TW201035312A

TW201035312A - Gene amplification method for enhancing gene expression

Info

Publication number: TW201035312A
Application number: TW98109106A
Authority: TW
Inventors: jing-tai Lin
Original assignee: Genetai Inc
Priority date: 2009-03-20
Filing date: 2009-03-20
Publication date: 2010-10-01
Also published as: TWI464263B

Abstract

The invention relates to an expression carrier that can enhance gene expression in mammal cells and comprising a gene to be expressed, a nuclear anchoring element, and at least an inverted repeat IP element. The invention further relates to a method for enhancing the gene expression by transfecting the expression carrier on mammal cells such as human.

Description

201035312 六、發明說明：【發明所屬之技術領域】本發明係一種能在哺乳動物細胞中進行增強基因表現之表現載體，其包括有—欲表現之基因、—㈣定（_iear anchoring)元件、及至少一反向重覆（inverted j幻元 •件>。本發明另關於一種藉由將前述表現載體轉染（transfect) 於諸如人類等哺乳類細胞以加強基因表現的方法。〇 ❹ 【先前技術】夕數的基因表現方法係於原核表現系統中表現人類重組蛋白貝。然而，大多數的人類重組蛋白質需要進行轉譯後或轉譯時修飾作用（p〇st_ and/〇r peri translati〇nai modifications) ’ 諸如醣化作用（glyc〇silati〇n)、g 羧化作用 (g-carboxyUtion)或 g_羥化作用（g_hydr〇xylati〇n)。因此，一個關於原核表現系統的已知問題係原核表現系統無法實行哺乳類表現系統所提供之轉譯後或轉譯時的修飾作用。於是在原核表現系統中，將無法正常地表現需要進行轉譯後或轉譯時修飾作用才能發揮正常功能之蛋白質。另一被廣泛用以表現前述人類重組蛋白之既有方法係採用一哺乳類表現系統。哺乳類表現系統使用哺乳類細胞以利用其實行轉譯後或轉譯時修飾作用之能力。尤其是中國倉鼠印巢（Chinese hamster ovary，CH0)細胞已經常規化地被用以表現生物醫藥蛋白以供治療與診斷。一於哺乳類細胞中加強基因表現之既有方法包括有— 以提高選擇劑（selection agent)濃度來進行逐步選擇之步 3 201035312 驟。此於喷乳類細胞中加強基因表現之既有方法之一•於例係採用中國倉鼠卵巢細胞，並以甲氨嗓呤(一 e二 MTX ’或谓「滅殺除癌鍵」）逐步地選擇二葉酸還原酶 (Dihydr0f0late reductase，卿R)的基因擴增。可參閱〇刪a， 2002，J. Biosci. Bioeng· 94:600-605。 ❹ 然而，前述逐步選擇之方式既繁項又耗時。其過程需經數月才能達到讓細胞内基因大量表現之目的，更何況在非人類之哺乳類細胞中的轉譯後修飾肖人類細胞所實行者不盡相同。且此利用基因擴增（gene ampUficati(>n)之既有加強基因表現方法，並未被廣泛地用於人類細胞中以生產生物醫藥蛋白以供治療與診斷。一般而言，在人類細胞中產出之人類蛋白質應較以其他非人類系統所生者更具醫療優勢。研九者已觀察到一種錯配修復（mismatch repair，系統’其於人類細胞中強效地壓抑基因擴增。可參照Lin et al.，2001，Nucleic Acids Res 29，3304-3310; Chen et al., 2001，Pr〇c Natl Acad Sci USA 98, 13802- 13807。因此藉由基因擴增而加強的基因表現在錯配修復系統正常（MMr_ normal/MMR-proficient，或記為MMR+)細胞中亦受壓抑。 HCT116細胞係導因於hMLHl基因突變之錯配修復系統缺陷（MMR-deficient，或記為MMR-)細胞。已知HCT116細胞能夠藉由基因擴增加強基因表現。相對之下， HCT116 + Ch3(藉由引入含有野生型hMLHl基因之第3對染色體於其中而具備正常錯配修復系統）則壓抑藉由基因擴增而加強的抗藥基因表現。 4 201035312 【發明内容】為克服前述在哺乳類細胞中（尤其是人類細胞中）既有基因表現方法與既有加強基因表現方法的缺點本發明之 • 目的在於提供一種能夠去除或改善前述問題之方法。 • _到前述目& ’本發明所採用之一種技術手段係提供一種表現載體（expression vect〇r),其包括有至少一基 ❹ 因、一核錨定（nuclear anchoring)元件、以及至少一反向重覆元件（inverted repeat’ IR)，其中該反向重覆元件具有二側面核酸序列（iateral nucleic acid sequences)，各側面核酸序列係互補（Complementary)於另一側面核酸序列。又，前述表現載體係-位於前述哺乳類細胞内之染色體外載體 (episomal vector)且係得用以轉染一哺乳類細胞者。較佳的是，前述反向重覆元件進一步包括有一中介序列（central seqUence)，其位於前述二側面核酸序列之間，〇且相異於前述：側面核酸序列之中的任―側面核酸序列。又，較佳的是，前述表現載體係如pGTOhGFp包括有反向重覆元件，更佳的是，前述表現載體係如pGT〇3_(}Fp 或PGT04-ID &括有二反向重覆元件。在前述包括有二反向重覆元件之情況下，前述二反向重覆元件可為彼此相同者或彼此相異者。。4述表現載體之基因可為一編碼一蛋白質之基因或一可被轉錄（transcribe)為一非編碼 RNA(n〇n_c〇ding RNA)之土口由#述基因所編碼之蛋白質可為但不限於訊息胜肽 (ignal peptuie)、生長因子（gr〇wth fact〇〇、激素（h〇rm〇ne)、 5 201035312 細胞介素（cytokine)、趨化因子（chemokine)、神經胜肽 (neuropeptide)、抗原（antigen)、抗體（antibody)、酵素 (enzyme，酶）、凝結因子（clotting factor)、抗血管生成因子（anti-angiogenic factor)、血管生成前趨因子（pro_ angiogenic factor)、運送蛋白（transport protein)、受體 (receptor)、配位子（ligand)、調控蛋白（regulatory protein)、結構蛋白（structural protein)、報導蛋白（reporter protein)、轉錄因子（transcription factor)、核糖酵素（ribozyme)、融合蛋白（fusion protein)或抗藥蛋白（drug-resistance protein)。前述蛋白質可為一天然蛋白質（protein of natural origin)或一經人工修改（artificially modified)之蛋白質，例如作為報導蛋白之強化綠榮光蛋白（enhanced green fluorescent protein，EGFP)。前述非編碼RNA可為但不限於tRNA、rRNA、反義 RNA(antisense RNA)、微小 RNA(micro-RNA)或雙股 RNA(double stranded RNA)。較佳的是，前述哺乳類細胞係一人類細胞，且可為一錯配修復糸統缺陷（mismatch repair_deficient，MMR-)細胞或一錯配修復糸統正常（mismatch repair-proficient，MMR+) 細胞。前述錯配修復系統缺陷細胞可為但不限於一 HCT116(hMLHl-)細胞或一 DLD-l(hMSH6-)細胞。前述錯配修復系統正常細胞可為但不限於一 HCT116 + ch3細胞或一 HCT116 + hMLHl 細胞。較it的疋’剞述核I苗定元素（nuciear_anch〇ring element) 包括有一編碼EB病毒（Epstein_Barr Virus , EBV)核蛋白 6 201035312 EBNA-l之EB病毒基因以及一 EB病毒複製起點（〇rigin 〇f replication)oriP。又，前述表現載體係得用以於哺乳類細胞中加強前述基因之表現者。本發明所採用之另一技術手段係提供一種構成 (organized as)反向雙聚體（inverted dimer，ID)之表現載體，其包括有一具有二長序列（long sequence)之環形核酸分子 (circular nucleic acid molecule)，前述二長序列係被二反向 © 重覆元件所分隔；其中’前述二長序列係彼此互補，且各長序列包括有至少一基因以及一核錨定元件，· 又’前述各反向重覆元件具有二側面核酸序列，各側面核酸序列係互補於另一側面核酸序列，前述二反向重覆疋件係彼此相同或相異，而前述表現載體係得用以轉染一哺乳類細胞者；且前述表現載體係一位於前述哺乳類細胞内之染色體外載體。〇此外本發明另採用之又一技術手段係提供一種於一哺礼類細胞t表現一基因之方法，包括有：以前述之表現載體轉染前述哺乳類細胞以製成一轉染細胞（transfected cell);培養（culturing)前述轉染細胞以令前述轉染細胞製造剛述基因所編碼之蛋白質；以及獲取前述蛋白質等步驟。 ^前述表現載體可以採用電穿孔法（eleCtroporation)送入前述哺孔類細胞。為更進一步描述本發明之標的、改善及創新技術將配合圖式詳述於後。 7 201035312 【實施方式】定義 Ο 用語「表現載體（expression vector)」係於此定義為— 載體’尤為一染色體外載體（episomal vector);其中所謂载體者’一般係為一質體（plasmid)，其係用以被引入（introduce) 一標的細胞（target cell)並於該標的細胞内表現一特定基因。前述表現載體可被用以製造大量之mRNA。當前述表現載體位於前述標的細胞内時，可藉由細胞之轉錄 (transcription)及轉譯（translation)機制而製造一由前述特定基因所編碼之蛋白質。前述質體係具高效啟動子（pr〇in〇ter) 以製造大量之mRNA。一染色體外載體則得被用以於其宿主細胞（host cell)中自主地（autonomously)自我複製（seif_ replicating) ° Ο 用》口基因（gene)」係於此定義為一組核酸（nucieic 即段（segments)，其包括有為藉由轉錄過程（transcripti〇n process)調控並製造一功能性RNA產品所需之資訊 (mformation)。藉此，前述RNA即可被直接使用（例如： tRNA、rRNA、snRNAS及其他如SRp RNA等非編碼 RNA(non-coding RNA)、反義抓八⑽“㈣e rna)或微小 RNA(miCr〇-RNA))，抑或是用以引導（direct)蛋白質之合成。在「-基因編碼一蛋白質」或「一蛋白質為一基因所編碼」等文句中’其義為前述基因係得被轉錄為—㈣職，而該 mRNA得進而被轉譯為— 貝且可忐進一步包括有哺礼I田胞之轉譯後及/或轉譯時修飾作用（叫 8 201035312 translational modifications)。可透過前述表現載體所提供的一種加強複製系統（enhanced replicati〇n system)於哺乳類細胞中表現之蛋白質可為但不限於訊息胜肽、生長因子、激素、細胞介素、趨化因子、神經胜肽、抗原、抗體、酵素、凝結因+、抗血管生成因子、血管生成前趨因子、運送蛋白、受體、配位子、調控蛋白、結構蛋白、報導蛋白、轉錄因子、核糖酵素、融合蛋白或抗藥蛋白。又前述蛋白貝可為一天然蛋白質或一經人工修改之蛋白質。用語「報導基因（reporter gene)」係於此定義為一基因，其結合於另一在細胞培養（cell culture)、動物或植物中欲使用之基因（another gene 〇f interest)。某些基因之所以被選為報導基因係因其易於檢測，或可用作選別標幟 (selectable marker)。報導基因主要被用以判斷是否前述欲使用基因是否已進入細胞或是否已表現於細胞或有機體群落（organism populati〇n)中。常用之報導基因包括有但不限於編碼水母綠螢光蛋白（GFP)而能夠使細胞在紫外光下發出螢光者、編碼螢光素酶〇uciferase)而能夠催化螢光素 (lucifedn)發光者、編碼半乳糖以讓宿主細胞呈現藍色之lacZ基因、以及可對於氯黴素產生抗藥性之氣黴素乙醯轉移酶（chl〇ramphenic〇1 aCetyltransferase，CAT)基因。為報導基因所編碼之蛋白質被稱為「報導蛋白」。用語「反向重覆（inverted repeat，IR)元件」係於此定義為一核酸分子，其包括有二側面核酸序列㈦…心nucieic acid sequences)，且可進一步包括有一中介序列 9 201035312 sequence)。