end and be beneficial to it and assist virus to pack in vitro transcription and after the PER.C6 cell is advanced in the transfection of Xi Nika paddy viral RNA.At first insert a SwaI restriction enzyme site, thereby in vitro transcription discharges the 3 ' end of Xi Nika paddy virus cDNA before from the plasmid skeleton again in the downstream of polyA tail.With p nucleotide X-04 is template, utilizes primer Xi Nika paddy virus 6056 (SEQIDNO:222) and SVVSwaRev (SEQ ID NO:226) to carry out pcr amplification and introduce restriction enzyme site.Reverse primer SVV3SwaRev contains 58 nucleotide sequences can representing Xi Nika paddy virus sequence 3 ' end, and contains SwaI and SphI restriction enzyme site.The PCR product that gets carries out double digestion with BamHI and SphI and replaces p the respective segments among the nucleotide X-04, generates p nucleotide X-06.Secondly in p nucleotide X-06 carrier, extra 4 nucleotide that are positioned between T7 promoter transcription initiation site and Xi Nika paddy virus cDNA5 ' the end preface have been removed with annealing oligonucleotide two dimensional method.Contain recognition site, the T7 core promoter of KpnI in the two-way oligonucleotide and in 17 nucleotide of Xi Nika paddy virus sequence 3 ' end (SEQ ID NO:227) beginning, contain the Apal restriction enzyme site.Utilize KpnI and the ApaI restriction enzyme site oligonucleotide of will annealing to be used for replacing the appropriate section of p nucleotide X-06, generate p nucleotide X-07.From Xi Nika paddy virus cDNA, amplify the fragment that contains BamHI and SphI restriction enzyme site with PCR at last, be inserted in the corresponding site of p nucleotide X-07 carrier, recover the two pair bases of script, thereby generate p nucleotide X-09 in polymerase coding region disappearance.

In vitro transcription: at first with p nucleotide X-09 carrier of SwaI digestion, 3 ' of Xi Nika paddy virus sequence held from the plasmid skeleton, discharge, subsequently in the infectiousness of vitro detection transcribe rna.At the plasmid of external use T7 polymerase (Promega) in the in vitro transcription linearity.

RNA transfection PER.C6 cell with in vitro transcription:, PER.C6 is inoculated in 6 orifice plates cultivates in the previous day of transfection.At second day, utilize Lipofetamine transfection reagent (Invitrogen) at in-vitro transfection through transcribing the RNA (1.5 μ g) that obtains, the description that specific operation process provides with reference to supplier.Can observe after transfection that poison due to illness duplicates in 36 hours and the cellulotoxic effect (CPE) that causes.Thereby cells transfected is carried out freeze-thaw cycle preparation virolysis thing three times, the virolysis thing is further infected the PER.C6 cell to determine the efficiency of infection of viral RNA.Therefore, total length Xi Nika paddy virus cDNA clone is proved to be and has infectivity.

As mentioned above, can carry out reverse transcription according to the plasmid that the standard test step will contain Xi Nika paddy virus full-length gene group.The viral RNA that obtains (100ng) can be used for any cell strain that natural Xi Nika paddy virus is allowed of transfection.And can judge by rule of thumb that in numerous cell strains which cell strain is applicable to effective transfection of viral RNA.

Embodiment 16

The structure in RGD-Xi Nika paddy virus library

For seek the best that makes up Xi Nika paddy viral capsid proteins mutant insert the site (with oligonucleotide at random insertion make up mutant), should select a kind of simple model system (RGD).RGD (arginine-glycine-aspartic acid acid) is a small peptide part, can combine with the integration element.The RGD-Xi Nika paddy virus derived virus that successfully constructs should have following characteristics: the gene order that insert (1) can not change the distinctive and Ideal Characteristics of Xi Nika paddy virus; (2) the RGD-Xi Nika paddy virus-virus derived virus that successfully constructs can infect express alpha v β 5 and integrate plain cell strain.

The Xi Nika paddy virus particle that contains complete capsid protein coding region can carry out single cutting on the site at random, inserts the nucleotide sequence of coding rgd peptide subsequently in corresponding site.Shown in Figure 56 and 57, from Xi Nika paddy virus particle library, make up viral Xi Nika paddy virus-RGD library by adopting method commonly used.

The easy steps of inserting the cRGD oligonucleotide in the capsid coding region at random is as follows: the plasmid (referring to Figure 56, " p Xi Nika paddy viral capsid proteins ") that at first makes up coding 2.1Kb Xi Nika paddy viral capsid proteins zone.Utilize mutually deserved enzyme that " p Xi Nika paddy viral capsid proteins carrier " carried out single site random shearing.Used enzyme both can be CviJI, also can be restriction endonuclease V (referring to Figure 57), and concrete grammar will be introduced below.Separate obtaining behind the plasmid of single enzyme action, the RGD sequence is inserted into is built into p Xi Nika paddy viral capsid proteins-RGD library in the carrier.

Compare with the method for other random shearing (for example ultrasonic fracture or physics shear fracture), restricted enzyme CviJI has many advantages when carrying out random shearing.At first CviJI is a kind of flush end restriction endonuclease, and the enzyme action end does not need to repair; Secondly the shearing of CviJI has randomness, the situation of enzyme action focus can not occur.Whole enzyme action process is simple and fast, the concentration of CviJI and enzyme action time is shortened gradually up to most of DNA plasmids take place to shear and linearisation (single enzyme action).The enzyme action result can observe in the standard agarose gel electrophoresis, referring to Figure 57.Separate the purpose band, be connected with the RGD oligonucleotide behind the purification.

The method of another random shearing DNA be restriction endonuclease V method (Kiyazaki, K., Nucleic Acids Res..2002,30 (24): e139).Restriction endonuclease V can contain on the DNA of uracil second and the 3rd phosphate bond of 3 ' of fracture uracil.This method equally also can be used to carry out random shearing DNA, and the frequency of shearing depends on the concentration of adjusting dUTP in the PCR reaction.Although shearing site is usually located at 2 to 3 base places, thymus pyrimidine (being replaced by uracil) downstream, to compare with other method, this method can generate less hot breakpoint or cold breakpoint.

