TWI354022B - Methods and compositions relating to expression fa - Google Patents

Methods and compositions relating to expression fa Download PDF

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TWI354022B
TWI354022B TW94100659A TW94100659A TWI354022B TW I354022 B TWI354022 B TW I354022B TW 94100659 A TW94100659 A TW 94100659A TW 94100659 A TW94100659 A TW 94100659A TW I354022 B TWI354022 B TW I354022B
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pump
cell
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candida
species
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TW200523364A (en
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yun liang Yang
Chiageun Chen
Hsiujung Lo
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Nat Health Research Institutes
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Description

1354022 九、發明說明: 【發明所屬之技術領域】1354022 IX. Description of the invention: [Technical field to which the invention belongs]

本發明係有關於數種表現因子。調控表現因子之表現 或活性可調節如藥物排除量(drug efflux)、微生物毒性及/ 或細胞生長等作用。表現因子包括轉錄因子、轉譯因子、 抑制因子及排除泵浦(efflux pumps)表現因子等。表現因 子本身之聚核苷酸序列中可能包含一或多個的中程孢子 生成序列(mid-sporulation element, MSE),又或表現因子 可能與目標聚核苷酸中的中程孢子生成序列結合,以調控 自身或目標聚核苷酸序列的表現。可藉著調控表現因子與 中程孢子生成序列之間的結合、調控藥物排除泵浦之表 現、或調控表現因子自身之表現或活性,來調控細胞之藥 物排除量、微生物毒性及/或細胞生長。 【先前技術】The present invention is directed to several performance factors. The performance or activity of the regulatory expression factors can be adjusted, such as drug efflux, microbial toxicity, and/or cell growth. Performance factors include transcription factors, translation factors, inhibitors, and efflux pumps. The polynucleotide sequence of the expression factor itself may contain one or more mid-sporulation elements (MSEs), or the expression factors may bind to the mid-range spore-forming sequences in the target polynucleotide. To regulate the performance of a self or target polynucleotide sequence. Cellular drug elimination, microbial toxicity and/or cell growth can be regulated by binding between regulatory expression factors and mid-range spore-forming sequences, regulating drug excluding pump performance, or regulating the performance or activity of the expression factor itself. . [Prior Art]

因各種用來治療及預防疾病之抗微生物劑的泛用(許 多病例中,會於病癒後再持續治療一段時間),故微生物 所產生之抗藥性已使大多數的藥物失效(see e.g·, ex ei al., Antimicrob. Agents and Chemother. 39:1 -8, 1 995;Because of the widespread use of various antimicrobial agents used to treat and prevent disease (in many cases, it will continue to be treated for a period of time after recovery), the drug resistance produced by microorganisms has rendered most drugs ineffective (see eg· , ex ei al., Antimicrob. Agents and Chemother. 39:1 -8, 1 995;

Vanden Bos sc he et a 1., Trends Microbiol. 2:393-400, 1 994)。引起抗藥性的最普遍機轉是與藥物相關之生化路 徑中的目標酵素(target enzyme)或其他酵素發生變異或突 變所致。例如,在臨床嗤類藥物(a ζ ο 1 e s)抗藥性培養試驗 中已鑑定出 ERG11 基因單點突變種的白色念珠菌 5 1354022 {Candida albicans) (White, Antimicrob. Agents Chemother 41:1488-14 94, 1997)。亦有文獻報導在臨床培養過程中已 在至少兩種白色念珠菌中找到造成抗藥性的五Λ 基因突 變種(尺e//少 ei a/·, FEBS Lett. 400:80-82,1 997)。Vanden Bos sc he et a 1., Trends Microbiol. 2:393-400, 1 994). The most common mechanism for causing drug resistance is caused by mutations or mutations in target enzymes or other enzymes in the biochemical pathways associated with drugs. For example, Candida albicans 5 1354022 {Candida albicans) (White, Antimicrob. Agents Chemother 41:1488-14) has been identified as a single point mutant of the ERG11 gene in a drug-resistant culture test for clinical steroids (a ζ ο 1 es). 94, 1997). It has also been reported in the literature that in the clinical culture process, five Λ gene mutations have been found to cause drug resistance in at least two species of Candida albicans (feet e// less ei a/·, FEBS Lett. 400:80-82,1 997 ).

然而,在許多案例中發現造成抗藥性的原因是抗藥性 細胞内部之藥物累積量下降所致(see, e.g·, Fanc/ert Bossche et al., Trends Microbiol. 2:393 -400, 1 9 9 4; Odds, J. Antimicrob. Chemother. 31: 463-471,1993)。細胞不論 是減少其攝入的藥物量及/或增加胞内藥物的排除速度均 可降低細胞内的藥物累積量。However, in many cases, the cause of drug resistance was found to be due to a decrease in drug accumulation within drug-resistant cells (see, eg, Fanc/ert Bossche et al., Trends Microbiol. 2:393-400, 1 9 9). 4; Odds, J. Antimicrob. Chemother. 31: 463-471, 1993). Whether the cell reduces the amount of drug it ingests and/or increases the rate of elimination of the intracellular drug, it reduces the amount of drug accumulation in the cell.

細胞膜上的運輸蛋白(membrane transporter proteins) 或稱排除泵浦(efflux pumps)與活躍的藥物排除作用有 關。從細菌至哺乳動物的細胞中普遍存在該些排除泵浦 (如文獻 Higgins,Annu. Rev. Cell Biol. 8:67- 1 1 3, 1 992 提 供相關資訊之回顧)。已知真核細胞具有兩種與抗藥性有 關的排除泵浦,分別是ATP結合區運輸蛋白(ATP binding cassette (ABC) transporters)與主要促進蛋白(major fac i 1 it at o rs) (Marge r and Saier, Jr. Trends Biochem. Sci. 18:13-20, 1 9 9 3; Michaelis and Berkower, Cold SpringMembrane transporter proteins or efflux pumps are associated with active drug elimination. These exclusion pumps are ubiquitous in bacteria-to-mammal cells (for a review of the information provided by Higgins, Annu. Rev. Cell Biol. 8:67- 1 1 3, 1 992). Eukaryotic cells are known to have two drug-related exclusion pumps, ATP binding cassette (ABC) transporters and major fac i 1 it at o rs (Marge r And Saier, Jr. Trends Biochem. Sci. 18:13-20, 1 9 9 3; Michaelis and Berkower, Cold Spring

Harbor Symp. Quant. Biol. 60:291-309,1995)。ATP 結合區 運輸蛋白係藉著水解ATP以獲得能量的方式來運作,而主 要促進蛋白則透過質子轉移來運作》 文獻顯示,過量表現細胞中的排除泵浦有助於細胞的 坑藥性(Kara baba et al., Antimicrob. Agents Chemother. 6 1354022 48:3064-3079, 2 0 0 4; Ma rger and Saier, Jr., TrendsHarbor Symp. Quant. Biol. 60:291-309, 1995). The ATP-binding region transport protein works by hydrolyzing ATP to obtain energy, while the main promoting protein operates through proton transfer. The literature shows that the exclusion of the pump in the excess expression cell helps the cell's potency (Kara baba) Et al., Antimicrob. Agents Chemother. 6 1354022 48:3064-3079, 2 0 0 4; Ma rger and Saier, Jr., Trends

Biochem. Sci. 18:1 3 -20, 1 993; Michaelis and B e r kow e r,Biochem. Sci. 18:1 3 -20, 1 993; Michaelis and B e r kow e r,

Cold Spring Harb. Symp.Quant.Biol. 60:29 1 -307, 1995)。 例如,白色念珠菌的CD/?/基因即可編碼出一個ATP結合 區排除泵浦(/Vasad er fl/‘, Curr_ Genet. 27:320-329, 1 995)。若基因發生突變,會造成ATP結合區排除 泵浦的表現量減少,進而增加白色念珠菌對β坐類藥物的敏 感性(Sanglard et al., Antimicrob. Agents Chemother. 40:23 00-23 05,1 996)。亦觀察到,過量表現CZ)iH基因有 助於增進白色念珠菌之抗藥性ei 〇/., Antimicrob. Agents Chemother. 42:2932-2937, 1 998; Yang and Lo,J Microbiol Immunol Infect 34:79-86,2001) 〇 此類排除泵浦的表現係受順式(cis)與反式(trans)-調控 因子調節。例如,文獻曾報導CD·/?/基因啟動子(promoter) 上的 AP-1 位址(AP-1 site)與藥物應答序列(Drug Responsive Element, DRE)是順式-調控序歹ij(de eiCold Spring Harb. Symp.Quant. Biol. 60:29 1 -307, 1995). For example, the CD/?/ gene of Candida albicans encodes an ATP binding region to exclude the pump (/Vasad er fl/', Curr_Genet. 27:320-329, 1 995). If the gene is mutated, it will result in a decrease in the ATP-binding region's exclusion of pumping, which in turn increases the sensitivity of Candida albicans to beta-based drugs (Sanglard et al., Antimicrob. Agents Chemother. 40:23 00-23 05, 1 996). It has also been observed that overexpression of the CZ)iH gene contributes to the resistance of Candida albicans ei 〇/., Antimicrob. Agents Chemother. 42:2932-2937, 1 998; Yang and Lo,J Microbiol Immunol Infect 34:79 -86, 2001) The performance of such excluded pumps is regulated by cis and trans-regulators. For example, the literature reports that the AP-1 site and the Drug Responsive Element (DRE) on the CD//?/gene promoter are cis-regulatory sequences. Ei

al., Mol. Microbiol. 43:1197-1214, 2002; Puri et al., FEMS Microbiol.Lett. 180:213-219,1999)。亦有文獻暗 示,細胞中具有(:£>/?/基因·的反式-調控因子(户wrz· ei fl/., FEMS Microbiol.Lett. 180:213-21 9, 1999)。 因此,若能了解調控排除泵浦之表現過程中的分子機 轉與各基因之間的相互關聯性,將有助於未來藥物的設計 與研發。 7 1354022Al., Mol. Microbiol. 43: 1197-1214, 2002; Puri et al., FEMS Microbiol. Lett. 180: 213-219, 1999). There is also literature suggesting that there are trans-regulatory factors in the cell (: £>/?/gene· (Well wrz·ei fl/., FEMS Microbiol. Lett. 180:213-21 9, 1999). If you can understand the correlation between the molecular mechanism and the genes in the process of regulating and eliminating the pump, it will help the design and development of future drugs. 7 1354022

【發明内容】 本文係根據本案發明人所發現之可調控排除泵 現的表現因子所做之發明。此外,本案發明人亦發現 現因子另外參與其他不涉及排除泵浦的訊息傳遞路徑 如,本發明之一表現因子的聚核苷酸中含有一或多個 程孢子生成序列(MS E),或該表現因子可與一目標聚 酸的中程孢子生成序列結合,以調控該表現因子自身 核苷酸或目標聚核苷酸的表現。因此,調控本發明之 因子活性及/或表現會對細胞造成各種不同影響,該 響包括抑制細胞的藥物排除反應、毒性及/或生長。 因子包含轉錄因子、轉譯因子、抑制因子及排除泵浦 因子等。 本發明之方向在於提供一種藉由抑制細胞中至 表現因子的活性或表現以抑制細胞生長、發育的方法 且此方法更包含使上述細胞與至少一種藥物接觸。本 之另一方向在於提供一種藉著使一細胞與至少一種 因子抑制劑接觸,以抑制該細胞生長的方法,且該方 包含令該細胞與至少一藥物接觸。本發明一較佳實 中,表現因子可能是一排除泵浦表現因子,表現因子 劑則可能是一種排除系浦表現因子抑制劑。因此,本 亦提供一種藉著使細胞與至少一排除泵浦表現因子 劑及至少一種藥物接觸,以提高該藥物活性的方法。 本發明另一方向在於提供一種藉著抑制細胞中 一排除泵浦聚核苷酸的表現以抑制細胞生長的方法, 浦表 該表 〇例 的中 核苷 之聚 表現 些影 表現 表現 少一 ,並 發明 表現 法更 施例 抑制 發明 抑制 至少 且此 8 1354022 方法更包括使上述細胞與至少一種藥物接觸。本發明亦提 供一種藉著抑制細胞中至少一排除泵浦聚核苷酸之表 現,並使該細胞與至少一藥物接觸以提高藥物活性的方 法。 另一種抑制細胞生長的方法包含抑制至少一表現因 子與中程孢子生成序列結合。該方法更包含使細胞接觸至 少一種藥物。SUMMARY OF THE INVENTION This document is based on the invention found by the inventors of the present invention to control the performance of the pump. In addition, the inventors of the present invention have also found that the current factor additionally participates in other message delivery paths that do not involve the exclusion of the pump. For example, the polynucleotide of one of the present invention has one or more spore generating sequences (MS E), or The expression factor can bind to a mid-range spore-forming sequence of a target polyacid to modulate the expression of the expression factor's own nucleotide or target polynucleotide. Thus, modulation of the activity and/or expression of a factor of the invention can have various effects on the cell, including inhibition of drug rejection, toxicity, and/or growth of the cell. Factors include transcription factors, translation factors, inhibitors, and exclusion of pumping factors. The present invention is directed to a method for inhibiting cell growth and development by inhibiting the activity or expression of a factor in a cell and further comprising contacting said cell with at least one drug. Another aspect of the present invention is to provide a method of inhibiting the growth of a cell by contacting a cell with at least one factor inhibitor, and the method comprises contacting the cell with at least one drug. In a preferred embodiment of the invention, the performance factor may be an exclusion of the pump performance factor and the performance factor agent may be an exclusion factor. Accordingly, there is also provided a method of increasing the activity of a drug by contacting the cell with at least one drug expelling agent and at least one drug. Another aspect of the present invention is to provide a method for inhibiting cell growth by inhibiting the expression of a pumping polynucleotide in a cell, and the nucleoside aggregation of the surface of the sample shows that the expression of the nucleoside is less than one. The invention of the invention inhibits the inhibition of the invention at least and the 8 1354022 method further comprises contacting the cells with at least one drug. The invention also provides a method of increasing the activity of a drug by inhibiting at least one of the cells from inhibiting the expression of the pumped polynucleotide and contacting the cell with at least one drug. Another method of inhibiting cell growth comprises inhibiting binding of at least one of the expression factors to a mid-range spore-forming sequence. The method further comprises contacting the cell with at least one drug.

本發明亦提供一種藉著抑制至少一表現因子的活性 或表現,以抑制一細胞毒性的方法。該方法可包含使上述 細胞與至少一表現因子抑制劑接觸,更可包含使該細胞與 至少一藥物接觸。The invention also provides a method of inhibiting a cytotoxicity by inhibiting the activity or expression of at least one expression factor. The method can comprise contacting the cell with at least one expression factor inhibitor, and more preferably contacting the cell with at least one drug.

本發明又提供一種治療患有疾病、機能失調或感染之 患者的方法。上述方法包括對該患者施用至少一有效劑量 之表現因子抑制劑,更包括對該患者施用至少一有效劑量 之藥物。本發明另一方向在於提供一種藥學組合物,其包 含至少一種表現因子抑制劑,且更可包含至少一種藥物。 上述藥學組合物更可包含一種藥學上可接受之載劑。 本發明又提供一種抑制細胞生長的組合物,此組合物 包含至少一種表現因子抑制劑,且更可包含至少一種藥 物。本發明還提供一種抑制微生物細胞之毒性的組合物, 其包含至少一種表現因子抑制劑,更可包含至少一種抗微 生物藥劑。 本發明更提供一種篩選可編碼出排除泵浦表現因子 之聚核苷酸的方法。該方法包含下列步驟:將一個受排除 9 1354022The invention further provides a method of treating a patient suffering from a disease, disorder or infection. The above method comprises administering to the patient at least one effective amount of a performance factor inhibitor, and further comprising administering to the patient at least one effective dose of the drug. Another aspect of the present invention is to provide a pharmaceutical composition comprising at least one expression factor inhibitor, and more preferably at least one drug. The above pharmaceutical compositions may further comprise a pharmaceutically acceptable carrier. The invention further provides a composition for inhibiting cell growth, the composition comprising at least one expression factor inhibitor, and more preferably at least one drug. The present invention also provides a composition for inhibiting the toxicity of a microbial cell, comprising at least one expression factor inhibitor, and more preferably at least one anti-microbial agent. The invention further provides a method of screening for a polynucleotide that encodes a pump performance factor. The method consists of the following steps: one is excluded 9 1354022

泵浦啟動子控制的報導基因導入一宿主細胞内;將一 排除泵浦表現因子之聚核苷酸植入該宿主細胞中;以 測上述報導基因的表現情形。該方法亦可用來篩選一 因庫(genomic library)或 cDNA 庫(cDNA library)中的 泵浦表現因子聚核苷酸。 【實施方式】 本發明提供一種藉著抑制一細胞中至少一種表現 的活性或表現,以抑制該細胞生長的方法。表現因子 包括轉錄因子、轉譯因子、抑制因子及排除泵浦表現g 本發明亦提供一種藉著抑制一細胞中至少一排除泵 核苷酸之表現且使該細胞接觸至少一種藥物,以抑制 胞生長的方法。 上述表現因子可能影響細胞之毒性,因此抑制表 子的活性或表現便可能抑制細胞毒性。故本發明提供 藉著抑制一細胞中至少一種表現因子之活性或表現 制該細胞毒性的方法。可藉著使一細胞與至少一種表 子抑制劑接觸,且更可使該細胞與一藥物接觸,而得 制該細胞的毒性。另一方面,上述表現因子可能藉著 中程孢子生成序列而影響細胞生長。中程孢子生成序 能調控一表現因子之聚核苷酸的表現,及/或調控該 因子之目標聚核苷酸的表現。因此,本發明提供一種 抑制表現因子與一中程孢子生成序列之結合作用以 細胞生長的方法,且此方法更包括使該細胞與至少一 候選 及偵 體基 排除 因子 至少 子。 浦聚 該細 現因 一種 以抑 現因 以抑 影響 列可 表現 藉著 抑制 藥物 10 1354022 接觸。 在一較佳實施例中,上述細胞可為植物細胞'細菌細 胞、酵母菌細胞或哺乳動物細胞。另一較佳實施例中,酵 母菌細胞為真菌細胞,且選自於由念珠菌屬 species)、曲真菌屬species)及隱球菌屬 (Ο少〆ococc μ species)所構成之群組中。念珠菌屬可選自 於由白色念珠菌(C. 克柔念珠菌(C. A:rwsez·)、熱The reporter gene controlled by the pump promoter is introduced into a host cell; a polynucleotide excluding the pump expression factor is implanted into the host cell; and the expression of the reporter gene is measured. This method can also be used to screen for pump expression factor polynucleotides in a genomic library or cDNA library. [Embodiment] The present invention provides a method for inhibiting the growth of a cell by inhibiting the activity or expression expressed by at least one of the cells. Expression factors include transcription factors, translation factors, inhibitory factors, and exclusion of pump performance. The present invention also provides a method for inhibiting cell growth by inhibiting at least one of the cells to exclude the expression of pump nucleotides and contacting the cells with at least one drug. Methods. These performance factors may affect the toxicity of the cells, and thus inhibiting the activity or performance of the epitope may inhibit cytotoxicity. Therefore, the present invention provides a method for inhibiting the activity of at least one expression factor in a cell or expressing the cytotoxicity. The toxicity of the cells can be obtained by contacting a cell with at least one of the epitope inhibitors and more contacting the cells with a drug. On the other hand, the above expression factors may affect cell growth by a mid-range spore-forming sequence. The mid-range spore formation sequence can regulate the expression of a polynucleotide of a performance factor and/or the performance of the target polynucleotide that regulates the factor. Accordingly, the present invention provides a method of inhibiting the binding of a performance factor to a mid-range spore-forming sequence for cell growth, and the method further comprises at least one candidate and a detector-based exclusion factor. Pu Ju is the result of a kind of inhibition of the effect of the inhibition of the drug by the inhibition of the drug 10 1354022 contact. In a preferred embodiment, the above cells may be plant cells 'bacterial cells, yeast cells or mammalian cells. In another preferred embodiment, the yeast cells are fungal cells and are selected from the group consisting of Candida species, Aspergillus species, and Cryptococcus ococc μ species. Candida can be selected from Candida albicans (C. a. rwsez), heat

帶念珠菌(C. iro/j/ca/b)及光滑念珠菌(C.容/aftraia)所構成 之群組中。曲真菌屬可能為薰煙色麴菌(Wipe /wm/gfliws)或黑麴菌niger)。隱球菌屬則可為 新型隱球菌(CV少prococcwj· «eo/orman·?)。亦可使用他種為 習知技藝者所熟知的真菌細胞。哺乳動物細胞則包括齧齒 類、人類、非人之靈長類、馬類、犬科、貓科、牛亞科、 豬科、羊屬、兔類等動物之細胞。In a group consisting of Candida (C. iro/j/ca/b) and Candida glabrata (C. 容/aftraia). The fungus genus may be Wipe sphaeroides (Wipe / wm / gfliws) or sorghum niger). Cryptococcus can be a new type of cryptococci (CV less prococcwj·eo_orman·?). It is also possible to use fungal cells known to those skilled in the art. Mammalian cells include cells of rodents, humans, non-human primates, equine, canine, feline, bovine subfamily, porcine, sheep, and rabbits.

在另一較佳實施例中,排除泵浦聚核苷酸為CDi?/、 或其同源體(homolog)或變異體(variant)。並 可藉著抑制排除泵浦聚核苷酸的轉錄與/或轉譯作用來調 控排除泵浦的表現。在一較佳實施例中,係利用一排除泵 浦表現因子來調控該些排除泵浦聚核苷酸的表現^在一較 佳實施例中,排除泵浦表現因子為 CaNdt80p。而在另一 較佳實施例中,排除泵浦表現因子是排除泵浦調節因子1 (Regulator of Efflux Pump 1,REP 1)。故本發明提供一種 藉著抑制至少一排除泵浦表現因子之活性或表現以抑制細 胞生長的方法。本發明之另一方向包括使細胞與至少一種 11 1354022 排除泵浦表現因子接觸,且更包括使該細胞與至少一種藥 物接觸。 上述較佳實施例及文中所用之專業術語,均僅作敘述 範例之用,並非用來限定本發明。該些未於文中敘述,但 習知技藝者可根據本發明揭露内容而實施之其他較佳實施 例亦為本發明範圍所涵蓋。In another preferred embodiment, the pumped polynucleotide is excluded from CDi?/, or a homolog or variant thereof. The performance of the excluded pump can be modulated by inhibiting the transcription and/or translation of the pumped polynucleotide. In a preferred embodiment, an exclusion of pump performance factor is utilized to modulate the performance of the excluded pumped polynucleotides. In a preferred embodiment, the pump performance factor is CaNdt80p. In yet another preferred embodiment, the exclusion of the pump performance factor is the elimination of the Regulator of Efflux Pump 1, REP 1 . The present invention therefore provides a method for inhibiting cell growth by inhibiting at least one activity or performance that excludes pump expression factors. Another aspect of the invention includes contacting the cells with at least one of the 13 1354022 exclusion pump expression factors, and further comprising contacting the cells with at least one drug. The above-described preferred embodiments and the technical terms used herein are for illustrative purposes only and are not intended to limit the invention. Other preferred embodiments that can be implemented in accordance with the present disclosure are also encompassed by the scope of the present invention.

文中,聚核苷酸之名稱均以斜體字型標示。文中之「聚 核苦酸(polynucleotide) j、「核苦酸(nucleotide) j 及「核酸 (nucleic acid)」等用語係指DNA或RNA。由於突變種聚 核苷酸未以小寫字形標示,故野生種聚核苷酸以大寫標示 以作區分。此外,如文中 之「Ca」係指在白色 念珠菌中與啤酒酵母菌(S1· 之基因同源In this context, the names of the polynucleotides are indicated in italics. The terms "polynucleotide", "nucleotide" and "nucleic acid" refer to DNA or RNA. Since the mutant polynucleotide is not indicated in the form of a small letter, the wild type polynucleotide is indicated in upper case to distinguish it. In addition, "Ca" as used herein refers to a gene homologous to S. cerevisiae (S1·) in Candida albicans.

的同源體(homolog)。文中之蛋白質不採斜體字型,並將 蛋白質名稱之第一個字母大寫。部分蛋白質名稱後方會加 註一小寫字母「p」,以標示其為一蛋白質分子。此處用來 區分一分子是野生種或突變種、是聚核苷酸或蛋白質之命 名規則均為習知技藝者所能清楚瞭解。 本發明係有關於一表現因子或其同源體或變異體之 調控作用。文中 「調控(modulate)」、「調控作用 (modulation)」係指提高或降低一表現因子的活性或表 現。「活性(a c t i v i t y)」一詞係指一個分子所表現出來的功 能或一組活動,其可能包含分子與各自之目標分子的結合 反應、該分子自身之聚核苷酸或一目標聚核苷酸之轉錄與 /或轉譯作用的抑制或活化。「表現(e X p r e s s i ο η)」一詞係指 12 1354022 一聚核苷酸或一蛋白質的轉錄與/或轉譯作用。Homolog. The protein in the text does not use italics and capitalizes the first letter of the protein name. Some protein names will be followed by a lowercase letter "p" to indicate that it is a protein molecule. The rules used to distinguish between a molecule that is a wild species or a mutant, a polynucleotide or a protein are well known to those skilled in the art. The present invention relates to the regulation of a expression factor or a homolog or variant thereof. In the context, "modulate" or "modulation" refers to increasing or decreasing the activity or performance of a performance factor. The term "activity" refers to a function or group of activities exhibited by a molecule, which may involve the binding reaction of a molecule to its respective target molecule, the polynucleotide of the molecule itself or a target polynucleotide. Inhibition or activation of transcription and/or translation. The expression "e X p r e s s i ο η" refers to the transcription and/or translation of a 13 1354022 polynucleotide or a protein.

「抑制反應(inhibition)」或「抑制(inhibiting)j —詞 係指降低或停止一細胞之任何表現型變化。對細胞生長而 言,「抑制反應」係指降低或停止細胞的生長速率。例如, 降低一種微生物族群的生長速度。舉例來說,可根據添加 抑制劑或不添加抑制劑之液體培養菌液的混濁度差異來 監控抑制反應的程度。或是根據培養孤上塗有抑制劑或不 含抑制劑之區域中的菌落大小來觀察抑制反應的情況。此 外,尚有數種習知技藝者熟知的其他方法可用來監控抑制 反應。聚核苷酸表現的「抑制反應」係指降低或停止一聚 核苷酸的轉錄或轉譯作用。在含有抑制劑或不含抑制劑的 情況下,可利用如北方墨點法(Northern blot)或西方墨點 法(Western blot)等方法來彳貞測mRNA或基因產物的量,以 監控或偵測聚核苷酸表現的抑制反應。"Inhibition" or "inhibiting" - refers to any phenotypic change in a cell that is reduced or stopped. For cell growth, "inhibition reaction" refers to reducing or stopping the growth rate of a cell. For example, reducing the growth rate of a microbial population. For example, the degree of inhibition reaction can be monitored based on the difference in turbidity of the liquid culture broth with or without the addition of an inhibitor. Alternatively, the inhibition reaction may be observed depending on the size of the colony in the area where the inhibitor is coated with or without the inhibitor. In addition, there are several other methods well known to those skilled in the art to monitor inhibition reactions. The "inhibitory response" exhibited by a polynucleotide refers to the reduction or cessation of transcription or translation of a polynucleotide. In the case of inhibitors or no inhibitors, methods such as Northern blot or Western blot can be used to detect the amount of mRNA or gene product for monitoring or detection. The inhibition reaction of the expression of the polynucleotide is measured.