各側面核酸序列係互補（c〇mpiementary)於另一側面核酸序列。前述二側面核酸序列可形成為回文 (palindrome)結構。前述中介序列係一異於前述二侧面核酸序列之短核酸序列，其插置於前述二側面核酸序列之間，從而破壞前述二側面核酸序列所形成之回文結構。例如： 6又一核酸具有「5 -taatecgga tt tccggatta-3’」之序列，其中相當於二側面核酸序列者即分別係r 5，_taatccgga _3,」與「5,_ tccggatta-3’」，而相當於中介序列者則係「5Mt_3,」。 ❹ 為數眾多的反向重覆元件散見於人類基因體之不同部位。關於該等位於人類基因體之反向重覆元件之資訊係公眾可付而知者。例如：波士頓大學（Boston University)維護一反向重覆資料庫（Inverted Repeats Database，IRDB)並對外公開，其不僅提供關於基因體DNA中反向重覆元件之資訊’更包括有多種分析用工具。可參照網站：「https://tandem.bu.edu/cgi-bin/irdb/irdb.exe」，現提供反向重覆搜尋演算法（Inverted Repeats Finder algorithm)、查詢及篩選特定重覆(repeat)元件之功能、基於序列相似度 (similarity)之重覆叢集運算法（repeat elustering algorithms)、PCR引子（PCR primer)選用功能、以及數種格式之資料下載功能。此外’前揭反向重覆資料庫可作為一集中式的研究平臺（centralized research workbench)，其提供了供儲存分析結果之空間，且可在複數使用者之間共同分享資料與分析。又所述之反向重覆元件可見於Lyu、Lin 及 Llu 等人之著作（Lyu et al·, 1999, J. Mol. Biol.， 285:148 5-1501);且反向重覆元件亦可人為設計之。 201035312 用語「反向雙聚體（inverted dimer，ID)」係於此定義為一具有二長序列（long sequence)之環形核酸分子（eireular nucleic acid molecule) ’前述二長序列係被二反向重覆元件所分隔；其中’前述二長序列係彼此互補，且各長序列包括有至少一基因以及一核錨定元件。前述反向雙聚體可自一包括有二終端（terminal)反向重覆元件之直鍵形 DNA(linear DNA)受質（substrate)製成。理論上，前述具有 Ο Ο 二終端反向重覆元件之直鏈形DNA受質係藉由細胞中的外切核酸酶（eX〇nuclease)或螺旋酶/核酸酶 (helicase/nuclease)之作用轉變為一啞鈴狀之dna中間產物（intermediate)。藉由螺旋酶/核酸酶之特異性可產出二種類之環狀反向雙聚體。就第一種類而言，一針對雙股=行 5二端至3·端（5.—3，）或3.端至5,端（3,七,）之外切核酸酶可以前，二終端反向4覆元件在相對股之隱上外露為突出的單股DNA。在前述反向重覆元件處所形成終端髮夹_ 令前述DNA轉變成-個具有終端圈⑽i〇〇ps)的n亞铃狀中間產物。後續的DNA複製過程則將前述⑽狀中間產物轉變成環形反向雙聚體。 _ 組跣弟一種類而言，與前述相同或相異的外切核酸酶/螺旋醢 & 疋姆可將則述直鏈形DNA受皙轉變為單股DNA。可採用萤 .. 、_ 木用早一外切核酸酶或螺施酶自一端進行處理。反向重覆元件囍罝早如DNA黏合（annealing)將开多成具有終端圈的η亞鈐狀a 4达 DNA.後續的DNA複製過程月1J述啞鈴狀DNA轉變志严γ Ε 肝文成衣形反向雙聚體。請參閱參考文獻所載.Lin et al. 20(ΐι μ 3538。 ’ ，Nucleic Acids Res· 29: 3529- 201035312 用語「核錨定元件（nuclear anch〇ring eiement)」係於此定義為一核酸分子的核酸序列，其被保持（retain)在一細胞的細胞核中，尤其是在一人類細胞的細胞核中。此核酸分子’作為一具有所述核錨定元件之載體，係錨定於細胞核（nucleus)的細胞核基質（nuciear matrix)中。核|苗定元件之例示包括有但不限於一編碼EBV核蛋白之EBV基因（例如核抗原-l(nuclear antigen-l)[EBNA-l])及一 EBV 複製起點（例如EBV複製子（replicon)之oriP複製起點）、一乳多 ❹ 泡病毒（papovavirus)複製起點及一乳多泡病毒大τ抗原 (large T antigen)、或一多瘤病毒（polyomavirus, Py)之複製起點（PyOri)及大T(large T，LT)抗原之基因（PyLT)，請參閱 Heffernan and Dennis, 1991，Nucleic Acids Res· 19:85-92)。較受偏好之核錨定元件係前述EBV基因及EBV複製起點。前述EBV基因及EBV複製起點之錨定效果係導因於oriP序列之高親和性（high-affinity)基質附著（matrix attachment)(Jankelevich et al., 1992, EMBO J., 11, 1165- 〇 1176; Mattia et al., 1999，Virology 262，9-17)以及 oriP 與複製起點結合蛋白（origin binding protein)EBNA-l之間的交互作用（interaction)(Lupton and Levine，1985, Mol. Cell Biol. 5, 2533-2542; Polvino-Bodnar and Schaffer, 1992, Virology 187, 591-603; Yates et al., 1984, Proc. Natl. Acad. Sci. USA 81，3806-3810)。在本發明中，建構了包括有至少一反向重覆元件或形成為反向雙聚體之表現载體。這些載體係可被選擇以有效地提昇基因表現。當用以表現一對抗選擇劑之抗藥性基因 12 201035312 (resistance gene)時，可，、,七苗止 …… 步驟中採用高濃度之選擇別來選擇細胞°即使在錯配修復系統正常人類細胞之中，亦能夠迅速而有效地獲取由前述表現載體所表現之蛋白質。以在細胞中生產至少—蛋白質並包本發明之方法係用括有下列步驟：201035312 VI. Description of the Invention: [Technical Field] The present invention is a performance vector capable of enhancing gene expression in mammalian cells, which includes a gene to be expressed, a _iear anchoring element, and At least one inverted overlap (inverted j). The present invention further relates to a method for enhancing gene expression by transfecting the aforementioned expression vector into mammalian cells such as humans. The gene expression method of eves is expressed in the prokaryotic expression system to express human recombinant protein. However, most human recombinant proteins need to be modified after translation or translation (p〇st_ and /〇r peri translati〇nai modifications) 'such as saccharification (glyc〇silati〇n), g-carboxylation (g-carboxyUtion) or g_hydroxylation (g_hydr〇xylati〇n). Therefore, a known problem with prokaryotic expression systems is the prokaryotic expression system. It is impossible to implement the post-translation or translation modification provided by the mammalian performance system. Therefore, in the prokaryotic expression system, there will be no The method normally expresses a protein that needs to be modified after translation or translation to function as a normal function. Another method widely used to express the aforementioned human recombinant protein is to use a mammalian expression system. The mammalian expression system uses mammalian cells to utilize It has the ability to modify after translation or translation. Especially Chinese hamster ovary (CH0) cells have been routinely used to express biomedical proteins for treatment and diagnosis. Existing methods of performance include – stepwise selection to increase the concentration of the selection agent. 3 201035312. One of the existing methods for enhancing gene expression in mammalian cells • Chinese hamsters used in the case The ovarian cells are gradually selected for the amplification of the difolic acid reductase (Dihydr0f0late reductase, Qing R) by methotrexate (one e-two MTX' or "killing the cancer-free bond"). 2002, J. Biosci. Bioeng 94: 600-605. ❹ However, the aforementioned stepwise selection method is both complicated and time consuming. It takes several months to achieve the goal of allowing a large number of genes in cells to be expressed, not to mention the fact that post-translational modification of human cells in non-human mammalian cells is performed differently, and this utilizes gene amplification (gene ampUficati (&gt n) has both enhanced gene expression methods and has not been widely used in human cells to produce biomedical proteins for treatment and diagnosis. In general, human proteins produced in human cells should be more People born to human systems have a medical advantage. Researchers have observed a mismatch repair (system) that strongly suppresses gene amplification in human cells. See Lin et al., 2001, Nucleic Acids Res 29, 3304-3310; Chen et al 2001, Pr〇c Natl Acad Sci USA 98, 13802-13807. Therefore, the gene enhanced by gene amplification is also expressed in the normal mismatch repair system (MMr_ normal/MMR-proficient, or MMR+) cells. The HCT116 cell line is caused by a mismatch repair system defect (MMR-deficient, or MMR-) cell of hMLH1 gene mutation. It is known that HCT116 cells can enhance gene expression by gene amplification. In contrast, HCT116 + Ch3 (with a normal mismatch repair system by introducing a third pair of chromosomes containing the wild-type hMLH1 gene) suppresses the expression of drug resistance genes enhanced by gene amplification. 