Linearization plasmid at random can link to each other with the cRGD oligonucleotide, is increased and purification in the p Xi Nika paddy viral capsid library that obtains.The sequence that had both contained coding Xi Nika paddy viral capsid proteins in the numerous nucleotide sequences that obtain also contains the sequence (referring to Figure 57 and 58) of the cRGD that encodes.Subsequently above-mentioned nucleotide sequence is cloned in the Xi Nika paddy virus full length sequence carrier that lacks the capsid coding region, thereby generates the library (because the insertion at random of cRGD is arranged, so capsid protein has nothing in common with each other in the library) of containing Xi Nika paddy virus full length sequence.This library is transcribed into corresponding RNA, the RNA transfection that obtains is advanced to allow to produce in the cell strain the various Xi Nika paddy virus derived virus (referring to Figure 59) of different capsid proteins.Obtain RGD-Xi Nika paddy virus-virus granule (" RGD-SVV library ") in case can reclaim, just the some strain virus of random choose (i.e.10 or more) carry out sequencing analysis from these viruses.Determine by the insertion of RGD sequence and the multiformity of capsid protein by the sequencing analysis result.

The in-vitro screening in RGD-Xi Nika paddy virus library: the pattern of infection that whether has enlarged Xi Nika paddy virus by detection insertion site comes the SVV-RGD library is screened.Infect express alpha with RGD-Xi Nika paddy virus library _vβ ₅Integrate plain NSCLC cell strain (non-small cell lung cancer cell strain, for example A549 express alpha _vβ ₅).The Xi Nika paddy virus derived virus that have only those to have function and express RGD could infect the permission cell and in time multiplexed cell system.

Utilization high flux automatic analysis system (TECAN) carries out in-vitro screening.This system has the ability of liquid handling, can hatch 20 kinds of cells simultaneously and detect (referring to Figure 62 and 63) in 384 orifice plates.Infect after 30 hours, when observing complete cell-cytotoxic reaction (CPE), reclaim cell, centrifugal 10 minutes of 4 ℃ of 1500rpm, supernatant discarded.Cell precipitation is resuspended and carry out freeze-thaw cycle three times with cells and supernatant.4 ℃ of 1500rpm obtain clarifying supernatant after centrifugal 10 minutes.Two circulation CsCl density gradient centrifugation is purification Xi Nika paddy virus-virus granule effectively: first step density gradient centrifugation (CsCl density is respectively CsCl 1.24g/ml and 1.4g/ml) and back to back second is taken turns density gradient centrifugation (CsCl density is 1.33g/ml).Concentration with the virion behind the absorption spectrometry detection purification: A ₂₆₀Deng to be estimated as 9.5 x 10 at 1 o'clock ¹²Individual virion (Scraba, D.G.and Palmenberg, A.C., 1999).Above-mentioned steps can be repeated repeatedly until from α _vβ ₅Be recovered to the virus of sufficient amount in the cell.

The RGD-Xi Nika paddy virus derived virus that is recovered to is analyzed: the different RGD-Xi Nika paddy virus derived virus to a small amount of sample (approximately 10-50) are analyzed.Virus mixture is diluted so that the single virus granule is analyzed.Viral derived virus in each is detected, judge those express alpha of infection whether it can be effective and special _vβ ₅Cell.Sequencing analysis is carried out to determine the insertion site of RGD in the capsid zone of each viral derived virus with above-mentioned attribute.The cRGD-Xi Nika paddy that obtains virus derived virus should have following characteristics: the initial every characteristic of (1) virus is still complete; (2) because RGD can with integrate plain the combination, so derived virus has the ability that high expressed is integrated plain cell that infects.The purpose of this method is in order to identify which or which site is suitable for the insertion of RGD, and can therefore can enlarge the pattern of infection of viral derived virus, changes the tropism of Xi Nika paddy virus-virus derived virus.

According to integrate plain bonded ability different, will be numbered and classify through the viral derived virus of order-checking cRGD-Xi Nika paddy.In order to detect in conjunction with active, with the β of reorganization ₂Integrate and be fixed in 96 orifice plates after element is dissolved in PBS, wash twice,, hatch with RGD subsequently with the PBS sealing that contains 3%BSA with PBS.Natural Xi Nika paddy virus-virus (not having the peptide section to insert) is hatched the hole as negative control.Hatch after 1-5 hour with PBS and give a baby a bath on the third day after its birth at least time, have or not the combination of Xi Nika paddy virus subsequently with the antibody test of anti-Xi Nika paddy virus.Rgd peptide or the anti-antibody of integrating element have with RGD-Xi Nika paddy virus to be competed in conjunction with the ability of integrating element.

20 are analyzed with integrating plain cRGD-Xi Nika paddy virus-virus derived virus with very strong binding ability, determine that the oligonucleotide of those codings cRGD can insert successful site.These are inserted the site studies and can further understanding be arranged to the tropism of Xi Nika paddy virus.On the basis that insertion site and other known structure are analyzed, can determine which site is suitable for the insertion of random sequence (this method also can be used for making Xi Nika paddy virus derived virus), and insertion sequence also can be known array or random sequence.Except two kinds of extra and new methods, being built with random sequence, to insert the method for SVV virus derived virus basic identical with the method in top structure RGD-Xi Nika paddy virus library.For fear of introducing unnecessary termination codon or deleterious aminoacid (for example cysteine or proline) at coding region, Morphosys (Munich, Germany) invent the technology of TRIM (trinucleotide mutagenesis), can be used for generating the regional sequence that is inserted into the coding capsid protein.TRIM can destination locations introduce coded amino acid three nucleotide (Virnekas, people such as B., Nucleic Acids Res.1994,22 (25): 5600-5607).The multiformity of the rondom polypeptide that Xi Nika paddy virus shows is 10 ⁸, it is enough sufficient that this quantity is considered to, and expection can obtain the polypeptide that the energy specificity navigates to virus tumor tissues.According to the method for introducing previously external or establish the rondom polypeptide that in the mice body of tumor model Xi Nika paddy virus is shown and detect.