「活化反應(activation)」或「活化(activating)」一詞 係指提高或促進一細胞之任何表現型的變化。對細胞生長 而言,「活化反應」係指提高細胞的生長速率,或使細胞 進行分裂,並可參考上述方法來監控這類活化反應。聚核 苷酸表現的「活化反應」係指提高或啟動一聚核苷酸的轉 錄或轉譯作用。此外,亦可採用如上述之方法來監控或偵 測聚核苷酸表現的活化反應。 「微生物(microbe)」或「微生物細胞(microbial cell)」 係指極微小的有機生物,通常需在顯微鏡下或是該些微生 物堆積成群時方可觀察到該些有機生物。「微生物」或「微 13 1354022 生物細胞」包括細菌、藻類、真菌類及原生動物。「微生 物J或「微生物細胞J 一詞亦包含處於多核(p〇lynucleate) 或單細胞(single-cel丨ed)狀態之有機生物。 「藥物(drug)」一詞係指用於診斷、治療、減緩、處 理或預防一患者之疾病或症狀的化合物,以及/或可影響 身體或細胞之各種功能或結構的化合物(除食物之外)。在 本發明一較佳實施例中,所使用之藥物會抑制細胞生長。The term "activation" or "activating" refers to the promotion or promotion of any phenotypic change in a cell. For cell growth, "activation reaction" refers to increasing the growth rate of cells or dividing cells, and monitoring such activation reactions can be carried out by referring to the above methods. The "activation reaction" exhibited by a polynucleotide means an increase or initiation of transcription or translation of a polynucleotide. In addition, methods such as those described above can also be employed to monitor or detect the activation reaction exhibited by the polynucleotide. "Microbe" or "microbial cell" refers to very small organic organisms that are usually observed under a microscope or when the microbes are clustered. "Microorganisms" or "micro 13 1354022 biological cells" include bacteria, algae, fungi and protozoa. "Microbial J or "Microbial Cell J" also includes organic organisms in a p〇lynucleate or single-cel丨ed state. The term "drug" is used for diagnosis, treatment, A compound that slows, treats, or prevents a disease or condition of a patient, and/or a compound that affects various functions or structures of the body or cells (other than food). In a preferred embodiment of the invention, the drug used inhibits cell growth.

表現因子包括轉錄因子、轉譯因子及抑制因子,且並 不僅限於上述種類。在一特定較佳實施例中,表現因子為 排除果浦表現因子。文中「排除栗浦(effluxpump)」一詞 係指一個蛋白質組合體(proteinassembly),其藉著消耗能 量來將細胞質或胞質周緣區内的受質分子運輸至細胞 外。本發明一較佳實施例中,排除泵浦參與細胞的抗藥性 作用。參與抗藥性作用的細菌排除泵浦包括 QacA、 NorA(Bmr)、Smr、AcrAB 與 MexAB-OprM (ZoWj, Trends Biochem. Sci. 1 9:1 1 9- 1 23, 1 994; Nikaido, SciencePerformance factors include transcription factors, translation factors, and inhibitors, and are not limited to the above categories. In a particularly preferred embodiment, the performance factor is the exclusion of the fruit performance factor. The term "effluxpump" is used to refer to a protein assembly that transports the cytoplasmic or cytoplasmic peripheral molecules to the extracellular domain by consuming energy. In a preferred embodiment of the invention, the resistance of the pump to the cells is excluded. Bacterial exclusion pumps involved in drug resistance include QacA, NorA (Bmr), Smr, AcrAB and MexAB-OprM (ZoWj, Trends Biochem. Sci. 1 9:1 1 9- 1 23, 1 994; Nikaido, Science

25^:382-388, 1 9 9 4; Poole e t al., J. Bacteriol. 175:7363-7372, 1 9 9 3; Okusu e t al., J. Bacteriol. 178:3 06-3 08,1 996; Nikaido, J. Bacteriol. 1 7 8:5 85 3 -5 859, 1 996)。真核細胞之排除泵浦包括ATP結合區運輸蛋白與 主要促進蛋白(MF) (PF/z/ie ei a/., Clinical Microbiol. Reviews 11:382-402,1998),亦可在人類細胞中發現這類運 輸蛋白(see, e_g·, Z/iman ei a/., Cell Mol Life Sci. 58:931-59,2001)。至今為止,已在啤酒酵母菌中找到至少 14 1354022 30種ATP結合區運輪蛋白,並根據該些ATP結合區運輸 蛋白的序列相似性將其分成6大家族。目前已知PZ)及5、 與三個家族之成員在不同系統中扮演產 生抗藥性的角色。已有文獻敘述在白色念珠菌中發現 家族的成員,並將這些基因命名為CDi?。特別是CDi?/ 與CZ)i?2’已為大眾所知悉是參與白色念珠菌之唑類化合 物抗藥性的兩個排除泵浦(尸"〇1^£/6/〇/.,0;111^.〇61161;· 27:320-329, 1995; Fling et al., Mol. Molec. Genet.25^: 382-388, 1 9 9 4; Poole et al., J. Bacteriol. 175:7363-7372, 1 9 9 3; Okusu et al., J. Bacteriol. 178:3 06-3 08,1 996; Nikaido, J. Bacteriol. 1 7 8:5 85 3 -5 859, 1 996). Excluded pumps for eukaryotic cells include ATP-binding region transport proteins and major-promoting proteins (MF) (PF/z/ie ei a/., Clinical Microbiol. Reviews 11:382-402, 1998), also in human cells. Such transport proteins were found (see, e_g., Z/iman ei a/., Cell Mol Life Sci. 58:931-59, 2001). To date, at least 14 1354022 30 ATP-binding domain proteins have been found in S. cerevisiae, and they have been divided into 6 major families based on the sequence similarity of these ATP-binding region transport proteins. It is currently known that PZ) and 5, members of the three families play a drug-resistant role in different systems. It has been documented that members of the family are found in Candida albicans and these genes are named CDi?. In particular, CDi?/ and CZ)i?2' have been known to the public as two exclusion pumps involved in the resistance of Candida albicans to azoles (corporate "〇1^£/6/〇/.,0 ;111^.〇61161;· 27:320-329, 1995; Fling et al., Mol. Molec. Genet.

227:3 1 8-329, 1 99 1; Sangalrd et al., Antimicrob. Agents Chemother. 39:2378-2386, 1 9 9 5; Sanglard e t al.,227:3 1 8-329, 1 99 1; Sangalrd et al., Antimicrob. Agents Chemother. 39:2378-2386, 1 9 9 5; Sanglard e t al.,

Antimicrob. Agents and Chemother. 40:2300-2305, 1 9 9 6; Sanglard et al., Microbiology 143:405-4 1 6, 1 997)° 參與白 色念珠菌之抗藥性的主要促進蛋白家族成員則包括MD/? 7 與Fli/J,其中 MDR1又稱為 CaMDi?/,更早期貝|J被稱為 BENr)(Fling et al., Mol. Molec. Genet. 227:3 1 8-329, 1 99 1; C a / a 6/* e i e e ί a /., M i c r o b i o 1 o g y 14 6 :2 7 4 3 - 2 7 5 4,2 0 0 0)。Antimicrob. Agents and Chemother. 40:2300-2305, 1 9 9 6; Sanglard et al., Microbiology 143:405-4 1 6, 1 997)° The main promoting protein family members involved in the resistance of Candida albicans include MD/? 7 and Fli/J, where MDR1 is also known as CaMDi?/, and earlier Bay|J is called BENr) (Fling et al., Mol. Molec. Genet. 227:3 1 8-329, 1 99 1; C a / a 6/* eiee ί a /., M icrobio 1 ogy 14 6 : 2 7 4 3 - 2 7 5 4, 2 0 0 0).

本發明亦有關於排除泵浦之同源體或變異體。文中 「同源體(homolog)」一詞係指在不同有機生物中展現相 似功能的結構或作用(processes)。如,的同源體可 能表現出與 CZ)/? i相似的功能,例如展現排除泵浦的功 能。CZ)i?7同源體的聚核苷酸與CDA7之聚核苷酸可能具 有相似的一級或二級結構。且CZ)i? /同源體的聚核苷酸所 表現出的基因產物結構,可能與CD之聚核苷酸的基因 產物結構類似。文中「變異體(variant)」一詞係指一種天 15 1354022The invention also relates to the exclusion of pumped homologs or variants. In the text, the term "homolog" refers to the structure or process that exhibits similar functions in different organic organisms. For example, a homologue may exhibit functions similar to CZ)/?i, such as exhibiting the ability to exclude pumping. The polynucleotide of the CZ) i?7 homolog may have a similar primary or secondary structure to the polynucleotide of CDA7. And the nucleotide structure of the CZ)i?/homologous polynucleotide may be similar to the gene product structure of the CD polynucleotide. The term "variant" in the context refers to a day 15 1354022

然或人工合成的氨基酸序列,其氨基酸序列與原排除泵浦 大致相同’但可能因刪除、取代或插入一個或多個氨基 酸’而使得變異體具有一段與原排除泵浦不同的氨基酸序 列《同樣地’一排除泵浦變異體之聚核苷酸係指一段天然 或人工合成的核苷酸序列,其序列與原排除泵浦聚核苷酸 序列大致相同’但可能因刪除、取代或插入一個或多個核 苷酸,而使變異體具有一段與原排除泵浦聚核苷酸不同的 核苷酸序列。相較於原排除泵浦,排除泵浦變異體(或一 排除泵浦聚核苷酸變異體)可能保留排除泵浦的活性,或 具有更高的排除泵浦活性。Or a synthetic amino acid sequence whose amino acid sequence is substantially identical to the original exclusion pump but may be deleted, substituted or inserted with one or more amino acids, such that the variant has a different amino acid sequence than the original exclusion pump. A polynucleotide that excludes a pump variant refers to a natural or synthetic nucleotide sequence whose sequence is approximately identical to the original excluded pump polynucleotide sequence 'but may be deleted, replaced or inserted Or a plurality of nucleotides, such that the variant has a different nucleotide sequence than the original excluded pump polynucleotide. Excluding pump variants (or one that excludes pumped polynucleotide variants) may retain pumping activity or have higher exclusion pump activity than the original exclusion pump.

此處所用之序列「相似性(similarity)」及/或「一致 性(identity)」一詞係用來敘述兩個聚核苷酸或聚胜肽 (polypeptide)序列之間的相關程度。通常,「一致性」代 表兩或多個核苷酸或氨基酸之序列相符,且被拿來相互比 較的核苷酸或氨基酸是相同的。而「相似性」通常是指代 表兩或多個核苷酸或氨基酸的序列剛好相符,且被拿來相 互比較的核苷酸或氨基酸若不是相同就是具有相似的化 學及/或物理性質。可利用如Devereux等人所叙述之GAP 電腦程式第6版來比較序列,以測定序列的一致性或相似 性百分比12:387,1984),其中 GAP 電腦 程式第6版可自美國威斯康辛大學基因運算中心取得(the University of Wisconsin Genetics Computer Group, UWGCG)。GAP程式採用Needleman與Wunsch所創並經 Smith 與 Waterman 修改的排列方法(J_ Mo/· 5ίο/. 48 :443, 16 1354022 1 970 ; Adv. Appl. Mar/z 2 :482,1 98 1)。亦可使用其他習知程 式來計算兩序列之間的一致性或相似性。 為達成本發明目的,排除泵浦或排除泵浦聚核苷酸之 變異體或同源體與原排除泵浦之間具有至少2 0 %、3 0 % 或 40 %的核苷酸或氨基酸一致性。本發明中所使用之序 列則與原排除泵浦之間具有至少約5 0 %、6 0 %、7 〇 %、The term "similarity" and/or "identity" as used herein is used to describe the degree of correlation between two polynucleotide or polypeptide sequences. Generally, "consistency" refers to the sequence of two or more nucleotides or amino acids that are identical and are identical to each other. "Similarity" generally refers to a sequence that represents two or more nucleotides or amino acids that exactly coincide, and the nucleotides or amino acids that are compared to each other have similar chemical and/or physical properties if they are not identical. The GAP computer program version 6 as described by Devereux et al. can be used to compare sequences to determine sequence identity or similarity ratios 12:387, 1984), of which the GAP computer program version 6 can be derived from the University of Wisconsin genetic algorithm The University of Wisconsin Genetics Computer Group (UGGCG). The GAP program uses the arrangement method created by Needleman and Wunsch and modified by Smith and Waterman (J_Mo/· 5ίο/. 48:443, 16 1354022 1 970; Adv. Appl. Mar/z 2:482,1 98 1). Other conventional procedures can also be used to calculate the consistency or similarity between the two sequences. For the purposes of the present invention, variants or homologs that are excluded from pumping or exclusion of pumped polynucleotides are at least 20%, 30% or 40% identical in nucleotide or amino acid to the original excluded pump. Sex. The sequence used in the present invention has at least about 50%, 60%, 7%, and the original excluded pump,

8 0 %、9 0 %、9 5 %與9 8 %的核苷酸或氨基酸一致性。本 發明涵蓋各種天然的同源體或變異體,且本發明内容亦有 關於表現因子的同源體或變異體。80%, 90%, 915% and 98% nucleotide or amino acid identity. The present invention encompasses a variety of natural homologs or variants, and the present invention also relates to homologs or variants of expression factors.

「DNA結合區(DNA binding domain)」係指一蛋白 質、聚胜肽、胜肽或聚核苷酸中可與一特定DNA序列結 合的區域。例如,目前已找出 Ndt80p的 DNA結合區 (Μο«eί 〇/·,PN A S 99 :1 404 1 -6,2002)» Ndt8 Op 的 DNA 結合區包含一段長約 32-kDa且具有 beta-三明治式核心 (beta-sandwich core)的區域。此beta-三明治式核心具有7 個 beta 褶板(beta-sheet)及三個短 alpha -螺旋(alpha-helice) 結構。並如以下内容所述,曾發現Ndt80p的DNA結合區 與數種蛋白質中的DNA結合區相似。"DNA binding domain" refers to a region of a protein, a peptide, a peptide or a polynucleotide that binds to a particular DNA sequence. For example, the DNA binding region of Ndt80p has been identified (Μο«eί 〇/·, PN AS 99 :1 404 1 -6, 2002). The DNA binding region of Ndt8 Op contains a length of about 32-kDa and has a beta-sandwich. The area of the beta-sandwich core. This beta-sandwich core has 7 beta-sheets and three short alpha-helice structures. As described below, it has been found that the DNA binding region of Ndt80p is similar to the DNA binding region of several proteins.

CaNdt80p係為啤酒酵母菌 Ndt80p蛋白的白色念珠菌 同源體。Ndt80p是啤酒酵母菌的減數分裂特異轉錄因子 (meiosis specific transcription factor) (Chu e t al., Science 282:699-705, 1 9 9 8; Chu and Her show i tz, Mol.CaNdt80p is a Candida albicans homolog of the S. cerevisiae Ndt80p protein. Ndt80p is a meiosis specific transcription factor of S. cerevisiae (Chu e t al., Science 282: 699-705, 1 9 9 8; Chu and Her show i tz, Mol.

Cell 1:685-696,1998)。第 1 圖係比較 CaNdt80p 與 Ndt80p 的氨基酸序列》CaNdt80p長度為 592個氨基酸,Ndt80p 17 1354022 之長度則為672個氨基酸。已證實Ndt80p蛋白的第1至第 330個氨基酸對DNA結合來說是重要的,並曾在此段區域 中找到對DN A结合來說相當重要的兩段不同區域。Cell 1: 685-696, 1998). Figure 1 compares the amino acid sequences of CaNdt80p and Ndt80p. The length of CaNdt80p is 592 amino acids, and the length of Ndt80p 17 1354022 is 672 amino acids. The first to 330th amino acids of the Ndt80p protein have been shown to be important for DNA binding, and two different regions that are important for DN A binding have been found in this region.

Ndt8Op蛋白的第!至第58個氨基酸係參與特定序列辨Ndt8Op protein number! To the 58th amino acid line involved in specific sequence discrimination

識作用(sequence-specific recognition),而第 59 至第 330 個氨基酸序列中則包含DNA結合區。CaNdt80p蛋白的第 223至572個氨基酸與Ndt80p蛋白的第3至330個氨基酸 序列具有3 7.6 %的一致性與5 7 · 9 %的相似性(第1 A圖中的 深灰色區瑰),因此可假設CaNdt80p蛋白之該段區域亦可 能參與CaNdt80p蛋白與DNA之間結合。而CaNdt80p蛋 白的N端序列與Ndt8 Op蛋白的C端序列之間則無任何相 似性(第1 A圖中的淺黑色線段)。Sequence-specific recognition, while the 59th to 330th amino acid sequences contain a DNA binding region. The 223th to 572th amino acids of the CaNdt80p protein and the 3rd to 330th amino acid sequences of the Ndt80p protein have a 37.6% identity and a 57.9% similarity (dark gray region in Figure 1A), thus It can be assumed that this region of the CaNdt80p protein may also be involved in the binding of CaNdt80p protein to DNA. There is no similarity between the N-terminal sequence of the CaNdt80p protein and the C-terminal sequence of the Ndt8 Op protein (light black line in Figure 1A).

若分析Ndt80p蛋白,則可能根據CaNdt80p與Ndt80p 之間的相似性而有助於解釋CaNdt8Op蛋白的功能。Ndt8Op 蛋白係藉著其 DNA結合區域與一目標聚核苷酸中的中程 孢子生成序列(gNCRCAAAA/T)结合,而活化該目標基因 (Chu and H er show itz, Mol. Cell 1:685-696, 1 998;If the Ndt80p protein is analyzed, it may be helpful to explain the function of the CaNdt8Op protein based on the similarity between CaNdt80p and Ndt80p. The Ndt8Op protein activates the target gene by binding its DNA binding region to the mid-range spore-forming sequence (gNCRCAAAA/T) in a target polynucleotide (Chu and H er show itz, Mol. Cell 1:685- 696, 1 998;

Lamoureux et al., EMBO J. 21:572 1 -5732,2002)。Ndt80p 之聚核苷酸本身亦含有中程孢子生成序列,並曾有文獻顯 示 Ndt 8 Op可能藉著本身的中程孢子生成序列進行轉錄調 控,而誘導自身分子的合成反應(law owrewx ei β/.,EMBO J. 2 1:572 1 -5732, 2002) »當啤酒酵母進行無性生殖 (vegetative growth)時,細胞中Ndt80p的表現受到抑制(Xw et al., Mol. Cell Biol. 15:6572-658 1, 1 995; Pak and is 1354022Lamoureux et al., EMBO J. 21:572 1 -5732, 2002). The Ndt80p polynucleotide itself also contains a mid-range spore-forming sequence, and there have been reports that Ndt 8 Op may induce transcriptional regulation by its own mid-range spore-forming sequence, and induce its own molecular synthesis reaction (law owrewx ei β/ .EMBO J. 2 1:572 1 -5732, 2002) » When beer yeast is subjected to vegetative growth, the expression of Ndt80p is inhibited in cells (Xw et al., Mol. Cell Biol. 15:6572) -658 1, 1 995; Pak and is 1354022

Sega//,Mol.Cell Biol· 22:64 1 7-6429,2002)。並參考第 6 圖,不論在啤酒酵母或是 突變種中過量表現 #£>7^0,均不會改變其藥物敏感性》 有趣的是,過量表現聚核苷酸會降低藥物敏 感性。若藉著使聚核苷酸發生突變來調控 排除泵浦聚核苷酸的表現,則會提高細胞的藥物敏感性。 並且 啟動子具有三個可能的中程孢子生成序列Sega//, Mol. Cell Biol. 22:64 1 7-6429, 2002). Referring to Figure 6, the overexpression of #£>7^0 in S. cerevisiae or mutants does not alter its drug sensitivity. Interestingly, overexpression of polynucleotides reduces drug sensitivity. If the expression of the pumped polynucleotide is regulated by mutating the polynucleotide, the drug sensitivity of the cell is increased. And the promoter has three possible mid-range spore-forming sequences

(CRCAAA) ’該些MSE序歹ij分另,J位於從轉譯起始編碼ATG 開始算起上游第2 70bp、第43 8bp與第8 3 5 bp之位置上。 CaWDrSO亦在從其轉譯起始編碼(ATG)算起上游第572bp 之位置上含有一個完整的中程孢子生成序列 (gNCRCAAAA),以及在ATG上游第70bp '第I22bp與第 461bp之位置上分別具有三個可能的中程孢子生成序列 (CRCAAA)。第2圖係繪示CaWDrSO的核苷酸序列。(CRCAAA) 'The MSE sequence 歹 ij, J is located at the 2nd 70bp, 43rd bp and 83rd bp positions upstream from the translation start code ATG. CaWDrSO also contains a complete mid-range spore-producing sequence (gNCRCAAAA) at the 572 bp upstream from its translation initiation code (ATG), and has a 70 bp 'I22 bp and 461 bp positions upstream of the ATG. Three possible mid-range spore-forming sequences (CRCAAA). Figure 2 depicts the nucleotide sequence of CaWDrSO.

除上述Ndt80p與CaNdt80p蛋白之外,尚有來自如紅 麵包衡crassa) ' 黏菌(Dz’ceosie/z’wm discoideum)、線蟲(Caenorhabditis elegans)、果规 (Droxo/7/ιί/α /we/awogiisier)及人類等高等真核細胞的數種 蛋白分子中,含有相似於Ndt80p蛋白之DNA結合區的序 列(Mowiano et al., Proc. Natl. Acad. Sci. U.S.A 99:14041-14046,2002)。此外,曾發現在侵略性或轉移性 腫瘤中大量表現一種含有Ndt8Op之DNA結合區相似序列 的人類轉錄因子 Cllorf9 (A^’emer ei a/., Oncogene 20:6679-6688,2001)»因此,若抑制該些可與中程孢子生 19 1354022In addition to the above Ndt80p and CaNdt80p proteins, there are also from the red bread scale crassa) 'Dz'ceosie / z'wm discoideum, Caenorhabditis elegans, fruit (Droxo / 7 / ιί / α / we /awogiisier) and several human protein molecules of higher eukaryotic cells contain sequences similar to the DNA binding region of the Ndt80p protein (Mowiano et al., Proc. Natl. Acad. Sci. USA 99: 14041-14046, 2002) ). Furthermore, it has been found that a large number of human transcription factors Cllorf9 (A^'emer ei a/., Oncogene 20:6679-6688, 2001) containing a similar sequence of the DNA binding region of Ndt8Op are expressed in aggressive or metastatic tumors. If inhibited, it can be used with medium-range spores 19 1354022

成序列結合之轉錄因子的活性,便可能降低 的生長。此外,由於多種轉移性腫瘤遠較其 療藥物具有更強的抗藥性,且多種具抗藥性 不具抗藥性的親代細胞更具侵略性 Cancer Drug Targets. 2:257-277, 2002)。故 能調控人類細胞之排除泵浦表現的轉錄因子 化療藥物的活性。 故本發明之排除泵浦表現因子可能與一 之中程孢子生成序列結合。文中「排除泵浦表 pump expression factor)」一詞係指一種能調 聚核苷酸之表現的轉錄因子、抑制因子或轉 明一較佳實施例中,排除泵浦表現因子為 另一較佳實施例中,排除泵浦表現因子是一 子「(Regularoe of Efflux Pump 1 )」。在 及聚核苷酸的序列中編碼著一個推斷長 基酸的蛋白分子。過量表現及五/5/聚核苦 及AfD/Π啟動子之基因的表現量。第: 的核苷酸序列。 此處「患者(patient)」之定義係指任一個 之疾病、症狀或感染的人類或非人類動物, 受治療後能獲得益處的對象,其包括人類或 接受治療的非人類動物包含所有馴養或野生 明一較佳實施例中,接受治療的對象以正接 罹患癌症的對象為佳。 或抑制癌細胞 初代細胞對化 之腫瘸細胞較 et °l·, Curr. 若能抑制該些 ,便可能提高 目標聚核苷酸 現因子(efflux 控一排除泵浦 譯因子《本發 CaNdt80p。在 個新發現的分 白色念珠菌之 度為693個氨 酸會提高具有 3圖繪示i?五? 7 具有需要治療 或是任何在接 非人類動物。 的動物。本發 受藥物治療或 20 1354022The activity of a sequence-bound transcription factor may reduce growth. In addition, a variety of metastatic tumors are far more resistant than their therapeutic drugs, and a variety of drug-resistant, non-drug resistant parental cells are more aggressive. Cancer Drug Targets. 2:257-277, 2002). Therefore, it is possible to regulate the activity of transcription factors of human cells that exclude pumping performance of chemotherapeutic drugs. Therefore, the excluded pump expression factor of the present invention may be combined with a mid-range spore-forming sequence. The term "excluding pump expression factor" as used herein refers to a transcription factor, inhibitory factor or transcript that modulates the expression of a nucleotide. In a preferred embodiment, the exclusion of the pump expression factor is another preferred. In the embodiment, the pump performance factor is excluded as "Regularoe of Efflux Pump 1". A protein molecule that infers long acid is encoded in the sequence of the polynucleotide. Excessive performance and the performance of the genes of the five/5/polynuclear and AfD/Π promoters. The nucleotide sequence of the first:. The term "patient" as used herein refers to a human or non-human animal of any disease, condition or infection, a subject who is treated to benefit, including humans or treated non-human animals, including all domestic or In the preferred embodiment of the wild, the subject to be treated is preferably a subject who is suffering from cancer. Or inhibiting the cells of the primary cells of the cancer cells, compared with et °l, Curr. If this can be inhibited, it may increase the target factor of the target polynucleotide (efflux control-excluding the pump translation factor "this issue CaNdt80p. In the newly discovered degree of Candida albicans, the degree of 693 amino acids will increase with 3 figures showing i? 5? 7 animals with treatment or any non-human animals. The present treatment is treated with drugs or 20 1354022

本發明包括一種藉著抑制細胞中至少一種表現因子 活性或表現以抑制細胞生長的方法,且方法中更可包括 該細胞與至少一藥物接觸。 本發明更有關於一種藉著抑制一細胞中至少一種表 因子之活性或表現,以提高該細胞内一藥物之活性的 法,並且該方法更可包括使該細胞與該藥物接觸。此處 「提高(enhance或enhancing)」一詞係指增加上述藥物 該細胞的作用效果。可如上述内容所述,根據該細胞生 速度降低或停止的情況,或是根據細胞中一聚核苷酸之 錄或轉譯作用上升或停止的情況來偵測該藥物的作用 果。 本發明一較佳實施例中,本發明包含一種藉著抑制 真菌細胞中至少一表現因子的活性或表現量以抑制該真 細胞生長的方法,且該方法中更包括使該真菌細胞接觸 少一種抗真菌劑(antifungal agent)。在另一較佳實施 中,本發明包含一種藉著抑制一真菌細胞中至少一表現 子的活性或表現量,以提高該真菌細胞中一抗真菌劑的 性,且該方法更包括使該真菌細胞與該抗真菌劑接觸。 此處「真菌(fungus或fungi)」一詞係指習知技藝者 為較低等的一種真核有機微生物。白色念珠菌可產生酵 細胞主體(yeast)、 假菌絲(pseudohyphae)及真菌 (hyphae)。部分的常在菌叢中,白色念珠菌的生長方式 如同出芽生殖酵母菌(budding yeast)之生長方式。本發 文中所包含之「酵母菌(yeast)」一詞係指一種低等的真 之 使 現 方 之 對 長 轉 效 菌 至 例 因 活 認 母 絲 明 核 21 1354022 有機微生物,其具有單一細胞生長階段,並根據細胞結構、 繁殖機轉、核酸序列相似性或其他用來分類有機微生物的 特性’而將其歸類於真菌類。酵母菌之假菌絲與真菌絲型 態使其呈現多細胞之絲狀菌落,具有酵母菌型態與菌絲型 態交互變化的能力與白色念珠菌的毒性是息息相關。 真國類包含接合菌綱(Zygomyce/ei)、子囊菌綱The invention includes a method of inhibiting cell growth by inhibiting the activity or expression of at least one of the expression factors in a cell, and the method further comprises contacting the cell with at least one drug. More particularly, the present invention relates to a method for increasing the activity of a drug in a cell by inhibiting the activity or expression of at least one factor in a cell, and the method further comprises contacting the cell with the drug. Here, the term "enhancement or enhancement" refers to the effect of increasing the effect of the above-mentioned drugs on the cells. The effect of the drug can be detected as described above, depending on whether the cell growth rate is reduced or stopped, or depending on whether the recording or translation of a polynucleotide in the cell is increased or stopped. In a preferred embodiment of the present invention, the present invention comprises a method for inhibiting the growth of the true cell by inhibiting the activity or expression amount of at least one expression factor in the fungal cell, and the method further comprises contacting the fungal cell with one less An antifungal agent. In another preferred embodiment, the invention comprises a method of inhibiting the activity of an antifungal agent in the fungal cell by inhibiting the activity or amount of at least one of the expressions in a fungal cell, and the method further comprises: The cells are contacted with the antifungal agent. Here, the term "fungus or fungi" refers to a lower eukaryotic organic microorganism. Candida albicans produces yeast (yeast), pseudohyphae and hyphae. Part of the common flora, Candida albicans grows like the growth of budding yeast. The term "yeast" as used in this publication refers to a low-grade true-acting pair of long-acting bacteria to the case of a living mother's silk nucleus 21 1354022 organic microorganism, which has a single cell. The growth phase is classified into fungi based on cell structure, reproductive machinery, nucleic acid sequence similarity, or other properties used to classify organic microorganisms. The pseudohyphae and fungal silk patterns of yeast make it multi-cell filamentous colonies, and the ability to interact with yeast type and hyphae is closely related to the toxicity of Candida albicans. The true country includes Zygomyce/ei and Ascomycetes.