4 201035312 [Summary of the Invention] The invention has the advantages of both gene expression methods and existing methods for enhancing gene expression in mammalian cells (especially in human cells). The object of the present invention is to provide an ability to remove or improve the aforementioned questions. The method of the present invention is to provide a performance vector (expression vect〇r) comprising at least one base factor, a nuclear anchoring element, And at least one inverted repeat 'IR', wherein the inverted repeating element has iateral nucleic acid sequences, and each side nucleic acid sequence is complementary to the other side nucleic acid sequence. Further, the expression vector is an episomal vector located in the aforementioned mammalian cell and is used to transfect a mammalian cell. Preferably, the reverse overlapping element further comprises a mediation sequence (central seqUence), which is located between the aforementioned two-sided nucleic acid sequences, and differs from the aforementioned: a side-side nucleic acid sequence among the side nucleic acid sequences. Further, preferably, the aforementioned expression vector such as pGTOhGFp includes a reverse weight More preferably, the aforementioned performance carrier is such as pGT〇3_(}Fp or PGT04-ID & includes two inverted repeating elements. In the case of two inverted overlapping elements, the two inverted overlapping elements may be identical to each other or different from each other. 4. The gene representing the carrier may be a gene encoding a protein or a transcript (transcribe) is a non-coding RNA (n〇n_c〇ding RNA). The protein encoded by ##基因 can be, but is not limited to, ignal peptuie, growth factor (gr〇wth fact〇〇, Hormone (h〇rm〇ne), 5 201035312 interleukin (cytokine), chemokine, neuropeptide, antigen, antibody, enzyme, coagulation Clotting factor, anti-angiogenic factor, pro- angiogenic factor, transport protein, receptor, ligand, regulatory protein (regulatory protein), structural protein, reporter protein, transcription factor, ribozyme, fusion protein or Protein drugs (drug-resistance protein). The aforementioned protein may be a protein of natural origin or an artificially modified protein, for example, an enhanced green fluorescent protein (EGFP) as a reporter protein. The aforementioned non-coding RNA may be, but not limited to, tRNA, rRNA, antisense RNA, micro-RNA or double stranded RNA. Preferably, the aforementioned mammalian cell line is a human cell and may be a mismatch repair_deficient (MMR-) cell or a mismatch repair-proficient (MMR+) cell. The defective cells of the aforementioned mismatch repair system may be, but not limited to, an HCT116 (hMLH1-) cell or a DLD-1 (hMSH6-) cell. The normal cells of the aforementioned mismatch repair system may be, but not limited to, an HCT116 + ch3 cell or an HCT116 + hMLH1 cell. The nuciear_anch〇ring element includes an Epstein-Barr virus (EBV) nucleoprotein 6 201035312 EBNA-l EB virus gene and an Epstein-Barr virus replication origin (〇rigin 〇) f replication) oriP. Further, the aforementioned expression vector is used to enhance the expression of the aforementioned gene in mammalian cells. Another technical means employed by the present invention provides an expression vector organized as an inverted dimer (ID) comprising a circular nucleic acid molecule having a long sequence (circular nucleic acid) Acid molecule), the aforementioned two-length sequence is separated by two reverse-repetitive elements; wherein 'the aforementioned two long sequences are complementary to each other, and each long sequence includes at least one gene and one nuclear anchoring element, and Each of the reverse repetitive elements has a two-sided nucleic acid sequence, each side nucleic acid sequence is complementary to the other side nucleic acid sequence, and the two inverted repeating elements are identical or different from each other, and the expression vector is used for transfection a mammalian cell; and the aforementioned expression vector is an extrachromosomal vector located in the aforementioned mammalian cell. Further, another technical means for the present invention is to provide a method for expressing a gene in a feeding cell, comprising: transfecting the aforementioned mammalian cells with the aforementioned expression vector to produce a transfected cell (transfected cell). And culturing the aforementioned transfected cells such that the transfected cells produce the protein encoded by the gene; and obtaining the aforementioned protein. The aforementioned expression vector can be delivered to the aforementioned porcine cells by electroporation. The subject matter, improvements and innovations of the present invention will be described in more detail in the accompanying drawings. 