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      改進(jìn)的病毒純化方法

      文檔序號:9344184閱讀:1997來源:國知局
      改進(jìn)的病毒純化方法
      【技術(shù)領(lǐng)域】 本發(fā)明涉及從細(xì)胞培養(yǎng)物提取病毒的方法。更具體地,該方法可以用于提取適合在臨 床上施用給哺乳動物(包括人)的形式的感染性病毒。 參考文獻(xiàn) 2002年3月28日公開的美國專利申請公開號20020037576。 1999年2月25日公開的TO99/08692A1。 1988年2月25日公開的日本專利63044532A。 Berry et al. , Biotechnology and Bioengineering,"Production of Reovirus Type-1 and Type-3 from Vero Cells Grown on Solid and Macroporous Microcarriers",Biotechnology and Bioengineering 62 :12-19 (1999) ? Bos,J. L., " Ras Oncogenes in Human Cancer :A Review" ? Cane. Res. 49 (17): 4682-4689(1989). Chandron and Nibert," Protease cleavage of reovirus capsid protein mul and mul C is blocked by alkyl sulfate detergents,yielding a new type of infectious subvirion particle",J. of Virology 72(1) :467_75 (1998). Coffey,M. C.,et al., " Reovirus therapy of tumors with activated Ras pathway" ,Science 282:1332-1334(1998). Davis,et al.,Microbiology,Lippincott,Philadelphia(1990) ? Drastini,Y. et al., " Comparison of eight different procedures for harvesting avian reoviruses grown in Vero cells" ? J.Virological Methods 39: 269-278(1992). Drayna D. and Fields B.N. /^Biochemical studies on the mechanism of chemical and physical inactivation of reovirus'',Journal of Genetic Virology63 (Pt 1): 161-170(1982). Duncan et al.," Conformational and functional analysis of the C-terminal globular head of the reovirus cell attachment protein ",Virology 182(2): 810-9(1991). Estes M. K. et al,"Rotavirus stability and inactivation'',J. of Genetic Virology 43(2):403-409 (1979). Floyd R. and Sharp D. G. /^Aggregation of poliovirus and reovirus by dilution in water'',Applied and Environmental Microbiology 33(1):159-167 (1977). Floyd R. and Sharp D. G.,''Viral aggregation :quantitation and kinetics of the aggregation of poliovirus and reovirus'Applied and Environmental Microbiology 35(6):1079-1083(1978). Floyd R. and Sharp D.G.,"Viral aggregation :effects of salts on the aggregation of poliovirus and reovirus at low pH'',Applied and Environmental Microbiology 35(6):1084-1094(1978).
      [0017] Floyd R. and Sharp D. G.,"Viral aggregation :buffer effects in the aggregation of poliovirus and reovirus at low and high pH'',Applied and Environmental Microbiology 38(3):395-401 (1979). Fields,B. N. et al.,F(xiàn)undamental Virology,3rd Edition,Lippincott-Raven(1996) ? Mah et al.," The N-terminal quarter of reovirus cell attachment protein sigma 1 possesses intrinsic virion-anchoring function ",Virology 179(1): 95-103(1990). McRae? M. A. and Joklik? ff. K. ? " The nature of the polypeptide encoded by each of the 10 double-stranded RNA segments of reovirus type 3",Virology,89 : 578-593(1979). Nibert et al., " Reovirus and their replication " ? in Fields et al., Fundamental Virology,3rd Edition,Lippincott-Raven(1996) ? Remingtonr s Pharmaceutical Sciences?Mack Publishing Company?Philadelphia Pa. 19. sup. th ed. (1995). Smith,R. E.,et al.," Polypeptide components of virions,top component and cores of reovirus type3!f? Virology? 