、擔子菌綱(万、不完全綱 (Dewierowyceiei)與卵菌綱。念珠菌屬為子囊 囷綱的成員之一。已知目前已分離之克柔念珠菌(C. 、熱帶念珠菌(c. 、光滑念珠菌(Caw心“ g/a6raia) '喷酸性念珠菌(CiZAii/fi/a 與葡萄牙Basidiomycetes (Dewierowyceiei) and Oomycetes. Candida is one of the members of the Ascomycetes. It is known that Candida krusei (C., Candida tropicalis) has been isolated. , Candida glabrata (Caw heart "g/a6raia" 'spray acid Candida (CiZAii/fi/a with Portugal

假絲念珠菌/wdMniare)等均是造成念珠菌感染 的來源’其中白色念珠菌為數量最豐富且顯著的菌種。曾 有報告指出,近年來由非白色念珠菌種如由光滑念珠菌與 克柔念珠菌造成的感染案例有增加之趨勢fl/., Clin. Infect. Dis. 2 4:1 1 22- 1 1 28, 1997; Aisner et al., Am. J. Med. 61:23-28, 1976; Arif et al., J. Clin. Microbiol. 34:2205-2209,1 996)。其他真菌類種類還包括曲真菌屬 (ζί·ϊ;7β"^/7/ωί species)、隱球菌屬(Crypiococcw·? spp),及其 他習知技藝者所熟知的菌種。 此處「抗真菌劑(antifungal agent)」一詞係指一種能 抑制真菌生長的藥物。抗真菌劑之活性係指此藥物基本上 能使真菌細胞停止生長,但不殺死真菌細胞,或此藥物能 先使其停止生長,之後再殺死真菌細胞。為了對抗白色念 22 1354022 珠菌及其他真菌所造成之感染,科學家們致力於發展抗真 菌劑,並找出真菌細胞之細胞膜、細胞壁中的必要組成。 與細菌不同但與其他真核生物相同的是,真菌的細胞膜上 含有固醇類(sterols),其幾乎是所有真菌細胞存活的必要 組成。固醇類對於保持細胞膜之流動性與完整性及維持多 種膜結合型酵素之適當功能來說相當重要,而細胞膜的完 整性與流動性、以及使膜結合型酵素保持適當功能對於細 胞生長與分裂來說極為重要awe/Candida albicans/wdMniare) are the sources of Candida infections. Among them, Candida albicans is the most abundant and significant strain. It has been reported that in recent years there has been an increase in infections caused by non-white Candida species such as Candida glabrata and Candida krusei. Clin. Infect. Dis. 2 4:1 1 22- 1 1 28, 1997; Aisner et al., Am. J. Med. 61:23-28, 1976; Arif et al., J. Clin. Microbiol. 34:2205-2209,1 996). Other fungal species include the genus Fungi (ζί·ϊ; 7β"^/7/ωί species), Crypiococcw® spp, and other species well known to those skilled in the art. The term "antifungal agent" as used herein refers to a drug which inhibits the growth of fungi. The activity of the antifungal agent means that the drug substantially stops the growth of the fungal cell, but does not kill the fungal cell, or the drug can stop growing first and then kill the fungal cell. In order to counter the infection caused by the white fungus 22 1354022 and other fungi, the scientists are committed to the development of anti-bacterial agents and to find out the necessary components in the cell membrane and cell wall of fungal cells. Unlike bacteria, but identical to other eukaryotes, fungal cell membranes contain sterols, which are essential components for the survival of almost all fungal cells. Sterols are important for maintaining the fluidity and integrity of cell membranes and for maintaining the proper function of a variety of membrane-bound enzymes, while cell membrane integrity and fluidity, as well as membrane-bound enzymes, maintain proper function for cell growth and division. Extremely important awe/

FEMS Microbiol. Lett. 149:14 卜 149,1997)。在真菌細胞膜 中主要的固醇類為麥角固醇(ergosterol)與酵母固醇 (zymosterol),而在哺乳動物細胞膜中則為膽固醇 (cholesterol)。科學家們為了發展抗真菌劑而研究各種固 醇組成的差異’並主要專注於發展能與真菌固醇形成複合 物’但不與人類固醇發生反應的抗真菌劑。這些抗真菌劑 的作用是在保持人類細胞膜完整的條件下,同時在真菌細 胞膜上造成穿扎》FEMS Microbiol. Lett. 149:14 Bu 149, 1997). The main sterols in the fungal cell membrane are ergosterol and zymosterol, while in mammalian cell membranes it is cholesterol (cholesterol). Scientists have studied the differences in various sterol compositions in order to develop antifungal agents and have focused primarily on the development of antifungal agents that form complexes with fungal sterols but do not react with human sterols. These antifungal agents act to prevent puncture on the fungal cell membrane while maintaining the integrity of the human cell membrane.

目前使用的抗真菌劑有兩性徽素B (amphotericin B) 與寧畜定(nystatin) ’其為一種多巨環内酯類抗生素 (polyene macrolide antibiotic)。相較於哺乳動物細胞膜上 的膽固醇,該些藥物對真菌細胞膜上的麥角固醇表現出較 高的親和力。該些藥物會插入細胞膜並在細胞膜上形成通 道,使細胞内的組成,特別是鉀離子,通過這些通道而流 出細胞’並因此破壞細胞膜内外的質子濃度梯度(proton gradient)( w Bossche et al ·, Trends Microbiol. 23 1354022The antifungal agents currently used are amphotericin B and nystatin, which is a polyene macrolide antibiotic. These drugs exhibit a higher affinity for ergosterol on the fungal cell membrane than cholesterol on mammalian cell membranes. These drugs are inserted into the cell membrane and form channels on the cell membrane, allowing intracellular components, especially potassium ions, to flow out of the cell through these channels' and thus destroy the proton gradient inside and outside the cell membrane (w Bossche et al. , Trends Microbiol. 23 1354022

2:393-400, 1994) °2:393-400, 1994) °

其他如丙醢胺類(allylamines)、硫代胺曱酸鹽 (thiocarbamates)、嗅類(azoles)與嗎嚇類(morpholines)等 抗真菌劑均作用在麥角固醇的生合成作用(biosynthesis) ϋ,Br. J. Dermatol. 126 Suppl 39:2-7,1992)。例 如氟康峻(fluconazole)、伊曲康嗤(itraconazole)及明康啥 (ketoconazole)等唑類藥物會與羊毛脂醇去甲基酶 (lanosterol demethylase)結合,並抑制羊毛脂醇去f基酶之 活性。羊毛脂醇去甲基酶是一種細胞色素 P-450酵素 (cytochrome P-450 enzyme),其通常將 14-α -曱基-固醇 (14-a-methyl-sterols)轉化成麥角固醇(5aag anrf Antimicrob. Agents Chemother. 32:1-8, 1988; Hitchcock, Biochem. Soc. Trans. 19:782-787, 1991)。 其他用來對抗真菌感染的抗真菌劑還包括5_氟胞嘧啶Other antifungal agents such as allylamines, thiocarbamates, azoles and morpholines act on the biosynthesis of ergosterol. Hi, Br. J. Dermatol. 126 Suppl 39: 2-7, 1992). For example, oxaconazoles such as fluconazole, itraconazole, and ketoconazole bind to lanosterol demethylase and inhibit lanolin alcohol to f-based enzymes. active. Lanolinol demethylase is a cytochrome P-450 enzyme that typically converts 14-a-methyl-sterols to ergosterol. (5aag anrf Antimicrob. Agents Chemother. 32: 1-8, 1988; Hitchcock, Biochem. Soc. Trans. 19: 782-787, 1991). Other antifungal agents used to combat fungal infections include 5-fluorocytosine

(5-fluey to sine,5-FC)。5-氟胞嘧啶的作用機轉是抑制DN A(5-fluey to sine, 5-FC). The action of 5-fluorocytosine is to inhibit DN A

與RNA合成。真菌細胞内的胞嘧啶去胺酶(cyt〇sine deaminase)會將 5-氟胞鳴咬轉化成 5-氟尿喷咬 (5-fluorouracil,5-FU)。哺乳動物細胞中的胞嘧啶去胺酶活 性較低’因此5 -氟胞嘧啶在人逋中表現出較低的毒性。% 氟尿胞嘲咬隨後會被轉化成5_氟尿苷酸(5_nu〇r〇uridilic acid),當5-氟尿苷酸被磷酸化後會成為5_氟-三磷酸尿嘧 啶(5-fluoro-UTP),並被合成在Rna中。當RNA中含有 5 -氟-二磷酸尿嘧啶時’會干擾蛋白質的合成作用,並抑制 真菌細胞生長。5-氟尿嘧啶亦可被轉化為5•氟-去氧三磷酸 24 1354022 尿嘧啶(5-fluoro-dUTP),其可能抑制細胞中的胸線嘴咬合 成酶(thymidilate synthase),而胸線嘴咬合成每疋DNA合 成作用中的一種必要酵素fl’·,Clin. Microbiol· Rev. 11:382-402,1 998) ° 對於抗真菌劑的討論可參考文獻心,J· Antimicrob· Chemother. 31: 463-47 1, 1 993, and Yang and Lo, J.With RNA synthesis. The cytosine deaminase in the fungal cell converts the 5-fluorocell bite into a 5-fluorouracil (5-FU). Cytosine deaminase activity in mammalian cells is low' so 5-fluorocytosine exhibits lower toxicity in human mites. % Fluoride urinary tract will be converted to 5-fluorouridine (5_nu〇r〇uridilic acid), which will become 5-fluoro-triphosphate uracil when 5-fluorouridine is phosphorylated (5- fluoro-UTP) and synthesized in Rna. When RNA contains 5-fluoro-diphosphate uracil, it interferes with protein synthesis and inhibits fungal cell growth. 5-fluorouracil can also be converted to 5 • fluoro-deoxytriphosphate 24 1354022 uracil (5-fluoro-dUTP), which may inhibit the thymidilate synthase in the cell, while the chest bite bites Synthesis of an essential enzyme in the synthesis of DNA per fl). Clin. Microbiol Rev. 11:382-402, 1 998) ° For the discussion of antifungal agents, please refer to the literature, J. Antimicrob Chemother. 31: 463-47 1, 1 993, and Yang and Lo, J.

M i c r o b i o 1 I m m u n o 1 · I n f e c t · 3 4 : 7 9 - 8 6, 2 0 〇 1。此處「抗真囷 劑(antifungal agent)」一詞係指上述内容所討論之任何一 種或多種抗真菌劑,且除上述抗真菌劑外’尚有他種為習 知技藝者所熟悉之抗真菌劑。M i c r o b i o 1 I m m u n o 1 · I n f e c t · 3 4 : 7 9 - 8 6, 2 0 〇 1. The term "antifungal agent" as used herein refers to any one or more of the antifungal agents discussed above, and in addition to the above-mentioned antifungal agents, there is still an antibiotic known to those skilled in the art. Fungal agent.

本發明亦有關於微生物細胞毒性。此處「毒性 (virulence)」一詞係指一病原體製造具感染性之各種型態 病原體的能力及其感染、傷害宿主細胞的能力。可藉著抑 制一微生物細胞中之表現因子的活性或表現,以抑制微生 物細胞的毒性。因此,本發明内容之「抑制毒性(inhibiting v 1 r u 1 e n c e )」一詞係指降低或停止一微生物細胞感染、傷害 宿主細胞的能力’以及降低或停止該微生物細胞製造具有 感染性之各種型態病原體的能力。 在本發明另一較佳實施例中’可藉著使一細胞接觸至 少一種排除泵浦表現因子抑制劑,以抑制該細胞生長。在 一較佳實施例中更可包括使該細胞與至少一種藥物接觸。 亦可藉著使一細胞與至少一種表現因子抑制劑接觸,以抑 制該細胞之毒性,且該方法更可包括使該細胞與至少一種 藥物接觸。在又一較佳實施例中,可藉著使一細胞接觸至 25 1354022 少一種排除泵浦表現因子抑制劑,以提高一藥物的活性, 且此方法更可包括使該細胞接觸至少一種藥物。在一特定 較佳實施例中’上述細胞可為一個真菌細胞,以及上述藥 物可為一種抗真菌劑。The invention also relates to microbial cytotoxicity. The term "virulence" as used herein refers to the ability of a pathogen to produce infectious forms of various pathogens and their ability to infect and injure host cells. The toxicity of the microbial cells can be inhibited by inhibiting the activity or expression of a performance factor in a microbial cell. Therefore, the term "inhibiting v 1 ru ence" in the context of the present invention refers to the ability to reduce or stop a microbial cell infection, to injure a host cell, and to reduce or stop the microbial cell manufacturing from being infectious. The ability of pathogens. In another preferred embodiment of the invention, the growth of the cells can be inhibited by contacting one cell with at least one inhibitor of the pump expression factor. In a preferred embodiment, the method further comprises contacting the cell with at least one drug. The cell can also be inhibited by contacting one cell with at least one expression factor inhibitor to inhibit toxicity of the cell, and the method further comprises contacting the cell with at least one drug. In yet another preferred embodiment, one of the cells can be contacted to 25 1354022 to eliminate a pump expression factor inhibitor to increase the activity of a drug, and the method further comprises contacting the cell with at least one drug. In a particularly preferred embodiment, the above cells may be a fungal cell, and the above drug may be an antifungal agent.

「表現因子抑制劑(expression factor inhibitor)」係指 一種能干擾一表現因子之活性或表現的物質。故表現因子 抑制劑可能干擾如上述表現因子之目標分子的活性或表 現,亦可能干擾上述表現因子與其目標分子之間的結合作 用,或干擾細胞將一物質或化合物輸出胞外的作用,例如 干擾細胞將一抗真菌劑輸出胞外的作用。表現因子抑制劑 的的範例包含如反義寡聚核苷酸 (antisense oligonucleotides)、RNAi 或干擾 RNA(interfering RNA) ' 化學物質以及各種天然、人工合成或重組的胜肽 (peptide)、 聚胜狀(polypeptide)、 蛋白質、多醋類 (polysaccharides)、小分子或其他設計用來降低或抑制一 表現因子之活性或表現的化合物。其中反義寡聚核苷酸可 與一個或多個編碼著一表現因子的核酸序列發生專一性 雜合(specifically hybridize)。表現因子抑制劑可能抑制一 表現因子與本身聚核苷酸、一排除泵浦聚核苷酸或任一目 標聚核苷酸上之中程孢子生成序列的結合作用。因此,本 發明亦提供一種藉著抑制一表現因子與一中程孢子生成 序列結合,以抑制細胞生長的方法。 本發明一較佳實施例中的表現因子抑制劑是一排除 泵浦表現因子抑制劑。此處「排除泵浦表現因子抑制劑 26 1354022 (efflux pump expression factor inhibitor)」是一種能藉著 抑制一排除泵浦表現因子的活性或表現,以干擾一排除泵 浦聚核苷酸之表現的物質。因此,該排除泵浦表現因子抑 制劑可能干擾如抗真菌劑等物質或其他化合物自細胞中 排出的作用。"Expression factor inhibitor" means a substance that interferes with the activity or expression of a performance factor. Therefore, the expression factor inhibitor may interfere with the activity or expression of the target molecule such as the above-mentioned expression factor, and may also interfere with the binding between the above-mentioned expression factor and its target molecule, or interfere with the action of the cell to export a substance or compound to the extracellular, such as interference. The cells export an antifungal agent for extracellular action. Examples of performance factor inhibitors include, for example, antisense oligonucleotides, RNAi or interfering RNA 'chemicals, as well as various natural, synthetic or recombinant peptides, polysymmetry (polypeptide), protein, polysaccharides, small molecules or other compounds designed to reduce or inhibit the activity or expression of a performance factor. The antisense oligonucleotide can be specifically hybridized with one or more nucleic acid sequences encoding a expression factor. A performance factor inhibitor may inhibit the binding of a performance factor to its own polynucleotide, to a pumped polynucleotide, or to a mid-range spore-forming sequence on any of the target polynucleotides. Accordingly, the present invention also provides a method for inhibiting cell growth by inhibiting binding of a performance factor to a mid-range spore-forming sequence. A performance factor inhibitor in a preferred embodiment of the invention is an exclusion pump performance factor inhibitor. Here, "efflux pump expression factor inhibitor" is an activity or expression that inhibits the exclusion of a pumping expression factor by inhibiting the activity or performance of a pumping performance factor. substance. Therefore, the exclusion of the pump performance factor inhibitor may interfere with the action of substances such as antifungal agents or other compounds excreted from the cells.

「反義募聚核苦酸(antisense oligonucleotide)」係指 一核酸(DNA或RN A),其能調控一個在序列上編碼著一蛋 白質的核酸分子,進而調控此蛋白質的產量。係藉著提供 一種能與一或多個編碼著一蛋白質之核酸發生專一性雜 合反應的反義寡聚核苷酸,來達到調控蛋白質產量的目 的。此處「目標核酸(target nucleic acid)」與「編碼著一 蛋白質之核酸(nucleic acid encoding a protein)」係包括編 碼有一蛋白質之 DNA、從該 DNA轉錄出的 RNA(包括 pre-mRNA與mRNA)、以及由該RNA所得出的cDNA » — 反義寡聚核苷酸與其目標核酸之間的專一性雜合反應會 干擾該目標核酸之正常運作。通常將該些能與一目標核酸 進行專一性雜合之化合物對該目標核酸之功能所做的調 控作用稱為「反義(antisense)」。可藉著干擾如DNA複製 與/或轉錄作用來干擾DNA功能。以及透過干擾RNA之 所有生命功能,例如干擾RNA在細胞中移動至蛋白質轉 譯區的作用、剪切RNA以形成一或多種mRNA之作用、 以及與RNA相關或由RNA促進之催化活性,而達到干擾 RNA功能的目的《使用反義寡聚核苷酸的技術均為習知技 藝者所熟悉,並可參考下列文獻://oh ei a/.,Mol. Cell 27 1354022"Antisense oligonucleotide" refers to a nucleic acid (DNA or RN A) that regulates a nucleic acid molecule that encodes a protein in sequence, thereby regulating the production of this protein. The purpose of regulating protein production is achieved by providing an antisense oligonucleotide that specifically reacts with one or more nucleic acids encoding a protein. Here, "target nucleic acid" and "nucleic acid encoding a protein" include DNA encoding a protein and RNA transcribed from the DNA (including pre-mRNA and mRNA). And the specific hybridization between the cDNA from the RNA and the target nucleic acid interferes with the normal operation of the target nucleic acid. The regulation of the function of the target nucleic acid by a compound which is specifically hybridized with a target nucleic acid is generally referred to as "antisense". DNA function can be interfered by interference such as DNA replication and/or transcription. And interference by interfering with all life functions of RNA, such as interfering with the action of RNA moving through the cell to the protein translation region, cleavage of RNA to form one or more mRNAs, and RNA- or RNA-promoted catalytic activity Purpose of RNA function "The technique of using antisense oligonucleotides is well known to those skilled in the art and can be referred to the following documents: oh ei a/., Mol. Cell 27 1354022

Biol. 8:963-973, 1988 ; Anf os si et al., Proc. Natl. Acad. Sci. USA 86:3 3 79-3 3 83, 1 989; Wickstrom et al., Proc. Nat. Acad. Sci. USA 8 5:1 028- 1 032, 1 988, Higgins et al ; Proc. Nat. Acad. Sci. USA 90:990 1 -9905, 1993; Kitaj im a et al., Science 258:1792-1795,1992; Li et al., Clin. Cancer Res. 8:3 5 70-3 5 7 8, 2002;及 Are« ei αΛ, Gut 51:529-535,2002 °Biol. 8: 963-973, 1988; Anf os si et al., Proc. Natl. Acad. Sci. USA 86:3 3 79-3 3 83, 1 989; Wickstrom et al., Proc. Nat. Acad. Sci. USA 8 5:1 028- 1 032, 1 988, Higgins et al ; Proc. Nat. Acad. Sci. USA 90:990 1 -9905, 1993; Kitaj im a et al., Science 258:1792-1795 , 1992; Li et al., Clin. Cancer Res. 8:3 5 70-3 5 7 8, 2002; and Are« ei αΛ, Gut 51:529-535, 2002 °

諸如此類干擾目標核酸功能之動作所造成的整體影 響即為對聚核苷酸表現之調控作用。本發明内容中的「聚 核苷酸表現之調控作用(modulation of the expression of the polynucleotide)」一詞係指一聚核普酸表現的提高或 降低。在本發明一較佳實施例中,係將反義寡聚核苷酸設 計成能與一表現因子聚核苷酸或與該表現因子之目標聚 核苷酸上的令程孢子生成序列發生專一性雜合反應。本發 明另一較佳實施例中的反義寡聚核苷酸為一種核醣核酸 酵素(ribozyme),其為一種能與一目標RNA發生專一性雜 合反應並專一性地切斷該目標RNA分子之磷酸雙醋鍵骨 架的反義RNA分子。該核醣核酸酵素亦能催化自身RNA 或其他目標RNA發生斷裂。 本發明所使用之反義寡聚核苷酸可利用方便且制式 的習知固相合成技術(solid-phase synthesis)製造之。並且 如 Applied Biosystems(Foster City,CA)等數家代理商均 販售上述技術之合成設備。尚有其他設備或方法可用來進 行上述之合成反應,且寡聚核苷酸的實際合成反應均為習 知技藝者所熟悉。需明白可使用相似技術來製備他種募聚 28 1354022The overall effect of such actions as interfering with the function of the target nucleic acid is the regulation of the expression of the polynucleotide. The term "modulation of the expression of the polynucleotide" in the context of the present invention refers to an increase or decrease in the performance of a polynucleotide. In a preferred embodiment of the invention, the antisense oligonucleotide is designed to be specific to a spRNA generating sequence on a expression factor polynucleotide or to a target polynucleotide of the expression factor. Sexual heterozygous reaction. The antisense oligonucleotide in another preferred embodiment of the present invention is a ribozyme which is a specific hybridization reaction with a target RNA and specifically cleaves the target RNA molecule. An antisense RNA molecule of a double acetaminophen backbone. The ribonuclease also catalyzes the cleavage of its own RNA or other target RNA. The antisense oligonucleotides used in the present invention can be produced by a conventional solid-phase synthesis technique which is convenient and standard. And synthetic equipment of the above technology is sold by several agents such as Applied Biosystems (Foster City, CA). There are other devices or methods that can be used to carry out the above-described synthetic reactions, and the actual synthesis of oligonucleotides is well known to those skilled in the art. It is important to understand that similar techniques can be used to prepare other species. 28 1354022

核苦酸’例如其硫代碟酸醋衍生物(phosphorothioates)與 院化衍生物(alkylated derivatives)(Zon, Pharm. Res. 5:5 3 9-549, 198 8)。亦需明白可使用類似技術、市售修飾核 音酸及可控微孔玻璃產品(control led-pore glass product, CGP)來合成出具有螢光素標記、生物素修飾或他種修飾的 券聚核苷酸,如膽固醇修飾寡聚核苷酸 (cholesterol-modified oligonucleotides)。且所用之市售修 飾核苦酸可為經過生物素(biotin)、螢光素(flurescein)、 吖啶(acridine)或補骨酯素(psoralen)等修飾的核苷酸。本 發明之反義寡聚核苷酸,其長度大致為至少7個核苦酸, 通常為至少12個核苷酸,更可為至少20個核苦酸,甚至 超過5 00個核苷酸之長度。但通常以不超過約5〇個核苷 酸為主’並以長度不超過約35個核苷酸為佳,其中反義 寡聚核苷酸的長度受抑制效率' 專一性、不發生交叉反應 等因素控制。曾有文獻提到長度約為7至8個驗基的短募 聚核苷酸是有效且具選擇性的基因表現抑制劑(…gnerThe nucleotide acid 'e.g., its phosphorothioates and alkylated derivatives (Zon, Pharm. Res. 5: 5 3 9-549, 198 8). It is also important to understand that a similar technology, commercially available modified nucleic acid and controlled-pore glass product (CGP) can be used to synthesize a fluorophore label, biotin modification or other type of modification. Nucleotides, such as cholesterol-modified oligonucleotides. And the commercially available modified puthic acid used may be a modified nucleotide such as biotin, flurescein, acridine or psoralen. An antisense oligonucleotide of the invention having a length of at least about 7 nucleotides, usually at least 12 nucleotides, more preferably at least 20 nucleotides, or even more than 50,000 nucleotides length. However, it is usually no more than about 5 nucleotides in length and preferably no more than about 35 nucleotides in length, wherein the length of the antisense oligonucleotide is inhibited by efficiency 'specificity, no cross reaction And other factors control. It has been mentioned in the literature that short nucleotides of about 7 to 8 lengths are effective and selective gene expression inhibitors (...gner