7 201035312 [Embodiment] Definition The term "expression vector" is defined herein as - the carrier is especially an episomal vector; the so-called carrier is generally a plasmid. It is used to introduce a target cell and express a specific gene in the target cell. The aforementioned expression vector can be used to produce a large amount of mRNA. When the aforementioned expression vector is located in the aforementioned target cells, a protein encoded by the aforementioned specific gene can be produced by the transcription and translation mechanisms of the cells. The aforementioned mass system has a highly efficient promoter to produce a large amount of mRNA. An extrachromosomal vector is used to autonomously self-replicate (seif_replicate) in its host cell. The term "gene" is defined herein as a set of nucleic acids (nucieic Segments, which include information required to regulate and manufacture a functional RNA product by a transcripti〇n process, whereby the aforementioned RNA can be used directly (eg, tRNA, rRNA, snRNAS and other non-coding RNAs such as SRp RNA, antisense octopus (10) "(tetra) e rna) or microRNA (miCr〇-RNA)), or used to direct protein synthesis In the words "-gene coding a protein" or "a protein is encoded by a gene", the meaning of the gene is transcribed into a (four) position, and the mRNA is further translated into - It further includes post-translation and/or translational modification of the feeding I field cell (called 8 201035312 translational modifications). The protein expressed in the mammalian cells through an enhanced replicati system provided by the aforementioned expression vector may be, but not limited to, a message peptide, a growth factor, a hormone, an interleukin, a chemokine, and a nerve victory. Peptides, antigens, antibodies, enzymes, coagulation factors, anti-angiogenic factors, angiogenesis precursors, transport proteins, receptors, ligands, regulatory proteins, structural proteins, reporter proteins, transcription factors, ribozymes, fusion proteins Or anti-drug protein. Further, the protein shell may be a natural protein or a manually modified protein. The term "reporter gene" is defined herein as a gene that binds to another gene (another gene 〇f interest) to be used in cell culture, animal or plant. Some genes have been selected as reporters because they are easy to detect or can be used as a selectable marker. The reporter gene is mainly used to determine whether the aforementioned gene to be used has entered the cell or has been expressed in a cell or organism population (organism populati〇n). Commonly used reporter genes include, but are not limited to, those encoding the jellyfish green fluorescent protein (GFP), which enable cells to emit fluorescence under ultraviolet light, encoding luciferase, and catalyzing luciferdn luminescence. The lacZ gene encoding galactose to give the host cell a blue color, and the chl〇ramphenic 〇1 aCetyltransferase (CAT) gene which is resistant to chloramphenicol. The protein encoded by the reporter gene is called a "reporter protein." The term "inverted repeat (IR) element" is defined herein as a nucleic acid molecule comprising a two-sided nucleic acid sequence (seven), and may further comprise a mediator sequence 9 201035312 sequence). Each side nucleic acid sequence is complementary to the other nucleic acid sequence. The aforementioned two-sided nucleic acid sequence can be formed into a palindrome structure. The aforementioned intermediate sequence is a short nucleic acid sequence which differs from the aforementioned two-sided nucleic acid sequence and is interposed between the two-sided nucleic acid sequences to disrupt the palindrome formed by the two-sided nucleic acid sequence. For example: 6 another nucleic acid has a sequence of "5-taatecgga tt tccggatta-3'", wherein the two side nucleic acid sequences are respectively r 5, _taatccgga _3," and "5, _ tccggatta-3'", and The equivalent of the mediation sequence is "5Mt_3,". ❹ A large number of inverted repetitive elements are scattered in different parts of the human genome. Information about these reverse repetitive elements located in the human genome is publicly available. For example, Boston University maintains an Inverted Repeats Database (IRDB) and publishes it, which not only provides information on reverse-repetitive components in genomic DNA, but also includes a variety of analytical tools. . Refer to the website: "https://tandem.bu.edu/cgi-bin/irdb/irdb.exe", now provide Inverted Repeats Finder algorithm, query and filter specific repeats (repeat) The function of the component, the repeat elustering algorithms based on the sequence similarity, the selection function of the PCR primer, and the data download function of several formats. In addition, the former reverse re-repository database can be used as a centralized research workbench, which provides space for storing analysis results and can share data and analysis among multiple users. Further, the reverse repeating elements can be found in the works of Lyu, Lin, and Llu et al. (Lyu et al., 1999, J. Mol. Biol., 285:148 5-1501); and the reverse repeating elements are also Can be artificially designed. 201035312 The term "inverted dimer (ID)" is defined herein as a long sequence of eireular nucleic acid molecules. The aforementioned two long sequences are reversed by two. Separated by the covering elements; wherein 'the aforementioned two long sequences are complementary to each other, and each long sequence includes at least one gene and one nuclear anchoring element. The aforementioned reverse dimer can be made from a linear DNA substrate comprising a terminal reverse repetitive element. Theoretically, the linear DNA-receptor having the reversed-repetitive elements of the 终端2 terminal is transformed by the action of an exonuclease (eX〇nuclease) or a helicase/nuclease in the cell. It is a dumbbell-shaped dna intermediate product (intermediate). Two types of cyclic reverse dimers can be produced by the specificity of the helicase/nuclease. In the first category, one for double strands = row 5 ends to 3 end (5.