Embodiment 17: serum research

Pig is the admissibility host from the isolating virus isolated strain of USDA.Thereby the virus isolated strain that is numbered MN88-36695 is inoculated into generation antiserum (GP102) in the aseptic pig body.This antiserum can combine with other all USDA separated strains and Xi Nika paddy virus.This antiserum can not combine with 24 kinds of common swine diseases poison, shows that it has excellent specificity.Equally also detected of the neutralizing antibody effect of this porcine blood serum with respect to 1278 (nurse island, pula viruses).Gather serum in the U.S., in 29 serum samples, have 8 positive, and titre at 1:57 to 1:36, between 500.

Whether in order to detect pig is the natural host of Xi Nika paddy virus, gathers serum sample in various animal bodies, detects them and allows cell and the ability of a neutralizing antibody at Xi Nika paddy viral infection.Serum neutralization test carries out according to following step: dilute various serum (1) according to the ratio of 1:2 and 1:4; (2) with 100 viral TCID ₅₀Mix mutually; (except Xi Nika paddy virus, other any virus also can be used to detect serum its course of infection that whether can neutralize); Hatched 1 hour for (3) 37 ℃; (4) said mixture is joined 1 x 10 ⁴PER.

In the cell (or other allows cell); Hatched 3 days for (5) 37 ℃; (6) detect CPE with the MTS method.In and the definition of titre be can 100% Zhong with the highest titre of Xi Nika paddy virus (or other interested virus) course of infection.

The result of serum neutralization test shows for people and Primate colony, exists a small amount of in the body or do not have neutralizing antibody.In a test, neither one shows in 22 serum samples has neutralizing effect to Xi Nika paddy virus.And in another test, have only 1 to contain neutralizing antibody in 28 serum samples.In test for the third time, there is not an example to show neutralizing effect in 50 serum samples from Amish peasant.And in another test, do not have an example to show neutralizing effect in 52 serum samples from 4 ethnic groups yet.

The result of serum neutralization test shows that the farming animals animal can generally produce the SVV neutralizing antibody.In the test, there are 27 in the serum sample from 71 kinds of farming animals animals and show neutralizing effect, and in another test, from the 30 routine serum samples that gather on anosis farm, have 4 examples to show neutralizing effect.In another test, there are 10 examples to show neutralizing effect in the 50 routine Ox blood serum samples, and from 35 serum samples that field rodent is gathered, have 5 examples to show neutralizing effect.

To carry out serum neutralization test at the antiserum of MN88-36694, see whether it can play neutralization (embodiment 2) to Xi Nika paddy virus.The aseptic porcine blood serum of anti-MN 88-36694 can Zhong with the course of infection of Xi Nika paddy virus (among the SVV and titre be 1:100) as previously mentioned, this antiserum can combine with its USDA separated strain and Xi Nika paddy virus.Because adopt IIF method to prove that these USDA virus isolated strains are with existing serological cross reaction between the Xi Nika paddy virus, so think that these virus isolated strains belong to Xi Nika paddy virus sample picornavirus.

These data show that Xi Nika paddy virus is associated in hereditism and serology with pig USDA virus isolated strain.

Embodiment 18: Xi Nika paddy virus and Xi Nika paddy virus sample picornavirus

Detect following virus isolated strain with indirect immunofluorescence assay: MN 88-36695, NC88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197 and SC363649.GP102 is the antiserum at MN 88-36695, and the back produces in the aseptic pig body by MN 88-36695 is inoculated into.This antiserum can combine with above-mentioned 12 kinds of virus isolated strains, points out to have dependency between them in serology.

The neutralization test that the GP102 antiserum is used for Xi Nika paddy virus.In this test, with the Xi Nika paddy virus (100TCID of the dilution antiserum of difference with known dose ₅₀) mix mutually.Place 37 ℃ to hatch 1 hour in mixture.Take out a part subsequently and join 1 x 10 ⁴Individual PER.

(or other Xi Nika paddy virus allows cell strain) is placed on and hatched in 37 ℃ 3 days in the cell.Detect subsequently and whether occur the cellulotoxic effect (CPE) that causes by virus in the hole.If contain neutralizing antibody in the serum, then can neutralize and known viruse infection PER.

Cell.Detect CPE, the number of the reacting condition living cells of the light absorption value of dyestuff with the dyestuff that contains tetrazolium salts.The result accounts for the percentage that does not infect control cells with serum living cells under the different pairs dilution factor and recently represents (shown in Figure 93).Data show that Xi Nika paddy virus is associated in hereditism and serology with pig USDA virus isolated strain.

In addition, in four kinds of different cell strains, detect the cytotoxicity (as shown in table 4) of MN88-36695 virolysis thing.NCI-H446 is identical with its admissibility with respect to Xi Nika paddy virus to the admissibility of MN 88-36695 with the HEK293 cell, and NCI-H460 and S8 then can not be infected by MN 88-36695.In addition, MN88-36695 with Xi Nika paddy virus equally to PER.

Cell has cytotoxicity.During the anti-Xi Nika paddy virus polyclonal antibody that extracts from mice also has and the ability of MN 88-36695, the result is shown in Figure 94.Can be neutralized to such an extent that MN 88-36695 has dependency in the serology and between Xi Nika paddy virus and other separated strain by the serum of anti-Xi Nika paddy virus.