αΛ’ Nature Biotechnol. 14:840-844,1996)。需明白各種自 然或非天然之寡聚分子均為本發明上述内容所囊括。 可利用具有所有或部分種類之目標聚核聲酸序列的適 當載體以體内表現方式來製造出各種反義分子,其中所製 造出來的反義股是一 RNA分子《反義RNA序列會與其目 標聚核苷酸的RN Α互補’並抑制該目標聚核苷酸的產物 表現。反義分子係透過各種不同的機轉,如降低轉譯用的 mRNA產量、活化核醣核酸酶H (RNAse H)、成造成立體 29 1354022 空間阻礙等機轉來達到抑制基因表現的目的。 或可利用一適當載體使所有或部分目標聚核苷酸序列 進行體外轉錄反應,以製造出上述反義分子,且所製造出 的反義分子是一 RNA分子。可利用含有沙門氏桿菌嗟菌體 SV 6 {S aim o ne 11 a typhimur ium bacterophage SP6)、或大腸桿 菌嘆菌體 T7 與 Τ3(£. eo/ί T7 and T3)之啟動αΛ’ Nature Biotechnol. 14: 840-844, 1996). It is to be understood that various natural or non-natural oligomeric molecules are encompassed by the above teachings of the present invention. Various antisense molecules can be produced in vivo using a suitable vector having all or a portion of the target polynuclear acid sequence, wherein the antisense strand produced is an RNA molecule and the antisense RNA sequence will be associated with its target. The RN of the polynucleotide is complementary to 'and inhibits the product performance of the target polynucleotide. Antisense molecules can inhibit gene expression through a variety of different mechanisms, such as reducing mRNA production for translation, activating ribonuclease H (RNAse H), and causing steric 29 1354022 steric hindrance. Alternatively, all or a portion of the polynucleotide sequence of interest may be subjected to an in vitro transcription reaction using an appropriate vector to produce the above antisense molecule, and the antisense molecule produced is an RNA molecule. Use of SV 6 {S aim o ne 11 a typhimur ium bacterophage SP6), or E. coli T7 and Τ3 (£. eo/ί T7 and T3)

子’並在該啟動子上游處具有多嫁接區(polycloning site) 的載體(vector)來構築質體(plasmid),以方便用來體外合成 單股 RNA(Gree« ei a/·,Cell 32:68 1,1 983 ;The child's vector with multiple polycloning sites upstream of the promoter to construct a plasmid for convenient synthesis of single-stranded RNA in vitro (Gree« ei a/·, Cell 32: 68 1,1 983 ;

Rosenberg, J . Mol. Bio. 1 53:503, 1981 ; D av anl ο o et al., Proc. Natl. Acad. Sci. USA 81 :203 5, 1 984 ; Tabor and Richardson, Proc. Natl. Acad. Sci. USA 82:1 074, 1 985)。 噬菌體各自編碼的 D ΝΑ 依賴型 RNA 聚合酶 (DNA-dependent RNA polymerases)能專一性地辨識其同 源啟動子,而不會使用到質體中為其他聚合酶所辨識的啟 動子,例如他種噬菌體、細菌或真核細胞的啟動子。因此, 若於體外將線型質體(linearized pi amid)與適當的DNA依 賴型 RN A 聚合酶及四種 rNTP(核醣核苷三磷酸 ribonucleotide tripho sphates)混合並反應,便會從質體上 所選擇的噬菌體啟動子開始進行RNA合成反應。亦可利用 各種習知技術來執行體外轉錄反應,該些習知技術可參閱 下述文獻資料:Sambrook et al., Mo/ecw/iir C/onZ Laboratory Manual, Vo Is 1-3 (2d ed. 1 989), Cold Spring Harbor Laboratory Press ; Miligan et al., Nucl. Acids Res. 30 1354022 15.8783, 19 8 7 ; Milligan and Uhlenbeck, Meth. Enzymol. 180:51,1989。亦可使用如 pr〇rnega(Madison, WI)公司所販 售商品名為 Riboprobe® Transcription Systems 等 市售試劑組合來進行上述體外轉錄反應。Rosenberg, J. Mol. Bio. 1 53:503, 1981 ; D av anl ο o et al., Proc. Natl. Acad. Sci. USA 81 :203 5, 1 984 ; Tabor and Richardson, Proc. Natl. Acad Sci. USA 82:1 074, 1 985). The phage-encoded D-DNA-dependent RNA polymerases can specifically recognize their homologous promoters without using promoters recognized by other polymerases in the plastid, such as other species. Promoter of bacteriophage, bacteria or eukaryotic cells. Therefore, if a linearized pi amid is mixed and reacted with an appropriate DNA-dependent RN A polymerase and four rNTPs (ribonucleotide tripho sphates) in vitro, it will be selected from the plastid. The phage promoter begins the RNA synthesis reaction. In vitro transcription reactions can also be performed using a variety of conventional techniques, which are described in Sambrook et al., Mo/ecw/iir C/onZ Laboratory Manual, Vo Is 1-3 (2d ed. 1 989), Cold Spring Harbor Laboratory Press; Miligan et al., Nucl. Acids Res. 30 1354022 15.8783, 19 8 7 ; Milligan and Uhlenbeck, Meth. Enzymol. 180:51,1989. The above in vitro transcription reaction can also be carried out using a commercially available reagent combination such as Riboprobe® Transcription Systems, which is commercially available from pr〇rnega (Madison, WI).

此處「RNAij 或「干擾 RNA(interfering RNA)」一詞 係指一種與目標核酸分子部分相似且部分雙股或完全雙股 的RNA。當RNAi進入細胞中,會誘導一個令與RNAi序 列相同之外來或内生雙股RNA(dsRNA)及單股RNA(ssRNA) 發生降解(degradation)的細胞作用。因此,RNAi對目標核 酸分子之功能所造成的整體影響是降低或抑制該目標基因 產物(如蛋白質或核醣核酸酵素)的表現。其概略内容可參 考文獻 Cell 107:415-418, 2001; no«,Here, the term "RNAij" or "interfering RNA" refers to an RNA that is partially similar to a target nucleic acid molecule and partially double-stranded or completely double-stranded. When RNAi enters a cell, it induces a cellular action that causes the same or the degradation of endogenous double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA) as the RNAi sequence. Thus, the overall effect of RNAi on the function of a target nucleic acid molecule is to reduce or inhibit the performance of the target gene product (such as a protein or ribonuclease). The general content can be found in the literature Cell 107:415-418, 2001; no«,

Nature 418:244-251,2002。為達成本發明之目的,可設計 RNAi分子使其針對目標核酸分子序列中的任一部位造成 影響’例如使RNAi攻擊一核醣核酸酵素。在一較佳實施 例中,RNAi鎖定之目標部位可包括一表現因子聚核苷酸 或該表現因子之目標聚核苷酸上的中程孢子生成序列。故 本發明之RNAi可降低或抑制該表現因子及/或該表現因子 之目.標聚核苷酸的表現。美國專利案5,5 0 6,5 5 9號中曾揭 露製造及使用RNAi的概略方法。 在一較佳實施例中,所使用之RNAi含有一段與目標聚 核苷酸之部分序列1 00 %相同的核苷酸序列,例如RNAi 若與含有排除泵浦表現因子編碼之基因的部分序列完全相 同。在另一較佳實施例中,所使用之RNAi與該目標聚核 31 1354022Nature 418: 244-251, 2002. For the purposes of the present invention, an RNAi molecule can be designed to effect an effect on any part of the sequence of a target nucleic acid molecule', e.g., to cause RNAi to attack a ribonuclease. In a preferred embodiment, the target site for RNAi targeting may comprise a performance factor polynucleotide or a mid-range spore-forming sequence on the target polynucleotide of the expression factor. Therefore, the RNAi of the present invention can reduce or inhibit the expression of the expression factor and/or the expression factor of the target. A general method of making and using RNAi has been disclosed in U.S. Patent No. 5,506,5,5,9. In a preferred embodiment, the RNAi used contains a nucleotide sequence that is 100% identical to the partial sequence of the polynucleotide of interest, eg, RNAi is completely identical to the partial sequence containing the gene encoding the pumping expression factor. the same. In another preferred embodiment, the RNAi used and the target polynuclear 31 1354022

苷酸之部分序列具有大於9 0 %的一致性。又一較佳實施例 中,所使用之RN A i與該目標聚核苷酸之部分序列的一致 性大於8 0 %、7 0 %、6 0 0/〇或5 0 % »習知技藝者可利用序 列比對及排列演算法(alignment algorithm)來得到最佳的 序列一致性,並利用如BESTFIT軟體及如美國威斯康辛大 學基因運算中心所提供之預設參數來執行 Smith-Waterman演算法,以計算出核苷酸序列之間的差異 性百分比,亦可使用他種習知方法來執行上述運算。上述 核苷酸序列之長度至少約為25、50、100、200、300或400 個驗基對。 上述RNAi可包含一條或多條聚核苷酸,且可在RNAi 的核苷酸(nucleoside)或鱗酸醣骨架(phosphate-suger backThe partial sequence of the nucleotide has a consistency of greater than 90%. In still another preferred embodiment, the consistency of the RN A i used with the partial sequence of the target polynucleotide is greater than 80%, 70%, 600%, or 50%. Sequence alignment and alignment algorithms can be used to obtain optimal sequence identity, and the Smith-Waterman algorithm can be performed using preset parameters such as BESTFIT software and the Genetic Algorithms Center of the University of Wisconsin, USA. The percentage difference between the nucleotide sequences is calculated, and the above-described operations can also be performed using other conventional methods. The above nucleotide sequences are at least about 25, 50, 100, 200, 300 or 400 base pairs in length. The above RNAi may comprise one or more polynucleotides and may be in the nucleoside or glucosamine backbone of the RNAi.

bond)上進行各種修飾。雙股結構之RNAi可由自身互補之 單股 RNA(single self-complementary RNA strand)或相互 補的兩條RN A所構成。不論於細胞外或細胞内均可形成雙 股纏繞之 RNA。且該些 RN A不需進行多 A修飾反應 (polyadenylate,即不需加上polyA tail),也不需利用細胞 的轉譯系統將其轉譯成聚胜肽。可利用上述製造反義寡聚 核苷酸的方法於體外或體内合成RNA。或是利用化學或酵 素方法以手動或自動的方式來合成RNA。 本發明之表現因子抑制劑包括各種可降低或抑制細月包 内表現因子之活性或表現的化學物質及天然、重組或人工 合成的胜肽、聚胜肽、蛋白質、多醣類、小分子與其他化 合物。上述表現因子抑制劑可能藉著如干擾一表現因子之 32 1354022Various modifications are made on the bond). The double-stranded RNAi can be composed of single self-complementary RNA strands or two RN A complement each other. Double-stranded RNA can be formed either extracellularly or intracellularly. Moreover, the RN A does not need to be polyadenylate (that is, without adding polyA tail), and does not need to be translated into a polypeptide by a cell translation system. RNA can be synthesized in vitro or in vivo using the methods for producing antisense oligonucleotides described above. Alternatively, RNA can be synthesized manually or automatically using chemical or enzymatic methods. The expression factor inhibitor of the present invention includes various chemicals which can reduce or inhibit the activity or expression of the expression factor in the fine moon package, and natural, recombinant or synthetic peptides, polypeptides, proteins, polysaccharides, small molecules and Other compounds. The above-mentioned performance factor inhibitor may be caused by interference such as a performance factor 32 1354022

活化或表現路徑,來降低或抑制該表現因子的活性。本發 明之表現因子抑制劑亦可降低或抑制該些受表現因子所 調控之聚核苷酸或蛋白質的表現—或活性,其中表現因子係 藉著與聚核苷酸或蛋白質直接結合來調控該聚核苷酸或蛋 白質。舉例來說,根據本發明一較佳實施例,表現因子抑 制劑可與一排除泵浦基因或排除泵浦表現因子基因上的中 程孢子生成序列結合。該排除泵浦表現因子抑制劑可能與 該排除泵浦表現因子的中程孢子生成序列相似,使得該排 除泵浦表現因子抑制劑得以與該排除泵浦基因上的中程孢 子生成序列結合,卻不會活化該排除泵浦基因的表現。在 另一較佳實施例中,表現因子抑制劑會阻止該表現因子的 磷酸化或去磷酸化反應。Activating or expressing a pathway to reduce or inhibit the activity of the expression factor. The expression factor inhibitor of the present invention may also reduce or inhibit the expression or activity of the polynucleotide or protein regulated by the expression factor, wherein the expression factor is regulated by direct binding to the polynucleotide or protein. Polynucleotide or protein. For example, in accordance with a preferred embodiment of the invention, the performance factor inhibitor can bind to a mid-spore-producing sequence that excludes a pump gene or excludes a pump expression factor gene. The exclusion pump performance factor inhibitor may be similar to the mid-range spore-producing sequence excluding the pump expression factor such that the exclusion pump expression factor inhibitor binds to the mid-range spore-forming sequence on the excluded pump gene, but The performance of the excluded pump gene is not activated. In another preferred embodiment, the performance factor inhibitor prevents the phosphorylation or dephosphorylation of the expression factor.

此外,可根據習知方法來製備能抑制一表現因子之活 性或表現的胜肽、聚胜肽與蛋白質。例如使用噬菌體胜肽 表現庫(phage peptide display libraries)來表現大量胜 肽,並以體外方法從該些胜肽中篩選出對該表現因子具有 專一性結合力,或能抑制該表現因子活性的胜肽。噬菌體 表現技術可用來表現各種隨機序列或選擇性隨機序列的 胜肽。各種噬菌體表現方法與製備不同胜肽的方法為習知 技藝者所熟悉。例如L a d n e r等人(美國專利5,2 2 3,4 0 9號) 曾敘述噬菌體表面上各種結合胜肽的製備方法。Ladner 等人曾敘述數種用來製備一噬菌體表現庫的噬菌體載體 (phage vector),以及數種用來篩選可能的結合胜肽及製造 隨機胜肽或選擇性突變之結合胜肽的方法。通常利用已純 33 1354022 化的目標分子來進行體外噬菌體表現庫的蒒選。係收集該 些能舆目標分子結合的噬菌體,並將收集到的噬菌體作個 別培養後偵測每一噬菌體中所表現的胜肽。同樣地,Smith and Scott 亦曾在文獻 2 1 7:228-2 5 7,1 993,Further, peptides, polypeptides and proteins which inhibit the activity or expression of a performance factor can be prepared according to a conventional method. For example, a phage peptide display library is used to express a large number of peptides, and an in vitro method is used to select a specific binding force for the expression factor from the peptides, or to suppress the activity of the expression factors. Peptide. Phage display techniques can be used to represent peptides of various random or selective random sequences. Various phage display methods and methods for preparing different peptides are well known to those skilled in the art. For example, L a d n e r et al. (U.S. Patent No. 5,222,409) describes the preparation of various binding peptides on the surface of phage. Ladner et al. describe several phage vectors used to prepare a phage display library, as well as several methods for screening possible binding peptides and binding peptides that produce random peptides or selective mutations. Selection of in vitro phage expression libraries is typically performed using target molecules that have been purified 33 1354022. The phage which binds to the target molecule are collected, and the collected phage are individually cultured to detect the peptide expressed in each phage. Similarly, Smith and Scott were also in the literature 2 1 7:228-2 5 7,1 993,

and Science 249:3 86-390, 1 990中敘述製備噬菌體胜肽表 現庫的方法、載體以及表現各種不同胜肽的方法(W0 91/0 7141與WO 91/0 7149)。在各種胜肽製備方法中,以 噬菌體表現技術別具效率,例如當配合編碼子突變法來執 行噬菌體表現技術時,便可製造出隨機序列的胜肽或隨機 變異或特定變異的胜肽(請參考美國專利案 5,264,563 號)。上述或其他習知方法均可用來製造噬菌體表現庫, 以供進行體外篩選與鑑定出能與一表現因子結合的胜 肽、聚胜肽或蛋白質。或從自然來源中分離出各種能與一 表現因子結合,或抑制一表現因子活性的胜肽、聚胜肽、 蛋白質、多醣類或其他類似功能的分子,隨後對該些分子 進行某些處理(如剪切胜肽,peptide digestion),或利用如 固相合成法、重組基因表現法等習知技藝者所熟悉的傳統 技術來製備上述分子(參考如Sambrook et al·,Mo/ecw/ar Cloning: A Laboratory Manual, Vols 1-3 (2d ed. 1989), Cold Spring Harbor Laboratory Press 等文獻)。可利用如 Applied Biosystems (Foster City, California)等製造商所 生產之設備來進行自動胜肽合成,且自動胜肽合成的方法 已建立。又可在如酵母菌、昆蟲細胞或哺乳動物細胞等真 核細胞、或如噬菌體或細菌等原核細胞中製造重組蛋白 34 1354022 辦iJl ί如免之)正替換ij ------ ---- 質,或利用習知的無細胞體外表現系統(cell-free z*« v/iro systems)來製造蛋白質。And Science 249:3 86-390, 1 990 describe methods for the preparation of phage peptide peptide expression libraries, vectors and methods for expressing various peptides (WO 91/0 7141 and WO 91/0 7149). In various peptide preparation methods, phage display technology is highly efficient, for example, when the phage display technology is performed in conjunction with the coding sub-mutation method, a peptide of random sequence or a random variation or a specific variation peptide can be produced (please See U.S. Patent No. 5,264,563). These and other conventional methods can be used to create phage display libraries for in vitro screening and identification of peptides, polypeptides or proteins that bind to a performance factor. Or separating from the natural source, various peptides, polypeptides, proteins, polysaccharides or other similar functional molecules capable of binding to a performance factor or inhibiting the activity of a performance factor, and then performing some treatment on the molecules (eg, peptide digestion), or by conventional techniques familiar to those skilled in the art, such as solid phase synthesis, recombinant gene expression, and the like (see, for example, Sambrook et al., Mo/ecw/ar). Cloning: A Laboratory Manual, Vols 1-3 (2d ed. 1989), Cold Spring Harbor Laboratory Press, etc.). Automated peptide synthesis can be performed using equipment manufactured by manufacturers such as Applied Biosystems (Foster City, California), and an automated peptide synthesis method has been established. It is also possible to produce recombinant protein 34 in a prokaryotic cell such as a yeast, an insect cell or a mammalian cell, or a prokaryotic cell such as a bacteriophage or a bacterium, or to replace it with ij ------ - Protein, or using known cell-free z*« v/iro systems to make proteins.

該些可與一表現因子結合或抑制一表現因子活性的 胜肽、聚胜狀或蛋白質與一非同源胜肽(heterologous peptide)結合而成為一融合蛋白。可在上述胜肽、聚胜肽 或蛋白質的氨基尾端(N-端)、羧基尾端(C-端)或同時於兩 尾端接上一個非同源胜肽。並視需要使上述融合蛋白中出 現數個非同源胜肽。或是使該些胜肽、聚胜肽或蛋白質接 上數個同一種非同源胜肽,例如在N端或C-端各接一個 相同的非同源蛋白。有些非同源蛋白能提高融合胜肽、聚 胜肽或蛋白質的半衰期,進而增加這些融合分子在體内的 治療效果(參考美國專利案5,876,969與5,565,335號)。 還可根據各種化學物質及天然、重組表現或人工合成 之胜肽、聚胜肽、蛋白質或多醣類抑制一表現因子之活性 或表現的能力來篩選其他表現因子抑制劑。The peptide, polymorph or protein which binds to or inhibits the activity of a performance factor binds to a non-homologous peptide to form a fusion protein. A non-homologous peptide can be attached to the amino terminus (N-terminus), the carboxyl terminus (C-terminus) of the above peptide, polypeptide or protein, or both ends. Several non-homologous peptides are also present in the above fusion protein as needed. Alternatively, the peptides, polypeptides or proteins may be ligated to several identical non-cologous peptides, for example, one at the N-terminus or at the C-terminus. Some non-homologous proteins increase the half-life of the fusion peptide, polypeptide or protein, thereby increasing the therapeutic effect of these fusion molecules in vivo (see U.S. Patent Nos. 5,876,969 and 5,565,335). Other performance factor inhibitors can also be screened for the ability of various chemical substances and natural, recombinant or synthetic peptides, polypeptides, proteins or polysaccharides to inhibit the activity or expression of a performance factor.

可藉著在一宿主細胞中導入一個受目標聚核苷酸之 啟動子控制的報導基因及一候選表現因子聚核苷酸,並偵 測該報導基因之表現情況來篩選表現因子,以找出預期能 與該目標聚核苷酸結合,並調控該目標聚核苷酸之表現的 該些表現因子。在本發明一較佳實施例中,該宿主細胞為 一真菌宿主細胞。在另一較佳實施例中,該目標基因為 ,以及該啟動子是匸乃及7排除泵浦基因之啟動子。 「報導基因(reporter gene)」包含序列上編碼著可偵測 產物的任一聚核苷酸’該報導基因可運作式地連接至一啟 35 1354022The expression factor can be screened by introducing a reporter gene controlled by a promoter of the target polynucleotide and a candidate expression factor polynucleotide in a host cell, and detecting the expression of the reporter gene. Such performance factors are expected to bind to the polynucleotide of interest and to modulate the expression of the polynucleotide of interest. In a preferred embodiment of the invention, the host cell is a fungal host cell. In another preferred embodiment, the gene of interest is , and the promoter is a promoter that excludes the pump gene. A "reporter gene" comprises any polynucleotide encoding a detectable product in sequence. The reporter gene is operably linked to a primordial 35 1354022

動子。「可運作式連接(operably linked)」係指報導基因以 能讓RNA聚合酶結合至調控區域之啟動子上,並轉錄該報 導基因之核苷酸序列的方式來與該啟動子連接。常用之報 導基因包括該些序列上編碼著点-半乳糖苷酶、綠色螢光蛋 白(green fluorescent protein, GFP)及抗新黴素基因 (neomycin-resistance)之聚核苦酸等。同時,债測該些報導 基因產物的方法為習知技藝者所熟知。本發明之方法可用 來篩選控制基因表現的抑制因子與活化因子(activators)。 可構築一表現載體,使其包含一種或多種編碼著一報 導基因、表現因子或一表現因子抑制劑的核酸序列。例如, 所構築之適當表現載體中可包含:一複製起點(origin of replication, ori)以在宿主細胞内進行自體複製、一個或多 個選擇標記(selective markers)、有限數量之可用限制酶位 址、具高複製數之能力以及一個或多個有力的啟動子。表 現載體可源自各種來源,如病毒、質體或如酵母菌及哺乳 動物細胞等高等有機微生物細胞》Mover. "Operably linked" refers to a reporter gene that is ligated to a promoter that allows RNA polymerase to bind to a regulatory region and transcribe the nucleotide sequence of the reporter gene. Commonly used reporter genes include polynucleic acid such as dot-galactosidase, green fluorescent protein (GFP), and neomycin-resistance. At the same time, methods for reporting these reported gene products are well known to those skilled in the art. The methods of the invention can be used to screen for inhibitors and activators that control gene expression. An expression vector can be constructed to contain one or more nucleic acid sequences encoding a reporter gene, a performance factor or a performance factor inhibitor. For example, a suitable expression vector constructed can include: an origin of replication (ori) for autologous replication in a host cell, one or more selective markers, and a limited number of available restriction enzyme sites. Address, the ability to have a high number of copies, and one or more powerful promoters. Expression vectors can be derived from a variety of sources, such as viruses, plastids, or higher organic microbial cells such as yeast and mammalian cells.