-3,) or 3. end to 5, end (3, seven,) exonuclease can be before, two The terminal reverse 4 overlay element is exposed as a prominent single strand of DNA on the opposite strand. The terminal hairpin is formed at the aforementioned reversely repetitive element - the aforementioned DNA is converted into an n-subring intermediate having a terminal ring (10) i 〇〇 ps). Subsequent DNA replication processes convert the aforementioned (10) intermediate to a circular reverse dimer. In the case of a class, the same or different exonuclease/spiral oxime & 疋m can convert the linear DNA into a single-stranded DNA. It can be processed from one end using firefly .., _ wood using early exonuclease or spirozyme. Reverse repetitive elements as early as DNA bonding (annealing) will open into a η sub-shaped a4 with terminal ring to reach DNA. Subsequent DNA replication process 1J described dumbbell-like DNA transformation Zhiyan γ Ε liver file Reverse complexes. Please refer to the reference. Lin et al. 20 (ΐι μ 3538. ', Nucleic Acids Res. 29: 3529- 201035312, "nuclear anch〇ring eiement" is defined herein as a nucleic acid molecule. a nucleic acid sequence that is retained in the nucleus of a cell, particularly in the nucleus of a human cell. This nucleic acid molecule acts as a vector with the nuclear anchoring element and is anchored to the nucleus (nucleus). In the nuciear matrix. Examples of nuclear | seedling elements include, but are not limited to, an EBV gene encoding EBV nuclear protein (eg, nuclear antigen-l [EBNA-l]) and a EBV origin of replication (eg, the oriP origin of replication of the EBV replicon), the papavirus origin of replication, and a large T antigen, or a polyomavirus , Py) The origin of replication (PyOri) and the gene for large T (LT) antigen (PyLT), see Heffernan and Dennis, 1991, Nucleic Acids Res 19:85-92). The more preferred nuclear anchoring elements are the aforementioned EBV genes and EBV origins of replication. The anchoring effect of the aforementioned EBV gene and EBV origin of replication is due to the high-affinity matrix attachment of the oriP sequence (Jankelevich et al., 1992, EMBO J., 11, 1165-〇1176) Mattia et al., 1999, Virology 262, 9-17) and the interaction between oriP and the originating binding protein EBNA-1 (Lupton and Levine, 1985, Mol. Cell Biol. 5, 2533-2542; Polvino-Bodnar and Schaffer, 1992, Virology 187, 591-603; Yates et al., 1984, Proc. Natl. Acad. Sci. USA 81, 3806-3810). In the present invention, an expression carrier comprising at least one inverted repeating element or forming a reversed dimer is constructed. These vectors can be selected to effectively enhance gene expression. When used to express a resistance gene 12 201035312 (resistance gene), can,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Among them, the protein expressed by the aforementioned expression vector can also be obtained quickly and efficiently. To produce at least a protein in a cell, the method of the invention comprises the following steps:

()背備表現載體，其包括有至少—核錨定元件、至少一基因、以及至少一反向重覆元件； (b)轉染前述表現載體於一哺乳類細胞以獲取一轉染細胞； (0培養前述轉染細胞以讓前述轉染細胞製造一為前述基因所編碼之蛋白質；以及 (d)獲取前述蛋白質。别述用以表現前述表現載體之基因的哺乳類細胞較佳係但不限於：中國倉鼠卵巢細胞、人類胚胎腎臟細胞 (HEK293)細胞、hMLHl-/-之人類大腸腫瘤（HCT116)細胞、引入第3對染色體之HCT116細胞（HCT116 + ch3 cell)、引入 hMLHl 基因之 HCT1 16 細胞（HCT1 16 + hMLHl cell)、人類結腸癌（DLD-1 (hMSH6-))細胞。以下的實驗設計係用以描述本發明而非意圖限定本發明之範圍。對所屬領域中具有通常知識者而言，可採行之合理變化調整將不逸脫本發明之範圍。實施例1 13 201035312 建構（Construction)並轉染表現載體如第1A圖所示，一商業上可購得之pEGFpm質體 (Clontech, CA)、一源於商業上可購得pDR2質體（cl〇ntech， CA)且基於EB病毒之p220.2質體、以及一商業上可購得之pMAMneo質體（Clontech，CA)係被用以構築一 pGT〇1_ GFP質體。前述pEGFP-Nl質體具有複數選殖位（ci〇ning sites)，〇包括有一 Bgln、一 BamHI、以及一 Xhol限制酶切位 (restriction sites)。前述pEGFP-Nl質體係先以Bglll限制酶與BamHI限制酶處理，以自前述複數選殖位之中排除前述Xh〇I限制酶切位，並獲取一直鏈形pEGFp_m DNA。在經過瓊脂凝() a back-up performance vector comprising at least - a nuclear anchoring element, at least one gene, and at least one inverted repetitive element; (b) transfecting the aforementioned expression vector into a mammalian cell to obtain a transfected cell; 0 cultivating the transfected cells to allow the transfected cells to produce a protein encoded by the aforementioned gene; and (d) obtaining the aforementioned protein. The mammalian cells for expressing the gene of the aforementioned expression vector are preferably, but not limited to: Chinese hamster ovary cells, human embryonic kidney cells (HEK293) cells, hMLH1-/- human colorectal tumors (HCT116) cells, HCT116 cells (HCT116 + ch3 cells) introduced into the third pair of chromosomes, and HCT1 16 cells into which the hMLH1 gene was introduced ( HCT1 16 + hMLH1 cell, human colon cancer (DLD-1 (hMSH6-)) cells. The following experimental design is intended to describe the invention and is not intended to limit the scope of the invention. The reasonable variation of the adjustments will not fall within the scope of the invention. Example 1 13 201035312 Construction (construction) and transfection performance carrier as shown in Figure 1A, a business The commercially available pEGFpm plastid (Clontech, CA), a commercially available pDR2 plastid (cl〇ntech, CA) and an Epstein-Barr virus based p220.2 plastid, and a commercially available product The pMAMneo plastid (Clontech, CA) was used to construct a pGT〇1_GFP plastid. The aforementioned pEGFP-Nl plastid has ci〇ning sites, including a Bgln, a BamHI, and a Xhol restriction sites are restricted. The aforementioned pEGFP-Nl system is first treated with Bglll restriction enzyme and BamHI restriction enzyme to exclude the aforementioned Xh〇I restriction enzyme cleavage site from the above multiple selection sites, and obtain a straight chain. Shaped pEGFp_m DNA. After agar condensation

膠（agarose gel)純化後’再讓前述直鏈形pEGFP-Nl DNA 進行自我連接（self-ligation)，且以EcoRI限制酶之截切After the purification of the agarose gel, the above-mentioned linear pEGFP-N1 DNA was self-ligated and cut with EcoRI restriction enzyme.

