CN1654665A - Attenuated vaccinia virus Tiantan strain vector and its preparation and application - Google Patents
Attenuated vaccinia virus Tiantan strain vector and its preparation and application Download PDFInfo
- Publication number
- CN1654665A CN1654665A CN 200510018129 CN200510018129A CN1654665A CN 1654665 A CN1654665 A CN 1654665A CN 200510018129 CN200510018129 CN 200510018129 CN 200510018129 A CN200510018129 A CN 200510018129A CN 1654665 A CN1654665 A CN 1654665A
- Authority
- CN
- China
- Prior art keywords
- virus
- vaccinia virus
- cell
- attenuated
- attenuated vaccinia
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention discloses one attenuated vaccinia virus Tiantan plant carrier and its preparation process and application, and the attenuated vaccinia virus is obviously attenuated TT.WHU.pZCI, CCTCC: V200416. The preparation process includes the following steps: A. constituting shuttle plasmid containing flanking sequence homogenous with the late promoter sequence or two flanking sequences of the vaccinia virus Tiantan plant HA gene; and B. homogenously recombining the shuttle plasmid and the vaccinia virus Tiantan plant to deactivate vaccinia virus HA gene part for obviously attenuating the vaccinia virus Tiantan plant. The attenuated vaccinia virus Tiantan plant carrier is used in preparing vaccine for treating or preventing infectious diseases.
Description
Technical field
The present invention relates to a kind of attenuated vaccinia virus Tiantan strain vector, they are derived from vaccinia virus Tiantan strain, and their feature is to lose or reduced breeding replication in zooblast, has reduced the toxicity of pair cell; The distinctive neurotoxicity of the characteristic that has shown remarkable attenuation in animal body, especially vaccinia virus significantly reduces.The present invention also relates to the preparation method of attenuated vaccinia virus Tiantan strain vector simultaneously.Remarkable attenuated vaccinia virus carrier of the present invention will have higher security than wild vaccinia virus Tiantan strain, can be used for making up human or animal's live vector vaccine; This attenuated vaccinia virus carrier also can be further used for developing the medicine of therapy of tumor or prevention; Or as expression of exogenous gene carrier, scale operation target protein; Or be used to express foreign pathogens antigen prepd pathogen detection and diagnostic reagent.
Background technology
Vaccinia virus (Vaccinia Virus) belongs to the Poxviridae orthomyxovirus and belongs to, once be used as the vaccine that the prevention smallpox infects and worldwide be used widely, make smallpox become the pernicious transmissible disease (Riva that first is become extinct fully by the mankind, G.et al., 1979.A milestone in the history of mankind.Eradication of smallpox.MMW Munch Med Wochenschr, 121 (50), 1669-70).Recent two decades comes, fast development along with genetic engineering technique, vaccinia virus is used to the research and design expression vector, or the research (Moss of the live-virus vaccine that is used to recombinate, B.1996.Genetically engineeredpoxviruses for recombinant gene expression, vaccination, and safety.ProcNatl Acad Sci USA 93 (21), 11341-8; Paoletti, E.et al., 1996.Applications ofpox virus vectors to vaccination:an update.Proc Natl Acad Sci USA 93 (21), 11349-53).Existing multiple vaccinia virus recombinant vaccine can successfully be expressed the antigen of viruses such as rabies virus, japanese encephalitis virus and is used for zoonotic control (Kieny, M.P.et al., 1984.Expressionof rabies virus glycoprotein from a recombinant vaccinia virus.Nature312 (5990), 163-6; Konishi, E.et al., 1992.A highly attenuated hostrange-restricted vaccinia virus strain, NYVAC, encoding the prM, E, and NS1genes of Japanese encephalitis virus prevents JEV viremia in swine.Virology190 (1), 454-8).Human reorganization live-virus vaccine is also obtained many impressive progresses, existing measles, EBV, HAV, HBV, HPV, HIV and the rabies virus etc. of comprising have carried out preliminary human immunity observation (Guo Fei etc., 2001, the non-vaccinia virus recombinant the Temple of Heaven strain C-K disappearance of duplicating is distinguished the structure of expression vector and the research of recombinant virus biological character.Virus journal 17 (1), 24-28; McMichael, A.et al., 2002.The quest for anAIDS vaccine:is the CD8+T-cell approach feasible? Nat Rev Immunol 2 (4), 283-91; Moss, engineered poxviruses for recombinantgene expression B.1996.Genetically, vaccination, and safety.Proc Natl Acad Sci USA 93 (21), 11341-8; Zhu, J.H.et at., 1996.Immunogenicity and relative attenuation ofdifferent vaccinia-rabies virus recombinants.Arch Virol 141 (6), 1055-65).
Vaccinia virus Tiantan strain (Tian Tan, TT) be Chinese distinctive vaccinia virus strain, the infectious vaccine strain of Ceng Zuowei prevention smallpox used in a large amount of crowd of the China midium or long term, having weak, the safe in utilization characteristics of relative virus force, is that development is applicable to the genetically engineered virus carrier of China's application and the desirable strain of live recombined vaccines.Yet, although rare but severe side effect very that vaccinia virus produces, be the important security obstacle (Wollenberg that vaccinia virus is used as carrier, A.at al., 2004.Smallpox, vaccinationand adverse reactions to smallpox vaccine.Curr Opin Allergy Clin Immunol4 (4), 271-5.), the method of using gene engineering is to the further attenuation of the Temple of Heaven strain, with reduce it in zooblast in (in Vitro) and the animal body toxicity (virulence) of (in Vivo) be the fundamental way that addresses this problem.
(Hemagglutinin is a kind of glycosylation Nonstructural Protein of virus HA) to the hemagglutinin of vaccinia virus, and molecular weight is 85KD.The expression of HA gene (A56R) is subjected to the control of two promotors in early stage and late period; but the 85KD albumen that the blood clotting function is arranged of its expression accumulates usually in (Brown in the late period of viral gene expression; C.K.et al.; 1991.Molecular characterization of the vaccinia virus hemagglutiningene.J Virol 65 (7), 3598-606).HA albumen mainly is expressed in the surface of infected cell, also appear at simultaneously born of the same parents' peplos virus (Extracellular enveloped virus of vaccinia virus, EEV) (Payne on the adventitia, L.G.et al., 1979.Identification of the vaccinia hemagglutininpolypeptide from a cell system yielding large amounts of extracellularenveloped virus.J Virol 31 (1), 147-55; Payne, L.G.et al., 1985.Extracellularrelease of enveloped vaccinia virus from mouse nasal epithelial cells in vivo.JGen Virol 66 (Pt3), 643-6).The HA gene is the dispensable gene that vaccinia virus is duplicated in cell, be proved the insertion site that can be used as foreign gene, the foreign gene that inserts this position can both obtain stable expression (Antoine with external in vivo, G.et al., 1996.Characterization of the vacciniaMVA hemagglutinin gene locus and its evaluation as an insertion site forforeign genes.Gene 177 (1-2), 43-6).The HA gene is not fully aware of in the definite function of vaccinia virus in life cycle and effect, but there is experiment to show, inactivation HA gene can cause vaccinia virus WR strain attenuation (Flexner in animal body, C.et al., 1987.Prevention of vaccinia virus infectionin immunodeficient mice by vector-directed IL-2 expression.Nature 330 (6145), 259-62).Shida etc. also confirm, disappearance HA gene can cause vaccinia virus Lister strain (LO) or its deutero-LC16mO strain attenuation, but the degree of its attenuation is far below WR strain (Shida, H.et al., 1988.Effects and virulences of recombinant vaccinia viruses derived from attenuatedstrains that express the human T-cell leukemia virus type I envelope gene.JVirol 62 (12), 4474-80).This shows that the difference of genetic background might cause the huge difference on the attenuation effect between different vaccinia virus strains system.
The genome sequencing of China's vaccinia virus Tiantan strain was finished in 1997, it is for studying and illustrating vaccinia virus Tiantan strain unique biological characteristic and the genetic engineering modified important background information (Jin Qi etc. that provide are provided, 1997, the analysis of the full genome structure characteristics of vaccinia virus Tiantan strain.Chinese science (C collects) 27 (6), 562-567).To studies show that of the Temple of Heaven pnca gene structure, there are bigger difference in it and other vaccinia virus strain WR strain and Copenhagen strain etc., these differences mainly are distributed in its genomic LHA (B of Hind III and C fragment), on the A fragment, also find to have the phenomenon (Chen Nanhai etc. of great variation, 1992, the difference of China's vaccinia virus Tiantan strain and WR pnca gene group mainly is distributed in its LHA.Virus journal 8 (4), 303-308).The degree of these variations of the Temple of Heaven strain and scope are unexistent in several vaccinia viruss of being studied at present.Sequential analysis to the HA gene (A56R) of the Temple of Heaven strain is found, with respect to the WR strain, there is the variation of 1.2% structure gene in it on nucleotide level, 2.5% aberration rate (Li Zhiliang etc., 1989. China's vaccinia virus Tiantan strain hemagglutinin gene complete sequence analysis are arranged on amino acid levels.Virus journal 5 (1), 2-9).The variation of the Temple of Heaven pnca gene group structure has shown that it is different from the genetic background of the uniqueness of other vaccinia virus strain, and the genetic background of this uniqueness might cause comprising the variation of the various biological characteristicses of virulence.