可將本發明之載體置於試管中進行體外表現因子活性 試驗。亦可透過表現載體將如反義寡聚核苷酸、RNAi及 編碼著一胜肽、一聚胜肽之聚核苷酸或一蛋白質等表現因 子抑制劑導入一宿主細胞t。導入表現載體的方法包含病 毒感染法或非病毒感染法,或使用文中所述之其他方法或 習知技藝者熟悉之技術,其中非病毒感染法如微脂粒轉染 法(lipofection)、配位子-DNA結合物法及裸露DNA直接 注射法,其相關内容可參考美國專利案6,140,484號。 36 1354022The vector of the present invention can be placed in a test tube for in vitro performance factor activity assay. A expression factor inhibitor such as an antisense oligonucleotide, RNAi, and a polynucleotide encoding a peptide, a polypeptide or a protein can also be introduced into a host cell through a performance vector. The method of introducing the expression vector comprises a viral infection method or a non-viral infection method, or using other methods described herein or techniques familiar to those skilled in the art, wherein non-viral infection methods such as lipofection, coordination The sub-DNA conjugate method and the naked DNA direct injection method can be referred to the U.S. Patent No. 6,140,484. 36 1354022

亦可篩選各種聚核苷酸來找出具有一個或多個中程孢 子生成序列的聚核苷酸。啤酒酵母菌之基因及白色 念珠菌相似基因 CaNDT80令均可找到中程孢子生成序 列。因此,中程抱子生成序列可能在藥物敏感性及細胞毒 性的機轉申扮演重要角色。同時可藉著鑑定其他聚核苷酸 中是否具有中程孢子生成序列來作為可能用來治療的分 子。可利用取自細胞的體基因庫或cDNA庫來進行聚核苷 酸篩選。習知技藝者均熟知製備體基因庫或cDNA庫的方 法。當欲篩選之聚核苷酸、蛋白質或小分子之數量龐大時, 使用生物資訊技術可大幅提高篩選效率,或利用DNA微陣 列晶片來篩選含有中程孢子生成序列的聚核苷酸。亦可使 用微陣列晶片來篩選該些能與含有中程孢子生成序列之聚 核苷酸結合的蛋白質。有關DNA微陣列晶片之相關資料可 參考以下文獻,如 Γαίσ/7 e/ a/., Science 289:1757-1760, 2000 ' Reichert et al., Anal. Chem. 72:6025-6029, 2000、 Lockhart et al., Nature 405:827-836, 2000 、 The Chipping Forecast, Nature Genetics 2:Supp., Jan. 1999。而此處之表 現因子抑制劑係指能干擾含有中程孢子生成序列之聚核苷 酸分子運作的該些物質。 本發明亦有關於藉著施用至少一有效劑量之表現因子 抑制劑,來治療一患者或使該患者獲得益處。亦可對患者 施用至少一有效劑量之藥物,且使用該藥物時可同時或接 續使用該表現因子抑制劑。此處「有效劑量(effective amount)」係指能有效抑制一患者體内一聚核苦酸之表現的 37 1354022Various polynucleotides can also be screened to find polynucleotides having one or more mid-range spore generating sequences. The S. cerevisiae gene and the Candida albicans-like gene CaNDT80 can find a sequence of mid-range spore formation. Therefore, the mid-range cuddling sequence may play an important role in the drug-susceptibility and cytotoxicity of the machine. It is also possible to identify molecules that may be used for treatment by identifying whether other polynucleotides have a mid-range sporulation sequence. Polynucleotide screening can be performed using a bulk gene bank or cDNA library taken from the cells. Those skilled in the art are familiar with methods for preparing bulk gene libraries or cDNA libraries. When the number of polynucleotides, proteins, or small molecules to be screened is large, bioinformatics can be used to greatly increase the efficiency of screening, or DNA microarray wafers can be used to screen for polynucleotides containing mid-range spore-forming sequences. Microarray wafers can also be used to screen for proteins that bind to polynucleotides containing mid-range spore-forming sequences. For information on DNA microarray wafers, refer to the following documents, eg Γαίσ/7 e/ a/., Science 289:1757-1760, 2000 'Reichert et al., Anal. Chem. 72:6025-6029, 2000, Lockhart Et al., Nature 405: 827-836, 2000, The Chipping Forecast, Nature Genetics 2: Supp., Jan. 1999. The expression factor inhibitor herein refers to those substances which interfere with the operation of a polynucleotide molecule containing a medium-range spore-forming sequence. The invention also relates to treating a patient or benefiting a patient by administering at least one effective amount of a performance factor inhibitor. The patient may also be administered at least one effective dose of the drug, and the performance factor inhibitor may be used simultaneously or sequentially when the drug is used. Here, "effective amount" means an effective inhibition of the performance of a polynucleic acid in a patient. 37 1354022

劑量,以降低聚核苷酸或蛋白質的活性,進而缓 症狀、消除患者之微生物感染情形或抑制細胞生 本發明亦提供實施本發明方法的組合物。該 能包含至少一表現因子抑制劑。在另一較佳實施 組合物可包含至少一表現因子抑制劑及至少一藥 物可能是一種抗微生物劑或抗真菌劑。本發明之 可配合他種藥學活性化合物一併使用。下述方法 作示範之用,非用以限制本發明。 口服組合物可單獨使用,或配合適當添加劑 劑、粉末、顆粒或膠囊,例如可配合使用乳醣、甘 玉米澱粉或馬鈐薯澱粉等傳統添加劑,或如結 素、纖維素衍生物、阿拉伯膠、玉米澱粉或明膠筹 又如玉米澱粉、馬鈐薯澱粉或羧基曱基纖維素i carboxymethylcellulose)等崩散劑、又或是如滑石 酸鎂等潤滑劑。如有需要,亦可添加稀釋劑、缓 潤劑、防腐劑及香料等。 本發明之組合物可視需要而包含生理上可接 性之載劑,其可以是各種常用於動物或人類用藥 溶劑。通常這些載劑為生理可接受之物質,即該 影響組合物之生物活性。此類載劑可為蒸顧水、 衝生理食鹽水、林格氏液(Ringer's solution)、葡 (dextrose solution)及漢克平衡鹽溶液(Hank's salt solution)。此外,組合物中亦可包含如i (p r e 〇 g 1 y c a n s)等載劑分子。在特定範例中,載劑 和患者之 長。 組合物可 例中,該 物。該藥 組合物亦 及輔劑僅 以製成錠 露糖醇、 晶狀纖維 黏著劑, 内(sodium 粉、硬脂 衝劑、濕 受且無毒 成分中的 些載劑不 磷酸鹽缓 萄糖溶液 balanced 瑭蛋白類 分子包括 38 1354022 睹胺多黯類(glycosaminoglycans),如硫酸肝素(heparin sulfate)、玻展酸(或稱聽搭酸,hyaluronic acid)、硫酸角 蛋白(keratin-sulfate) 、 4-硫酸軟骨素(chondroitin 4-sulfate)、6-硫酸軟骨素(chondroitin 6-sulfate)、硫酸角 蛋白與硫酸皮膚素昆合物(heparin sulfate and dermatin sulfate)、基底族蛋白聚醣(perlecan)及戊聚醣聚硫酸鹽 (pento polysulfate)。此外,本發明組合物可能包含其他輔 藥、佐劑或無毒性、無治療效果且非免疫性之穩定劑等等。Dosage to reduce the activity of a polynucleotide or protein, thereby alleviating symptoms, eliminating microbial infections in a patient, or inhibiting cell growth. The invention also provides compositions for practicing the methods of the invention. The energy can comprise at least one performance factor inhibitor. In another preferred embodiment the composition may comprise at least one performance factor inhibitor and at least one drug may be an antimicrobial or antifungal agent. The present invention can be used in combination with other pharmaceutically active compounds. The following methods are illustrative and are not intended to limit the invention. The oral composition may be used alone or in combination with suitable additives, powders, granules or capsules, for example, conventional additives such as lactose, corn maize starch or horse yam starch may be used together, or as a vegetarian, cellulose derivative, gum arabic Corn starch or gelatin is also a disintegrating agent such as corn starch, horse starch or carboxymethyl cellulose, or a lubricant such as magnesium talcate. Thinners, demulsifying agents, preservatives and fragrances may also be added if necessary. The compositions of the present invention may comprise a physiologically acceptable carrier, as desired, which may be a variety of solvents commonly used in animals or humans. Typically these carriers are physiologically acceptable materials, i.e., which affect the biological activity of the composition. Such carriers can be distilled water, physiological saline, Ringer's solution, dextrose solution, and Hank's salt solution. In addition, carrier molecules such as i (p r e 〇 g 1 y c a n s) may also be included in the composition. In a particular example, the carrier and the patient are long. The composition can be, for example, the substance. The pharmaceutical composition and the auxiliary agent are only made into a lozenitol, a crystalline fiber adhesive, and the carrier (sodium powder, hard fat granule, wet carrier and non-toxic component, some carrier non-phosphate slow sugar solution) Balanced prion protein molecules include 38 1354022 glycosaminoglycans, such as heparin sulfate, hyaluronic acid, keratin-sulfate, 4- Chondroitin 4-sulfate, chondroitin 6-sulfate, heparin sulfate and dermatin sulfate, perlecan and pentane In addition, the compositions of the present invention may contain other adjuvants, adjuvants or non-toxic, non-therapeutic and non-immune stabilizers and the like.

本發明之組合物可溶解、懸浮或乳化於生理上可接受 之載劑中,以作注射之用》所使用之載劑包括各種滅菌液 體,如水、油類及甘油類,並可添加或不添加介面活性劑。 所使用之油類可為各種石油衍生物、動物或植物之油脂或 合成油脂,如花生油、大豆油及礦物油等。甘油類包括丙 二醇(propylene glycol)與聚乙二醇(polyethylene glycol)。組合物中亦可包含傳統添加物,如助溶劑 (solubilizer)、等張壓劑(isotonic agents)、使懸浮劑The carrier of the present invention can be dissolved, suspended or emulsified in a physiologically acceptable carrier for injection. The carrier used includes various sterilizing liquids such as water, oils and glycerol, and may or may not be added. Add an surfactant. The oil to be used may be various petroleum derivatives, animal or vegetable fats or synthetic oils such as peanut oil, soybean oil and mineral oil. Glycerols include propylene glycol and polyethylene glycol. The composition may also contain conventional additives such as solubilizers, isotonic agents, suspending agents.

(suspending agents)、乳化劑(emulsifying agents)、安定劑 (stabilizers)與防腐劑(preservatives)等。亦可將本發明之 組合物製備成持續釋放劑型,如製備成可透過積存注射、 植入或利用滲透泵浦供藥等方式來用藥之劑型’以達到持 續釋放活性成分的目的。 可將本發明之組合物製備成喷霧型組合物,透過吸入 肺部來用藥。並可將本發明組合物混入如二氣二氟曱烷 (dichlorodifluoromethane)、氮氣(nitrogen)及其他壓縮推進 39 1354022(suspending agents), emulsifying agents, stabilizers, and preservatives. The compositions of the present invention may also be prepared in a sustained release dosage form, such as a dosage form prepared to be administered by injection, implantation or by osmotic pump delivery, for the purpose of sustained release of the active ingredient. The composition of the present invention can be prepared into a spray type composition for administration by inhalation into the lungs. The compositions of the present invention may be incorporated into, for example, dichlorodifluoromethane, nitrogen, and other compression propulsions 39 1354022

劑中配合使用。 可利用包括如非腸胃道注射等各種便利的方法來 本發明組合物,並可對局部或全身進行用藥。同時, 過如口服、頰腔、腸胃道、非腸胃道、腹腔、皮内、 膚(transdermal)、氣管内、脊髓内、鼻腔内、胃、肌肉 顱内及皮下等管道來使用本發明之組合物。用藥後, 成分可能會遍布全身,或是藉著局部用藥、體内用藥 用一種能將活性成分保留在植入區域的植入物而令活 分作用於局部部位。 可提供各種用於口服或直腸用藥的單劑形式, 漿、錠劑及懸浮液體等,其中如一茶匙、一餐匙、一 —栓劑等各種劑量單位中含有一預定量之組合物。 地,各種用於注射或靜脈滴注用藥的單劑形式中可包 發明之組合物,並採將組合物溶於滅菌水、一般生理 水、或其他藥學上可接受之載劑中之形式來使用。 習知技藝者可輕易地根據特定組合物之作用、症 重程度、患者對副作用的敏感程度來調整本發明之劑 此外,本發明之部分特定組合物可能較其他組合物更 效。習知技藝者可根據不同方法來決定一特定組合物 用劑量,例如利用習知方法來測量一指定組合物相對 一組合物的生理效力,並根據其結果來調整組合物之旁 除了上述實施例或他處内容所記載者,需明白所 示數量、组成、反應條件及本申請案所使用之其他數 為概略值。因此,除非另有指示,本申請案上述之數 使用 可透 經皮 内、 活性 或使 性成 如糖 鍵或 同樣 含本 食鹽 狀嚴 量。 具功 之使 於另 Η量。 有表 據均 據參 40 1354022 數為近似值,且可根據本發明所欲得到之性質而加以改 變。最後,各項均等物為本案之申請專利範圍所涵蓋,故 各項數據參數需根據所使用之技術及輸出的數據來解釋。 儘管本發明上述之數據範圍及參數為近似值,然本說 明書特定較佳實施例之數據已盡可能地精確記載。然而由 於各項試驗均有其標準偏差,故各項數據值中必包含部分 誤差值。Used in combination with the agent. The composition of the present invention can be utilized by various convenient methods including, for example, parenteral injection, and can be administered locally or systemically. At the same time, the combination of the present invention is used, such as oral, buccal, gastrointestinal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, intraspinal, intranasal, gastric, intramuscular, and subcutaneous. Things. After administration, the ingredients may be distributed throughout the body, or by topical or in vivo administration, with an implant that retains the active ingredient in the implanted area, allowing the active action to be applied to the localized area. A variety of single-dose forms for oral or rectal administration, such as a syrup, a lozenge, a suspension liquid, and the like, may be provided, wherein a predetermined amount of the composition is contained in various dosage units such as one teaspoon, one serving spoon, and one suppository. The compositions of the invention may be included in a single dosage form for injection or intravenous drip administration, and may be dissolved in sterile water, general physiological water, or other pharmaceutically acceptable carrier. use. A person skilled in the art can readily adjust the agent of the present invention depending on the action of the particular composition, the degree of severity, and the sensitivity of the patient to side effects. Further, some of the specific compositions of the present invention may be more effective than other compositions. The skilled artisan can determine the dosage for a particular composition according to various methods, for example, using conventional methods to measure the physiological efficacy of a given composition relative to the composition, and adjusting the composition according to the results. Or the contents described in the other contents, it is necessary to understand that the quantity, composition, reaction conditions and other numbers used in the present application are approximate values. Therefore, unless otherwise indicated, the above-mentioned number of the present application can be used to be dermally permeable, active or toxic, such as a sugar bond or the same salt. The power is made to make another difference. Some of the data are approximated according to the number of reference numerals, and can be changed according to the properties of the present invention. Finally, the equivalents are covered by the scope of the patent application in this case, so the data parameters should be interpreted according to the technology used and the output data. Although the above-described data ranges and parameters of the present invention are approximate, the data of the specific preferred embodiments of the present invention have been described as accurately as possible. However, since each test has its standard deviation, some data values must contain partial error values.

除非另行定義,此處所使用之所有技術性或科學性用 詞均與本發明之習知技藝者所熟知之意義相同。文中所敍 述之示範方法及材料僅作表達本發明之目的,各種與文中 所述内容相似或均等之方法及材料亦可用來實施本發明。All technical or scientific terms used herein have the same meaning as commonly understood by those skilled in the art, unless otherwise defined. The exemplary methods and materials described herein are merely illustrative of the invention, and various methods and materials similar or equivalent to those described herein may be used to practice the invention.

本發明所提及之所有公開文獻均完整納入參考,以揭 露並敘述該些公開文獻之相關方法及/或材料。此外,文中 所提及之公開文獻僅揭露先於本發明申請日以前的習知技 術,然本發明中未有任何内容聲稱本發明晚於該些習知技 術。再者,文中所提供之文獻公開日期可能非為實際公開 曰期,故需個別確認之。 需明白,除非說明書内容中作其他明確定義,否則文 中及後附申請專利範圍所使用之單數用語「一(a)」、「以及 (and)」與「該(the)」均包括其複數型態。故舉例來說,參 考文中「一種抗微生物劑(anntimicrobialagent)」一詞係 包含數種抗微生物劑,以及「一真菌細胞(a f u n g a丨c e 1丨)」 一詞係包含一種或多種真菌細胞及習知技藝者所熟知的其 他均等物。 41 1354022 用來實施本發明的方法、技術及/或程序(通稱方法)並 不僅限於本說明書中所記載之特定較佳實施例的方法,任 何可達成相同目的之習知方法均為本發明所涵蓋。此外, 雖已於本說明書之特定内容中敘述部分方法,然該些方法 並非用來限制本發明内容。 以下數個較佳實施例係用來顯示本發明,而非限制本 發明之用。All publications mentioned in the present specification are hereby incorporated by reference in their entirety to the extent of the disclosure of the disclosure of In addition, the publications referred to herein merely disclose prior art prior to the filing date of the present invention, and nothing in the present invention claims that the present invention is later than the prior art. Furthermore, the date of publication of the literature provided in the text may not be the actual disclosure period, so it needs to be confirmed separately. It is to be understood that the singular terms "a", "and" and "the" are used in the singular and state. Thus, for example, the term "an antimicrobial agent" in the text contains several antimicrobial agents, and the term "a fungus cell (afunga丨ce 1丨)" contains one or more fungal cells and Other equals known to the skilled artisan. 41 1354022 The methods, techniques, and/or procedures (generally referred to as methods) used to practice the present invention are not limited to the methods of the specific preferred embodiments described in the specification, and any conventional method that achieves the same objectives is the present invention. Covered. In addition, some of the methods are described in the specific content of the specification, and the methods are not intended to limit the invention. The following preferred embodiments are intended to illustrate the invention and not to limit the invention.

較佳實施例1 1 築 Leu2+ 矣規杯鞞 構築一個含有/czcZ基因及啟動子的表現 載體,該/flcZ基因會表現冷-半乳糖苦酶(β-ga丨actosidase 或稱 β-ga 卜 Μ少 er αΛ,Gene 45:299-310,1986)以作為報 導基因(reporter gene),且該/acZ基因受啟動子控 制,使得該表現載體可用來監控/啟動子的表現情形。Preferred Embodiment 1 1 Construct a Leu2+ 矣 鞞 鞞 鞞 construct a expression vector containing the /czcZ gene and a promoter, and the /flcZ gene will express cold-galactosidase (β-ga丨actosidase or β-ga dip) Less er αΛ, Gene 45:299-310, 1986) is used as a reporter gene, and the /acZ gene is under the control of a promoter, so that the expression vector can be used to monitor/promoter performance.

利用聚合酶鏈鎖反應法將白色念珠菌SC5314菌株之體 基因中一段長約 1.2 kb且包含 基因之轉譯起始碼 ATG與啟動子的 DNA片段數量加以放大。係利用引子 HJL21[5,d(TTTCCCGGGGGATCCTCGTTACTCAA)l 及弓| 子 HJL22 ( 5, dfCCCAAGCTTGCATAATTTTTTTCTTTTTGACCT)來製備 上述PCR片段。且上述兩引子分別在該PCR片段上引入 一個5,端的尤mill及一個3,端的//ΜίΠΠ限制酶位址(標示 底線之序列)》將所得之PCR片段插入ΥΙΡ3 63質體上的 42 1354022A population of about 1.2 kb in the somatic gene of Candida albicans SC5314 strain and the number of DNA fragments containing the translation start code ATG and the promoter were amplified by polymerase chain reaction. The above PCR fragment was prepared using the primers HJL21 [5, d (TTTCCCGGGGGATCCTCGTTACTCAA) l and the bow | sub-HJL22 (5, dfCCCAAGCTTGCATAATTTTTTTCTTTTTGACCT). And the above two primers respectively introduce a 5, a terminal, and a 3, a contiguous restriction enzyme site (the sequence indicating the bottom line) on the PCR fragment, and insert the obtained PCR fragment into the ΥΙΡ3 63 plastid 42 1354022

Zwal與Ζ/?_«ίΠΙΙ兩個限制酶位址之間,使此段含有 啟動子的 PCR片段能符合編碼規則地與/flcZ基因相連 接,而產生LOB42質體。Zwal and Ζ/?_«ίΠΙΙ between the two restriction enzyme sites, so that the PCR fragment containing the promoter can be ligated to the /flcZ gene in accordance with the coding rules, and the LOB42 plastid is produced.

使用引子 HJL44(5, d(TTTTCCCGGOCAGCAGTTTA GAAGCAAT))及引子 HJL45(5,drCCCCCCCGGGTGATTTG TCTTAACATT))來放大 基因之 37bp 至 1 648bp 的 DNAUse the primer HJL44 (5, d(TTTTCCCGGOCAGCAGTTTA GAAGCAAT)) and the primer HJL45 (5, drCCCCCCCGGGTGATTTG TCTTAACATT) to amplify the DNA from 37 bp to 1 648 bp of the gene.

片段。以與融合基因相反向的方式,將乂Z)£3 基因之該DNA片段插入LOB42質體中的Xwal位址,而構 築出LOB43質體。以限制酶切斷LOB43質體之 基因轉譯起始碼ATG下游第3 3 7bp之位置,使LOB43質 體成為線狀後,再將線狀的LOB43質體轉型至啤酒酵母菌 10560-2B菌株中(參考第4圖)。透過同源重組,使LOB43 質體上的融合基因插入細胞染色體上的 基因座,而製造出 Leu2 +轉型株(trsformant),並標示為 SLO 1。YIp363質體上的野生種基因係作為篩選標記 之用。Fragment. The DNA fragment of the 乂Z)£3 gene was inserted into the Xwal site in the LOB42 plastid in a manner opposite to the fusion gene to construct the LOB43 plastid. The LOB43 plastid was linearized by the restriction enzyme to cleave the LOB43 plastid starter code ATG downstream of the 3rd 3 7bp, and then the linear LOB43 plastid was transformed into the Saccharomyces cerevisiae 10560-2B strain. (Refer to Figure 4). Through the homologous recombination, the fusion gene on the LOB43 plastid is inserted into the locus on the chromosome of the cell, and the Leu2 + transformant (trsformant) is produced and labeled as SLO 1. The wild-type gene line on the YIp363 plastid is used as a screening marker.

較佳實施例2 構築一白色念珠諂艚基因庫並篩選之 利用具有南複製數(COpy number)的哮酒酵母載體 來構築白色念珠菌體基因庫μ a/·,Science 266: 1 723- 1 726,1 994)。根據較佳實施例1所敘述之方法, 利用標準技術將此體基因庫轉型至SL01菌株十。並將 2μ-ί/7Μ5控制載體轉型至SL〇1菌株中,以產生控制組菌 43 1354022 株SL02。使用/acZ基因作為報導基因’以監控0£>Λ/啟 動子的活性,並利用上述濾紙分析法及/或液體分析法來偵 測β -半乳糖苷酶(β-gal)之活性Current Protocols in Molecular Biology, 2 , pp. 3.6.2-3.6.5. John Wiley & Sons, New York, NY, 1997; Mosch et al., 93:53 52-53 56,1 996)。PREFERRED EMBODIMENT 2 A white rosé gene bank was constructed and screened using a roten yeast vector having a COpy number to construct a Candida albicans gene library μ a/·, Science 266: 1 723- 1 726,1 994). This somatic gene pool was transformed to SL01 strain X using standard techniques according to the method described in the preferred embodiment 1. The 2μ-ί/7Μ5 control vector was transformed into the SL〇1 strain to produce the control group 43 1354022 strain SL02. The /acZ gene was used as a reporter gene to monitor the activity of the £/promoter and to detect the activity of β-galactosidase (β-gal) by using the above filter paper analysis and/or liquid analysis. Protocols in Molecular Biology, 2, pp. 3.6.2-3.6.5. John Wiley & Sons, New York, NY, 1997; Mosch et al., 93:53 52-53 56,1 996).

使基因庫轉型株(每15〇 mm之培養皿上約500個菌落) 生長3天後’再將濾紙覆蓋在洋菜膠培養孤上,使菌落複 印在濾紙上。與僅含有2仁_ 3控制載體之控制組比較, 並將濾紙上呈現升高之冷-gal活性的URA+菌落挑選出 來’以作更進一步篩選。將一菌落細胞中之基因庫質體移 除後’若其沒•半乳糖苷酶活性回到基礎程度,則表示此菌 落具有一個CD/?/基因之反式調控因子的候選質體。使上 述細胞分別在YPD培養液中生長兩天,以去除該些候選菌 落細胞中的基因庫質體。隨後以液體分析法來比較該些候The gene bank transformed strain (about 500 colonies per 15 mm of culture dish) was grown for 3 days, and then the filter paper was overlaid on the agar resin culture so that the colonies were replicated on the filter paper. URA+ colonies with elevated cold-gal activity on the filter paper were selected for further screening compared to control groups containing only 2 kernels _3 control vector. Removal of the genoplasts in a colony cell, if its galactosidase activity returns to a basal level, indicates that the colony has a candidate plastid of a CD/?/gene trans-regulatory factor. The above cells were separately grown in YPD medium for two days to remove the gene bank plastids in the candidate colony cells. Then compare these with liquid analysis

選菌落及已去除基因庫質體之該些候選菌落的半乳糖 普酶活性。 利用上述方法製備出約24000種不同的基因庫轉型 株’其包含的白色念珠菌體基因序列數量約為白色念珠菌 體基因大小的3倍。並且在最初次篩選出的74株候選菌株 中’有16株菌株於第二次濾紙篩選試驗中表現出升高之沒 -半乳糖苷酶活性。此16株候選菌株中’又有5株之 半乳糖苦酶活性高於其他候選菌株,並對此5株候選菌株 進行進一步分析。在此五株候選菌株中,判斷有兩株菌株 44 1354022The galactosidase activity of the colonies and the candidate colonies of the gene bank plastids were selected. About 24,000 different gene bank transformation strains were prepared by the above method, and the number of the Candida albicans gene sequences contained was about three times that of the Candida albicans gene. And among the 74 candidate strains screened out at the first time, 16 strains showed elevated galactosidase activity in the second filter paper screening test. Among the 16 candidate strains, 5 of them had higher galactose activity than other candidate strains, and further analysis was carried out on the 5 candidate strains. Among the five candidate strains, two strains were judged 44 1354022

之染色體上發生突變’因為在移除該兩候選菌株中的基因 庫質體後’其仍表現出同樣強度的半乳糖苷酶活性。反 之’當移除另外三株候選菌株之基因庫質體後,其β·半乳 糖苷酶活性則降低至基礎程度。在這三株候選菌株中,兩 菌株中所含之質體相同,並將該質體標示為LOB 44,而該 含有LOB44質體的URA +候選菌株則標示為SL03。對 LOB44質體進行分析,顯示其含有一段長約5kb的插入序 列,該此插入序列中包含兩個完整的開放讀碼區(open reading frame,ORFs),其一為 CaNdt80p 之開放讀碼區, 另一為or f6.1265的開放讀碼區。〇Γ f6.1265可能是一個長 度為106個氨基酸的短蛋白質。從白色念珠菌種SC5314 之體基因中取出長度為2.8 kb的基因片段來構築 LOB45質體,並與LOB44質體中相同的片段作 活性比較。該構築方法係利用引子 HJL72(5’ d(CGG_GATCC.TTGTGGCGATTTTCACTTTC))及 HJL73(5, d(CCGGATCCTCAATGGGGGTGGATTGA'))來放大 SC5314 菌株中的白色念珠菌基因,以構築LOB45質體。 並且HJL72與HJL73兩引子會在放大出來的基 因片段上製造出限制酶位址(標示底線之序列)以作 選殖之用。放大的DNA片段序列係從基因之起 始碼ATG上游第5 7 8bp起至終止碼TAA之下游第479bp 為止。以限制酶來剪切放大的DNA片段後,將該 DNA片段插入pRS426載體上的位置中,以製迤出 LOB45 質體(Christianson et al·, Gene 110:119-122, 45 1354022Mutations occur on the chromosomes because they still exhibit the same intensity of galactosidase activity after removal of the gene library in the two candidate strains. In contrast, when the gene bank plastids of the other three candidate strains were removed, the β-galactosidase activity was reduced to a basal level. Among the three candidate strains, the plastids contained in the two strains were identical, and the plastid was designated as LOB 44, and the URA + candidate strain containing the LOB44 plastid was designated as SL03. Analysis of the LOB44 plastid revealed that it contained an insertion sequence of about 5 kb in length, which contained two complete open reading frames (ORFs), one of which was the open reading frame of CaNdt80p. The other is the open reading area of or f6.1265. 〇Γ f6.1265 may be a short protein of 106 amino acids in length. A 2.8 kb gene fragment was extracted from the body gene of Candida albicans SC5314 to construct a LOB45 plastid and compared with the same fragment in the LOB44 plastid. In the construction method, the Candida albicans gene in the SC5314 strain was amplified by the primers HJL72 (5'd(CGG_GATCC.TTGTGGCGATTTTCACTTTC)) and HJL73(5, d(CCGGATCCTCAATGGGGGTGGATTGA')) to construct the LOB45 plastid. And the two primers HJL72 and HJL73 will create a restriction enzyme address (the sequence indicating the bottom line) on the amplified gene fragment for colonization. The amplified DNA fragment sequence is from the 578 bp upstream of the start code of the gene to the 479 bp downstream of the termination code TAA. After the amplified DNA fragment is cleaved by a restriction enzyme, the DNA fragment is inserted into a position on the pRS426 vector to produce a LOB45 plastid (Christianson et al., Gene 110: 119-122, 45 1354022).