0 (digest)確認Xhol限制酶切位已遭排除，進而獲取一無Xh〇I 限制酶切位之pEGFP-Nl質體。藉由以ΑΠΙΙ與ApaLI限制酶截切前述無Xh〇I限制酶切位之pEGFP-Nl質體，可獲取一強化綠螢光基因（EGFP gene)以及一驅動（driving)前述強化、，，彔螢光蛋白基因之細胞巨大病毒（Cyt〇megal〇viruS， CMV)啟動子。其中，前述強化綠螢光基因具有一編碼強化綠螢光蛋白之強化綠螢光蛋白編碼序列（c〇ding sequence) ° 如第1A圖、第1B圖及第ic圖所示，一 pGTOl-GFP 貝體具有一核錯定元件、一濕黴素B(hygroniycin B)抗藥性 14 201035312 基因、一安比西林（ampicilHn)抗藥性基因、一 HindIII限制酶切位、一 Aflll限制酶切位、一 PvuII限制酶切位、/ Xhol限制轉切位、以及一限制酶切位。前述強化、、’彔螢光基因係作為一基因被插入在前述BamHI限制酶切位。其核錯定元件包括有一編碼EBV核蛋白EBNA-1之EBV 基因以及一 EBV之orip複製起點。前述pEGFP-Nl質體係主要源自於前述p22〇2質體、以及前述pMAMneo質體之BamHI-Hindlll片段。 D — HPH片段係一反向重覆元件，且包括有位於二端之二Η段落（segment)與位於中間之一 p段落。前述HpH片段係取自一 PHPH質體。前述pHPH質體係自pBR322質體所製成（Bi and Liu，1996，Proc. Natl. Acad· Sci. USA 93, 819-823)。前述 HPH 片段作為一 HPH/tet 片匣（cassette)存在於前述pHPH質體上’其係一基因開關（genetic swhch)，藉由則述P段落之指向（orientati〇n)來調控具有功能的 (functional)四環黴素（tetracycline)基因轉錄 (transcription) 〇削短的（shortened)前述HPH片段可藉由NruI限制酶截切而自 PHPH 質體（Lin et al.，1 997，Nucleic Acids Res. 25, 3009-3016)取得。 pGT02-GFP質體係藉由選殖⑻⑽丨叫）前述 HPH片段至削鈍的（blUnted)BamHI限制酶切位作為第一反向重覆元件，並藉由選殖一強化綠螢光基因至Afln限制酶切位而製成。一 PGT03-GFP質體係更進一步選殖前述HpH片段至 15 201035312 前述PGT02-GFP質體之PvuII限制酶切位作為第二反向重覆元件，且令前述第二反向重覆元件之p段落與前述第— 反向重覆元件之P段落具有相反之指向。藉由限制酶截切的方式可以決定前述第一反向重覆元件p段落與前述第二反向重覆元件P段落間相對指向的關係。請配合參照第2圖，前述PGT03-GFP質體係以Xh〇I 限制酶截切’並經過凝膠純化（gel_purificati〇n)以獲取一直鏈形 pGT03-GFP DNA。前述直鏈形PGT03-GFP DNA具有二端，前述第一反向重覆元件與前述第二反向重覆元件係分別位於前述二端。前述直鏈形PGT03-GFP DNA被變性（denatured)以獲取一變性的（denatUred)DNA溶液’溶液内含單股（single stranded)直鏈形PGT03-GFP DNA。可採用相當於前述DNA 溶液容積（a volume)且最後濃度為0.1 N氫氧化鈉（NaOH) 溶液對前述雙股直鏈形pGT03-GFP DNA進行鹼性變性。等容積（an equal volume)的0.1 N鹽酸（HC1)溶液與十分之一容積的 1M 三羥甲基胺基甲烷 (tris(hydroxymethyl)aminomethane，簡稱 Tris)緩衝液（pH 7.8) 被加入前述DNA溶液進行中和並停止（qUench)變性。被中和的DNA溶液暫時置於攝氏3 7度1 0分鐘後再移至冰上，以讓第一反向重覆元件各Η段落係相互黏合，並讓第二反向重覆元件各Η段落亦相互黏合。於前述單股直鏈形pGT03-GFP DNA二端形成類髮夾結構（hairpin-like structure)，以獲取一自我黏合（self- 16 201035312 annealing)之單股直鏈形PGT03-GFP DNA。如第2圖所示，在前述自我黏合之單股直鏈形pGTO3-GFP DNA的二端之間形成一間隙（gap)。可採用DNA聚合酶（DNA polymerase) 與連接酶（ligase)處理前述自我黏合之單股直鏈形pGT03-GFP DNA以封閉前述間隙並獲取一啞鈴形狀之自我黏合之單股直鍵形 pGT03-GFP DNA(Lin et al., 1997, Nucleic Acids0 (digest) confirmed that the Xhol restriction enzyme cleavage site has been excluded, thereby obtaining a pEGFP-Nl plastid without Xh〇I restriction enzyme cleavage site. By pluting the aforementioned pEGFP-N1 plastid without Xh〇I restriction enzyme cleavage with ApaLI restriction enzyme, an enhanced green fluorescent gene (EGFP gene) and a driving of the aforementioned enhancement, 彔The fluorescent protein gene cell giant virus (Cyt〇megal〇viruS, CMV) promoter. Wherein, the enhanced green fluorescent gene has a green fluorescent protein coding sequence encoding a enhanced green fluorescent protein (c〇ding sequence), as shown in FIG. 1A, FIG. 1B and ic, a pGTO1-GFP The shell has a nuclear misidentification element, a hygromycin (B), resistance gene 14 201035312 gene, an ampicillin (ampicilHn) drug resistance gene, a HindIII restriction enzyme cleavage site, an Aflll restriction enzyme cleavage site, a PvuII Restriction enzyme cleavage site, /Xhol restriction cleavage site, and a restriction enzyme cleavage site. The aforementioned enhanced, 彔-fluorescent gene line is inserted as a gene in the aforementioned BamHI restriction enzyme cleavage site. The nuclear misidentification element includes an EBV gene encoding the EBV nuclear protein EBNA-1 and an orip origin of replication of an EBV. The aforementioned pEGFP-Nl system is mainly derived from the aforementioned p22〇2 plastid, and the BamHI-Hindlll fragment of the aforementioned pMAMneo plastid. D — The HPH segment is a reverse-repetitive component and includes a two-segment segment at the two ends and a p segment at the middle. The aforementioned HpH fragment was taken from a PHPH plasmid. The aforementioned pHPH system was prepared from pBR322 plastids (Bi and Liu, 1996, Proc. Natl. Acad. Sci. USA 93, 819-823). The aforementioned HPH fragment is present as a HPH/tet cassette on the pHPH plastid, which is a genetic swhch, which is regulated by the orientation of the P paragraph (orientati〇n). Functional) tetracycline gene transcription The shortened aforementioned HPH fragment can