Before more than ten years, the Temple of Heaven strain be studied be used to be transformed into expression vector express different foreign genes (peak etc., 1992, the structure of hepatitis A vaccinia virus recombinant (VMS11HAV25) is some biological characteristics extremely.Virus journal 8 (2), 110-117; Horse Helen etc., 1999, express the structure of the non-replicating vaccinia virus recombinant of various exogenous genes simultaneously.Virus journal 15 (1), 21-28; Xushui the moon etc., 1998, Measles virus fusion rotein (F) and the expression of hemagglutinin (HA) in vaccinia virus.Virus journal 9 (4), 301-307; Gu Shuyan etc., 1988, express the establishment of hepatitis B virus surface antigen and antigenic pair of valency vaccinia virus of Epstein-Barr viromembrane.Virus journal 4 (1), 1-8; Ruan Li etc., 1992, respiratory syncytial virus glycoprotein F and the G expression in same vaccinia virus recombinant.Virus journal 8 (2), 101-109; Guo Fei etc., 2001.The non-vaccinia virus recombinant the Temple of Heaven strain C-K disappearance of duplicating is distinguished the structure of expression vector and the research of recombinant virus biological character.Virus journal 17 (1), 24-28).The site that is used to insert foreign gene mainly comprise thymidine kinase gene (thymidine kinase, TK), C～K fragment district of HA gene and Hind III etc.Wherein, the gene of existing multiple HIV-1 is inserted into the HA gene locus of the Temple of Heaven strain, experimental result shows that the foreign gene that inserts this site of the Temple of Heaven strain all can obtain effective and stable expression (Wang Yuhong etc. with external in vivo, 2000, coexpression HIV-1 env and hL-6 gene in the vaccinia virus recombinant.China Amphixenosis magazine, 16 (4), 22-24; Wang Yuhong etc., 1999, the coexpression research in vaccinia virus recombinant of HIV-1env and hL-2 gene.Journal of Immunology 15 (4), 217-219; Mao Chunsheng etc., 1996, the structure of HIV-1 gag gene recombination vaccinia virus and efficiently expressing.China's animal doctor journal 16 (6), 562-569), but relevant recombinant virus does not have the toxicity of organism and its security as potential vaccine use and studies and experimental results show that.Simultaneously, any other experimental result or research are not arranged so far yet, show by inactivation or the disappearance HA can cause vaccinia virus Tiantan strain in vivo with external attenuation.
In order to obtain the safer vaccinia virus vector that originates from the Temple of Heaven strain, we are at the HA gene of vaccinia virus Tiantan strain, and the variation at its late promoter sequence area introducing genetic sequence causes the HA Gene Partial to lose function,, obtained the Temple of Heaven vaccinia virus body of remarkable attenuation.We also simultaneously, systematic research for the first time the biological characteristics that in zooblast, duplicates of attenuation the Temple of Heaven vaccinia virus vector, and the toxicity of (in the animal model) in vivo, important and foundation science of this attenuated virus as the carrier safe handling is provided thus.Significantly the Temple of Heaven vaccinia virus of attenuation will be safer, ideal virus vector, can be widely used in the live vector recombinant vaccine that makes up humans and animals, and comprise the gene therapy of the human diseases of cancer; In addition, this carrier also can be used as the expression of exogenous gene carrier, the scale operation target protein, or be used to express foreign pathogens antigen, preparation pathogen detection and diagnostic reagent.
Summary of the invention
The object of the present invention is to provide a kind of attenuated vaccinia virus Tiantan strain vector, this carrier has limited host range, comprises showing in the humanization cell that the breeding replication reduces in most cells, and diffusion levels descends, and the toxicity of pair cell alleviates.This carrier is also shown in the decline of animal toxicity in vivo simultaneously, and especially the distinctive neurotoxicity of vaccinia virus greatly descends.Therefore, this carrier is safer more reliable than wild the Temple of Heaven virus, can effectively prevent and reduce the side effect that vaccinia virus produces, and comprises the generation of postvaccinal encephalitis; Also can prevent simultaneously the STOCHASTIC DIFFUSION of virus vector in crowd and environment, not inoculate the crowd and prevent the pollution of the environment with protection.
Another object of the present invention is to provide a kind of method for preparing attenuated vaccinia virus Tiantan strain vector, and this method is easy, and is easy to operate, is easy to obtain the vaccinia virus vector of safety, remarkable attenuation.Simultaneously, the attenuated vaccinia virus carrier that obtains with this method has also shown the high stability that foreign gene inserts and expresses in continuous passage.
The invention still further relates to the application of attenuated vaccinia virus Tiantan strain vector in the vaccine of preparation treatment or infection prevention characteristic of disease disease.
The invention still further relates to the application of attenuated vaccinia virus Tiantan strain vector in the medicine of preparation treatment or prophylaxis of tumours.
The invention still further relates to the application of attenuated vaccinia virus Tiantan strain vector in expression alien gene.
The invention still further relates to the application of attenuated vaccinia virus Tiantan strain vector in expressing foreign pathogens antigen prepd pathogen detection and diagnostic reagent.
In order to realize above-mentioned task, provide new vaccinia virus in the preferred embodiment of the present invention, it is derived from vaccinia virus Tiantan strain.Vaccinia virus Tiantan strain in the applicating histories of state-owned decades, China begins to produce Smallpox Vaccine since the twenties in last century, uses the Temple of Heaven strain to make the bovine vaccine seed culture of viruses always.The original vaccinia virus seed culture of viruses that the present invention uses is the vaccine for man strain the Temple of Heaven 761 that China produces.New vaccinia virus provided by the invention because there is the change at least one place gene order in the late promoter sequence of the HA gene in its genome, causes HA gene function part inactivation; New vaccinia virus has the limited replication of reduction in the most animals cell, or loses replication fully, and/or has reduced the toxicity to zooblast; It shows the feature of remarkable attenuation in the animal pattern mouse, this attenuated vaccinia virus shows that also neurotoxicity declines to a great extent, and it has also shown the high stability that foreign gene inserts and expresses in continuous passage.The present invention also provides the method for the Temple of Heaven vaccinia virus that can obtain remarkable attenuation, the carrier that is further used as a kind of security with this attenuated vaccinia virus is applied to the structure of human or animal's vaccine, or be applied to genetic treatment of tumor as the gene transfer vector of security, or as expression of exogenous gene carrier scale operation target protein, or be used to express foreign pathogens antigen prepd pathogen detection and diagnostic reagent.
The new vaccinia virus vector that will obtain: remarkable attenuated vaccinia virus (TT.WHU.pZCI), depositary institution: Chinese typical culture collection center, address: China. Wuhan. Wuhan University, preservation date: on September 17th, 2004, deposit number: CCTCC NO:V200416.TT.WHU.pZCI is abbreviated as pZCI in this article.
The present invention also finds, different loci at vaccinia virus Tiantan strain HA gene late promoter is introduced the extraneous nucleotide sequence, or make the late promoter disappearance, and all can cause the part inactivation of HA gene function, obtain the remarkable attenuated vaccinia virus identical with the pZCI feature.Therefore, the late promoter of HA gene is carried out genetic modification, make all can obtain remarkable attenuated vaccinia virus by HA gene function part inactivation.
Term " genetic modification " is meant, by engineered method the nucleotide sequence of original species increased, reduces or change operations such as genetic sequence.
The copy feature in cell for the ease of research and description vaccinia virus is at first carried out following introduction to several specific targets of experiment.
The replication of virus is meant the breeding replication of virus, calculates in accordance with the following methods usually: the virus quantity that produces within a certain period of time behind the virus infected cell and the ratio of the input virus quantity of cells infected are the propagation multiple of virus in cell.The present invention with virus infected cell after 72 hours output (virus titre, titre) than the input virus quantity of last cells infected as the standard of judging virus replication in this cell, and with the method pair cell of the Moss B (Carroll that classifies, M.W., and Moss, B.1997.Host rangeand cytopathogenicity of the highly attenuated MVA strain of vaccinia virus:propagation and generation of recombinant viruses in a nonhumanmammalian cell line.Virology 238 (2), 198-211), that is:
Nonpermissive cell (nonpermissive cell, NP):＜1 times virus replication increases multiple, and virus is at this
Lack the reproductive ability replication in the cell;
(semipermissive cell, SP): 1～25 times virus replication increases multiple, virus to semipermissive cell
In this cell, have limited reproductive ability and duplicate energy
Power;
(permissive cell, P):＞25 times virus replication increases multiple to allow cell.Virus is at this cell
In have the reproductive ability replication.
Virus is that virus is duplicated a kind of key property of growth in cell in the ability of iuntercellular diffusion.Measure wild the Temple of Heaven strain virus and the velocity of diffusion of deutero-attenuation the Temple of Heaven virus vector in zooblast thereof with the method for immunostaining, further obtained attenuated vaccinia virus is different from wild the Temple of Heaven virus in zooblast duplication characteristic.The velocity of diffusion of virus in cell done following regulation, so that better describe and distinguish the difference of virus diffusibility in cell: with 0.01MOI (Multiplicity of infection, MOI) virus quantity cells infected, stop infecting at different time point (24 hours, 48 hours and 72 hours), pair cell carries out immunostaining, observes the process that virus infects and spreads in cell.The method classification of Moss B is pressed in the diffusion of virus in cell, promptly in 72 hours behind the virus infection, can painted cell count judge viral diffusibility (Carroll, M.W., and Moss, B.1997.Host range andcytopathogenicity of the highly attenuated MVA strain of vaccinia virus:propagation and generation of recombinant viruses in a nonhumanmammalian cell line.Virology 238 (2), 198-211):
Do not have cell to be colored in-72 hours, this cell can not be infected,
+＜5 cells are colored, and virus only has very low diffusibility,
++ 5～25 cells are colored, and virus has limited diffusibility in cell,
25 cells of +++＞are colored, and virus has higher diffusibility in cell.
The virus toxicity that pair cell produces when duplicating in cell be called as cytopathic effect (cytopathiceffects, CPE).The toxicity of virus pair cell is the key property that virus is duplicated in cell, directly related with virus toxicity in animal body.The virus that produces less CPE at cell levels is lower to animal toxicity (virulence) usually.Therefore, in the velocity of diffusion of research virus, observed the ability of virus formation CPE in cell, comprised that virus forms the ability of plaque (plaque) in cell.Simultaneously, also observed virus especially under high infection multiplicity (5.0MOI), in zooblast, formed the situation of CPE, and described by following method:
Do not have cell to be colored in-72 hours, this cell can not be infected,
+＜5 cells are colored, and virus only has very low diffusibility,
++ 5～25 cells are colored, and virus has limited diffusibility in cell,
25 cells of +++＞are colored, and virus has higher diffusibility in cell.
The present invention has studied the duplication characteristic of attenuated vaccinia virus carrier pZCI in zooblast that is derived from vaccinia virus Tiantan strain, comprises kinetic curve, the velocity of diffusion of virus in cell and the toxicity of viral pair cell etc. of virus replication; To its comparing property of parent's vaccinia virus Tiantan strain experiment, obtained preferred attenuation the Temple of Heaven virus is different from the uniqueness of wild the Temple of Heaven virus in zooblast duplication characteristic thus under the same conditions.
For the copy feature of clearer and more definite understanding improvement the Temple of Heaven virus, at first summarize the duplication characteristic of vaccinia virus Tiantan strain in zooblast.Its characteristic is:
1. vaccinia virus Tiantan strain has host range (host range) widely, and it all has good breeding replication in 11 kinds of zooblasts being tested, and these cells all are the permission cells that vaccinia virus Tiantan strain duplicates.