The cellulotoxic effect result of table 11:MN88-36695

Cell strain	TCID 50(pfu/ml)	The result
			NCI-H446	1.6?x?10-6	Admissibility
HEK293	1.3?x?10-2	Taking in property
			NCI-H460	0	Non-admissibility
S8
		0	Non-admissibility

A plurality of USDA separated strains are carried out portion gene group sequence analysis find that these separated strains and Xi Nika paddy virus have very high race (referring to the sequence comparative result of Figure 87-89).What table 12 showed is Xi Nika paddy virus and 6 percentage ratios that the Strain consensus sequence is shared.Found that these viruses are at genome 3 ' end coding 3D ^PolReach 95-98% (referring to Figure 89) with the degree of consistency of 3 ' UTR zone (being about 460 nucleotide).Every strain USDA virus all has characteristics, and is 95-98% with the concordance of Xi Nika paddy virus on the nucleotide acid level.

Table 12: Xi Nika paddy virus and 6 shared percentage ratios of USDA Strain consensus sequence

1	2	3	4	5	6	7		Virus Name
										96.5	99.1	97.2	97.0	97.4	97.0	1	NJ?90-10324
		97.0	95.7	94.8	95.0	98.3*	2	CA?13195
												97.6	97.2	97.6	97.2	3	IA?89-47752
				95.4	96.1	96.3	4	IL?92-48963
														98.9	95.2	5	MN?88-36695
						95.4	6	NC?88-23626
																7	Xi Nika paddy virus-001

After carrying out sequencing analysis, the portion gene to the P1 (shown in Figure 87) of two strain virus and 2C (shown in Figure 88) further determined to have very high race between they and the Xi Nika paddy virus.For other known viruse, (comprise cardiovirus), the ethnic Geng Gao of these USDA virus isolated strains and Xi Nika paddy virus.The sequence and the Xi Nika paddy virus of these USDA virus several regions are compared and make up contiguous cladogram (shown in Figure 95 A and 95B).The cladogram analysis result that makes up further confirms height correlation between these viruses, and the race of proof CA131395 and Xi Nika paddy virus recently.

Sequence table

<110>Neotropix，Inc.

Hallenbeck，Paul?L.

Police，Seshidar?Reddy

Hales，Laura?M.

＜120〉based on the compositions of Xi Nijia paddy virus and the method for treatment disease

<130>287037.128W01

<140>TBD

<141>2006-03-13

<150>60/664,442

<151>2005-03-23

<150>60/726,313

<151>2005-10-13

<150>11/335,891

<151>2006-01-19

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＜221〉base of Xiu Shiing

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＜223〉variable amino acid

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＜213〉Xi Nika paddy virus

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<211>14

<212>PRT

＜213〉Xi Nika paddy virus

<400>10

<210>11

<211>384

<212>DNA

＜213〉Xi Nika paddy virus

<400>11

<210>12

<211>128

<212>PRT

＜213〉Xi Nika paddy virus

<400>12

<210>13

<211>966

<212>DNA

＜213〉Xi Nika paddy virus

<400>13

<210>14

<211>322

<212>PRT

＜213〉Xi Nika paddy virus

<400>14

<210>15

<211>270

<212>DNA

＜213〉Xi Nika paddy virus

<400>15

<210>16

<211>90

<212>PRT

＜213〉Xi Nika paddy virus

<400>16

<210>17

<211>66

<212>DNA

＜213〉Xi Nika paddy virus

<400>17

<210>18

<211>22

<212>PRT

＜213〉Xi Nika paddy virus

<400>18

<210>19

<211>633

<212>DNA

＜213〉Xi Nika paddy virus

<400>19

<210>20

<211>211

<212>PRT

＜213〉Xi Nika paddy virus

<400>20

<210>21

<211>1389

<212>DNA

＜213〉Xi Nika paddy virus

<400>21

<210>22

<211>462

<212>PRT

＜213〉Xi Nika paddy virus

<400>22

<210>23

<211>2292

<212>PRT

＜213〉encephalomyocarditis virus (encephalomyocarditis virus)

<400>23

<210>24

<211>2292

<212>PRT

＜213〉encephalomyocarditis virus

<400>24

<210>25

<211>2292

<212>PRT

＜213〉encephalomyocarditis virus

<400>25

<210>26

<211>2292

<212>PRT

＜213〉encephalomyocarditis virus

<400>26

<210>27

<211>2292

<212>PRT

＜213〉encephalomyocarditis virus

<400>27

<210>28

<211>2292

<212>PRT

＜213〉encephalomyocarditis virus

<400>28

<210>29

<211>2293

<212>PRT

＜213〉encephalomyocarditis virus

<400>29

<210>30

<211>2301

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>30

<210>31

<211>2303

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>31

<210>32

<211>2303

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>32

<210>33

<211>2307

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>33

<210>34

<211>930

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<220>

<221>MOD_RES

<222>(557)