be cleaved from the PHPH plastid by NruI restriction enzymes (Lin et al., 1 997, Nucleic Acids Res. 25, 3009-3016) Acquired. The pGT02-GFP system by breeding (8) (10) nicknames the aforementioned HPH fragment to the blunt (blUnted) BamHI restriction enzyme cleavage site as the first inverted repetitive element, and by cultivating a potentiating green fluorescent gene to Afln It is made by limiting the cleavage site. A PGT03-GFP system further clones the aforementioned HpH fragment to 15 201035312, the PvuII restriction enzyme cleavage site of the aforementioned PGT02-GFP plastid is used as the second reverse repetitive element, and the p segment of the second reverse repetitive element is It has the opposite orientation to the P paragraph of the aforementioned first-reverse repeating element. The relationship between the aforementioned first reversely repetitive element p and the second reversely repetitive element P can be determined by limiting the enzyme cut. Referring to Fig. 2, the PGT03-GFP system was cleaved by Xh〇I restriction enzyme and gel-purified (gel_purificati〇n) to obtain the all-chain pGT03-GFP DNA. The linear PGT03-GFP DNA has two ends, and the first reverse overlapping element and the second reverse overlapping element are respectively located at the two ends. The aforementioned linear PGT03-GFP DNA was denatured to obtain a denatured (denatUred) DNA solution' solution containing single stranded linear PGT03-GFP DNA. The above-mentioned double-stranded linear pGT03-GFP DNA can be alkaline denatured by using a solution corresponding to the aforementioned DNA solution volume (a volume) and finally having a concentration of 0.1 N sodium hydroxide (NaOH). An equal volume of 0.1 N hydrochloric acid (HC1) solution and one tenth of a volume of 1 M tris (hydroxymethyl) aminomethane (Tris) buffer (pH 7.8) were added to the aforementioned DNA. The solution was neutralized and stopped (qUench) denatured. The neutralized DNA solution is temporarily placed at 37 degrees Celsius for 10 minutes and then transferred to ice so that the first inverted repeating elements are bonded to each other and the second inverted overlapping elements are each The paragraphs also stick to each other. A hairpin-like structure was formed at the two ends of the single-stranded linear pGT03-GFP DNA to obtain a self-adhesive (self- 16 201035312 annealing) single-stranded linear PGT03-GFP DNA. As shown in Fig. 2, a gap is formed between the two ends of the self-adhesive single-stranded linear pGTO3-GFP DNA. The self-adhesive single-stranded linear pGT03-GFP DNA can be treated with a DNA polymerase and a ligase to block the gap and obtain a dumbbell-shaped self-adhesive single-stranded pGT03-GFP. DNA (Lin et al., 1997, Nucleic Acids

Res. 25, 3009-3016; Lin et al., 2001, Nucleic Acids Res. 29: 3529-3538)。前述°亞鈐形狀之自我黏合之單股直鏈形pGT03-GFP DNA係被引入至一 DH5〇： (RecA-)大腸桿菌（Ε· coli)之中’以進行DNA複製（DNA replication)並自前述β亞鈴形狀之自我黏合之單股直鏈形pGT03-GFP DNA產生一雙股 DNA質體（請參照第2圖）。前述雙股DNA質體係以Hindlll 限制酶與Notl限制酶截切並進行選擇以獲取一 pGT04/ID 反向雙聚體。被建構之pGTOl-GFP質體、pGT02-GFP質體、 PGT03-GFP質體、以及pGT〇4/ID反向雙聚體，係作為表現載體，藉由電穿孔（electr〇p〇rati〇n)以轉染至細胞中。前述電穿孔可採用諸如商業上可得之Nuleofect〇r system (Amaxa，Germany)，依據其製造者提供之指引實行。轉染細胞（transfected ceU)經24至48小時培養後，加入5〇以 g/ml之濕黴素B以選擇穩定的殖系（cl〇ne)。本發明之主要目的在於加強轉染細胞的基因表現。為此，前述可在人類細胞中複製的表現載體被設計為包括有核錯定it件’以便渡過長時間的培養期間。在本實施例中， 17 201035312 前述核錯定元件包括有編碼EBV核蛋白職A]之Ebv 基因以及EBV之。riP複製起點。-包括有編碼EBV核蛋白EBNA-1之EBV基因以及刪之〇rip複製起點之載體，或-包括有編碼EBV核蛋白EBNA」之謂基因以及咖之oriP複製起點之表現載體皆可被稱為載體。一載體藉由包括有oriP序列之高親和性基質附著區域錯定於細胞核基質（jankelevich et al，1992, EMB〇 1Π6; Mattia et al·, 1999, Virology, 262, 9-17)。