2. vaccinia virus Tiantan strain all shows the speed of the diffusion of iuntercellular faster and the toxicity of quite serious pair cell in the zooblast of being tested.
Significantly attenuated vaccinia virus has shown and the diverse duplication characteristic of wild the Temple of Heaven virus, and its essential characteristic of duplicating in zooblast is: significantly the vaccinia virus of attenuation has limited host range in zooblast; In the most animals cell, (comprise in the humanization cell) and have levels of replication limited or that reduce; Has lower cell to intercellular diffusibility and/or littler cytotoxicity; Only in African green monkey kidney cell Vero and COS-7, has toxicity to the pair cell of the replication of wild the Temple of Heaven virus par, similar iuntercellular diffusion levels and certain degree.
Significantly the copy feature of attenuated vaccinia virus in zooblast specifically describes as follows:
1. have limited host range (host range) and in most cells, comprise reproductive ability levels of replication limited or that reduce in the humanization cell:
The vaccinia virus vector of attenuation can only carry out limited duplicate or losing replication fully in multiple zooblast, thereby has than the more limited host range of wild the Temple of Heaven virus.Concrete, significantly the vaccinia virus pZCI of attenuation is to lose the reproductive ability replication fully among the RK-13 (ATCC CCL-37) at rabbit kidney cell, its virus multiplication multiple in the RK-13 cell is less than 0.01, and it is low more than 40000 times to duplicate the propagation multiple than wild the Temple of Heaven vaccinia virus in this cell.RK-13 is the nonpermissive cell of attenuated virus pZCI.
Attenuated vaccinia virus only has limited breeding replication in multiple zooblast, these cells comprise that Madin-Darby canine kidney(cell line) (MDCK) is MDCK (ATCC CCL-34), mouse glioma brain tumour clone C6 (ATCCCCL-37), porcine kidney cell line PK (15) (ATCC CCL-33) and hamster kidney cell line BHK-21 childhood (ATCC CCL-10).Concrete, significantly attenuated vaccinia virus pZCI only has in MDCK, C6, PK (15) and BHK-21 greater than 1 times but is lower than 25 times replication, and its replication is respectively 8.3%, 1.9%, 1.2% and 16.9% of wild the Temple of Heaven virus.Thereby MDCK, C6, PK (15) and BHK-21 are the semipermissive cells of pZCI.
Preferred attenuated vaccinia virus has also shown the levels of replication of obvious decline in two kinds of humanization clone 293T (ATCC CRL11268) and MRC-5 (ATCC CCL-171), although the propagation multiple that duplicates of virus shows the permission cell that these two kinds of clones are attenuated virus.PZCI in human embryonic kidney cell line 293T duplicate multiple have only the Temple of Heaven strain in 293T 36.8%, and in people's embryo hepatic cell line MRC-5 duplicating propagation multiple have only 14.7% of the Temple of Heaven strain.
Attenuated vaccinia virus is at former generation chick-embryo cell (Chick embryo fibriblasts, CEF, self-control), human cervical carcinoma cell is HeLa (ATCC CCL-2), two kinds of African green monkey kidney cell Vero compare with wild the Temple of Heaven strain with the replication among the COS-7 does not have significant difference, more than three kinds of permission cells that cell is wild the Temple of Heaven virus and attenuated vaccinia virus.
2. has lower cell to intercellular diffusion levels
After tested, wild the Temple of Heaven virus all has very strong diffusibility in 11 kinds of zooblasts of test, all has greater than 25 cells in its 72 hours to be colored, and therefore the velocity of diffusion of wild the Temple of Heaven virus in these cells all counted +++.
Significantly attenuated vaccinia virus pZCI only shows the single cell that is colored in the RK-13 cell, shows that pZCI does not have diffusibility in this cell; In MDCK and PK (15), preferred attenuated virus pZCI only is less than 5 cells and is colored, and shows that pZCI only has very low diffusibility in these two kinds of cells.
In other two kinds of cell C6 and HeLa, the velocity of diffusion of attenuated vaccinia virus pZCI also is lower than wild the Temple of Heaven virus, and it is infected all only to be less than 25 cells in 72 hours, shows pZCI only limited diffusibility (++) in above two kinds of clones.
Although in BHK-21, MRC-5 and former generation CEF cell, preferred attenuated vaccinia virus pZCI all has higher diffusibility (+++), but in the identical time, the cell count that pZCI infects still obviously is less than the cell of wild the Temple of Heaven virus infection, shows that the velocity of diffusion of attenuated vaccinia virus pZCI in BHK-21, MRC-5 and CEF still obviously slow down than wild virus.
Attenuated vaccinia virus pZCI only has the velocity of diffusion similar to wild virus in HEKC 293T and African green monkey kidney cell Vero, COS-7, show that Vero, COS-7 and 293T all can effectively support the reproductive ability of wild the Temple of Heaven virus and improvement the Temple of Heaven virus to duplicate and spread.
Vaccinia virus Tiantan strain and the attenuated vaccinia virus propagation multiple that duplicates in zooblast sees Table 1.The diffusion in zooblast of vaccinia virus Tiantan strain and attenuated vaccinia virus sees Table 2.
Table 1 vaccinia virus Tiantan strain and attenuated vaccinia virus carrier duplicating in zooblast
| Clone | Kind | Tissue | Cellular form | Virus duplicate increment multiple * | |
| ?pZCI | ????TT | ||||
| ??CEF ??293T ??BHK-21 ??C6 ??COS-7 ??HeLa ??MDCK ??MRC-5 ??PK(15) ??RK13 ??Vero | ?Chick?embryo ?Human ?Hamster，Syrian ?Rat ?African?green?monkey ?Human ?Canine ?Human ?Pig ?Rabbit ?African?green?monkey | ?Assorted ?Kidney ?Kidney ?Brain；glial?cell；glioma ?Kidney ?Cervix ?Kidney ?Lung ?Kidney ?Kidney ?Kidney | ?Fibroblast ?Epithelial ?Fibroblast ?Fibroblast ?Fibroblast ?Epithelial ?Epithelial ?Fibroblast ?Epithelial ?Epithelial ?Epithelial | ?196.88(P) ?25.45(P) ?11.67(SP) ?2.97(SP) ?125(P) ?67.42(P) ?12(SP) ?27.5(P) ?1.16(SP) ?0.0031(NP) ?500(P) | ?128.13(P) ?69.09(P) ?62.50(P) ?156.67(P) ?160(P) ?63.64(P) ?145(P) ?187.50(P) ?100.83(P) ?131.25(P) ?562.5(P) |
*0.05MOI virus infection zooblast in blocks, collected sample at 0,24,48 and 72 hour respectively, in the Vero cell, measure the titre of virus.The propagation multiple of virus is the input virus quantity with cells infected on 72 hours the rate ratio behind the virus infected cell.Among the figure, cell is divided into nonpermissive cell, semipermissive cell and permission cell:
The virus replication of NP＜1 times increases multiple
The virus replication that SP is 1～25 times increases multiple
The virus replication of P＞25 times increases multiple
Table 2 vaccinia virus Tiantan strain and the diffusion of attenuated vaccinia virus carrier in zooblast
| Cell or clone | Kind | Tissue | Cellular form | The diffusion * of virus in cell | |
| ????pZCI | ????TT | ||||
| CEF 293T BHK-21 C6 COS-7 HeLa MDCK MRC-5 PK(15) RK13 Vero | ?Chick?embryo ?Human ?Hamster，Syrian ?Rat ?African?green?monkey ?Human ?Canine ?Human ?Pig ?Rabbit ?African?green?monkey | ?Assorted ?Kidney ?Kidney ?Brain；glial?cell；glioma ?Kidney ?Cervix ?Kidney ?Lung ?Kidney ?Kidney ?Kidney | ?Fibroblast ?Epithelial ?Fibroblast ?Fibroblast ?Fibroblast ?Epithelial ?Epithelial ?Fibroblast ?Epithelial ?Epithelial ?Epithelial | ????+++ ????+++ ????+++ ????++ ????+++ ????++ ????+ ????+++ ????+ ????+ ????+++ | ????+++ ????+++ ????+++ ????+++ ????+++ ????+++ ????+++ ????+++ ????+++ ????+++ ????+++ |
*With the virus infection of 0.01MOI zooblast in blocks, stopped infecting at 24,48 and 72 hours respectively, pair cell carries out immunostaining.The diffusion of cell is according to can painted cell count in 72 hours:
There is not cell to be colored in-72 hours
+＜5 cells are colored
++ 5～25 cells are colored
25 cells of +++＞are colored
3. the toxicity (Cytopathicity) that has littler pair cell
Under the high infection multiplicity (5.0MOI), attenuated vaccinia virus pZCI has than wild the Temple of Heaven virus CPE still less in the tested zooblast of majority, and this shows among CEF, BHK-21, C6, MDCK, RK13, PK (15) and the 293T; The wild the Temple of Heaven virus of time ratio that CPE perhaps occurs is more late, as in humanization cell HeLa.Show that attenuated vaccinia virus has the toxicity of lower pair cell than wild the Temple of Heaven virus.
In MRC-5 and African green monkey kidney cell Vero, COS-7, attenuated vaccinia virus does not show the difference with wild the Temple of Heaven virus significant cytotoxicity, and they all can form the CPE phenomenon to the similar severity of wild the Temple of Heaven virus in these three kinds of cells.
The cytotoxicity that attenuated vaccinia virus reduces also shows in the formation of plaque.The result of immunostaining shows, in former generation CEF, C6, MDCK and RK13, wild the Temple of Heaven virus all can produce tangible plaque, especially in the neuroglial cytoma C6 cell and rabbit kidney cell RK13 of mouse, wild the Temple of Heaven virus can form typical plaque in early days virus infection, behind 48 hours of virus infection, all infected and death that comes off in a large number of nearly all cell; And attenuated vaccinia virus pZCI does not all form tangible plaque in CEF, MDCK, C6 and RK13 cell, only show form through immunostaining by Foci of virus infection (CEF, MDCK, C6) or infected individual cells (RK13), show that attenuated vaccinia virus obviously reduces the toxicity of above-mentioned cell.Among this external 293T and the BHK-21, it is also late than wild the Temple of Heaven virus that pZCI forms the time of plaque.
Vaccinia virus Tiantan strain and the CPE of attenuated vaccinia virus pZCI in zooblast see Table 3.