39:791-800 (1969). Spinner M.L.and DiGiovanni G. D.,"Detection and identification of mammalian reoviruses in surface water by combined cell culture and reverse transcription-PCR'',Applied and Environmental Microbiology 67(7): 3016-3020(2001). Strong? J. E. and P. ff. Lee? " The v-erbV oncogene confers enhanced cellular susceptibility to reovirus infection" ? J. Virol. 70:612~616 (1996). Strong,J. E.,et al.," Evidence that the Epidermal Growth Factor Receptor on Host Cells Confers Reovirus Infection Efficiency ",Virology 197(1): 405-411(1993). Strong,J. E.,et al.," The molecular basis of viral oncolysis usurpation of the Ras signaling pathway by reovirus",EMB0 J. 17 :3351_3362(1998). Taber et al., " The selection of virus-resistant Chinese hamster ovary cells" ,Cell 8:529-533(1976). Taylor D.H.and Bosmann H.B. ,"Measurement of the electrokinetic properties of vaccinia and reovirus by laser-illuminated whole-particle microelectrophoresis",J. of Virology Methods 2(5) :251_260(1981). Turner and Duncan," Site directed mutagenesis of the C-terminal portion of reovirus protein sigmal :evidence for a conformation-dependent receptor binding domain" ,Virology 186(1) :219-27(1992). ZerdaK.S.etal,"Adsorptionofvirusestocharge-modifiedsilica'',Applied andEnvironmentalMicrobiology49(1) :91~95 (1985). 【背景技術(shù)】 由于病毒會造成非常多的疾病,病毒學(xué)已經(jīng)成為被集中研究的領(lǐng)域??偸谴嬖谟行?地生產(chǎn)病毒的需求,以便分離和純化病毒蛋白、制備疫苗、或提供用于實(shí)驗(yàn)室研究的感染性 病毒。近年來,病毒治療的新發(fā)展還需要有效率地生產(chǎn)感染性病毒。 呼腸孤病毒治療是病毒治療的一個(gè)實(shí)例。呼腸孤病毒是能結(jié)合許多種細(xì)胞的雙鏈RNA病毒。但是,大多數(shù)細(xì)胞對呼腸孤病毒感染不敏感,呼腸孤病毒向它的細(xì)胞受體的結(jié)合不會 在這些細(xì)胞中導(dǎo)致病毒復(fù)制或病毒顆粒生產(chǎn)。這可能是尚不清楚呼腸孤病毒與任何特定疾 病有關(guān)的原因。 用ras癌基因轉(zhuǎn)化的細(xì)胞會變得對呼腸孤病毒感染敏感,而它們的未轉(zhuǎn)化的對應(yīng)物則 不然(Strongetal.,1998)。例如,當(dāng)用激活的Ras或Sos(-種會激活Ras的蛋白)轉(zhuǎn)化 呼腸孤病毒-抗性的NIH3T3細(xì)胞時(shí),會增強(qiáng)呼腸孤病毒感染。類似地,在用EGF受體基因 或v-erbB癌基因(它們二者均會激活ras途徑)轉(zhuǎn)染后,對呼腸孤病毒感染有抗性的小鼠 成纖維細(xì)胞變得敏感(Strongetal.,1993;Strongetal.,1996)。因而,呼腸孤病毒可 以選擇性地感染具有激活的Ras途徑的細(xì)胞,并在其中復(fù)制。 ras癌基因是高比例的哺乳動物腫瘤的原因。激活ras基因自身的突變,發(fā)生在約 30%所有人腫瘤中(Bos,1989),主要是胰腺癌(90% )、散發(fā)的結(jié)腸直腸癌(50% )和肺癌 (40%),以及髓細(xì)胞白血?。?0%)。在ras途徑中,在ras上游或下游的因子的激活,也 與腫瘤有關(guān)。例如,ffiR2/Neu/ErbB2或表皮生長因子(EGF)受體的過表達(dá)常見于乳腺癌 (25-30%),血小板衍生的生長因子(PDGF)受體或EGF受體的過表達(dá)在神經(jīng)膠質(zhì)瘤和成膠 質(zhì)細(xì)胞瘤中很普遍(40-50% )。已知EGF受體和TOGF受體二者在結(jié)合它們各自的配體后 都會激活ras,v-erbB會編碼缺少胞外域
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