1992)〇將LOB44質體轉型至SL01菌株中,並標示為SL05 菌株。利用定量液體分析法來測試SL03菌株之;S -半乳糖 苷酶活性(參考第5圖)。結果數據顯示SL03菌株(B直行) 之冷-半乳糖苷酶活性是作為控制組之SL02菌株(A直行) 的六倍。當移除SL03菌株中的LOB44質體後(C直行), SL0 3菌株的召·半乳糖苷酶活性會降低至基礎程度(參考a 直行)。再次將LOB44質體導入親代之SL01菌株中,可 使SL〇 1菌株的冷-半乳糖苷酶活性恢復至先前的強度(參 考D直行)。此外,具有LOB45質體之SL05菌株的冷-半 乳糖苷酶活性與SL03菌株所表現出來的活性相等。該些 結果顯示基因與啤酒酵母菌中插入 基因之泠-半乳糖苷酶活性的提高有密切關連》 較佳實施例31992) The LOB44 plastid was transformed into the SL01 strain and labeled as SL05 strain. The SL03 strain was tested by quantitative liquid analysis; S-galactosidase activity (refer to Fig. 5). The results showed that the cold-galactosidase activity of the SL03 strain (B straight) was six times that of the control group SL02 strain (A straight). When the LOB44 plastid in the SL03 strain was removed (C straight), the galactosidase activity of the SL0 3 strain was reduced to a basal level (see a straight). The introduction of the LOB44 plastid into the parental SL01 strain again restored the cold-galactosidase activity of the SL〇 1 strain to the previous intensity (Ref. D). In addition, the cold-galactosidase activity of the SL05 strain having the LOB45 plastid was equivalent to that exhibited by the SL03 strain. These results show that the genes are closely related to the increase in the 泠-galactosidase activity of the inserted gene in S. cerevisiae.

過量袅現蛋白質(CaNdt80p)會降低藥物Μ感性 利用 Etest 抗真菌劑試片(AB BIODISK,Solna,Sweden) 來比較過量表現CaNdt80p及其啤酒酵母菌相似物Ndt80p 對藥物敏感性的影響。ei α/·,J. Antimicrob. Chemother. 47:521-526, 2 0 0 1; Chang et al., J. Clin.Excessive protein (CaNdt80p) reduces drug susceptibility. Etest antifungal test strips (AB BIODISK, Solna, Sweden) were used to compare the effects of excess performance of CaNdt80p and its S. cerevisiae analog Ndt80p on drug sensitivity. Ei α/·, J. Antimicrob. Chemother. 47:521-526, 2 0 0 1; Chang et al., J. Clin.

Microbiol 39:1328-1333, 2001)。啤酒酵母菌之 Ndt80p 蛋 白為一個減數分裂特定轉錄因子(meiosis specific transcription factor)(C/jw et al., Science 282:699-705, 1 9 9 8; Chu and Herskowitz, Mol. Cell 1:685-696, 1 998)。 Ndt 8 Op蛋白會進行自我調控,同時透過其目標分子上的中 46 1354022 程孢子生成序列(MSE·· gNCRCAAAA/T)來活化該目標基因 (Chu and Herskowitz, Mol. Cell 1:685-696, 1 998)。並且, 在無性生殖的過程中,#£>7^0基因的表現受到抑制。 α/·, Mol. Cell Biol. 15:6572-658 1,1 995; Pak and Segall, Mol.Cell Biol. 22:64 1 7-6429, 2002)。Microbiol 39: 1328-1333, 2001). The Ndt80p protein of S. cerevisiae is a meiosis specific transcription factor (C/jw et al., Science 282: 699-705, 1 9 9 8; Chu and Herskowitz, Mol. Cell 1:685 -696, 1 998). The Ndt 8 Op protein is self-regulated and the target gene is activated by the mid-46 1354022 spore-producing sequence (MSE··gNCRCAAAA/T) on its target molecule (Chu and Herskowitz, Mol. Cell 1:685-696, 1 998). Moreover, in the process of asexual reproduction, the performance of the #£>7^0 gene was suppressed. α/·, Mol. Cell Biol. 15:6572-658 1,1 995; Pak and Segall, Mol. Cell Biol. 22:64 1 7-6429, 2002).

首先’自已培養一個晚上的SD培養皿(SD plate)上挑 出數個菌落且均勻混合後移至0.85 %氣化鈉溶液中,成 為細胞濃度為5xl06 cells/ml的懸浮液。取一滅菌棉花棒 沾取上述接種用之懸浮液後,使用沾有菌液的棉花棒在一 SD培養皿之整個洋菜膠表面上均勻畫線。待SD培養皿將 多餘水分完全吸收後,在上述培養皿上放置E test藥物試 片(AB B10DISK,Solna,Sweden)。將培養皿置於 30 °C 之 壤境下培養三天後,拍攝其細胞生長狀況。使用之Etest 藥物試片係用來試驗細胞對酮康唑(0 ·002-3 2 μg/m 1)及氟 康唑(0.0 1 6-2 5 6 pg/ml)的敏感性。根據抗真菌劑試片周圍 是否產生抗菌區域來判斷藥物敏感性。並根據環形抗菌區 域的大小來判斷藥物敏感性的程度。接受測試之菌株分別 如下:接受 2p-UJiA3 載體與 CEN-LEU2質體轉型之 NDT80/NDT80野生種啤酒酵母菌(標示為 SL016菌株, Christianson et al., Gene 110:119-122, 1992; Sikor ski and /f/eier, Genetics 122:19-27, 1989,且參考第 6 圖之 直行)、接受載體轉型之ndt80/ndt80同型 接合子突變種(標示為 SL014菌株,並參考第 6圖之 ilndt80/ndt80 2pvector” 直行)、接受 2 μ-URA 3 - ND:T8 0 質體 47 1354022 (LOB46)轉型之 《心同型接合子突變種(標示為 SL013菌株,並參考第6圖之直 行)、以及接受 2μ-ί//^3-〇3Λί£)Γδ0 質體(LOB44)轉型之 «心抑/«心抑同型接合子突變種(標示為SL015菌株,並參 考第6圖之“/7心50/«心占0 2 0-€^#£»7'<^”直行)。First, several colonies were picked from the SD plate which had been cultured overnight, and uniformly mixed, and then transferred to a 0.85 % sodium carbonate solution to obtain a suspension having a cell concentration of 5 x 106 cells/ml. Take a sterilized cotton swab. After picking up the suspension for inoculation, use a cotton swab dipped in bacteria to evenly draw a line on the surface of the whole acacia. After the SD culture dish completely absorbed the excess water, an E test drug test piece (AB B10DISK, Solna, Sweden) was placed on the above culture dish. After the culture dish was placed in a soil at 30 ° C for three days, the cell growth was photographed. The Etest drug test strips used were used to test the sensitivity of cells to ketoconazole (0·002-3 2 μg/m 1 ) and fluconazole (0.0 1 6-2 5 6 pg/ml). Drug sensitivity is judged based on whether or not an antibacterial area is produced around the antifungal test piece. The degree of drug sensitivity is judged based on the size of the annular antibacterial region. The tested strains were as follows: Nt80/NDT80 wild-type S. cerevisiae (2) designated by the 2p-UJiA3 vector and CEN-LEU2 plastid (marked as SL016 strain, Christianson et al., Gene 110: 119-122, 1992; Sikor ski And /f/eier, Genetics 122:19-27, 1989, and refer to the straight line of Figure 6), accepting vector-transformed ndt80/ndt80 homozygous mutants (labeled SL014 strain, and refer to ilndt80/ in Figure 6) Ndt80 2pvector" straight), accept 2 μ-URA 3 - ND:T8 0 plastid 47 1354022 (LOB46) transformation of the heart-type zygote mutant (labeled SL013 strain, and refer to Figure 6 straight), and accept 2μ-ί//^3-〇3Λί£) Γδ0 plastid (LOB44) transformation of the «heart suppression / « heart suppression homozygous mutated mutant (labeled SL015 strain, and refer to Figure 6 "/7 heart 50 / «心占0 2 0-€^#£»7'<^" goes straight).

係利用聚合酶鏈鎖反應來放大 PRS 3 05質體(SMowH and //ze,er, Genetics 1 22:1 9-27,1 9 89)中的 I五ί/2 DNA 片 段,其前後兩端段分別具有WDTSO基因長度約65bp的5’ 端非轉譯區(untranslated region,UTR)及 3’端的非轉譯 區,並利用此段DNA片段來製備《心^/«心50同型接合子 啤酒酵母菌突變種。將上述 DNA片段轉型至啤酒酵母菌 1 0 5 6 0 - 2 B 菌株中(ra A/iJ.’/ZiiiG wraJ-5·? 以 產生 ηί/Μ0._.·Ι£:ί/2 菌株,並將其標示為 SL06(M/ra «ciίS0.. .·Z£ 2 /π’·ϊ3 .· ·· ΛίsG wra3 -52 /e w2: ··办z_?C?J。隨後使 S LΟ6The polymerase chain reaction is used to amplify the I ί/2 DNA fragment in the PRS 307 plastid (SMowH and //ze, er, Genetics 1 22:1 9-27,1 9 89). The 5D-untranslated region (UTR) and the 3'-end non-translated region of the WDTSO gene are approximately 65 bp in length, and the DNA fragment is used to prepare the "heart ^/«心50 isoform zygomycetes" Mutant species. The above DNA fragment was transformed into S. cerevisiae 1 0 5 6 0 - 2 B strain (ra A/iJ.'/ZiiiG wraJ-5·? to produce ηί/Μ0._.·Ι£:ί/2 strain, And mark it as SL06 (M/ra «ciίS0.. ..Z£ 2 /π'·ϊ3 .··· ΛίsG wra3 -52 /e w2: ··do z_?C?J. Then make S LΟ6

菌株與 10560-5B 後森(MAT a trpl::hisG ura3-52 /ew_2 ::¾/·?(?)交配,而分別產生 SL07 菌株(MX ndt80:: LEU2 /η·5·/.·.·Λζ·5·〇 Μ,α3-·5_2 /ew2··.. WsG)、SL08 菌株 (ΜΑ T a ndt8 0:: LEU2 trp 1:: hisG ur a 3 -5 2 leu2:; hisG) ' S L O 9 菌株(Λ/Α ra ir/»·/.·.. /2/5G wraJ-52 /ewU/eG)以及 SLO 1 0 菌株(MAT α,/ii5i .· .·Λί·ϊ6, ur a 3 - 5 2, /e .·.. Λ !··? G)。再 令 SL07 菌株與 SL08 菌株交配以產生二倍體菌株 SLOl 1 {MAT&/MAT a ndt80:: LEU2/ ndt80: :LEU2 Μ^·α3-·5·2/ΐί,β3-·52 /ew2:.. DG//ew2···· ft z.sG)。又令 SL09 菌株 與 SLO10 菌株配對以製造出二倍體菌株 48 1354022 SLO 1 2(ΜΑΤ&/ΜΑ Τα ura3-52/ura3-52 leu2:: hisG/leu2:: Z/bG)。將含有基因且長度約為3.6kb之PCR片段 才垂入 2 μ- URA3 載體中(ρΚ·8426)((Γ/ι/·ί·5/ί·α«·ϊ0« e t al., Gene 110:119-122,1 992),以製造出 質體,並 標示為 LOB46。分別將 質體(LOB46)、載The strain was mated with 10560-5B Housen (MAT a trpl::hisG ura3-52 /ew_2 ::3⁄4/·?(?), and the SL07 strain (MX ndt80:: LEU2 /η·5·/.. ·Λζ·5·〇Μ, α3-·5_2 /ew2··.. WsG), SL08 strain (ΜΑ T a ndt8 0:: LEU2 trp 1:: hisG ur a 3 -5 2 leu2:; hisG) ' SLO 9 strains (Λ/Α ra ir/»·/.../2/5G wraJ-52 /ewU/eG) and SLO 10 strains (MAT α,/ii5i .· .·Λί·ϊ6, ur a 3 - 5 2, /e .·.. Λ !··? G). The SL07 strain is mated with the SL08 strain to produce the diploid strain SLOl 1 {MAT&/MAT a ndt80:: LEU2/ ndt80: :LEU2 Μ ^·α3-·5·2/ΐί,β3-·52 /ew2:.. DG//ew2···· ft z.sG). The SL09 strain was further paired with the SLO10 strain to produce a diploid strain 48 1354022 SLO 1 2 (ΜΑΤ&/ΜΑ Τα ura3-52/ura3-52 leu2:: hisG/leu2:: Z/bG). The PCR fragment containing the gene and having a length of about 3.6 kb was dropped into the 2 μ- URA3 vector (ρΚ·8426) ((Γ/ι/·ί·5/ί·α«·ϊ0« et al., Gene 110) : 119-122, 1 992), to produce the plastid, and labeled as LOB46. The plastid (LOB46), respectively

體(pRS426)及 2\i-URA3-CaNDT80 質體(LOB44)轉型至 SL011菌株令以製造出SL013、SL014及SL015菌株。並 將 2μ·ί/ϋΑ3 質體(pRS426)及 2\i-HIS3 質體(pRS315, ί Λ 〇/* j Α: ί α n d e f e r,G e n e t i c s 1 2 2 : 1 9 - 2 7,1 9 8 9 )—同轉型至 SL012菌株中,以獲得SL016菌株。 如第6圖所示,不論是過量表現啤酒酵母菌之WDrSO 基因(ndt80/ndt80 2μ NDT80)良 基因之突變種The plastids (pRS426) and 2\i-URA3-CaNDT80 plastids (LOB44) were transformed into SL011 strains to produce SL013, SL014 and SL015 strains. And 2μ·ί/ϋΑ3 plastid (pRS426) and 2\i-HIS3 plastid (pRS315, ί Λ 〇/* j Α: ί α ndefer, Genies 1 2 2 : 1 9 - 2 7,1 9 8 9) - The same transformation into the SL012 strain to obtain the SL016 strain. As shown in Figure 6, the mutant of the WDrSO gene (ndt80/ndt80 2μ NDT80) gene of the S. cerevisiae is overexpressed.

(/7 A 5 0/«心5 0 2μ vector),均不會改變其對藥物的敏感性》 且該結果與啤酒酵母菌二倍體細胞之減數分裂過程中 Ndt80p蛋白呈不活化狀態之事實相符合。反之,過量表現 CaNDT80基因會降低啤酒酵母菌對酮康唑的藥物敏感性 (比較第 6 圖之 NDT80/NDT80 與 ndt80/ndt80 2 μ CaNDT80~)。 在另一實驗中,對一含有LOB44質體且含基因 的菌株進行Etest測試。第7圖顯示’相較於僅含控制載 體之菌株,過量表現CaiV£»r<?i?基因會降低啤酒酵母菌對氟 康唑及酮康唑的敏感性。當細胞過量表現CaTVDrSO基因 時,氟康°坐之最低抑制濃度(minimum inhibitory concentration, MIC)會從 24 pg/ml 提高到 64 pg/ml(參考第 49 1354022 7B圖)。同樣地,當細胞過量表現基因時,闺康 唑之最低抑制濃度則由 6 pg/ml提高至等於或高於 32 pg/ml(參考第7d圖)。含有LOB45質體之菌株對抗真菌劑 的敏感性則類似於含有LOB44質體之菌株。上述實驗數據 暗示白色念珠菌之CaNdt80p蛋白能活化該些參與啤酒酵 母菌藥物敏感性的基因。 較佳實施例4(/7 A 5 0/«heart 5 0 2μ vector), neither change its sensitivity to drugs" and this result is inactivated with Ndt80p protein during meiosis of diploid cells of S. cerevisiae The facts are in line. Conversely, overexpression of the CaNDT80 gene reduced the drug sensitivity of S. cerevisiae to ketoconazole (compare NDT80/NDT80 and ndt80/ndt80 2 μ CaNDT80~ in Figure 6). In another experiment, a strain containing a LOB44 plastid containing a gene was tested for Etest. Figure 7 shows that excessive expression of the CaiV£»r<?i? gene reduces the sensitivity of S. cerevisiae to fluconazole and ketoconazole compared to strains containing only control vectors. When the cells overexpress the CaTVDrSO gene, the minimum inhibitory concentration (MIC) of fluconazole increases from 24 pg/ml to 64 pg/ml (see Figure 49 1354022 7B). Similarly, when the cells overexpressed the gene, the minimum inhibitory concentration of meconazole was increased from 6 pg/ml to 32 pg/ml or more (see Figure 7d). The strain containing the LOB45 plastid is more sensitive to antifungal agents than the strain containing the LOB44 plastid. The above experimental data suggest that the CaNdt80p protein of Candida albicans can activate these genes involved in the drug sensitivity of the beer yeast. Preferred embodiment 4

CaNDT80之失活舍提高藥物敗氮性 製備同型接合子突變種,並測試其對 抗真菌劑的藥物敏感性。係根據習知的基因干擾法(gene disruption method)來構築 同型接合子突Inactivation of CaNDT80 improves drug septic activity Prepare homozygous mutants and test their drug sensitivity to antifungal agents. Constructing a homozygous zygote according to the conventional gene disruption method

^ ft {Wilson e t al., Yeast 16: 65-70,2 0 0 0; Gerami-Nejad et al., Yeast 18:859-864, 2001; Wilson et al., J Bacteriol. 181:1868-1874,199 9),且於第8圖中顯示其基因架構。使 用含有URA3-dpI 200區段的pDDB57與pGFP-Zi?G4質體 作為聚合酶鍵鎖反應中的模版股(tempi ate),分別構築出 CaNDT80的異型接合子突變種與同型接合子突變種 (ΡΠ/sow ei i?/·,Yeast 16: 65-70,2000)。所得到的聚合酶鍵 鎖反應產物含有GF尸-/ΛG4基因月段,且該片段兩端分別 具有一段約長70bp的CfliV£>7^0相似區,並將此聚合酶鏈 鎖反應產物轉型至 BWP17 菌株中 ur a 3 A :: Ximm 4 3 4 his 1:: hisG/his J :: hisG ar g 4:: hi s G /^ ft {Wilson et al., Yeast 16: 65-70, 2 0 0 0; Gerami-Nejad et al., Yeast 18: 859-864, 2001; Wilson et al., J Bacteriol. 181:1868-1874, 199 9), and its genetic architecture is shown in Figure 8. The pDDB57 and pGFP-Zi?G4 plastids containing the URA3-dpI 200 segment were used as the templating strands in the polymerase-locking reaction to construct the heterozygous mutant and the homozygous mutant of CaNDT80, respectively. ΡΠ/sow ei i?/·, Yeast 16: 65-70, 2000). The obtained polymerase-binding reaction product contains a GF cadaver-/G4 gene segment, and each of the two ends of the fragment has a CfliV£>7^0 similar region of about 70 bp, and the polymerase chain reaction product is obtained. Transition to BWP17 strain ur a 3 A :: Ximm 4 3 4 his 1:: hisG/his J :: hisG ar g 4:: hi s G /

,以剔除從基因從轉擇起始碼ATG 50 1354022 下游第283bp至轉譯终止碼下游第3 59bp之間的區段。轉 型株為 ARG+ ,並標示為菌株 YL0131To exclude a segment from the gene from the 283 bp downstream of the transfer initiation code ATG 50 1354022 to the 3 59 bp downstream of the translation termination code. The transgenic strain is ARG+ and is labeled as strain YL0131

CaNDT80/Candt80: :GFP]RG4),矣為 CaiVD Γ5 0 基因之異CaNDT80/Candt80: :GFP]RG4), which is the difference between CaiVD Γ5 0 gene

型接合子突變種。再將含有C/h J -办7 2 0 0基因片段,且該 片段兩端分別具有一段約長70bp的相似區的聚 合酶鏈鎖反應產物轉型至 YL0131菌株中,以剔除 基因組中另一半的基因,其剔除從 (^#£>7^0基因之轉譯起始磉八丁〇下游第1〇15?至轉譯終止 碼上游第1 48bp之間的區段。並將所得之Cani/iSO/Ca«心50 同型接合子突變種標示為YL0132菌株。 YL0133菌株是一株 啟動子受到破壞的Type zygote mutant. The polymerase chain reaction product containing the C/h J-run 7200 gene fragment and having a similar region of about 70 bp in length at both ends of the fragment was transformed into the YL0131 strain to eliminate the other half of the genome. The gene, which excludes the segment from the translation of the (^#£>7^0 gene from the first 〇15 downstream of the octopus to the first 48 bp upstream of the translation termination code. The resulting Cani/iSO/ The Ca«xin 50 homozygous mutant is indicated as the YL0132 strain. The YL0133 strain is a promoter that is disrupted.

Candt80/Candt80同型接合子突變種。係將一段會被dccl 限制酶分解之 pTTtetR-ZZ/Si 片段(d ccl-digested pTltetR-HIS 1 , Nakayama e t al., Infect. Immun.Candt80/Candt80 homozygous mutant. Is a pTTtetR-ZZ/Si fragment that is decomposed by dccl restriction enzymes (d ccl-digested pTltetR-HIS 1 , Nakayama e t al., Infect. Immun.

68:6712-6719,2000)插入YL0132菌株的£#(97啟動子中以 製造出 YL0133 菌株。彳/ wrflJzi··..乂imw萃3岑 his 1:: hisG/his 1:: hisG arg4:: hisG/ arg4: :hisG68:6712-6719, 2000) Inserted the YL0132 strain of £# (97 promoter to produce the YL0133 strain. 彳/ wrflJzi··..乂imw extraction 3岑his 1:: hisG/his 1:: hisG arg4: : hisG/ arg4: :hisG

Candt80: :GFP-ARG4/Candt80:: URA3-dpi 2 00 EN01/enolp::tetR-HISl) 製備YL0137菌株以恢復同型接合子突變種YL0132之 其中一股CaWDrSO基因。以限制酶從LOB45質體 上切下一段含有野生種基因的DNA片段,並將 該 DNA 片段插入 pGEM-//7S7 載體中,以製造 pGEM-HISl-CaNDT80 質體,並將其標示為 LOB49( Whon 51 1354022 "α/·,J. Bacteriol. 181:1868-1874,1999)。隨後利用 限制酶將LOB49質體自其啟動子上的印el限制 酶位址切開後,再將成為線形的 LOB49質體轉型至 YL0132 菌株中,以構築出菌株 YL0137Candt80: :GFP-ARG4/Candt80:: URA3-dpi 2 00 EN01/enolp::tetR-HISl) The YL0137 strain was prepared to restore one of the CaWDrSO genes of the homozygous mutant YL0132. A DNA fragment containing the wild-type gene was excised from the LOB45 plastid by restriction enzymes, and the DNA fragment was inserted into the pGEM-//7S7 vector to produce pGEM-HISl-CaNDT80 plastid and labeled as LOB49 ( Whon 51 1354022 "α/·, J. Bacteriol. 181:1868-1874, 1999). Subsequently, the restriction enzyme was used to cut the LOB49 plastid from the im-restricted enzyme site on its promoter, and then the linear LOB49 plastid was transformed into the YL0132 strain to construct the strain YL0137.

URA3A:: λ imm434 his l: :hisG/his 1:: hisGURA3A:: λ imm434 his l: :hisG/his 1:: hisG

arg4:: his G/arg 4:: his GArg4:: his G/arg 4:: his G

Candt80: :GFP-ARG 4/Candt80:: URA -dp 12 00::Candt80: :GFP-ARG 4/Candt80:: URA -dp 12 00::

CaNDT80: :HIS1、。CaNDT80: :HIS1.

於30 °C下,使白色念珠菌SC5314(野生種)、YL0133 菌株(Ca«心同型接合子突變種)及YL0137菌株 (恢復其中之一 基因的YL0133菌株)分別在含有Candida albicans SC5314 (wild species), YL0133 strain (Ca«cardiogenic zygote mutant) and YL0137 strain (recovering one of the genes YL0133 strain) were contained at 30 °C

或不含100 pg/ml 0米康唾(miconazole,Sigma)的培養液中生 長1小時。隨後利用反轉錄聚合酶鏈鎖反應(RT-PCR)來偵 測上述各菌株細胞中 TEF3(內部對照組)、與 CaNDT80基因的mRNA產量。第9圖顯示各菌株的CD;?/ 與mRNA表現量,且圖中之數據均以野生菌種之 與 mRNA 數據加以標準化(normalized)» 第 2-4與第11- 13直行顯示YL0133菌株(同型接合子突變種) 之mRNA表現量,第5-7與14-16直行顯示出YL0137菌 株(拯救後突變種)之mRNA表現量,以及第8-10與17-19 直行係顯示野生種SC5314菌株之mRNA表現量。第2、5、 8、1 1、1 4與1 7直行係為基因之mRNA表現量的俄 測結果(内部對照組);第3、6、9、1 2、1 5與1 8直行為 基因之mRNA表現量的偵測結果;以及第4、7、1 〇、 52 1354022Or grow in culture medium containing 100 pg/ml 0 m. (miconazole, Sigma) for 1 hour. Subsequently, reverse transcription polymerase chain reaction (RT-PCR) was used to detect mRNA production of TEF3 (internal control) and CaNDT80 gene in each of the above strains. Figure 9 shows the CD;?/ and mRNA expression of each strain, and the data in the figure are normalized with the wild strain and mRNA data» The 2-4 and 11-13 straight lines show the YL0133 strain ( mRNA expression of homozygous mutants, 5-7 and 14-16 showed direct mRNA expression of YL0137 strain (post-rescue mutant), and lines 8-10 and 17-19 showed wild species SC5314 The amount of mRNA expression of the strain. The 2nd, 5th, 8th, 1st, 14th, and 1st straight lines are the Russian test results of the mRNA expression of the gene (internal control group); the 3rd, 6th, 9th, 1st, 1st, 5th, and 18th straight behaviors Detection of the mRNA expression of the gene; and 4, 7, 1 〇, 52 1354022

1 3、1 6與1 9直行則是之mRNA表現量的偵測結 果。如預期般,無法在同型接合子突變種中偵測到 CaNDT80 mRNA(^ 9圖之第4與1 3直行)。對照哞酒酵母 菌中的相似基因之表現情形,發現可在野生種菌株 的無性生殖過程中偵測到mRNA的表現(如第9 圖,第10與19直行)。而拯救後突變種之mRNA 的表現量低於野生種菌株,但高於其同型接合子突變種菌 株之mRNA的表現量(參考第9圖,第7與16直 行)。並且比較接受或未經咪康唑處理之各菌株的 mRNA 圖形。在同型接合突變種菌株中,編碼著排除泵浦的CD/?/ 基因之mRNA表現量下降(比較第9圖之第3與第9直行 及比較第1 2與第1 8直行)。該結果顯示表現CaWDrSO基 因可增進排除泵浦的表現。由於在同型接合子突變 種中仍可偵測到基因的mRNA(參考第9圖之第3與 12直行),因此(7蛋白可能僅是控制基因表 現之該些因子中的一員。此外,利用 LightCycler試劑中 的 LC FastStartDNA Master SYBR Green I 試劑組(Cat-No.2239264, Roche, Germany)進行熱啟動式聚合酶鏈鎖反 應(real-time hot-start PCR)亦可得到一致的結果。 分另|J取白色念珠菌 SC5314(野生種)、YL0133 (〇:以心50/<^«心50同型接合子突變種)及丫[〇137(恢復 其中之一(:《///)7^0基因的丫1^〇13 3)三種菌株進行£1651測 試,以偵測該些菌株對五種藥物的藥物敏感性。所使用之 五種抗真菌劑试片(AB BIODISK,Solna,Sweden)分別為 53 濃度 0.002.u1 3, 1 6 and 1 9 straight is the detection result of the mRNA expression. As expected, CaNDT80 mRNA could not be detected in homozygous mutants (Figs. 4 and 13 are straight). In contrast to the performance of similar genes in Alcoholic yeast, it was found that mRNA expression was detected during asexual reproduction of wild strains (see Figure 9, straight lines 10 and 19). The mRNA expression of the mutant species after rescue was lower than that of the wild strain, but higher than that of the homozygous mutant strain (see Fig. 9, lines 7 and 16). And compare the mRNA profiles of each strain that received or was not treated with miconazole. In the homozygous mutant strain, the amount of mRNA encoding the CD/?/ gene excluding the pump was decreased (compare the 3rd and 9th straight lines in Fig. 9 and compare the 1st and 1st straight lines). This result shows that the performance of the CaWDrSO gene enhances the performance of the excluded pump. Since the mRNA of the gene can still be detected in the homozygous mutant (refer to paragraphs 3 and 12 of Figure 9), (7 proteins may be only one of the factors controlling the expression of the gene. In addition, use The LC FastStartDNA Master SYBR Green I reagent set (Cat-No. 2239264, Roche, Germany) in the LightCycler reagent also yielded consistent results for hot-start hot-start PCR. |J Take Candida albicans SC5314 (wild species), YL0133 (〇: heart 50/<^«心50 homozygous mutated mutant) and 丫[〇137 (restore one of them (:///)7 01^〇13 of the ^0 gene 3) Three strains were tested for £1651 to detect the drug susceptibility of the strains to the five drugs. Five antifungal test strips used (AB BIODISK, Solna, Sweden) ) respectively, the concentration of 53 is 0.002.u

Pg/ml 之兩性黴素 b(AP)、〇·〇〇2_32 μ§/ιη1 之 5-氟胞喷咕 & (FC)、0.0 1 6-256 pg/ml 之氟康唑(FL)、 0.002-32 μ〇/ηΊΐ 之伊曲康唑(IT)與0.002-32 Mg/ml之酮康 喷(ΚΕ)β使毒娃 ^裡囷株在SD培養皿中生長一個晚上’並將 培養皿上的窗 各混合後移至濃度為0 · 8 5 %的氯化鈉溶液 中,形成細胎_ a吸度為5Μ06 cells/ml的懸浮液。取已滅囷 之棉花棒沾取_ & Λ 1乍為接種源的懸浮液,並以沾有液的棉才匕Pg/ml amphotericin b (AP), 〇·〇〇2_32 μ§/ιη1 5-fluorocytosine & (FC), 0.016-256 pg/ml fluconazole (FL), 0.002-32 μ〇/ηΊΐ of itraconazole (IT) and 0.002-32 Mg/ml of ketocon spray (ΚΕ)β to grow the poisonous worms in the SD culture dish for one night' and the culture dish The upper windows were mixed and transferred to a sodium chloride solution having a concentration of 0 · 8 5 % to form a suspension of fine _ a absorbance of 5 Μ 06 cells/ml. Take the smashed cotton swab and take _ & Λ 1乍 as the inoculation source and use cotton with liquid

棒在一 SDiit矣 « 。香皿之洋菜膠表面上均勻晝線。待培養i將 過多的水分人Stick in a SDiit矣 « . The surface of the acacia gel is evenly twisted on the surface. I will train too much water people

疋王吸收後,於每個培養班上放置五種不同的 Etest藥物試H 月。隨後使該些培養孤於3 5 °C之環境下生長 兩天後拍摄4立¥ 。養皿敏上各菌株的生長情形。抗真菌劑試片 周圍界線凊楚的抑菌區域說明各種菌株對該藥物的敏感 !±並根據抑菌區域的大小來判斷菌株對該藥物的敏感程 度。After the absorption of the king, five different Etest drugs were placed on each training class to test the month of H. Subsequently, the cultures were allowed to grow in an environment of 35 ° C for two days and then photographed 4 ¥. The dish was sensitive to the growth of each strain. Antifungal test strips The bacteriostatic areas around the boundaries indicate the sensitivity of various strains to the drug! ± and determine the sensitivity of the strain to the drug based on the size of the zone of inhibition.