Table 3 vaccinia virus Tiantan strain and the CPE of attenuated vaccinia virus carrier in zooblast
*
| ????pZCI ??? CPE | Wild the Temple of Heaven virus TT CPE | |
| Clone CEF 293T BHK-21 C6 COS-7 HeLa MDCK MRC-5 PK (15) RK13 Vero | ????12h????24h ????+??????+ ????+??????++ ????-/+????+ ????+??????++/+++ ????++++???++++ ????++?????++++ ????++?????+++ ????++++???++++ ????+??????++/+++ ????++?????+++ ????+++????++++ | ????12h?????24h ????++??????+++ ????++++????++++ ????++??????+++ ????++++????++++ ????++++????++++ ????++++????++++ ????++++????++++ ????++++????++++ ????++??????+++/++++ ????++++????++++ ????++++????++++ |
*With the virus infected cell of 5.0MOI, observed the situation that occurs CPE in the inoculating cell respectively at 12 and 24 hours:
The control cells of+inoculating cell and uninfecting virus is as broad as long
++ CPE appears in＜25% cell
++ CPE appears in+25%～50% cell
++ ++ CPE appears in 50%～75% cell
Virus vector must have stable biological character safely and effectively, and its foreign gene insertion and the stability of expressing are particularly important.The remarkable attenuated vaccinia virus that will insert external source GFP gene (green fluorescence protein gene) carries out continuous six times with 0.01MOI and goes down to posterity in the Vero cell.With the first-generation and hexabasic attenuated vaccinia virus vero cells infection, at first under fluorescent microscope, the plaque that produces green fluorescence is counted after 48 hours, then the cells infected in the culture dish is carried out immunostaining, the plaque that shows virus infection is counted once more, compared with the plaque number that produces green fluorescence.Experiment finds, count up to through observed plaque of immunostaining and the plaque that shows green fluorescence identical entirely, show foreign gene (GFP) but stablely insert also continuous passage and the expression of attenuated vaccinia virus carrier.
The genetic stability that foreign gene GFP inserts in the attenuated vaccinia virus
Table 4
| Virus algebraically | The plaque that shows green fluorescence | The plaque that immunostaining shows | The plaque of the plaque/immunostaining of green fluorescence (%) |
| ???P1 ???P6 | ????????77 ????????83 | ?????????77 ?????????83 | ??????????????????100 ??????????????????100 |
With the first-generation and hexabasic attenuated vaccinia virus with the 5.0MOI vero cells infection, results infected cells and supernatant liquor after 24 hours, the Vero cell of Gan Raning is not as negative control.The sample of getting equal volume carries out SDS-polyacrylamide gel electrophoresis protein isolate, change film after Western blot with the expression output of the viral GFP of the different algebraically of the antibody test of anti-GFP.The result shows, the similar (see figure 3) of expression amount of six front and back GFP that go down to posterity.But show the exogenous object protein that remarkable attenuated carrier stably express inserts.
In a word, significantly attenuated vaccinia virus has limited host range in zooblast, in most cells, comprise having shown the breeding replication that reduces in the humanization cell, and have lower cell, and/or have the toxicity of littler pair cell to intercellular diffusion levels.Attenuated vaccinia virus these characteristics in zooblast will help control virus in target animal or intravital levels of replication of people and velocity of diffusion, reduce virus to organism toxicity, simultaneously also for preventing that the STOCHASTIC DIFFUSION of virus in crowd and environment from providing effective barrier.In addition, the remarkable attenuated vaccinia virus carrier that the present invention obtains has also shown the high stability that foreign gene inserts and expresses, for the further application of this carrier provides effective guarantee.
In a preferred embodiment, attenuated vaccinia virus carrier of the present invention also has in vivo the significantly interior feature of the body of attenuation.The feature of attenuation comprises in vivo: 1) intranasal inoculation approach (intranasal route, attenuation IN); 2) cranial cavity route of inoculation (intracranail route, attenuation IC).' attenuation in vivo ' is meant, virus can be carried out toxicity test in other inbred lines closed colony BALB/c mouse of SPF level, and by the intranasal inoculation approach time, can significantly reduce the decline of immune mouse body weight; By the cranial cavity route of inoculation time, can significantly reduce the mortality ratio of immune mouse.Concrete feature is as follows:
1) attenuation of nasal:
BALB/c mouse to 5 ages in week inoculates 10 respectively
6The wild the Temple of Heaven virus of PFU, preferably improve the Temple of Heaven virus pZCI, in 10 days observation period, can cause mouse weight loss 28.6% and 13.7% respectively at most.Inoculation 10
5The wild the Temple of Heaven virus of PFU, preferably improve the Temple of Heaven virus pZCI, in the observation period, can cause mouse weight loss 19.2% and 13.3% at most.Show that improvement the Temple of Heaven virus pZCI detects the body internal characteristic with remarkable attenuation through nasal.
2) attenuation of cranial cavity route of inoculation:
Vaccinia virus is a neurotropic virus, and the cranial cavity inoculation can directly cause the murder by poisoning of virus to central nervous system, and can cause the generation of acute encephalitis thus, and this is comparatively one of severe complications that the inoculation vaccinia virus causes.Thereby mouse intracranial injection medial lethal dose (IC LD
50) very sensitive reflect viral neurovirulent power.
The BALB/c mouse in 3 ages in week is inoculated the wild the Temple of Heaven virus of serial dilution and improved the Temple of Heaven virus, in the observation period of two weeks (14 days), observe and write down and respectively organize mortality of mice, press the medium lethal dose IC LD that the Reed-Muench method is calculated the cranial cavity approach
50After tested, the IC LD of wild the Temple of Heaven virus, improvement the Temple of Heaven virus pZCI
50Be respectively 10
3.46PFU (3.46Log
10Unit) and 10
5.89PFU (5.89 Log
10Unit), the LD of improvement the Temple of Heaven virus pZCI
50Than the increase of the wild the Temple of Heaven virus 2.4Log
10Unit, the neurotoxicity of attenuated virus pZCI descends about 240 times than wild the Temple of Heaven virus.
Thus, no matter be the intranasal inoculation approach, or the cranial cavity route of inoculation, all confirm the improvement the Temple of Heaven virus pZCI body internal characteristic of remarkable attenuation in vivo.Therefore, more than improvement the Temple of Heaven virus will be safer carrier, when development that further is applied to newtype drug or reagent or live vector developing vaccines, have than the higher security of wild the Temple of Heaven virus.
In a word, preferred improvement the Temple of Heaven virus of the present invention has following seven specific characters:
1) has limited host range and in the most animals cell, have the reproductive ability replication lower than wild the Temple of Heaven virus;
2) in the most animals cell, have the cell lower and arrive intercellular diffusion levels than wild the Temple of Heaven virus;
3) in the most animals cell, has the toxicity of the pair cell littler than wild the Temple of Heaven virus;
4) in African green monkey kidney cell Vero and COS-7, have and the replication of wild the Temple of Heaven virus similar level, similar iuntercellular diffusion levels and similar cytotoxicity;
5) has the via intranasal application approach inoculation interior feature of body of remarkable attenuation in vivo;
6) have through cranial cavity inoculation and show feature in neurotoxicity is than the significantly reduced body of wild the Temple of Heaven virus;
7) the attenuated vaccinia virus carrier has the stable feature of inserting and expressing of foreign gene simultaneously.
The known remarkable attenuation the Temple of Heaven virus that how to obtain to have above feature of researchist of the present invention.The method that obtains remarkable attenuation the Temple of Heaven virus comprises the steps:
1) make up shuttle plasmid, this plasmid comprises the specific promoter sequence of vaccinia virus (as pSYN and pH5).This plasmid contains the flanking sequence with vaccinia virus Tiantan strain HA gene late promoter sequence or its outside sequence homology, can insert heterologous nucleotide sequence (as marker gene GFP) between this plasmid flanking sequence, and it is under the control of vaccinia virus promotor;
2) (as Vero or CEF) makes shuttle plasmid and vaccinia virus Tiantan strain homologous recombination (homologous recombination can use lipofectamine box (Qiagen) to carry out transfection in the permission cell of vaccinia virus, method is seen the test kit specification sheets), so that vaccinia virus HA gene late promoter destroys, the HA Gene Partial loses function, reaches the purpose of vaccinia virus being carried out remarkable attenuation;
3) by selection markers (as the green fluorescence of GFP), recombinant virus is carried out single spot screening, obtain pure remarkable attenuated vaccinia virus to separate;
Attenuation vaccinia virus recombinant to above acquisition can further carry out cytology and zoological evaluation.
Be included in the zooblast and analyze the duplication characteristic of determining recombinant virus, as velocity of diffusion in cell of virus replication kinetic curve, virus, the CPE of virus in cell etc., with the checking recombinant virus at the cell levels attenuation.By different route of inoculation, as nasal, encephalic approach, Analysis and Identification recombinant virus virulence in animal body is to verify the recombinant virus feature of attenuation in vivo in animal model.
Term " recombinant virus " is meant the nucleotide sequence that contains at least a " allos " in the viral genome, promptly is not the arbitrary combination with the closely-related nucleotide sequence of described virus usually.
This research invention the attenuated vaccinia virus carrier can be used for making up candidate vaccine, prevent and/or treat the communicable disease of the mankind or animal; This attenuated vaccinia virus carrier also can be further used for developing the medicine of therapy of tumor or prevention; This carrier also can be used as the expression of exogenous gene carrier, and is safer in scale operation; In addition, attenuated vaccinia virus can be used for expressing foreign pathogens antigen prepd pathogen detection and diagnostic reagent.
1. attenuated vaccinia virus is used to make up candidate vaccine
1) make up the shuttle plasmid contain the purpose exogenous DNA array, the flanking sequence homology of the flanking sequence of its both sides and the shuttle plasmid that obtains attenuated vaccinia virus pZCI of being used to recombinate, and exogenous DNA array is under the control of vaccinia virus promotor.
2) (as African green monkey kidney cell Vero) makes shuttle plasmid and attenuated vaccinia virus Tiantan strain pZCI homologous recombination in the permission cell of vaccinia virus, make the marker gene (as GFP) among the purpose exogenous DNA array replacement attenuated vaccinia virus pZCI, so that external source target DNA sequence is inserted into the predetermined position of the HA gene of vaccinia virus.
3) in allowing cell (as the Vero cell), screen the recombinant virus that loses specific markers (as losing GFP fluorescence), acquisition can be expressed the recombinant virus of remarkable attenuation of purpose exogenous DNA array as candidate vaccine, is used to prevent and treat the communicable disease of the mankind or animal.