＜223〉variable amino acid

<400>34

<210>35

<211>142

<212>PRT

＜213〉Xi Nika paddy virus

<400>35

<210>36

<211>239

<212>PRT

＜213〉Xi Nika paddy virus

<400>36

<210>37

<211>259

<212>PRT

＜213〉Xi Nika paddy virus

<400>37

<210>38

<211>14

<212>PRT

＜213〉Xi Nika paddy virus

<400>38

<210>39

<211>128

<212>PRT

＜213〉Xi Nika paddy virus

<400>39

<210>40

<211>322

<212>PRT

＜213〉Xi Nika paddy virus

<400>40

<210>41

<211>90

<212>PRT

＜213〉Xi Nika paddy virus

<400>41

<210>42

<211>22

<212>PRT

＜213〉Xi Nika paddy virus

<400>42

<210>43

<211>211

<212>PRT

＜213〉Xi Nika paddy virus

<400>43

<210>44

<211>462

<212>PRT

＜213〉Xi Nika paddy virus

<400>44

<210>45

<211>6

<212>PRT

＜213〉Xi Nika paddy virus

<400>45

<210>46

<211>6

<212>PRT

＜213〉Xi Nika paddy virus

<400>46

<210>47

<211>6

<212>PRT

＜213〉Xi Nika paddy virus

<400>47

<210>48

<211>6

<212>PRT

＜213〉Xi Nika paddy virus

<400>48

<210>49

<211>22

<212>PRT

＜213〉the prompt Shen virus (Porcine teschovirus) of pig

<400>49

<210>50

<211>22

<212>PRT

＜213〉the prompt Shen of pig virus

<400>50

<210>51

<211>22

<212>PRT

＜213〉the prompt Shen of pig virus

<400>51

<210>52

<211>22

<212>PRT

＜213〉the prompt Shen of pig virus

<400>52

<210>53

<211>22

<212>PRT

＜213〉the prompt Shen of pig virus

<400>53

<210>54

<211>22

<212>PRT

＜213〉the prompt Shen of pig virus

<400>54

<210>55

<211>22

<212>PRT

＜213〉the prompt Shen of pig virus

<400>55

<210>56

<211>22

<212>PRT

＜213〉the prompt Shen of pig virus

<400>56

<210>57

<211>22

<212>PRT

＜213〉the prompt Shen of pig virus

<400>57

<210>58

<211>22

<212>PRT

＜213〉the prompt Shen of pig virus

<400>58

<210>59

<211>22

<212>PRT

＜213〉the prompt Shen of pig virus

<400>59

<210>60

<211>22

<212>PRT

＜213〉the prompt Shen of pig virus

<400>60

<210>61

<211>19

<212>PRT

＜213〉foot and mouth disease virus (Foot and mouth disease virus)

<400>61

<210>62

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>62

<210>63

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>63

<210>64

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>64

<210>65

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>65

<210>66

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>66

<210>67

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>67

<210>68

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>68

<210>69

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>69

<210>70

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>70

<210>71

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>71

<210>72

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>72

<210>73

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>73

<210>74

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>74

<210>75

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>75

<210>76

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>76

<210>77

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>77

<210>78

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>78

<210>79

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>79

<210>80

<211>19

<212>PRT

＜213〉foot and mouth disease virus

<400>80

<210>81

<211>22

<212>PRT

＜213〉horse rhinitis B virus (Equine rhinitis virus)

<400>81

<210>82

<211>22

<212>PRT

＜213〉horse rhinitis B virus

<400>82

<210>83

<211>22

<212>PRT

＜213〉horse rhinitis B virus

<400>83

<210>84

<211>22

<212>PRT

＜213〉encephalomyocarditis virus

<400>84

<210>85

<211>22

<212>PRT

＜213〉encephalomyocarditis virus

<400>85

<210>86

<211>22

<212>PRT

＜213〉mengo virus (Mengo virus)

<400>86

<210>87

<211>22

<212>PRT

＜213〉Xi Nika paddy virus

<400>87

<210>88

<211>22

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>88

<210>89

<211>22

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>89

<210>90

<211>22

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>90

<210>91

<211>22

<212>PRT

＜213〉rat plug Le Shi sample virus (Rat thei1er ' s like virus)

<400>91

<210>92

<211>22

<212>PRT

＜213〉Ljungan virus (Ljungan virus)

<400>92

<210>93

<211>22

<212>PRT

＜213〉Ljungan virus

<400>93

<210>94

<211>22

<212>PRT

＜213〉Ljungan virus

<400>94

<210>95

<211>22

<212>PRT

＜213〉Ljungan virus

<400>95

<210>96

<211>22

<212>PRT

＜213〉trypanosoma bocagei (Trypanosoma brucei)

<400>96

<210>97

<211>22

<212>PRT

＜213〉Ku Shi trypanosomicide (Trypanosoma Cruzi)

<400>97

<210>98

<211>22

<212>PRT

＜213〉bovine rota (Bovine rotavirus)

<400>98

<210>99

<211>22

<212>PRT

＜213〉Human reoviruslike agent (Human rotavirus)

<400>99

<210>100

<211>23

<212>PRT

＜213〉porcine rotavirus (Porcine rotavirus)

<400>100

<210>101

<211>22

<212>PRT

＜213〉DCV (Drosophila C virus)

<400>101

<210>102

<211>22

<212>PRT

＜213〉cricket paralysis virus (Cricket paralysis virus)

<400>102

<210>103

<211>22

<212>PRT

＜213〉acute Apis paralysis virus (Acute bee paralysis virus)

<400>103

<210>104

<211>22

<212>PRT

＜213〉cricket paralysis virus

<400>104

<210>105

<211>22

<212>PRT

＜213〉Ficus microcarpa Linn. f clearwing acute tonsillitis Microrna sample virus (Perina nuda picorna like virus)

<400>105

<210>106

<211>22

<212>PRT

＜213〉tea geometrid caryogram Microrna sample virus (Ectropis obliqua picorna like virus)

<400>106

<210>107

<211>22

<212>PRT

＜213〉Ficus microcarpa Linn. f clearwing acute tonsillitis Microrna sample virus

<400>107

<210>108

<211>22

<212>PRT

＜213〉tea geometrid caryogram Microrna sample virus

<400>108

<210>109

<211>22

<212>PRT

＜213〉vestigial wing virus (Deformed wing virus)

<400>109

<210>110

<211>22

<212>PRT

＜213〉Kakugo virus

<400>110

<210>111

<211>4

<212>PRT

＜213〉Xi Nika paddy virus

<400>111

<210>112

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: exemplary conservative motif

<220>

<221>MOD_RES

<222>(2)..(3)

＜223〉variable residue

<220>

<221>MOD_RES

<222>(5)