同型接合子突變種對氟康唑 '酮康唑及5 -氟胞嘧啶之 敏感性增加(比較第1 〇 A與1 0 B圖)。此外,相較於具有兩 個CflWDrSO基因的野生種菌株而言,僅具有一個 基因的拯救後菌株YLqu?對氟康唑更為敏感(比較第10A 與10C圖)’顯示出基因之劑量效應(dosage effect) ° 洋菜膠稀釋試驗則顯示出不論是或基 因其中之一發生突變,均會使白色念珠菌對抗真菌劑氟康 °坐與伏立康°坐(voriconazole)更為敏感。同型接 合子突變種源自 Sang/ard等人在 Amtimicrob. Agents 54 1354022The homozygous mutant has increased sensitivity to fluconazole 'ketoconazole and 5-fluorocytosine (compare the first 〇A and 10B plots). In addition, compared to wild-type strains with two CflWDrSO genes, the rescued strain YLqu? with only one gene is more sensitive to fluconazole (compare Figures 10A and 10C) to show the dose effect of the gene ( The dosage of the acacia powder showed that either the mutation of one of the genes or the gene caused the Candida albicans to be more sensitive to the antifungal agent Fluconazole and voriconazole. Homozygous mutants are derived from Sang/ard et al. at Amtimicrob. Agents 54 1354022

Chemother. 40·· 2300-2305,1996文獻中所述之方法構築。 隨後配製含有25gg/ml氟康°圭及lpg/ml伏立康啥的二甲 基亞 (dimethylsulfoxide, DMSO)的SD培養基。使白色念 珠菌細胞生長在不含藥物但含有二甲基亞 之SD培養基 中,作為控制組。將白色念珠菌之細胞稀釋成〇D 6 00值為 2的菌液(即細胞濃度約為2 X 1 07 ),並利用一複製元件 (replica device, Oxoid Inc.,Canada)將 0.5 μΐ 之上述菌液 點在含有氟康唑(25 pg/ml)或伏立康峻(1 pg/ml)的培養皿 上。並將上述菌液進行一連串的10倍稀釋後(如從104細 胞稀釋至1 0個細胞),以相同方法點在培養皿上。Chemother. 40·· 2300-2305, 1996 Method construction as described in the literature. SD medium containing 25 gg/ml fluconazole and lpg/ml voriconazole dimethylsulfoxide (DMSO) was then prepared. Candida albicans cells were grown in SD medium containing no drug but containing dimethyl amide as a control group. The cells of Candida albicans were diluted to a bacterial solution with a D 6 00 value of 2 (ie, a cell concentration of approximately 2×1 07), and 0.5 μΐ of the above using a replica element (Oxoid Inc., Canada) The bacterial solution was spotted on a petri dish containing fluconazole (25 pg/ml) or voriconine (1 pg/ml). The above bacterial solution was subjected to a series of 10-fold dilutions (e.g., diluted from 104 cells to 10 cells), and spotted on a petri dish in the same manner.

如第11圖所示,不含藥物的狀況下,培養皿上每一點 的細胞均可正常成長(第1 1 Α圖)。突變種 較野生種菌株(wt)與拯救後菌株(僅有一野生種CaNDT80 基因)對I康唾與伏立康。坐兩者皆較為敏感。此外, 突變種菌株比Ca«心別/Can心利突變種菌株對抗 真菌劑更為敏感。如第1 1 B與1 1 C圖所示,當細胞濃度約 為103與102時,細胞可在含有兩種藥物之培養 i上的生長數目少於Cani/iSO/Cani/iSO細胞在含有兩種藥 物之培養孤上的生長數目。 較佳實施例5 同剞接合子突變種不具喜性 白色念珠菌與啤酒酵母菌可從酵母菌體變成菌絲狀體 (filamentous form),絲狀體通常以假菌絲狀體或真菌絲狀 55 1354022As shown in Figure 11, cells at every point on the culture dish can grow normally without drug (Figure 1 1). Mutant species were compared to wild-type strains (wt) and rescued strains (only one wild species CaNDT80 gene) against I Kang saliva and voricon. Sitting on both is more sensitive. In addition, mutant strains are more sensitive to fungal agents than Ca «Heart/Can heart mutant strains. As shown in Figures 1 1 B and 1 1 C, when the cell concentration is about 103 and 102, the number of cells grown on the culture i containing the two drugs is less than that of the Cani/iSO/Cani/iSO cells. The number of growths in the culture of the drug. Preferred Embodiment 5 The homologous zygote mutant does not have a preference. Candida albicans and Saccharomyces cerevisiae can be changed from a yeast cell to a filamentous form, and the filamentous body is usually a pseudo-mycelium or a fungal filament. 55 1354022

體的形式存在(i〇 fl/.,Cell 90:939-949s 1 997)。酵母菌 的型體轉變,係受到包括Stel2p與Phdlp等調控蛋白所調 ^ (Lo et al., Cell 90:939-949,1997)。Stel2p 與 Phdlp 的 念珠菌同源蛋白分別是Cphlp與Efglp,並曾有文獻顯示 Cph 1 p與Efglp這兩個白色念珠菌同源蛋白調控型體轉變 (Lo et al., Cell 9 0:.9 3 9-949, 1 997) » 在 Cphlp 與 Efglp 這 兩個蛋白質的基因座(丨oci)上發生同型接合子突變,會使念 珠菌鎖定在酵母菌體的型態,且在小鼠模型中呈無毒性表 現([〇 ei d., Cell 90:939-949, 1997)。同樣測驗The form of the body exists (i〇 fl/., Cell 90: 939-949s 1 997). The yeast transformation is regulated by regulatory proteins including Stel2p and Phdlp (Lo et al., Cell 90: 939-949, 1997). The Candida homologous proteins of Stel2p and Phdlp are Cphlp and Efglp, respectively, and there have been reports that Cph 1 p and Efglp are two Candida homologous proteins that regulate the transformation (Lo et al., Cell 9 0:.9) 3 9-949, 1 997) » A homozygous mutation at the locus of Cphlp and Efglp (丨oci) causes Candida to lock into the yeast form and is in a mouse model. It is non-toxic ([〇ei d., Cell 90: 939-949, 1997). Same test

Candt80/Candt80同型接合子突變種在發芽管(germ tube) 形成及小鼠模型中的毒性情況。 於37 °C下,以含有10 %馬血清的 YPD培養液培養 Candt80/Candt80同型接合子突變種念珠菌2小時後,置 於顧微鏡下觀察。如第1 2圖所示,發現在上述測試條件 下’ 突變種無法形成發芽管與真菌絲,The toxicity of Candt80/Candt80 homozygous mutants in germ tube formation and mouse models. The Candt80/Candt80 homozygous mutant Candida was cultured in YPD medium containing 10% horse serum at 37 °C for 2 hours, and then observed under a microscopic microscope. As shown in Fig. 12, it was found that under the above test conditions, the mutant could not form a germinated tube and a fungal filament.

而CflWDrSO/CfliVDrSO野生種卻可形成發芽管與真菌絲。 並且,可藉箸在細胞中補入野生種CaNDT80基因來拯救 突變種,而使其形成發芽管與真菌絲。 因而證明 基因參與念珠菌由酵母菌體轉變至真 菌絲狀體的型體轉變過程》 亦根據fl/·,Cell 90:939-949,1997文獻所述之方 法來測驗突變種在小鼠模型中的毒性。 係取 BALB/c 小鼠(Charles River Laboratories, Wilmington, M A),由其尾端靜脈注射〇 · 1 m 1的待測念珠菌種(1 x 1 〇 6個 56 1354022 月Θει修(更)政」吳頁The wild species of CflWDrSO/CfliVDrSO can form germinated tubes and fungal filaments. Furthermore, the wild type CaNDT80 gene can be added to the cells to rescue the mutant species to form germinating tubes and fungal filaments. Thus, the gene was involved in the transformation process of Candida from yeast to fungal filaments. The mutants were also tested in a mouse model according to the method described in fl/., Cell 90: 939-949, 1997. Toxicity. BALB/c mice (Charles River Laboratories, Wilmington, MA) were injected with 〇·1 m 1 of Candida species to be tested by their tail veins (1 x 1 〇 6 56 1354022 months Θ ει修 (more) Wu page

細胞)’並每日追縱小鼠之存活數目以測驗細胞毒性。 如第13圖所示’注射野生念珠菌之小鼠於注射1〇天 後的存活數目低於原先數量的40%,顯示野生種念珠菌 (Ca从具致死之毒性。相對的,注射 Candt80/Candt80突變種之小鼠於η天後的存活率為1〇〇 %。Cells) and the number of surviving mice was followed daily to test for cytotoxicity. As shown in Figure 13, the number of mice injected with wild Candida was less than 40% of the original number after one day of injection, indicating that the wild species Candida (ca from the lethal toxicity. In contrast, the injection of Candt80/ The survival rate of the mice of the Candt80 mutant species after η days was 1%.

此較佳實施例顯示與念珠菌之毒性有關,可 能作為治療念珠菌感染之用。 較佳實施例6This preferred embodiment has been shown to be associated with the toxicity of Candida and may be used as a treatment for Candida infection. Preferred embodiment 6

構築及皙艚並將其整公黾細胞之染色艚中 構築五種含有不同長度之/啟動子的質體,這些 質體中的ΜΙ>Λ7啟動子分別是由之起始位置往上游 算起約300、600、900、1200及2700個鹼基對長度之片段。 並利用數組不同的引子(primer)以聚合酶鏈鎖反應(PCR) 來放大這些不同長度之从£>及/啟動子片段的數量。所有反 應中均使用寡聚核苷酸HJL305這段反置引子(reverse primer),其序列為 5’ - AACCC AAGCTTGr. A TTGTGAAGTTCTATGT - 3,。反置引子 HJL305 具有一個 /ihrflll限制酶位置,且該段引子之序列與AfD/J/啟動子從 + 4至-15位址的序列相互補,其中係將轉譯起始位置ATG t的A定義為+1。每個啟動子片段之放大反應中所使用的 前置引子(forward primer)分別顯示如下: LHL3 (與啟動子-310至-293之序列互補): 57 3,1354022Constructing and cultivating five plastids containing different lengths/promoter in the staining 整 of the whole 黾 cells, the ΜΙ>Λ7 promoters in these plastids are counted from the starting position to the upstream Fragments of about 300, 600, 900, 1200, and 2700 base pairs in length. The number of different lengths from £> and / promoter fragments is amplified by polymerase chain reaction (PCR) using different primers in the array. The oligonucleotide HJL305, a reverse primer, was used in all reactions and its sequence was 5' - AACCC AAGCTTGr. A TTGTGAAGTTCTATGT - 3 . The inverted primer HJL305 has a /ihrflll restriction enzyme position, and the sequence of the primer is complementary to the sequence of the AfD/J/promoter from the +4 to -15 position, wherein the A definition of the translation start position ATG t is defined. Is +1. The forward primers used in the amplification reaction of each promoter fragment are shown below: LHL3 (complementary to the promoter-310 to -293 sequence): 57 3,1354022

55 - CGCGGATCCACCAATTAATCACAACGG -55 - CGCGGATCCACCAATTAATCACAACGG -

LHL2 (與MD及/啟動子-644至-627之序列互補): 5’ - GCGGGATCCTCATGTAACCTTGCAATC - 3’ LHL1 (與啟動子_966至-947之序列互補): 5’ CGCGGATCCCTTAGACTTACTTATATCCG -3, HJL3 1 (與啟動子-1242至-1224之序列互補): 55 - CGCGGATCCGGCTTGCTAAACATTATCA - 3’ HJL29 (與MD及i啟動子_2ό9ό至-2073之序列互補): 55 - CGCGGATCCAGAGAATCCAGAAAAGAG - 3, 將數量放大後啟動子片段以符合編碼原則的方 (in-frame)插入/acZ融合質體ΥΕρ3 63與ΥΙρ363中,上 之/flcZ融合質體含有野生種基因以作為篩選標記 {Myers et al., Gene 45:299-3 1 0, 1 986)。YEp363 質體可 大腸桿菌與酵母菌中自行複製。YIp3 63質體則適合用來 上述質體序列整合至酵母菌宿主的染色體中(Myeri ei a Gene 45:299-3 1 0,1 986)。 透過同源重組的方法,將含有長度為2 7 00個鹼基對 啟動子片段的質體整合至啤酒酵母 的五J位址中。首先,利用下列寡聚核苷酸及聚合酶 式 述 〇 在 將 /·, 之 菌 鏈LHL2 (complementary to the sequence of MD and / promoter -644 to -627): 5' - GCGGGATCCTCATGTAACCTTGCAATC - 3' LHL1 (complementary to the sequence of promoter _966 to -947): 5' CGCGGATCCCTTAGACTTACTTATATCCG -3, HJL3 1 (with The sequence of promoter -1242 to -1224 is complementary): 55 - CGCGGATCCGGCTTGCTAAACATTATCA - 3' HJL29 (complementary to the sequence of MD and i promoters 2ό9ό to -2073): 55 - CGCGGATCCAGAGAATCCAGAAAAGAG - 3, the number of amplified promoter fragments is The in-frame insertion/acZ fusion plastids ΥΕρ3 63 and ΥΙρ363 in accordance with the coding principle, the /flcZ fusion plastid contains the wild species gene as a screening marker {Myers et al., Gene 45:299-3 1 0, 1 986). YEp363 plastids can replicate themselves in E. coli and yeast. The YIp3 63 plastid is suitable for integration of the above plastid sequence into the chromosome of the yeast host (Myeri ei a Gene 45: 299-3 1 0, 1 986). A plastid containing a length of 2700 base pair promoter fragment was integrated into the five J site of S. cerevisiae by homologous recombination. First, the following oligonucleotides and polymerases are used to describe the bacterium chain.

58 1354022 鎖反應來放大部分之基因,使用之寡聚核苷酸如下·· HJL44 (與基因中+547至+565之序列互補): 5、TTTCCGGGCAGCATTTAGAΑΡΓ Α AT - 3, HLJ45 (與基因中+2143至+ 2170之序歹互補): 5’ - CCCCCCCG^_GTGATTTGTCTTAACATT - 3,58 1354022 Lock reaction to amplify part of the gene, the oligonucleotide used is as follows: HJL44 (complementary to the sequence of +547 to +565 in the gene): 5, TTTCCGGGCAGCATTTAGAΑΡΓ Α AT - 3, HLJ45 (+2143 with the gene) Complementary to + 2170): 5' - CCCCCCCG^_GTGATTTGTCTTAACATT - 3,

在含有長度為 27 00個鹼基對之 AfDi?/啟動子的 MDi?/p-/acZ質體中插入長度為i.6kb的基因之DNA 片段’以得到一整合質體(integrative plasmid)。再將該整 合質體送入啤酒酵母菌細胞内,並插入酵母菌宿主染色體 之位址中。 較佳實施例7 篩選CZ^/基因之及式調控因子 Φ 將根據較佳實施例2所製備出的白色念珠菌體基因庫 轉型至含有CD;?質體的啤酒酵母菌細胞内,並選 殖出該些能活化/flcZ表現的基因。根據上述步驟鑑定出兩 個候選基因,分別命名為及£:/^(第3圖)與(:αΛ^Γ5 0(第2 圖),其中/?£户/全名為排除泵浦調控因子l(regulat〇r 〇f efflux pump 1)。隨後根據較佳實施例2對此兩個候選基因 進行測試,相較於控制組之冷-半乳糖苷酶活性,户/透 過CZ)/?7啟動子將冷-半乳糖苦酶活性提高6倍,且過量表 59 1354022 現及£尸/亦能經由MDi? /啟勤子使/5 -半乳糖苷酶活性提高 6倍。同樣與控制組作比較,Ca/V’DrSO能透過CZ)J?7啟動 子使/3 -半乳糖苷酶活性提高6倍,卻無法透過MDi? /啟動 子來提高;5-半乳糖苷酶活性。並發現過量表現 CaNDT80 基因可降低啤酒酵母菌對氟康唑與酮康唑的藥物敏感性, 且該結果與觀察到/排除泵浦會運輸氟康唑及酮康唑 之結果一致。A DNA fragment of the gene of i.6 kb in length was inserted into the MDi?/p-/acZ plastid containing the AfDi?/promoter of 27 00 base pairs to obtain an integrated plastid. The plastid is then introduced into the Saccharomyces cerevisiae cell and inserted into the site of the yeast host chromosome. Preferred Example 7 Screening CZ^/gene and Regulatory Factor Φ The Candida albicans gene bank prepared according to the preferred embodiment 2 was transformed into a Saccharomyces cerevisiae cell containing CD; These genes that activate /flcZ expression are colonized. According to the above steps, two candidate genes were identified, named as: £:/^ (Fig. 3) and (:αΛ^Γ5 0 (Fig. 2), where /?£ household/full name excludes pump regulatory factors l (regulat〇r 〇f efflux pump 1). The two candidate genes were subsequently tested according to the preferred embodiment 2, compared to the cold-galactosidase activity of the control group, household/through CZ)/?7 The promoter increased the cold-galactosidase activity by a factor of 6 and the excess of the table 59 1354022 and the corpse/can also increase the/5-galactosidase activity by a factor of 6 via MDi®/starter. Similarly, compared with the control group, Ca/V'DrSO can increase the /3-galactosidase activity by 6-fold through the CZ)J?7 promoter, but it cannot be improved by MDi?/promoter; 5-galactoside Enzyme activity. It was also found that overexpression of CaNDT80 gene reduced the drug sensitivity of S. cerevisiae to fluconazole and ketoconazole, and the results were consistent with the observation/exclusion of the results of pumping fluconazole and ketoconazole.

可根據說明書中引用之參考文獻來瞭解本說明書内 容,並將引用之文獻完整納入參考。本說明書内容提供數 個根據本發明之較佳實施例以作為示範,然其並非用以限 定本發明。習知技藝者需明白本發明尚包括多種其他較佳 實施例,且說明書内容及各種範例均僅作示範之用。本發 明實際範圍與精神係由後附申請專利範圍所界定。 【圖式簡單說明】The contents of this specification can be understood from the references cited in the specification, and the cited documents are fully incorporated by reference. The description provides several preferred embodiments in accordance with the present invention as exemplary, and is not intended to limit the invention. It will be apparent to those skilled in the art that the present invention is intended to cover various other preferred embodiments. The actual scope and spirit of the invention are defined by the scope of the appended patent application. [Simple description of the map]

第 1A 與 1B 圖顯示 CaNdt80p(SEQ ID NO: 1)及 N d t 8 0 p (S E Q ID Ν Ο : 2)之氨基酸序列的比較結果。 第2A與2B圖顯示CaiVDrSO (SEQ ID NO: 3)之核苷 酸序列。其顯示在起始編碼(+ 1)上游之第5 72個鹼基對上 有一個完整的中程孢子生成序列(MSE)。 第3A與3B圊顯示/?£P/(SEQ ID NO: 4)之核苷酸序 列。 60 1354022 第4圖顯示將含有CD/?7啟動子融合基 因的LOB43質體整合至啤酒酵母菌之J£)£J基因座中。水 平箭頭指示從/啟動子開始的轉錄方向。 第5圖為一條狀圖,分別顯示各啤酒酵母菌株的p-半乳糖苷酶相對活性,各啤酒酵母菌株分別為(A)轉型 2\i-URA3載體之控制組(標示為 SL02菌株)、(B)轉型Figures 1A and 1B show the results of comparison of the amino acid sequences of CaNdt80p (SEQ ID NO: 1) and N d t 8 0 p (S E Q ID Ν Ο : 2). Figures 2A and 2B show the nucleotide sequence of CaiVDrSO (SEQ ID NO: 3). It shows a complete mid-range spore-forming sequence (MSE) on the 5th 72th base pair upstream of the start coding (+ 1). Figures 3A and 3B show the nucleotide sequence of /?P/(SEQ ID NO: 4). 60 1354022 Figure 4 shows the integration of the LOB43 plastid containing the CD/?7 promoter fusion gene into the J£) £J locus of S. cerevisiae. The horizontal arrow indicates the direction of transcription from the /promoter. Figure 5 is a bar graph showing the relative activity of p-galactosidase of each S. cerevisiae strain. Each S. cerevisiae strain is (A) the control group of the transformation 2\i-URA3 vector (labeled as SL02 strain), (B) Transformation

質體(LOB44)(標示為 SL03 菌株)、(〇 移除LOB44質體的 SL03啤酒酵母菌(標示為 SL04菌 株)' 以及(D)將LOB44質體再次轉型至已移除LOB44質 體的SL03啤酒酵母菌中(標示為SL05菌株)。 第 6 圖為 Etest 試驗結果(AB BIODISK,Solna, Sweden),以顯示各菌株對抗真菌劑氟康唑(FL)(下圖)及酮 康唑(KE)(下圖)的敏感性。接受測試的菌株分別是Plastid (LOB44) (labeled SL03 strain), (〇SL03 Saccharomyces cerevisiae (labeled SL04 strain) removed, and (D) re-transformed LOB44 plastid to SL03 with LOB44 plastid removed In S. cerevisiae (labeled SL05 strain). Figure 6 shows the Etest test results (AB BIODISK, Solna, Sweden) to show the antifungal agents of each strain, fluconazole (FL) (bottom) and ketoconazole (KE) ) (bottom) sensitivity. The strains tested were

野生種啤酒酵母菌(WDrSWDrSO)、轉型 2μ-ί//Μ3控制載體的12心50/«心^同型接合子突變種哞酒 酵母菌(π心抑2μ-載體)、轉型2μ-質體 的同型接合子突變種啤酒酵母菌〇心別/«心抑 2μ-Α^Γ<?ί?)、以及轉型 2\i-URA3-CaNDT80 質體的 同型接合子突變種啤酒酵母菌(以/⑽/«心⑽ 2p-CaNDT80)。Wild-type brewer's yeast (WDrSWDrSO), transforming 2μ-ί//Μ3 control vector of 12-heart 50/«heart^ homozygous zygote mutant 哞 wine yeast (π heart suppression 2μ-vector), transformation 2μ-plastid Homozygous mutated mutant Saccharomyces cerevisiae / «Heart suppressor 2μ-Α^Γ<?ί?), and transformed 2\i-URA3-CaNDT80 plastid homozygous mutant S. cerevisiae (/(10) / «Heart (10) 2p-CaNDT80).

第7圖顯示轉型有2μ控制載體或2μ LOB44質體之啤 酒酵母菌對抗真菌劑的敏感性,第7 Α與7 Β圖分別為兩 啤酒酵母菌對氟康唑(FL)之敏感性測試結果,第7C與7D 61 1354022 圖則為兩啤酒酵母菌對酮康唑(KE)之敏感性測試結果。 第8圖缯·示一 null mutation的基因架構。 其顯示在兩個 CaNDT80基因複製體(空白部分)先後被 GFP-ARG4基因片段(實心部分)及 URA3-dpl 200-based cassette(斜線部分)所取代。Figure 7 shows the sensitivity of the S. cerevisiae with 2μ control vector or 2μ LOB44 plastid to antifungal agents. The 7th and 7th Β diagrams are the susceptibility test results of two S. cerevisiae to fluconazole (FL). , 7C and 7D 61 1354022 The plot is the result of the sensitivity test of two brewer's yeast against ketoconazole (KE). Figure 8 shows the genetic structure of a null mutation. It was shown that the two CaNDT80 gene replicas (blank portions) were replaced by the GFP-ARG4 gene fragment (solid part) and the URA3-dpl 200-based cassette (slashed part).