2. the attenuated vaccinia virus carrier that is used to make up transgenosis is applied to therapy of tumor
1) makes up the shuttle plasmid contain exogenous DNA array, the flanking sequence homology of the flanking sequence of its both sides and the shuttle plasmid that obtains attenuated vaccinia virus pZCI of being used to recombinate.This exogenous DNA array can place under the regulation and control of vaccinia virus promotor, to guarantee that the exogenous DNA array that needs to express can effective expression.This foreign DNA sequence is energy expression inhibiting or the dna sequence dna that helps the kill tumor cell, also can be the exogenous DNA array of not expressing, with the vaccinia virus gene transfer vector that obtains remarkable attenuation after the attenuated vaccinia virus Tiantan strain pZCI homologous recombination.
2) shuttle vectors of Gou Jianing (as Vero cell) and attenuated vaccinia virus Tiantan strain pZCI homologous recombination in the permission cell of vaccinia virus, make the marker gene (as GFP) among the purpose exogenous DNA array replacement attenuated vaccinia virus pZCI, so that external source target DNA sequence is inserted into the predetermined position of vaccinia virus gene group, but obtains expression inhibiting or help the attenuated vaccinia virus gene transfer vector of kill tumor cellular constituent.
3), further make thymidine kinase (thymidine kinase, TK) gene inactivation, the recombinant attenuated vaccinia virus of generation TK-of above attenuated vaccinia virus by engineered method.This recombinant virus is because can't the normal expression thymidine kinase, but in the vigorous tumour cell of division, the infecting and duplicate (McCart of highly selective, J.A.et al., 2001, Systemic cancer therapy with a tumor-selective vaccinia virusmutant lacking thymidine kinase and vaccinia growth factor genes.Cancer Research, 61 (24), 8751-7), then comparatively safe in normal cell owing to the remarkable attenuation of virus.Thus this attenuated vaccinia virus gene transfer vector can be high the sexy transfected tumor cell of target, suppress or the kill tumor cell, then comparatively safe to normal human tissue.
3. attenuated vaccinia virus is as the safely expressed carrier of foreign gene
1) make up the shuttle plasmid contain purpose foreign gene dna sequence dna, the flanking sequence homology of the flanking sequence of its both sides and the shuttle plasmid that obtains attenuated vaccinia virus pZCI of being used to recombinate, and external source target DNA sequence is under the control of vaccinia virus promotor.
2) in the permission cell of vaccinia virus, make shuttle plasmid and attenuated vaccinia virus Tiantan strain pZCI homologous recombination, make the marker gene (as GFP) among the purpose exogenous DNA array replacement attenuated vaccinia virus pZCI, so that external source target DNA sequence is inserted into the predetermined position of the HA gene of vaccinia virus.
3) screen the recombinant virus that loses specific markers (as losing GFP fluorescence) in allowing cell, acquisition can be expressed the recombinant virus of the remarkable attenuation of purpose exogenous DNA array, is used for expression of exogenous object protein and production.Because the remarkable attenuation of this carrier, thereby will have higher security when this vector expression foreign gene carries out target protein scale operation using, can reduce in the production process to the issuable potential hazard of contact personnel with to the pollution of environment.
4. attenuated vaccinia virus is used to express foreign pathogens antigen prepd pathogen detection and diagnostic reagent
1) makes up the shuttle plasmid that contains purpose foreign gene dna sequence dna, the flanking sequence homology of the flanking sequence of its both sides and the shuttle plasmid that obtains attenuated vaccinia virus pZCI of being used to recombinate, and the antigenic dna sequence dna of the foreign pathogens that will express is under the control of vaccinia virus promotor.
2) in the permission cell of vaccinia virus, make shuttle plasmid and attenuated vaccinia virus Tiantan strain pZCI homologous recombination, make the marker gene (as GFP) among the antigenic dna sequence dna replacement of the purpose foreign pathogens attenuated vaccinia virus pZCI, so that the antigenic dna sequence dna of foreign pathogens is inserted into the predetermined position of the HA gene of vaccinia virus.
3) screen the recombinant virus that loses specific markers (as losing GFP fluorescence) in allowing cell, acquisition can be expressed the recombinant virus of the antigenic remarkable attenuation of purpose foreign pathogens, is used for expression of exogenous object protein and production.Be used to prepare the detection and the diagnostic reagent of pathogenic agent.Because the vaccinia virus vector system has better antigenicity and immunogenicity than the product of other expression system (as bacterium or yeast) preparation, thereby will have higher specificity and susceptibility with the detection and the diagnostic reagent (detecting pathogen antigen or corresponding antibodies) of the expression product of this carrier preparation, can be widely used in the prevention and control of poultry, fowl, hydrobiont and people's etiology, epidemiology, diagnostics and pathogenic infection.Simultaneously, because the remarkable attenuation of this attenuated vaccinia virus carrier, thereby will have higher security when this vector expression foreign gene carries out target protein scale operation using, can reduce in the production process to the issuable potential hazard of contact personnel with to the pollution of environment.
The present invention compared with prior art has the following advantages:
1) genetic modification to the Temple of Heaven strain HA gene makes attenuated vaccinia virus duplicate reduction in zooblast, toxicity descends, remarkable attenuation in the animal pattern body, therefore no matter be to make up vaccine with this vaccinia virus vector, or as the therapy of tumor carrier, or with this carrier mass expressing external albumen with safer reliable.Especially the neurotoxicity of attenuated vaccinia virus declines to a great extent, and will increase the security that this virus is used as carrier, will prevent that effectively vaccinate or contactee from relevant complication of vaccinia virus such as encephalitis etc. taking place.In addition, use when this vector expression foreign gene carries out target protein scale operation and will have higher security, can reduce in the production process to the issuable potential hazard of contact personnel with to the pollution of environment.
2) vaccinia virus vector has bigger foreign gene capacity than the carrier of other type, can insert the foreign gene that total amount reaches 25Kb; There are a plurality of nonessential regions that comprise TK, HA, therefore can insert various exogenous genes simultaneously, realize the combination of multiple effective antigenic component or biotic component, improve the effect of vaccine immunity or therapy of tumor.
Description of drawings
Fig. 1: vaccinia virus Tiantan strain and the attenuated vaccinia virus duplicating dynamics in zooblast.The different virus of cell inoculation 0.05MOI was collected viral sample at 0,24,48 and 72 hour respectively, measured the titre of virus in the Vero cell.
Fig. 2: vaccinia virus Tiantan strain and the attenuated vaccinia virus diffusion in zooblast.The different virus of cell inoculation 0.01MOI infects termination in 24,48 and 72 hours respectively, and pair cell carries out immunostaining, observes the process that virus infects and spreads in cell, and takes pictures under inverted microscope.
Fig. 3: the stability of exogenous gene expression in the attenuated vaccinia virus carrier.The remarkable attenuated vaccinia virus that contains P1 generation of foreign gene GFP and P6 generation is with the 5.0MOI vero cells infection, gathers in the crops infected cell and supernatant in 24 hours.The Vero cell of Gan Raning is not as negative control.The sample of getting equal volume carries out SDS-PAGE gel electrophoresis protein isolate, change film after Western blot with the expression output of the antibody test GFP of anti-GFP.
Fig. 4: vaccinia virus Tiantan strain and the attenuated vaccinia virus virulence (nasal) in animal pattern.The BALB/c mouse in 5 ages in week, via intranasal application is inoculated 10 of 30ulPBS dilution respectively
6The virus of PFU (A) and 10
5The virus of PFU (B), the variation of record mouse body weight every day in 10 days observation period.
Fig. 5: the expression of reorganization env attenuated vaccinia virus in allowing cell Vero.With env vaccinia virus recombinant (A) and attenuated vaccinia virus pZCI (B) vero cells infection (0.01MOI), using the antiserum(antisera) from aids patient after 24 hours is an anti-immunostaining that carries out respectively.
Embodiment
Following examples will further illustrate the present invention.
The duplicating dynamics of attenuated vaccinia virus and virus replication correlation properties in zooblast.
1) duplicating dynamics of virus in zooblast:
In order to understand the replication of attenuated vaccinia virus in zooblast, selected zooblast from different sorts, different tissues, comprise clone from the people, carried out the dynamic (dynamical) research experiment of virus replication.Simultaneously, studied the duplicating dynamics curve of parental virus the Temple of Heaven strain in these cells under the same conditions, so that can compare with the duplication characteristic of attenuated vaccinia virus.
Virus relatively is: remarkable attenuated vaccinia virus pZCI, wild the Temple of Heaven vaccinia virus TT.Selected zooblast and kind thereof, source and cell type see Table 1.Its Central Plains for the self-control cell, is former generation although indicate in this article for chick-embryo cell CEF, in use is preceding triple-substituted passage cell.Vero and C6 cell draw from the Chinese Academy of Sciences typical case culture collection council (Type Culture Collection of ChineseAcademy of Sciences, CTCCCAS); 293T draw from U.S. cell culture preservation center (American Type Culture Collection, ATCC); COS-7, PK (15), RK13, HeLa, MDCK, MRC-5 and BHK-21 draw from Chinese typical culture collection center (China Centerfor Type Culture Collection, CCTCC).The cultural method of cells involved is all cultivated according to the requirement of U.S. ATCC and Chinese CCTCC and is gone down to posterity.
Below be the cultivation that example is introduced passage cell with COS-7:
Cell in the DMEM substratum that contains 10% new-born calf serum (NCS) incubation growth in flakes (37 ℃, 5%CO
2), sop up nutrient solution, wash 1 time with PBS.0.25% the trypsin digestion cell that adds proper volume.Treat that cell retraction gap becomes big, cell and begins to add fresh substratum when the bottle wall comes off, blow evenly, install in the new culturing bottle, place 37 ℃, 5%CO in 1: 3 ratio branch
2Incubator cultivate.
Preparation and the cultural method of former generation chick-embryo cell CEF are as follows:
The chicken embryo sterilization of getting 9～11 days is placed on the egg frame, knocks the blunt nosed of chicken embryo open with scissors, chooses shell membrane, takes out the chicken embryo and places aseptic plate, and removal head, wing, leg, internal organ etc. also shred.Wash twice with the substratum that does not contain serum, the 10ml syringe extracts the chicken embryo to becoming rotten shape.The pancreatin (precooling) that adds 100ml0.25% was in 37 ℃ of digestion 15 minutes.Filtered through gauze, filtrate add the NCS stopped reaction of 10% final volume.Remaining fragment of tissue repeats digestion once on the gauze.Merging filtrate, 4 ℃ of 1500rpm are centrifugal 10 minutes.Abandon supernatant, cell precipitation is resuspended with the substratum that contains 10%NCS, centrifugal 10 minutes of 1500rpm.Install to culturing bottle with the resuspended post precipitation branch of the fresh culture that contains 10%NCS, place 37 ℃ of 5%CO
2Cultivate in the incubator.Propagating method used before the three generations with the COS-7 cell.