＜223〉variable residue

<400>112

<210>113

<211>6

<212>PRT

＜213〉Xi Nika paddy virus

<400>113

<210>114

<211>4

<212>PRT

＜213〉Xi Nika paddy virus

<400>114

<210>115

<211>4

<212>PRT

＜213〉Xi Nika paddy virus

<400>115

<210>116

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic

The 6-His labelled peptide

<400>116

<210>117

<211>4

<212>PRT

＜213〉Xi Nika paddy virus

<400>117

<210>118

<211>4

<212>PRT

＜213〉Xi Nika paddy virus

<400>118

<210>119

<211>4

<212>PRT

＜213〉Xi Nika paddy virus

<400>119

<210>120

<211>4

<212>PRT

＜213〉Xi Nika paddy virus

<400>120

<210>121

<211>4

<212>PRT

＜213〉Xi Nika paddy virus

<400>121

<210>122

<211>4

<212>PRT

＜213〉Xi Nika paddy virus

<400>122

<210>123

<211>4

<212>PRT

＜213〉Xi Nika paddy virus

<400>123

<210>124

<211>4

<212>PRT

＜213〉Xi Nika paddy virus

<400>124

<210>125

<211>4

<212>PRT

＜213〉encephalomyocarditis virus

<400>125

<210>126

<211>4

<212>PRT

＜213〉encephalomyocarditis virus

<400>126

<210>127

<211>4

<212>PRT

＜213〉encephalomyocarditis virus

<400>127

<210>128

<211>4

<212>PRT

＜213〉encephalomyocarditis virus

<400>128

<210>129

<211>4

<212>PRT

＜213〉encephalomyocarditis virus

<400>129

<210>130

<211>4

<212>PRT

＜213〉encephalomyocarditis virus

<400>130

<210>131

<211>4

<212>PRT

＜213〉encephalomyocarditis virus

<400>131

<210>132

<211>4

<212>PRT

＜213〉encephalomyocarditis virus

<400>132

<210>133

<211>4

<212>PRT

＜213〉encephalomyocarditis virus

<400>133

<210>134

<211>4

<212>PRT

＜213〉encephalomyocarditis virus

<400>134

<210>135

<211>4

<212>PRT

＜213〉encephalomyocarditis virus

<400>135

<210>136

<211>4

<212>PRT

＜213〉encephalomyocarditis virus

<400>136

<210>137

<211>4

<212>PRT

＜213〉encephalomyocarditis virus

<400>137

<210>138

<211>4

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus (Theiler ' s encephalomyelitis virus)

<400>138

<210>139°

<211>4

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>139

<210>140

<211>4

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>140

<210>141

<211>4

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>141

<210>142

<211>4

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>142

<210>143

<211>4

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>143

<210>144

<211>4

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>144

<210>145

<211>4

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>145

<210>146

<211>4

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>146

<210>147

<211>4

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>147

<210>148

<211>4

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>148

<210>149

<211>4

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>149

<210>150

<211>4

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>150

<210>151

<211>4

<212>PRT

＜213〉plug Le Shi murine encephalomyelitis virus

<400>151

<210>152

<211>4

<212>PRT

＜213〉rat Theilo virus (Rat Theilovirus)

<400>152

<210>153

<211>4

<212>PRT

＜213〉rat Theilo virus

<400>153

<210>154

<211>4

<212>PRT

＜213〉rat Theilo virus

<400>154

<210>155

<211>4

<212>PRT

＜213〉rat Theilo virus

<400>155

<210>156

<211>4

<212>PRT

＜213〉rat Theilo virus

<400>156

<210>157

<211>4

<212>PRT

＜213〉rat Theilo virus

<400>157

<210>158

<211>4

<212>PRT

＜213〉rat Theilo virus

<400>158

<210>159

<211>4

<212>PRT

＜213〉rat Theilo virus

<400>159

<210>160

<211>4

<212>PRT

＜213〉rat Theilo virus

<400>160

<210>161

<211>4

<212>PRT

＜213〉rat Theilo virus

<400>161

<210>162

<211>4

<212>PRT

＜213〉rat Theilo virus

<400>162

<210>163

<211>4

<212>PRT

＜213〉Vilyuisk people's encephalomyelitis virus (Vilyuisk human encephalomyelitis virus)

<400>163

<210>164

<211>4

<212>PRT

＜213〉Vilyuisk people's encephalomyelitis virus

<400>164

<210>165

<211>4

<212>PRT

＜213〉Vilyuisk people's encephalomyelitis virus

<400>165

<210>166

<211>4

<212>PRT

＜213〉Vilyuisk people's encephalomyelitis virus

<400>166

<210>167

<211>4

<212>PRT

＜213〉Vilyuisk people's encephalomyelitis virus

<400>167

<210>168

<211>7310

<212>DNA

＜213〉Xi Nika paddy virus

<400>168

<210>169

<211>2181

<212>PRT

＜213〉Xi Nika paddy virus

<400>169

<210>170

<211>1560

<212>DNA

＜213〉Xi Nika paddy virus

<400>170

<210>171

<211>1560

<212>DNA

＜213〉picornavirus

<220>

<221>misc_feature

<222>(8)..(8)

＜223〉n is a, c, g or t

<220>

<221>misc_feature

<222>(51)..(51)

＜223〉n is a, c, g or t

<400>171

<210>172

<211>1560

<212>DNA

＜213〉picornavirus

<400>172

<210>173

<211>936

<212>DNA

＜213〉picornavirus

<400>173

<210>174

<211>936

<212>DNA

＜213〉picornavirus

<400>174

<210>175

<211>936

<212>DNA

＜213〉picornavirus

<400>175

<210>176

<211>936

<212>DNA

＜213〉picornavirus

<400>176

<210>177

<211>936

<212>DNA

＜213〉picornavirus

<400>177

<210>178

<211>1421

<212>DNA

＜213〉artificial sequence

<220>

＜223〉from SVV and SVV sample picornavirus VP2 (part), VP3, the coding region of VP1 and 2A (part)