第 9圖顯示各種啤酒酵母菌株之反轉錄聚合酶連鎖 反應產物的瓊酯凝膠電泳結果。圖中第11-19直行顯示經 過咪康唑(Sigma)處理之啤酒酵母菌的 RT-PCR產物,第 2-1 0直行則顯示不經咪康唑處理之啤酒酵母菌的RT-PCR 產物。第1直行為標準分子量標記(Invitrogen之lkbDNA ladder);第 2-4 直行與第 11-13 直行為 CtfnA⑽ (YL0133)同型接合子突變種菌株;第5-7直行與第14-16 直行為 Candt80/Candt80::CaNDT80 (YL0137)菌株;第 8-10 直行與第 17-19 直行為 CfliVDr50/CaA^r50(SC5314) 野生種菌株。第2'5、8、11、14與17直行為7^/^1111?^八 的測試結果(作為内部對照internal control);第3、6、9、 12、15與18直行為CD7U mRNA之測試結果;以及第4、 7、1〇、13、16與19直行為(^///)7^01111^八的測試結果》 第1 0圖顯示各菌種對於兩性黴素B(AP)、5 -氟胞嘧啶 (FC)、氟康唑(FL)、伊曲康唑(IT)與酮康唑(KE)五種抗真 菌劑之E t e s t (AB BIODISK,Solna, Sweden)藥物敏感性試驗的結 果。受測菌種為白色念珠菌之(A)野生種 CaNDT80/CaNDT80 (SC5314)、 (B)同型接合子突變種 (YL0133)、與(C)補入野生種 CaNDT80 62 1354022 基因的同型接合子突變種(YLO 137)。 第11圖為白色念珠菌的照片,顯示野生種白色念珠菌 SC5314(wt) 、 cdrl/cdrl 同型接合子 突變種Fig. 9 shows the results of agarose gel electrophoresis of the reverse transcription polymerase chain reaction product of various S. cerevisiae strains. The 11-19 straight lines in the figure show the RT-PCR products of S. cerevisiae treated with miconazole (Sigma), and the 2-1 0 straight line shows the RT-PCR product of S. cerevisiae without miconazole treatment. 1st direct behavior standard molecular weight marker (Invitrogen's lkbDNA ladder); 2-4 straight and 11-13 straight behavior CtfnA (10) (YL0133) homozygous mutant strain; 5-7 straight and 14-16 straight behavior Candt80 /Candt80::CaNDT80 (YL0137) strain; 8-10 straight and 17-19 straight behavior CfliVDr50/CaA^r50 (SC5314) wild strain. 2'5, 8, 11, 14 and 17 straight behavior test results of 7^/^1111?^8 (as internal control); 3, 6, 9, 12, 15 and 18 straight behavior CD7U mRNA Test results; and 4, 7, 1 , 13, 16 and 19 straight behaviors (^///) 7^01111^8 test results" Figure 10 shows each strain for amphotericin B (AP) , 5-Fluorocytosine (FC), fluconazole (FL), itraconazole (IT) and ketoconazole (KE) five antifungal agents E test (AB BIODISK, Solna, Sweden) drug sensitivity The result of the test. The tested strains were Candida albicans (A) wild species CaNDT80/CaNDT80 (SC5314), (B) homozygous mutant (YL0133), and (C) wild type CaNDT80 62 1354022 gene homozygous mutation Species (YLO 137). Figure 11 is a photograph of Candida albicans showing wild species Candida albicans SC5314(wt), cdrl/cdrl homozygous mutant

OSY^4Z(cdrl/cdrl) ' Candt80/Candt80 同型接合子突變種 YLOlZ3(Candt80/Candt80) ' 與 Candt80/Candi80 拯救後菌 種 YL01 37分別在(A)不添加 藥物、(B)添加25 pg/ml氟康唑、與(C)添加1 pg/ml伏立 康唑之洋菜膠培養皿上的細胞生長情形。 第12圖顯示白色念珠菌之菌種 (野生種)、Can心菌種(同型接合子突變種)、與 拯救後菌種之發芽管形成試 驗的照片。 第13圖為一圖表’顯示分別注射CaNDT80ICaNDT80 野生種與同型接合子突變種之小鼠存活 率’已說明小鼠模型中體内的白色念珠菌毒性。OSY^4Z(cdrl/cdrl) 'Candt80/Candt80 homozygous mutant YLOlZ3(Candt80/Candt80)' and Candt80/Candi80 post-rescue strain YL01 37 were added (A) without drug, (B) 25 pg/ Cell growth of ml fluconazole, and (C) addition of 1 pg/ml voriconazole to the culture dish. Fig. 12 is a photograph showing the formation test of the Candida albicans strain (wild species), the Can heart fungus species (the homozygous mutant species), and the germination tube formation after the rescue. Figure 13 is a graph showing the survival of mice inoculated with CaNDT80ICaNDT80 wild species and homozygous mutants respectively. The toxicity of Candida albicans in vivo in a mouse model has been demonstrated.

【元件代表符號簡單說明】 63[Simplified description of component symbol] 63

Claims (1)

1354022 _____ 公告本 十、申請專利範圍: 1. 一種於活體外抑制一細胞生長的方法,其至少包 括抑制該細胞之 排除泵浦表現因子,其中該 排除泵浦表現因子能與一目標聚核苷酸之中程 孢子生成序列(M S E)結合。1354022 _____ Announcement 10, the scope of the patent application: 1. A method for inhibiting the growth of a cell in vitro, comprising at least an inhibitory pump expression factor for inhibiting the cell, wherein the exclusion of the pump expression factor can be combined with a target polynucleoside Acid mid-spore generation sequence (MSE) binding. 2. 如申請專利範圍第1項所述之方法,其中該 CaNDT80能調控一排除泵浦,該排除泵浦係選自於由 CDJU、CDR2、MDJiJ所構成之群組中。 3. 如申請專利範圍第1項所述之方法,其中該細胞 係選自於由細菌細胞、眞菌細胞與酵母菌細胞所構成之群 組中。 4. 如申請專利範圍第3項所述之方法,其中該眞菌 細胞係選自於由念珠菌屬(Can山species)、曲真菌屬 /·//«《 species)、隱球菌屬(〇少piococcwi1 species)及 其混合物所構成之群組中。 5. 如申請專利範圍第4項所述之方法,其中該念珠 菌屬係選自於由白色念珠菌(c· α/6ζ·caws)、克柔念珠菌 /fcrwie/)、熱帶念珠菌(C. 、光滑念珠菌(C· g/Wraia)及其混合物所構成之群組中。 64 (S ) 1354022 6. 一種於活想外抑制一細胞生長的方法’該方法至 少包括抑制該細跑之灭五户7的表現或活性。 7. 如申請專利範圍第ό項所述之方法’其中五/> 7 可調節一排除泵浦之表現’該排除泵浦係選自於由 CDR1 ' CDR2 ' 7?/所構成之群組中。2. The method of claim 1, wherein the CaNDT 80 is capable of regulating an exclusion pump selected from the group consisting of CDJU, CDR2, MDJiJ. 3. The method of claim 1, wherein the cell line is selected from the group consisting of bacterial cells, sputum cells, and yeast cells. 4. The method of claim 3, wherein the sputum cell line is selected from the group consisting of Candida species, Aspergillus sp. /·//«" species, Cryptococcus (〇) Less piococcwi1 species) and its mixture. 5. The method of claim 4, wherein the Candida is selected from the group consisting of Candida albicans (c·α/6ζ·caws), Candida krusei/fcrwie/), Candida tropicalis ( C., Candida glabrata (C·g/Wraia) and a mixture thereof. 64 (S ) 1354022 6. A method for inhibiting the growth of a cell outside of the living's method, the method comprising at least inhibiting the run The performance or activity of the five households. 7. The method described in the third paragraph of the patent application 'the five/> 7 can adjust the performance of a excluded pump'. The excluded pump is selected from CDR1' CDR2 '7?/ is formed in the group. 8. 如申請專利範圍第6項所述之方法,其中該細胞 係選自於由細菌細胞、眞菌細胞與酵母菌細胞所構成之群 組中。 9. 如申請專利範圍第8項所述之方法,其中該眞菌 細胞係選自於由念珠菌屬species)、曲真菌屬 species)、隱球菌屬(〇兄pMcoccw·? species)及 其混合物所構成之群組中。8. The method of claim 6, wherein the cell line is selected from the group consisting of bacterial cells, sputum cells, and yeast cells. 9. The method of claim 8, wherein the sputum cell line is selected from the group consisting of Candida species, the fungus species, the genus Cryptococcus (Pmcoccw·? species), and mixtures thereof Among the groups formed. 10. 如申請專利範圍第9項所述之方法,其中該念珠 菌屬係選自於由白色念珠菌(C. 克柔念珠菌(C· hwid)、熱帶念珠菌(c_ iropicfl/b)、光滑念珠菌(C. 容及其混合物所構成之群組中。 11. _種抑制細胞生長的組合物,該組合物包括至少 一排除泵浦表現因子抑制劑,其中該排除泵浦表現因子抑 制劑係(ΓίΐΛ^ΓδΟ的反義DMA、反義RNA或干擾RNA[mi]’ 65 1354022 且該排除泵浦表現因子抑制劑會抑制能與一目標聚核苷 酸之中程孢子生成序列(MSE)結合的。 12. 如申請專利範圍第11項所述之組合物,其中該 排除泵浦表現因子可調控一排除泵浦的表現, 該排除泵浦係選自於由所構成之群 組中。10. The method of claim 9, wherein the Candida is selected from the group consisting of Candida albicans (C. hwid, C. iropicfl/b), Candida glabrata (C. commissariat and its mixture) 11. A composition for inhibiting cell growth, the composition comprising at least one inhibitory pump expression factor inhibitor, wherein the exclusion of pump expression factor inhibition The antisense DMA, antisense RNA or interfering RNA [mi]' 65 1354022 of the 剂ίΐΛ^ΓδΟ and the exclusion of the pump expression factor inhibitor inhibits the mid-range sporulation sequence (MSE) of a target polynucleotide 12. The composition of claim 11, wherein the exclusion of the pump performance factor modulates a performance of the excluded pump, the excluded pump being selected from the group consisting of . 13. 如申請專利範圍第11項所述之組合物,更包含 至少一藥物。 14.如申請專利範圍第11項所述之組合物,其中該 細胞係選自於由細菌細胞、眞菌細胞與酵母菌細胞所構成 之群組中。13. The composition of claim 11, further comprising at least one drug. 14. The composition of claim 11, wherein the cell line is selected from the group consisting of bacterial cells, sputum cells, and yeast cells. 15.如_請專利範圍第14項所述之組合物,其中該 眞菌細胞係選自於由念珠菌屬species)、曲真菌 屬species)、隱球菌屬(Oypiococcw·? species) 及其混合物所構成之群組中β 16. 如辛請專利範圍第15項所述之組合物,其中該 念珠菌屬係選自於由白色念珠菌(C. 克柔念珠 菌(C1. A:rwiez_)、熱帶念珠菌(C. "o/n’cfl/ίί)、光滑念珠菌(C. 及其混合物所構成之群組中。 66 1354022 17. —種抑制細胞生長的组合物,該組合物包括至少 一排除泵浦表現因子抑制劑,其中該排除泵浦表現因子抑 制劑係及五户/的反義DNA、反義RNA或干擾RNA,其能 抑制灭五尸7的表現或活性。15. The composition of claim 14, wherein the sputum cell line is selected from the group consisting of Candida species, the genus Aspergillus species, Oypiococcw® species, and mixtures thereof. The composition of the group of claim 16, wherein the Candida species is selected from the group consisting of Candida albicans (C. a. rwiez_) , Candida tropicalis (C. "o/n'cfl/ίί), Candida glabrata (C. and a mixture thereof). 66 1354022 17. A composition for inhibiting cell growth, the composition Included is at least one exclusion of a pump performance factor inhibitor, wherein the exclusion of a pump expression factor inhibitor line and a five-family/antisense DNA, antisense RNA or interfering RNA that inhibits the performance or activity of the genus. 18. 如申請專利範圍第 17項所述之組合物,其中 及£:户7能調控一排除泵浦之表現,該排除泵浦係選自於由 CDRJ、CDR2、MDR1所構成之群組中。 19. 一種於活體外提高一細胞中之藥物活性的方 法,該方法至少包括: a. 抑制一排除泵浦表現因子的表現或活性,該 排除泵浦表現因子能與一目標聚核苷酸之中 程孢子生成序列結合,其中該排除泵浦表現 西子為CaNDT80 ·,18. The composition of claim 17, wherein: and the household 7 is capable of regulating the performance of an excluded pump, the excluded pump is selected from the group consisting of CDRJ, CDR2, and MDR1. . 19. A method of increasing the activity of a drug in a cell in vitro, the method comprising at least: a. inhibiting the performance or activity of a pump-expressing factor that is capable of interacting with a target polynucleotide The mid-range spore-forming sequence is combined, wherein the exclusion pump exhibits a Western-like CaNDT80, b. 抑制一細胞中至少一排除泵浦聚核苷酸的表 現; c. 使該細胞與至少一藥物接觸;以及 d . 提高該藥物活性。 20. 如申請專利範圍第19項所述之方法,其中該排 除泵浦聚核苷酸係選自於由/所構成 之群組中。 67 C S ) 1354022b. inhibiting the expression of at least one of the excluded polynucleotides in a cell; c. contacting the cell with at least one drug; and d. increasing the activity of the drug. 20. The method of claim 19, wherein the draining of the pumping polynucleotide is selected from the group consisting of /. 67 C S ) 1354022 21.如申請專利範圍第19項所述之方法 胞係選自於由細菌細胞、眞菌細胞與酵母菌細 群組中。 22·如申請專利範圍第21項所述之方法 菌細胞係選自於由念珠菌屬species) species)、隱球菌屬 其混合物所構成之群組中。 23·如申請專利範圍第22項所述之方法 珠菌屬係選自於由白色念珠菌(C. α/δ/caw)、i (C· ^rMiei·)、熱帶念殊菌(C. iropica/ίί)、光滑 及其混合物所構成之群組中。 24·如申請專利範圍第19項所述之方法 物為A真菌劑,以及其中該抗真菌劑是一嗤 種於活趙外提高_細胞中之藥物 法,該方法至少包括: a·抑制及£户7的表現或活性; b • 抑制該細胞中至少一排除泵浦聚木 現; C · 使該細胞接觸至少一藥物;以及 其中該細 i所構成之 其中該眞 曲真菌屬 species)及 其中該念 ,柔念珠菌 念珠菌(C. ,其中該藥 類藥物。 活性的方 5苷酸的表 68 < S ) 1354022 d. 提高該藥物之活性。 26. 如申請專利範圍第25項所述之方法,其中該排 除泵浦聚核苷酸係選自於由所構成 之群組中。21. The method of claim 19, wherein the cell line is selected from the group consisting of bacterial cells, sputum cells, and yeast. 22. The method of claim 21, wherein the bacterial cell line is selected from the group consisting of a species of the genus Cryptococcus and a mixture of Cryptococci. 23. The method of claim 22, wherein the genus of the genus is from Candida albicans (C. α/δ/caw), i (C·^rMiei·), and tropical bacterium (C. Iropica/ίί), smooth and its mixture. 24. The method of claim 19, wherein the method of the invention is a fungicide, and wherein the antifungal agent is a pharmaceutical method in a booster cell, the method comprises at least: a The performance or activity of the household 7; b • inhibition of at least one of the cells to exclude the pumping of the polychlorinated; C · contacting the cell with at least one drug; and wherein the fine i consists of the tortuous fungus species) and Among the thoughts, Candida albicans candida (C., of which the drug is active. Table 68 of the active 5-glycoside) <S) 1354022 d. Increase the activity of the drug. 26. The method of claim 25, wherein the draining of the pumping polynucleotide is selected from the group consisting of. 27. 一種篩選細胞中可編碼有一排除泵浦表現因子 之聚核苷酸的方法,該方法至少包括: a. 將一受排除泵浦啟動子所控制之報導基因導 入至一宿主細胞中,其中該排除泵浦啟動子 係選自CDR1、CDR2或MDRI的啟動子·, b. 將一候選排除泵浦表現因子聚核苷酸導入至 步驟(a)之該宿主細胞中;以及 c. 偵測該報導基因的表現。27. A method of screening a cell encoding a polynucleotide having a pump expression factor, the method comprising at least: a. introducing a reporter gene under the control of a pump-inducing promoter into a host cell, wherein The exclusion pump promoter is selected from a promoter of CDR1, CDR2 or MDRI, b. a candidate exclusion pump expression factor polynucleotide is introduced into the host cell of step (a); and c. detection The performance of the reported gene. 28. 如_請專利範圍第27項所述之方法,其中該宿 主細胞係選自於由細菌細胞、眞菌細胞、酵母菌細胞與哺 乳動物細胞所構成之群組中。 29. 如申請專利範圍第28項所述之方法,其中該酵 母菌宿主細胞為一酵母菌種。 30. 一種使用 CaNDT80之良I DNA、反義RNA或 干擾RNA來製備用以治療罹患疾病或受感染患者之藥劑 69 1354022 的用途,其中該反義DNA、反義RNA或干擾RNA抑制能 與一目標聚核苷酸之中程孢子生成序列(MS E)結合的一排 除泵浦表現因子》 31. 如申請專利範圍第30項所述之用途,其中該感 染為細菌感染、真菌感染或酵母菌感染。28. The method of claim 27, wherein the host cell line is selected from the group consisting of a bacterial cell, a sputum cell, a yeast cell, and a mammal cell. 29. The method of claim 28, wherein the yeast host cell is a yeast species. 30. Use of CaNDT80-derived I DNA, antisense RNA or interfering RNA to prepare a medicament for the treatment of a disease or infected patient 69 1354022, wherein the antisense DNA, antisense RNA or interfering RNA inhibits energy The use of a medium-range spore-producing sequence (MS E) of a target polynucleotide to eliminate the use of a pump expression factor. 31. The use of claim 30, wherein the infection is a bacterial infection, a fungal infection or a yeast infection. 32. 如申請專利範圍第31項所述之用途,其中造成 該真菌感染之細胞係選自於由念珠菌屬(Ca «山‘心 species)、曲真菌屬(hpergi/Zws species)、隱球菌屬 (CV少piococcws species)及其混合物所構成之群組中。 33. 如申請專利範圍第32項所述之用途,其中該念 珠菌屬係選自於由白色念珠菌(C. α/Zu'ca^O、克柔念珠菌 (C. ArrwseO、熱帶念珠菌(c· wopfca/fj·)、光滑念珠菌(c. 及其混合物所構成之群組中。32. The use according to claim 31, wherein the cell line causing the fungal infection is selected from the group consisting of Candida (Ca «Mountain' heart species), H. genus (Hergi/Zws species), Cryptococcus Genus (CV less piococcws species) and its mixture. 33. The use of claim 32, wherein the Candida is selected from the group consisting of Candida albicans (C. α/Zu'ca^O, Candida krusei (C. ArrwseO, Candida tropicalis) (c· wopfca/fj·), Candida glabrata (c. and its mixture). 34. 如申請專利範圍第30項所述之用途,其中該排 除果浦表現因子為CaA^7^0[m2],其可調控一排除泵浦之 表現’該排除粟浦係選自於由CD/?/、所構 成之群組中。 35. 一種使用及丑P1之反義DNA、反義RNA或干擾 RNA來製備用以治療罹患疾病或受感染患者之藥劑的用 70 1354022 途,其中該能調控一排除泵浦的表現,且該藥物能 抑制i?五尸/的表現或活性。 36. 如申請專利範圍第35項所述之用途,其中該排 除泵浦的表現受到抑制,該排除泵浦係選自於由、 及 所構成之群組中。34. The use according to claim 30, wherein the exclusion factor is CaA^7^0[m2], which can regulate the performance of a pump exclusion. CD/?/, in the group formed. 35. An antisense DNA, antisense RNA or interfering RNA using ugly P1 for the preparation of a medicament for treating a diseased or infected patient, wherein the regulation can exclude the performance of the pump, and the The drug can inhibit the performance or activity of i? 36. The use of claim 16, wherein the exclusion pump is selected from the group consisting of and . 37. 一種藥學組合物,其包含至少一排除泵浦表現因 子抑制劑,其中該排除泵浦表現因子抑制劑為 CflWDr別 的反義DNA、反義RNA或干擾RNA,能抑制能與一目標 聚核苷酸之中程孢子生成序列結合的一排除泵浦表現因 子0 38.如申請專利範圍第3 7項所述之藥學組合物,其 中該排除泵浦表現因子為其可調控一排除泵浦 之表現,該排除泵浦係選自於及所構37. A pharmaceutical composition comprising at least one inhibitory pump expression factor inhibitor, wherein the exclusion pump expression factor inhibitor is CflWDr other antisense DNA, antisense RNA or interfering RNA, capable of inhibiting binding to a target A pharmaceutical composition according to the third aspect of the invention, wherein the exclusion of the pump performance factor is a regulatable pump Performance, the exclusion of the pump is selected from and constructed 39. 如申請專利範圍第3 7項所述之藥學組合物,更 包含至少一藥物。 4 0. 如申請專利範圍第3 7、3 8或3 9項所述之藥學組 合物,更包含一藥學上可接受之載劑。 71 S 1354022 4 41. 一種藥學組合物,其包含至少一排除果浦表現因 子抑制劑,其中該排除泵浦表現因子抑制劑為的反 義DNA、反義RNA或干擾RNA ’其能抑制之表現 或活性。 42.如申請專利範圍第41項所述之藥學組合物,其 更包含一藥學上可接受之載劑。39. The pharmaceutical composition of claim 37, further comprising at least one drug. The pharmaceutical composition of claim 3, 3, 8 or 39, further comprising a pharmaceutically acceptable carrier. 71 S 1354022 4 41. A pharmaceutical composition comprising at least one inhibitor of a fruit-producing factor inhibitor, wherein the anti-sense DNA, antisense RNA or interfering RNA that inhibits a pump expression factor inhibitor is capable of inhibiting expression Or active. 42. The pharmaceutical composition of claim 41, further comprising a pharmaceutically acceptable carrier. 43. 一種於活體外抑制一細胞生長的方法,該方法至 少包括: a. 抑制與一中程孢子生成序列結合; 以及 b. 抑制一細胞的生長。43. A method of inhibiting growth of a cell in vitro, the method comprising, at least: a. inhibiting binding to a mid-range spore-forming sequence; and b. inhibiting growth of a cell. 44. 如申請專利範圍第43項所述之方法,其中該細 胞係選自於由細菌細胞、真菌細胞與酵母菌細胞所構成之 群組中。 45. 如申請專利範圍第44項所述之方法,其中該真 菌細胞係選自於由念珠菌屬species)、曲真菌屬 species)、隱球菌屬(Cspecies)及 其混合物所構成之群組中。 46. 如申請專利範圍第45項所述之方法,其中該念 72 1354022 珠菌屬係選自於由白色念珠菌(C. 、克柔念珠議 (C. Arwsei)、熱帶念珠菌(C. 、光滑念珠菌(C· 及其混合物所構成之群組中》 47.如申請專利範圍第43項所述之方法,其中該中 程孢子生成序列(MSE)係來自一聚核苷酸,該聚椋苷酸係 選自於由CaNDT80、CDRJ' CDR2反MDRJ所構成之群组· 中044. The method of claim 43, wherein the cell line is selected from the group consisting of bacterial cells, fungal cells, and yeast cells. 45. The method of claim 44, wherein the fungal cell line is selected from the group consisting of a Candida species, a Fungus species, Cspecies, and mixtures thereof. . 46. The method of claim 45, wherein the genus 72 1354022 is derived from Candida albicans (C., C. Arwsei, Candida tropicalis (C. 47. The method of claim 41, wherein the mid-range spore-producing sequence (MSE) is derived from a polynucleotide, Polydecanoic acid is selected from the group consisting of CaNDT80 and CDRJ' CDR2 anti-MDRJ. 48. 如申請專利範圍第43項所述之方法,更包含使 該細胞接觸至少一藥物。 49. 一種於活體外抑制一細胞毒性的方法,該方法至 少包括: a. 使該細胞接觸至少一排除泵浦表現因子抑制 劑;以及48. The method of claim 43, further comprising contacting the cell with at least one drug. 49. A method of inhibiting a cytotoxicity in vitro, the method comprising at least: a. contacting the cell with at least one inhibitor of a pump expression factor; b. 抑制一細胞之毒性; 其中該排除泵浦表現因子抑制劑為 CaNDT80、 的反義DNA、反義RNA或干擾RNA,其抑制能 與一目標聚核苷酸之中程孢子生成序列結合的一排除泵 浦表現因子》 50·如申請專利範圍第49項所述之方法,其中該排 除泵浦表現因子CflWD 7^0,其可調控一排除泵浦的表現, 73 1354022 * 該排除泵浦係選自於由及所構成之群 組中。 5 1 . 如申請專利範圍第49項所述之方法,更包含使 該微生物細胞接觸至少一藥物。b. inhibiting the toxicity of a cell; wherein the exclusion of the pump expression factor inhibitor is CaNDT80, antisense DNA, antisense RNA or interfering RNA, the inhibition of which can bind to a target polynucleotide mid-range spore-forming sequence Excluding the pump performance factor 50. The method of claim 49, wherein the pump performance factor CflWD 7^0 is excluded, which can regulate the performance of a pump exclusion, 73 1354022 * The pump is excluded It is selected from the group consisting of and. 5 1. The method of claim 49, further comprising contacting the microbial cell with at least one drug. 52. 如申請專利範圍第49項所述之方法,其中該微 生物細胞係選自於由細菌細胞、真菌細胞及酵母菌細胞所 構成之群組中。 53. 如申請專利範圍第52項所述之方法,其中該真 菌細胞係選自於由念珠菌屬(Cawi/ii/a species)、曲真菌屬 (/spergi/Zw·? species)、隱球菌屬(Ο少piococcw·? species)及 其混合物所構成之群組中。52. The method of claim 49, wherein the microbial cell line is selected from the group consisting of bacterial cells, fungal cells, and yeast cells. 53. The method of claim 52, wherein the fungal cell line is selected from the group consisting of Cawi/ii/a species, /spergi/Zw·? species, Cryptococcus It is a group of genus (small piococcw?? species) and mixtures thereof. 54. 如申請專利範圍第53項所述之方法,其中該念 珠菌屬係選自於由白色念珠菌(C. α/6 i caw)、克柔念珠菌 (C.々rwsez·)、熱帶念珠菌(C. 、光滑念珠菌(C_ 及其混合物所構成之群組中。 74 (S )54. The method of claim 53, wherein the Candida is selected from the group consisting of Candida albicans (C. a/6 i caw), Candida krusei (C. 々rwsez), and the tropics Candida (C., Candida glabrata (C_ and its mixture). 74 (S)
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