The method of measuring the duplicating dynamics curve of virus in cell is as follows:
Infective virus precontract 24 hours an amount of different cells are connected to 24 orifice plates, treat that cell grows to individual layer, pair cell is counted in standby hole, removes substratum, insert various vaccinia viruss respectively by 0.05MOI, 24 orifice plates placed 37 ℃ of incubator viral adsorptions 90 minutes.From culture plate, remove supernatant, and the virus of not adsorbing with the substratum wash-out.Adding contains 3% foetal calf serum, and (FBS is GIBCO) with 1% couple of fresh culture DMEM, MEM that resists (penicillin, Streptomycin sulphate), in 37 ℃, 5%CO
2Incubator in cultivate.0 hour sample was promptly gathered in the crops after finishing virus absorption, after this collected sample respectively in 24,48 and 72 hours.The substratum that cell uses is introduced the requirement of unit by it, as U.S. ATCC and Chinese CCTCC or the CTCCCAS of the Chinese Academy of Sciences.The freezing of virus replication sample of the different time of results three times is with releasing virus.The virus of results is measured titre in the Vero cell.
The titration of virus:
In 24 orifice plates, insert the Vero cell, behind the individual layer to be grown to (about 24 hours), the sucking-off substratum, the viral sample of access serial dilution, 37 ℃ adsorbed 90 minutes.Absorption is removed supernatant after finishing from culture plate, and the virus of not adsorbing with the substratum wash-out.Add fresh semifixed substratum (it is the two anti-of 3% new-born calf serum NCS (GIBCO) and 1% that 2 times MEM substratum (GIBCO) and 1% agarose 1: 1, substratum contain final concentration), in 37 ℃, 5%CO
2Incubator in cultivated 48～72 hours.With Viola crystallina cells infected is dyeed, wash away Viola crystallina and agarose, the plaque counting that produces in the pair cell calculates viral titre according to plaque number and extension rate.
Virus is duplicated the classification of determining of multiple and cell in cell: 72 hours titre (PFU) with virus is removed viral input titre (Input), for multiple is duplicated in the breeding of virus in this cell.With reference to the method pair cell of the Moss B (Carroll that classifies, M.W., and Moss, B.1997.Hostrangeand cytopathogenicity of the highly attenuated MVA strain of vaccinia virus:propagation and generation of recombinant viruses in a nonhumanmammalian cell line.Virology 238 (2), 198-211), that is:
Nonpermissive cell (nonpermissive cell, NP):＜1 times virus replication increases multiple, and semipermissive cell (semipermissive cell, SP): 1～25 times virus replication increases multiple, (permissive cell, P):＞25 times virus replication increases multiple to allow cell.
Wild the Temple of Heaven virus and attenuated vaccinia virus pZCI duplicating in zooblast increase multiple and see table 1 for details, and the duplicating dynamics curve is seen Fig. 1.Experimental result shows, in 11 kinds of zooblasts, comprise former generation chick-embryo cell (CEF), 3 kinds of human cells (293T, Hela and MRC-5), 2 kinds of African green monkey kidney cells (Vero and COS-7), (MDCK, PK (15) and RK13) are to allow cell for the Temple of Heaven virus TT for 2 kinds of mouse cells (BHK-21 and C6) and three kinds of other animal nephrocytes.This result shows that the Temple of Heaven strain has host range extremely widely, can breed well in most zooblasts.
The attenuated vaccinia virus of test has shown duplicate and growth characteristics very different with wild TT in zooblast, they have littler host range, and the decline of the different levels of replication of degree is all arranged in most of test cell.PZCI has lost the reproductive ability replication in RK13, but has only lost the replication of part in MDCK, and MDCK is the semipermissive cell of pZCI.In other two kinds of mouse cells (BHK-21 and C6) and porcine kidney cell PK (15), duplicating of pZCI all has been subjected to restriction significantly, and it can only keep 1%～17% of wild TT replication usually in these cells.Although increasing multiple, virus replication shows that 293T and MRC-5 are the permission cells that attenuated vaccinia virus duplicates, but the levels of replication of attenuated vaccinia virus in them still shown reduction significantly, wherein the duplicate multiple of pZCI in 293T has only 36.8% of the Temple of Heaven strain, and the multiple that duplicates in people's embryo hepatic cell line MRC-5 has only 14.7% of the Temple of Heaven strain.In CEF, Hela and two kinds of African green monkey kidney cell Vero, COS-7, the replication of two kinds of attenuated vaccinia virus does not show with wild TT obvious difference, more than four kinds of cells can support that all attenuated vaccinia virus effectively duplicates.
2) cytotoxicity (CPE) and virus the diffusion in cell of virus in zooblast
Different cells are inoculated to forming individual layer (needing 24h approximately) in every hole in 6 porocyte culture plates, go into virus infected cell by the infection multiplicity access node of 0.01MOI, and absorption is 90 minutes in cell culture incubator.From culture plate, remove supernatant, and the virus of not adsorbing with the substratum wash-out.Add and contain 3%FBS and 1% pair of anti-substratum, place cell culture incubator to continue to cultivate, take out Tissue Culture Plate respectively at 24,48 and 72 hours, carry out immunostaining.Immuning dyeing method is as follows: the substratum supernatant in the sucking-off culture plate, PBS washes once.Methyl alcohol with 1: 1: isopropyl acetone solution fixed cell, add by the anti-vaccinia virus serum of the rabbit of the dilution proportion of necessarily tiring (one is anti-), effect is 2 hours under the room temperature.Sucking-off one is anti-, washes once with PBS, adds the two anti-albumen-A (HRP mark) by the dilution proportion of necessarily tiring, and effect is 1 hour under the room temperature.Sucking-off two is anti-, washes once with PBS, and (the adjacent benzene methyl oxyaniline of 10mlPBS+200ul is at the freshly prepared H of 100% alcoholic acid saturated solution+15ul to use freshly prepared staining fluid then
2O
2) colour developing.Wait to occur reddish-brown Foci clearly, sucking-off colour developing liquid with the reaction of PBS color development stopping, is taken the cellular invasion result with digital camera under inverted microscope.The method classification of Moss B is pressed in the diffusion of virus in cell, the cell count that is colored in promptly 72 hours:
-there is not cell to be colored, show that this cell can not infect
+＜5 cells are colored, and cell can be infected, but very low diffusibility is only arranged
++ 5～25 cells are colored, and have limited diffusibility in cell
25 cells of +++＞are colored, and have higher diffusibility in cell
Accompanying drawing 2 and table 2 are seen in the diffusion of virus in cell.
The toxicity of virus pair cell is called cytopathic effect (CPE).When carrying out immunostaining, observe the situation and the virus that under low infection multiplicity (0.01MOI), occur CPE in the cell and in cell, whether formed plaque.In addition, also observed the toxicity of viral pair cell under the high infection multiplicity.Experimental technique is as follows: infective virus precontract 24 hours an amount of different cells are connected to 24 orifice plates, treat that cell grows up to individual layer, remove substratum, insert wild the Temple of Heaven virus TT and improvement the Temple of Heaven virus pZCI by 5.0MOI, 24 orifice plates placed 37 ℃ of incubator viral adsorptions 90 minutes.From culture plate, remove supernatant, and the virus of not adsorbing with the substratum wash-out.Adding contains 3% foetal calf serum, and (FBS GIBCO) with 1% pair of fresh culture that resists (penicillin, Streptomycin sulphate), places 37 ℃, 5%CO
2Incubator in cultivate.The CPE that occurred in the observation of cell in 12 hours, 24 hours takes pictures under inverted microscope.With reference to the method for Moss B, the toxicity of viral pair cell is divided into five classes by the degree that CPE occurs:
The control cells of-inoculating cell and uninfecting virus is as broad as long
CPE appears in+＜25% cell
++ CPE appears in 25%～50% cell
++ CPE appears in+50%～75% cell
++ ++ CPE appears in＞75% cell
The CPE of virus in zooblast sees Table 2 under the high infection multiplicity.
The result of immunostaining shows, wild TT virus all has very strong diffusibility in 11 kinds of zooblasts of test, can promptly diffuse to adjacent cells from infected cells, slow slightly although it spreads in part clone, as in PK (15), 293T and Hela cell.Also find, wild the Temple of Heaven virus can not only be in cell rapid diffusion, and in most of zooblasts, can both form typical plaque, wherein in Vero, COS-7, RK13, C6, MDCK, can form very big plaque in early days what infect.Under this external high infection multiplicity (5.0MOI), wild the Temple of Heaven virus has all shown serious CPE phenomenon in the cell of all tests.
The preferred significantly diffusion between the attenuated vaccinia virus showed cell descends in the most animals cell.Wherein significantly attenuated vaccinia virus pZCI shows do not have diffusibility in the RK-13 cell; In MDCK and PK (15), only has very low diffusibility; In other two kinds of cell C6 and HeLa, have limited diffusibility, have in 72 hours that to be less than 25 cells infected.
Although in BHK-21, MRC-5 and former generation CEF cell, preferred attenuated vaccinia virus pZCI all has higher diffusibility (+++), but in the identical time, the cell count that pZCI infects still obviously is less than the cell of wild the Temple of Heaven virus infection, shows that the velocity of diffusion of attenuated vaccinia virus pZCI in BHK-21, MRC-5 and CEF still obviously slow down than wild virus.
Attenuated vaccinia virus pZCI only has the velocity of diffusion similar to wild virus in HEKC 293T and African green monkey kidney cell Vero, COS-7, show that Vero, COS-7 and 293T all can effectively support the reproductive ability of wild the Temple of Heaven virus and attenuation the Temple of Heaven virus to duplicate and spread.
Under the high infection multiplicity (5.0MOI), attenuated vaccinia virus pZCI has than wild the Temple of Heaven virus CPE still less in the tested zooblast of majority, and this shows among CEF, BHK-21, C6, MDCK, RK13, PK (15) and the humanized cell 293T; The wild the Temple of Heaven virus of time ratio that CPE perhaps occurs is more late, as in humanization cell HeLa, shows that attenuated vaccinia virus has lower cytotoxicity than wild the Temple of Heaven virus.