Concensus sequence in the sequence

<400>178

<210>179

<211>551

<212>DNA

＜213〉Xi Nika paddy virus

<400>179

<210>180

<211>551

<212>DNA

＜213〉picornavirus

<400>180

<210>181

<211>551

<212>DNA

＜213〉picornavirus

<400>181

<210>182

<211>523

<212>DNA

＜213〉artificial sequence

<220>

＜223〉from the concensus sequence at the part coding region sequence of 2C of SVV and SVV sample picornavirus

<400>182

<210>183

<211>460

<212>DNA

＜213〉Xi Nika paddy virus

<400>183

<210>184

<211>460

<212>DNA

＜213〉picornavirus

<220>

<221>misc_feature

<222>(19)..(19)

＜223〉n is a, c, g or t

<220>

<221>misc_feature

<222>(22)..(22)

＜223〉n is a, c, g or t

<400>184

<210>185

<211>460

<212>DNA

＜213〉picornavirus

<220>

<221>misc_feature

<222>(40)..(40)

＜223〉n is a, c, g or t

<220>

<221>misc_feature

<222>(388)..(388)

＜223〉n is a, c, g or t

<220>

<221>misc_feature

<222>(403)..(403)

＜223〉n is a, c, g or t

<400>185

<210>186

<211>460

<212>DNA

＜213〉picornavirus

<400>186

<210>187

<211>460

<212>DNA

＜213〉picornavirus

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉n is a, c, g or t

<400>187

<210>188

<211>460

<212>DNA

＜213〉picornavirus

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉n is a, c, g or t

<220>

<221>misc_feature

<222>(19)..(19)

＜223〉n is a, c, g or t

<220>

<221>misc_feature

<222>(28)..(28)

＜223〉n is a, c, g or t

<220>

<221>misc_feature

<222>(420)..(420)

＜223〉n is a, c, g or t

<220>

<221>misc_feature

<222>(422)..(422)

＜223〉n is a, c, g or t

<400>188

<210>189

<211>460

<212>DNA

＜213〉picornavirus

<400>189

<210>190

<211>460

<212>DNA

＜213〉picornavirus

<400>190

<210>191

<211>420

<212>DNA

＜213〉artificial sequence

<220>

＜223〉from consistent at the part coding region sequence in 3D polymerase and 3 ' UTR district of SVV and SVV sample picornavirus

Sequence

<400>191

<210>192

<211>4

<212>PRT

＜213〉picornavirus

<400>192

<210>193

<211>4

<212>PRT

＜213〉picornavirus

<400>193

<210>194

<211>4

<212>PRT

＜213〉picornavirus

<400>194

<210>195

<211>4

<212>PRT

＜213〉picornavirus

<400>195

<210>196

<211>4

<212>PRT

＜213〉picornavirus

<400>196

<210>197

<211>4

<212>PRT

＜213〉picornavirus

<400>197

<210>198

<211>4

<212>PRT

＜213〉picornavirus

<400>198

<210>199

<211>4

<212>PRT

＜213〉picornavirus

<400>199

<210>200

<211>4

<212>PRT

＜213〉picornavirus

<400>200

<210>201

<211>4

<212>PRT

＜213〉picornavirus

<400>201

<210>202

<211>4

<212>PRT

＜213〉picornavirus

<400>202

<210>203

<211>4

<212>PRT

＜213〉picornavirus

<400>203

<210>204

<211>4

<212>PRT

＜213〉picornavirus

<400>204

<210>205

<211>4

<212>PRT

＜213〉picornavirus

<400>205

<210>206

<211>4

<212>PRT

＜213〉picornavirus

<400>206

<210>207

<211>4

<212>PRT

＜213〉picornavirus

<400>207

<210>208

<211>4

<212>PRT

＜213〉picornavirus

<400>208

<210>209

<211>4

<212>PRT

＜213〉picornavirus

<400>209

<210>210

<211>4

<212>PRT

＜213〉picornavirus

<400>210

<210>211

<211>4

<212>PRT

＜213〉picornavirus

<400>211

<210>212

<211>4

<212>PRT

＜213〉picornavirus

<400>212

<210>213

<211>4

<212>PRT

＜213〉picornavirus

<400>213

<210>214

<211>4

<212>PRT

＜213〉picornavirus

<400>214

<210>215

<211>4

<212>PRT

＜213〉picornavirus

<400>215

<210>216

<211>4

<212>PRT

＜213〉picornavirus

<400>216

<210>217

<211>4

<212>PRT

＜213〉picornavirus

<400>217

<210>218

<211>4

<212>PRT

＜213〉picornavirus

<400>218

<210>219

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used for the primer sequence of Xi Nika paddy virus

<400>219

<210>220

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used for the primer sequence of Xi Nika paddy virus

<400>220

<210>221

<211>54

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used to generate the infectious plasmid clone's of Xi Nika paddy virus primer sequence

<400>221

<210>222

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used to generate the infectious plasmid clone's of Xi Nika paddy virus primer sequence

<400>222

<210>223

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used to generate the infectious plasmid clone's of Xi Nika paddy virus primer sequence

<400>223

<210>224

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used to generate the primer sequence of the infection clones of Xi Nika paddy virus

<400>224

<210>225

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used to generate the infectious plasmid clone's of Xi Nika paddy virus primer sequence

<400>225

<210>226

<211>76

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used to generate the infectious plasmid clone's of Xi Nika paddy virus primer sequence

<400>226

<210>227

<211>47

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used to generate the infectious plasmid clone's of Xi Nika paddy virus primer sequence

<400>227

Claims (37)

1. isolating nucleic acid comprises: with (i) SEQ ID NO:168 or (ii) the length of the SEQ ID NO:168 continuous part that is at least 20 nucleotide have at least 75% conforming nucleotide sequence.

2. the isolating nucleic acid of claim 1, wherein this nucleic acid is RNA or DNA.

3. isolating polypeptide comprises: with (i) SEQ ID NO:169 or (ii) the length of SEQ IDNO:169 be at least 10 amino acid whose continuous parts and have at least 75% conforming aminoacid sequence.