In MRC-5 cell and African green monkey kidney cell Vero, COS-7, attenuated vaccinia virus does not show the difference with wild the Temple of Heaven virus significant cytotoxicity, and they all can form the CPE phenomenon to the similar severity of wild the Temple of Heaven virus in these three kinds of cells.
The cytotoxicity that attenuated vaccinia virus reduces also shows in the formation of plaque.The result of immunostaining shows, in former generation CEF, C6, MDCK and RK13, wild the Temple of Heaven virus all can produce tangible plaque, especially in test cell C6 and RK13, wild the Temple of Heaven virus can form typical plaque in early days virus infection, behind 48 hours of virus infection, all infected and death that comes off in a large number of nearly all cell.And attenuated virus pZCI does not all form tangible plaque in CEF, C6, MDCK and RK13 cell, only show produce through immunostaining by Foci of virus infection (CEF, C6 and MDCK) or infected individual cells (RK13), show that attenuated vaccinia virus obviously reduces the toxicity of above-mentioned cell.Among this external 293T and the BHK-21, it is also late than wild the Temple of Heaven virus that pZCI forms the time of plaque.
The genetic stability that foreign gene inserts and expresses in the remarkable attenuated vaccinia virus
Virus vector must have stable biological character safely and effectively, and its foreign gene insertion and the stability of expressing are particularly important.Remarkable attenuated vaccinia virus (HA gene late promoter district has inserted external source GFP gene) is infected individual layer Vero cell with 0.01MOI, adsorb and remove substratum after 90 minutes, wash cell once with the DMEM substratum that contains 10%NCS, add contain 10%NCS fresh culture based on 37 ℃, 5%CO
2Incubator in cultivated 48 hours, the cell and the supernatant of results virus infection, freezing three times, sample is measured viral titre in the Vero cell.Then with same MOI vero cells infection.Make attenuated vaccinia virus in the Vero cell, carry out 6 continuous passages with this method.The first-generation and the infection of hexabasic attenuated vaccinia virus are incubated in the Vero cell of 350mm culture dish, at first under fluorescent microscope, the plaque that produces green fluorescence is counted after 48 hours, then the cells infected in the culture dish is carried out immunostaining (method is seen immunostaining), the plaque that shows virus infection is counted once more, compared with the plaque number that produces green fluorescence.Experimental result sees Table 4.Experiment finds, it is identical entirely that the plaque that immunostaining shows and the plaque that shows green fluorescence count up to, show foreign gene (GFP) but stablely insert the also continuous passage of attenuated vaccinia virus carrier, this further application for this carrier provides effective guarantee.
With the first-generation and hexabasic attenuated vaccinia virus with the 5.0MOI vero cells infection, 24 hours results cells infecteds and supernatant, the cell of Gan Raning is not as negative control.The sample of results was left the heart 10 minutes in 4000, and precipitation washes twice with PBS, resuspended being deposited among the PBS, through-70 ℃ with room temperature repeatedly after the freezing three times, sample retention is in-70 ℃.The different samples of getting equal volume carry out SDS-PAGE electrophoretic separation albumen (12% separation gel, 5% concentrated glue) through boiling water bath after 3 minutes.Obtain isolating protein gelatin and be transferred to the NC film, blockaded 2 hours with PBST (the PBS damping fluid that the contains 0.2% soil temperature 20) room temperature that contains 3% bovine serum albumin (BSA).PBST washes behind the film by the specific antibody (BD company) that adds anti-GFP at 1: 1000, and the room temperature effect was washed film after 2 hours.By two anti-(goat anti-rabbit antibodies of HRP mark) that add peroxidase labelling at 1: 5000, the room temperature effect was washed film after 1 hour.With DAB develop the color (9mlTris, 1ml NiCl
2, 6mg DAB, 10ul H
2O
2), treat special band colour developing back PBS stopped reaction.The result shows, the remarkable attenuated vaccinia virus carrier first-generation of inserting GFP with the 6th generation expressing viral the GFP protein content similar.But show the exogenous object protein that remarkable attenuated carrier stably express inserts.
The toxicity of attenuated vaccinia virus carrier and wild the Temple of Heaven virus relatively in the animal pattern mouse
In order to identify the attenuated vaccinia virus characteristic that reduces of virulence in vivo, adopt the BALB/c mouse model system, measured remarkable attenuated vaccinia virus pZCI and the virulence of the wild the Temple of Heaven strain of parental virus TT in mouse thereof respectively by two kinds of different approach of nasal cavity and cranial cavity.The BALB/c mouse system is widely used in the world animal model system, be proved vaccinia virus sensitivity (Betakova et al., 2000, The vaccinia virusA14.5L gene encodes a hydrophobic 53-amino-acid virion membrane proteinthat enhances virulence in mice and is conserved among vertebratepoxviruses.J Virol, 74 (9), 4085-92; Lee, M.S.et al., Molecular attenuation ofvaccinia virus:mutant generation and animal characterization.J Virol, 66 (5), 2617-30; Zhang.W.H.et al., Vaccinia virus F12L protein is required for actintail formation, normal plaque size, and virulence.J Virol, 74 (24), 11654-62), be used for the toxicity and the infectious experiment of vaccinia virus in a large number.
1) virulence experiment of intranasal inoculation approach
With the BALB/c mouse grouping in 5 ages in week, 5 every group, inoculate respectively with 10 of PBS dilution from nasal
6PFU and 10
5Improvement the Temple of Heaven virus pZCI and the wild the Temple of Heaven virus of PFU; Another group mouse of PBS (30ul) intranasal inoculation with equal volume is organized in contrast.In the continuous 10 days observation period, measure the mean body weight of mouse every day, and liken ordinate zou to observing the fate mapping with the percentage that the mouse mean body weight is equivalent to initial body weight.The toxicity of different virus in BALB/c mouse is seen Fig. 4, and wherein Fig. 4 A is inoculation 10
6PFU dosage virus is to the influence of mouse body weight, and Fig. 4 B is inoculation 10
5PFU dosage virus is to the influence of mouse body weight.
The mouse that inoculates any papova does not all have death at duration of test, but different body weight change occurred.Inoculation 10
6Serious weight loss takes place in the mouse of PFU TT virus, during by the 9th day, loses weight about 30%; In inoculation 10
5It is also fairly obvious to lose weight in the mouse of PFU TT, at postvaccinal the 8th day, weight loss about 20%.The mouse of the improvement the Temple of Heaven virus pZCI of inoculating two kinds dosage, weight loss is about 13%, shows that pZCI toxicity in vivo is than the obviously decline (Fig. 4 A, B) of wild the Temple of Heaven virus.
2) virulence experiment of cranial cavity route of inoculation
With the BALB/c mouse grouping in 3 ages in week, 6 every group, inoculate purifying improvement the Temple of Heaven virus and the wild the Temple of Heaven virus of 10ul respectively with the PBS serial dilution from the cranial cavity approach.Be not counted in experimental result the mouse of injecting death in 24 hours.In the continuous 14 days observation period, observe the reaction behind the mouse inoculation every day, and the death condition of record mouse, calculate the mld LD of virus with the Reed-Muench method
50
Term ' mld ' (LD
50) be meant, cause the dosage of the virus inoculation of 50% experimental subjects death.Usually calculate with the Reed-Muench method, formula is: mld (LD
50Logarithm+the distance of the high critical extent of dilution inverse of)=50% infection rate is than the logarithm of * dilution factor, and in the formula, distance is than=(50% high critical infection rate-50%)/(the low critical infection rate of 50% high critical infection rate-50%).
The IC LD of wild the Temple of Heaven virus, improvement the Temple of Heaven virus pZCI
50Be respectively 10
3.46PFU (3.46Log
10Unit) and 10
5.89PFU (5.89 Log
10Unit), improvement the Temple of Heaven virus pZCI increases LD
502.4Log
10Unit.Because vaccinia virus is to have a liking for nervosa virus, might cause severe complications such as postvaccinal acute encephalitis, the cranial cavity inoculation then can directly cause the murder by poisoning of virus to central nervous system, thereby mouse intracranial injection medial lethal dose (IC LD
50) closely related with the neurotoxicity of virus.The experimental result of cranial cavity approach virus inoculation shows that the neurotoxicity of attenuated virus pZCI descends about 240 times.
Experimental result confirms, no matter is intranasal inoculation, still carries out direct intracerebral injection, and significantly attenuated vaccinia virus pZCI toxicity in vivo significantly descends than wild TT, and its neurotoxicity is proved and is significantly less than wild the Temple of Heaven strain.Therefore, be vector construction live vector vaccine with remarkable attenuated vaccinia virus, or use it for gene therapies such as cancer and drug development will have better security than wild the Temple of Heaven virus.
The significantly application of attenuated vaccinia virus in the human candidate AIDS vaccine that makes up safety
Since Chinese first report acquired immune deficiency syndrome (AIDS) case in 1985, acquired immune deficiency syndrome (AIDS) has entered the quick rise period of acquired immune deficiency syndrome (AIDS) morbidity up till now in the popular ascendant trend year by year that is of China.Adopt an effective measure rapidly containment HIV-1 diffusion and AIDS patient effectively treated, be Chinese Government and the instant responsibility of medical research personnel.The life-span of duplicating and prolong AIDS patient that the efficient antiretroviral therapy that the foreign scholar proposes can suppress HIV-1 effectively, however its expensive expense makes developing country hang back.Meanwhile, the AIDS vaccine has great expectations the whole world as potential preventive means.The vaccinia virus of Ceng Zuowei prevention variola virus vaccine is owing to its many advantage is transformed into the live vector expression alien gene or makes up recombinant vaccine, the communicable disease of preventing and treating multiple human and animal.Vaccinia virus Tiantan strain once was extensive use of in China, good immunogenicity and comparatively safe feature are arranged, but because it still keeps certain toxicity to organism, may cause the various complication that comprise encephalitis, especially might be to immunosuppressant crowd, produce serious influence as acquired immune deficiency syndrome (AIDS) patient or HIV virus carrier, thereby be not suitable for directly as the carrier that makes up vaccine.
Attenuated vaccinia virus of the present invention, owing to the late promoter that destroys the HA gene causes remarkable attenuation, especially its neurotoxicity greatly reduces, and makes this carrier have higher security, can be used for making up treating AIDS or the preventative vaccine that uses at Chinese population.
1) to carrying out Molecule Epidemiology Investigation at the popular HIV-1 of Central China, determine that this geographic main epidemic strain is HIV-1 Thailand B hypotype strain (B ') (Su et al., 2003, HIV-1 subtype B ' dictates the AIDS epidemic among paid blood donors in the Henanand Hubei provinces of China.AIDS, 17 (17), 2515-20).
2) made up the full-length molecule clone who represents Central China's epidemic strain in view of the above, based on this, the corresponding gene in clone's epidemic strain is as the external source goal gene, to make up at prevention of this area's acquired immune deficiency syndrome (AIDS) popular and therapeutic vaccine.
3) structure contains the shuttle plasmid of the dna sequence dna of purpose foreign gene HIV-1 envelope protein Env, the flanking sequence that is positioned at its both sides is identical with the flanking sequence of the shuttle plasmid that obtains attenuated vaccinia virus pZCI of being used to recombinate, and exogenous DNA array is under the control of vaccinia virus promotor.
4) (as Vero) makes shuttle plasmid and attenuated vaccinia virus Tiantan strain pZCI homologous recombination in the permission cell of vaccinia virus, the env sequence inserted among the attenuated vaccinia virus pZCI replaced out marker gene GFP.
5) reverse screening loses the fluorescently-labeled recombinant virus of GFP in allowing cell Vero, and acquisition can be expressed the recombinant virus of the remarkable attenuation of Env, and enlarges and purifying in the Vero cell.
6) expression of env attenuation vaccinia virus recombinant in allowing cell Vero: the attenuation vaccinia virus recombinant that will contain the env gene is with 0.01MOI inoculation Vero cell in blocks, adsorbed 90 minutes, and changed the fresh DMEM substratum that contains 10%NCS and continue to cultivate 24 hours.Carry out immunostaining by the aforesaid method of this paper, wherein one anti-is AIDS patient's serum, obtains from one less than separating AIDS patient's blood sample of 20 years old.In the negative control experiment,, be an anti-immunostaining that carries out with identical AIDS patient's serum with the attenuation vaccinia virus recombinant pZCI inoculation Vero cell of identical MOI.The plaque that the env recombinant vaccinia virus infection produces all shows red-brown (Fig. 5 A), and the plaque that vaccinia virus recombinant pZCI infection produces can not be colored (Fig. 5 B).This result shows, env successfully recombinates in the genome of attenuated vaccinia virus, and can effective expression.
7) other nonessential gene regions of this recombinant virus can be further with the method for homologous recombination insert other one or more stimulate the gene of the effective constituent of immunity of organism from the gene of acquired immune deficiency syndrome (AIDS) Chinese epidemic strain or other, make up the multivalence acquired immune deficiency syndrome (AIDS) candidate vaccine of various combination, to improve the prevention or the result of treatment of vaccine.
Claims (6)
1, a kind of attenuated vaccinia virus Tiantan strain vector, vaccinia virus vector are remarkable attenuated vaccinia virus (TT.WHU.pZCl), CCTCC:V200416.
2, a kind of preparation method who is used to realize the described a kind of attenuated vaccinia virus Tiantan strain vector of claim 1, it comprises the following steps:
A. make up shuttle plasmid, this plasmid comprises the flanking sequence with vaccinia virus Tiantan strain HA gene late promoter sequence or its both sides sequence homology;
B. in the permission cell of vaccinia virus, make shuttle plasmid and vaccinia virus Tiantan strain homologous recombination,, reach purpose, obtain attenuated vaccinia virus with above feature to the remarkable attenuation of vaccinia virus so that vaccinia virus HA Gene Partial loses function;
C. by selection markers, recombinant virus is carried out single spot screening, obtain pure remarkable attenuated vaccinia virus to separate.
3, the application of a kind of attenuated vaccinia virus Tiantan strain vector in the vaccine of preparation treatment or infection prevention disease.
4, the application of a kind of attenuated vaccinia virus Tiantan strain vector in the medicine of preparation treatment or prophylaxis of tumours.
5, the application of a kind of attenuated vaccinia virus Tiantan strain vector in expression alien gene.
6, the application of a kind of attenuated vaccinia virus Tiantan strain vector in expressing foreign pathogens antigen prepd pathogen detection and diagnostic reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100181296A CN1295339C (en) | 2005-01-11 | 2005-01-11 | Attenuated vaccinia virus Tiantan strain vector and its preparation and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100181296A CN1295339C (en) | 2005-01-11 | 2005-01-11 | Attenuated vaccinia virus Tiantan strain vector and its preparation and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1654665A true CN1654665A (en) | 2005-08-17 |
| CN1295339C CN1295339C (en) | 2007-01-17 |
Family
ID=34894226
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100181296A Expired - Fee Related CN1295339C (en) | 2005-01-11 | 2005-01-11 | Attenuated vaccinia virus Tiantan strain vector and its preparation and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1295339C (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206679A (en) * | 2011-03-30 | 2011-10-05 | 中国人民解放军军事医学科学院军事兽医研究所 | Shuttle vector of vaccinia virus and its application |
| CN113481240A (en) * | 2021-06-18 | 2021-10-08 | 苏州工业园区唯可达生物科技有限公司 | Recombinant vaccinia virus vector with L1R gene knocked out |
| CN114032217A (en) * | 2021-11-02 | 2022-02-11 | 中国疾病预防控制中心性病艾滋病预防控制中心 | Novel coronavirus compound vaccine based on DNA vector and replicative vaccinia virus vector |
| CN114058643A (en) * | 2021-06-22 | 2022-02-18 | 苏州工业园区唯可达生物科技有限公司 | Recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibody existing in vivo |
| CN114058644A (en) * | 2021-06-22 | 2022-02-18 | 苏州工业园区唯可达生物科技有限公司 | Recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibody existing in vivo |
| CN114058645A (en) * | 2021-06-22 | 2022-02-18 | 苏州工业园区唯可达生物科技有限公司 | Recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibody existing in vivo |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100537773C (en) * | 2000-11-23 | 2009-09-09 | 巴法里安诺迪克有限公司 | The improvement vaccinia ankara virus variant |
| EP1489164A1 (en) * | 2003-06-18 | 2004-12-22 | Genelux GmbH | Recombinant vaccinia viruses with modified F3 genes, uses thereof |
| CN1560248A (en) * | 2004-03-10 | 2005-01-05 | 中国疾病预防控制中心性病艾滋病预防 | Tiantan remocined vaccine virus of IFN-alpha receptor gene (B8R) deletion and application thereof |
-
2005
- 2005-01-11 CN CNB2005100181296A patent/CN1295339C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206679A (en) * | 2011-03-30 | 2011-10-05 | 中国人民解放军军事医学科学院军事兽医研究所 | Shuttle vector of vaccinia virus and its application |
| CN102206679B (en) * | 2011-03-30 | 2013-03-13 | 中国人民解放军军事医学科学院军事兽医研究所 | Shuttle vector of vaccinia virus and its application |
| CN113481240A (en) * | 2021-06-18 | 2021-10-08 | 苏州工业园区唯可达生物科技有限公司 | Recombinant vaccinia virus vector with L1R gene knocked out |
| CN114058643A (en) * | 2021-06-22 | 2022-02-18 | 苏州工业园区唯可达生物科技有限公司 | Recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibody existing in vivo |
| CN114058644A (en) * | 2021-06-22 | 2022-02-18 | 苏州工业园区唯可达生物科技有限公司 | Recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibody existing in vivo |
| CN114058645A (en) * | 2021-06-22 | 2022-02-18 | 苏州工业园区唯可达生物科技有限公司 | Recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibody existing in vivo |
| CN114032217A (en) * | 2021-11-02 | 2022-02-11 | 中国疾病预防控制中心性病艾滋病预防控制中心 | Novel coronavirus compound vaccine based on DNA vector and replicative vaccinia virus vector |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1295339C (en) | 2007-01-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1056878C (en) | Recombinant herpesvirus of turkeys and live vector vaccines derived thereof | |
| CN1020752C (en) | Vaccines and immunoassays for acquired immune deficiency syndrome | |
| JP4138848B2 (en) | Immunodeficient recombinant poxvirus | |
| CN103923884B (en) | A kind of porcine pseudorabies virus gene-deleted strain, vaccine combination and its preparation method and application | |
| CN1295339C (en) | Attenuated vaccinia virus Tiantan strain vector and its preparation and application | |
| CN1292071C (en) | Attenuated vaccinia virus Tiantan strain vector and its preparation and application | |
| JP2004331673A (en) | Animal cell and process for replication of influenza virus | |
| CN1912112A (en) | Recombinant infectious non-segmented negative strand rna virus | |
| CN1646158A (en) | Modified vaccinia virus ankara for the vaccination of neonates | |
| CN1289663C (en) | Method for the recovery and purification of poxviruses from infected cells using high pressure homogenisation | |
| CN110218706B (en) | Construction and application of recombinant turkey herpesvirus expressing HA protein of H7N9 subtype highly pathogenic avian influenza virus | |
| CN1800374A (en) | Recombinant gene engineered strain of aujeszkys disease-porcine reproductive and respirator syndrome virus and its uses | |
| CN1742083A (en) | Immunizing fish against viral infection | |
| CN107828741B (en) | Pseudorabies virus double-gene deletion attenuated strain and application thereof | |
| US20220267713A1 (en) | Method for producing virus and harvest liquid composition | |
| CN1990042A (en) | SARS coronary virus resistant cell vaccine and its uses | |
| WO2015149387A1 (en) | Method for adapting rabies virus ctn-1 strain to primary chicken embryonic fibroblasts | |
| AU6880501A (en) | Method for growing or for removing circoviruses from biological material | |
| JPS637754B2 (en) | ||
| CN104726409B (en) | A kind of preparation method and application of the duck embryos hepatic cell line of immortalization | |
| CN1258596C (en) | Leporipox-based vector vaccines | |
| CN108055827B (en) | High-growth enterovirus 71 type virus strain and vaccine thereof | |
| CN1283788C (en) | Recombination pseudorabies virus for expressing pig breeding and respiration comproehensive oyndrome virus and application | |
| CN1911445A (en) | Grippe primary generation susliks kidney cell multivalent raccine and its preparation method | |
| CN111690688B (en) | Recombinant Marek's disease virus expressing CRISPR/Cas9 targeting REV and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070117 Termination date: 20100211 |