4. isolating Xi Nijia paddy virus or its derived virus or its correlated virus, it has following genome, and described genome comprises with SEQ ID NO:168 and has at least 95%, 90%, 85%, 80%, 75%, 70% or 65% conforming sequence.

5. the virus of claim 4, it has following characteristics: the replication capacity in tumor cell, tumor cell tropism and do not have lysis in normal cell.

6. the virus of claim 5, wherein said virus has replication capacity in the tumor cell type with neuroendocrine characteristic.

7. pharmaceutical composition, it comprises among the claim 4-6 of effective dose each virus and pharmaceutically acceptable carrier.

8. isolated antibody, it is specifically in conjunction with the polypeptide of claim 3, or claim 4 or 5 each the epi-positions of isolating virus.

9. the purposes of virus in treatment of cancer, described virus has following genome, and the continuous sequence that is at least 100 nucleotide that described genome comprises with SEQ ID NO:168 has at least 75% conforming sequence.

10. the method for claim 9 should virus be a picornavirus wherein.

11. the method for claim 10, wherein this picornavirus is a Cardioviruses.

12. the method for claim 11, wherein this Cardioviruses is selected from: vilyuisk people's encephalomyelitis virus, plug Le Shi murine encephalomyelitis virus and encephalomyocarditis virus.

13. the method for claim 12, wherein this picornavirus is the member of the genus under the Xi Nijia paddy virus.

14. the method for claim 10, wherein this picornavirus is a Xi Nika paddy virus.

15. the method for claim 10, wherein this picornavirus is a Xi Nika paddy virus sample picornavirus.

16. the method for claim 15, wherein this Xi Nika paddy virus sample picornavirus is selected from: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395, LA 1278, IL 66289, IL 94-9356, MN/GA 99-29256, MN99197 and SC 363649.

17. kill the method for the cell of abnormality proliferation, comprise this cell is contacted with each virus among the claim 4-6.

18. prepare the method for tumor cell cracking performance virus, this method comprises:

(a) the relatively genome sequence of Xi Nika paddy virus and the genome sequence of virus to be measured, the genome of its Chinese and Western Ni Kagu virus comprise with SEQ ID NO:168 and have at least 95% conforming sequence;

(b) identify at least first amino acid whose difference between the genome sequence encoded polypeptides of the genome sequence encoded polypeptides of Xi Nika paddy virus and virus to be measured;

(c) genome sequence of sudden change virus to be measured makes and to reduce 1 at least by the amino acid whose difference between the polypeptide of the genome encoding of the polypeptide of the group group sequential coding of virus to be measured and Xi Nika paddy virus;

(d) the genome sequence transfection of the virus to be measured after will suddenling change is advanced in the tumor cell; With

Whether (e) measure tumor cell is infected by the genome sequence cracking performance ground of the virus to be measured after the sudden change.

19. the method for claim 18, wherein this virus to be measured is picornavirus.

20. the method for claim 19, wherein this virus to be measured is Xi Nika paddy virus sample picornavirus.

21. the method for claim 18, wherein this aminoacid difference is the aminoacid difference between Xi Nika paddy viral capsid proteins and the viral capsid proteins to be measured.

22. the method for claim 18, the genome sequence of the virus to be measured of wherein suddenling change comprise that the cDNA to the genome sequence with virus to be measured suddenlys change.

23. the method for claim 18, wherein the genome sequence of the virus to be measured after the transfection sudden change comprises transfection RNA, and wherein RNA produces the cDNA of the genome sequence of the virus to be measured after having sudden change.

24. preparation has the mutant virus of the cell type tropism of change, this method comprises:

(a) create the virus mutant library of containing multiple nucleotide sequence, wherein create from following parental array in this virus mutant library, described parental array comprise and the length of the SEQ ID NO:168 continuous part that is at least 100 nucleotide have at least 75% conforming nucleotide sequence;

(b) with this virus mutant library transfection to allowing in the cell, thereby produce the various mutations precursor virus;

(c) separate the various mutations precursor virus;

(d) nonpermissive cell and isolating various mutations precursor virus are hatched; With

(e) reclaim the mutant virus that produces in the nonpermissive cell, preparation has the mutant virus of the tropism of change in view of the above.

25. the method for claim 24, it also comprises:

(f) mutant virus that reclaims is hatched in nonpermissive cell; With

(g) be recovered in the mutant virus that produces in the nonpermissive cell.

26. the method for claim 25, it also comprises repeatedly repeating step (f) and (g).

27. the method for claim 25 is wherein hatched in porous high flux platform, wherein this platform contains different nonpermissive cell types in each hole.

28. the method for claim 27, it comprises that also this platform of screening contains the hole of the mutant virus of cell killing with evaluation.

29. the method for claim 28 is wherein screened by the absorbance of analyzing each hole.

30. the method for claim 24, wherein nonpermissive cell is a tumor cell.

31. the method for claim 24 is wherein created the virus mutant library and is comprised:

(i) provide the polynucleotide that have with the corresponding to sequence of part of parental array;

(ii) these polynucleotide that suddenly change in order to generate multiple different mutant polynucleotide sequence; With

(iii) the polynucleotide of various mutations are connected in the carrier with the virus genome sequence except the contained virus genome sequence part of the polynucleotide of step (i), create the virus mutant library in view of the above.

32. the method for claim 31, wherein in step (i), this virus genome sequence is from picornavirus.

33. the method for claim 32, wherein picornavirus is a Xi Nijia paddy virus sample picornavirus.

34. the method for claim 31 is wherein carried out step sudden change (ii) by nucleotide is inserted at random in the polynucleotide.

35. the method for claim 34 is wherein carried out step sudden change (ii) in the capsid coding region of polynucleotide.

36. the method for claim 34 is wherein carried out the insertion at random of nucleotide by trinucleotide mutagenesis (TRIM).

37. the method for claim 36 is wherein inserted at least a portion coding epi-position label of the nucleotide in the polynucleotide.

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