〇此外，oriP與複製起點結合蛋白EBNA-1之間的交互作用對於EBV載體在靈長類細胞中之複製、分離 (segregation)、以及保留（retenti〇n)而言是必要的 and Levine, 1985, Mol. Cell Biol. 5, 2533-2542; Polvino-Res. 25, 3009-3016; Lin et al., 2001, Nucleic Acids Res. 29: 3529-3538). The self-adhesive single-stranded linear pGT03-GFP DNA line of the aforementioned yttrium shape is introduced into a DH5 〇: (RecA-) E. coli (Ε·coli) for DNA replication and self-binding The self-adhesive single-stranded linear pGT03-GFP DNA of the β-bell shape produces a double-stranded DNA plastid (see Figure 2). The aforementioned double-stranded DNA system was cut with a Hindlll restriction enzyme and a Notl restriction enzyme and selected to obtain a pGT04/ID reverse dimer. The constructed pGTO1-GFP plastid, pGT02-GFP plastid, PGT03-GFP plastid, and pGT〇4/ID reverse duplex were used as expression vectors by electroporation (electr〇p〇rati〇n ) to transfect into cells. The electroporation described above may be carried out using, for example, the commercially available Nuleufect(R) system (Amaxa, Germany) in accordance with guidelines provided by its manufacturer. After transfected ceU was cultured for 24 to 48 hours, 5 g of hyphamycin B in g/ml was added to select a stable colony (cl〇ne). The main object of the present invention is to enhance the gene expression of transfected cells. To this end, the aforementioned expression vector which can be replicated in human cells is designed to include a nuclear-corrected element' to allow for a long period of culture. In the present embodiment, 17 201035312 The aforementioned nuclear misidentification element includes an Ebv gene encoding EBV nuclear protein A] and EBV. riP copy starting point. - a vector comprising an EBV gene encoding EBV nucleoprotein EBNA-1 and a vector for deleting the rip origin of rip, or a gene comprising a gene encoding EBV nucleoprotein EBNA and an originator of oriP origin of coffee can be called Carrier. A vector is erroneously bound to the nuclear matrix by a high affinity matrix attachment region comprising the oriP sequence (jankelevich et al, 1992, EMB® 1Π6; Mattia et al, 1999, Virology, 262, 9-17). In addition, the interaction between oriP and the replication origin binding protein EBNA-1 is necessary for replication, segregation, and retention of EBV vectors in primate cells and Levine, 1985 , Mol. Cell Biol. 5, 2533-2542; Polvino-

Bodnar and Schaffer, 1992, Virology 187, 591-603; Yates et al·，1984, Proc. Natl, Acad. Sci. USA 81，3806-3810)。請再參閱苐1A圖、苐IB圖及第1C圖’前述包括有ebv元件與至少一反向重覆元件之表現載體之結構，是為了確認是否可以藉由至少一反向重覆元件所調控（mediate)之DNA 結構（DNA rearrangement)進而藉由被增加的藥物選擇與被增加的基因複本（copy)來加強基因表現。前述編碼強化綠螢光蛋白且受細胞巨大病毒啟動子所驅動之強化綠榮光基因係作為基因被選殖進入A述各表現載體中。請配合來閱第2圖，前述pGT04/ID反向雙聚體係藉由反向重覆元件調控的方式自前述pGT03-GFP質體製得。實施例2 18 201035312 齊備細胞 HCT116(hMLHl-/-之人類大腸腫瘤）細胞係來自Dr. C. R. Boland (UCSD, CA) (Koi et al., 1994, Cancer Res. 54, 4308-43 12; ATCC#CCL-247)。 pC9MLHWT質體包含有由細胞巨大病毒啟動子所控制野生型（wild-type)hMLHl 互補 DNA(complement DNA, cDNA)，其藉電穿孔轉染HCT1 1 6細胞以回復錯配修復功 Ο 能。前述 PC9MLHWT 質體係來自 Dr. B. Vogelstein (Lin et al.，Nucleic Acids Res. 29，3304-3310)。其表現 hMLHl 蛋白之 G418抗藥性殖系（G418-resistant clone)被稱為 HCT116+hMLHl。HCT116 + hMLHl 細胞係於含有 100 // g/ml G41 8之環境下培養。另一人類結腸癌細胞 DLD-l(hMSH6-)(ATCC#CCL-221) 係來自 Dr. Thomas A. Kunkel (NIEHS，NC)。DLD-1 細胞係於添加10%胎牛血清（fetal bovine serum)之完整RPMI培養 ® 基（complete RPMI medium)中培養。實施例3 高抗藥性測定在進行藥物選擇（drug selection)之24小時前’約1X104 之未匯合細胞被殖於（seed)含有完整RPMI-1640培養基或添加10%胎牛血清（可自Gibco商業獲得）DMEM培養基的 100x20 mm培養孤（culture dish)中。隨著培養基營養消耗 19 201035312 以及細胞廢棄物（ceU waste)之累 -^ Α δ 積，經過一定的期間即以新鮮的培養基更新老化的（aged) 脣基。在更新培養基後，在各盤（plate)中加入指定濃度之渴 ^ ^法 '、、做素B。刖述培養皿传培養於攝氏37度並添加5%二氧 · 、 ,. 灭之培養器（incubator) 中。ik者濕黴素B所帶來的細胞毒性 ’在處理2至4曰後，重新補充新鮮的培養基予細胞。存什在的群洛（0〇1〇117)在至Μ日後觀察其生長狀態。將細胞殖於培養盟中的生長 Ο 效率⑻aUng ef制ency)之量測方法係藉由將綱細胞殖入 100x20 mm培養皿中，样豆决j 1rk τ待具生長10曰後再計算其群落數目° 實施例4 (1) 測量綠螢光蛋白生產量以PBS清洗細胞、並進行胰蛋白酶處理使細胞懸浮於 PBS。接著使用一螢光激發細胞分選器（FACS Vantage cell sorter) (Becton Dickinson，Mountain View, CA)以激發波長 (excitation wavelength)480 nm 及放射波長（emission wavelength) 525 nm測定綠螢光蛋白之綠色螢光。 (2) 加強重組基因表現作為人類結腸癌細胞的OLD-1細胞、HCT1 1 6細胞、以及HCT116 + hMLHl細胞係轉染前述各種表現載體並以50 # g/ml濕黴素b選擇穩定殖系。於各穩定殖系均培養了複數穩定殖系細胞。各種人類結腸癌細胞採1 X1 〇4穩定殖系細胞殖於培養皿並於包括有4〇〇 "g/ml或1〇〇〇濕 20 201035312 黴素B的培養基中進行培養。如第3A圖、第3B圖、第3C圖及第3D圖所示，在1000 濕黴素B的選擇條件下，轉染pGT〇4/ID反向雙聚體的結腸癌細胞殖系DLD-l(MMR-)細胞、HCT116(MMR-) 與HCT1 16+hMLHl(MMR+)細胞比其他具有至少一反向重覆元件之細胞具尚於50倍的群落數目。請配合參閱第4A 圖’在400 #g/ml或10〇〇 #g/ml濕黴素b的選擇條件下’以流動式細胞測量術（fl〇w cytometry)測得增加的綠螯〇光蛋白產出量（yield)。實施例5 西方墨點法一蛋白酶抑制劑混合物（cocktail of protease inhibitors) 對於諸如絲胺酸（serine)、副胱胺酸（cysteine)及天冬胺酸蛋白酶（aspartic pro teases)與胺肽酶（aminopeptidases)等具有廣泛的特異性（specificity)。一蛋白酶抑制劑混合物可包括有 4-(2-氨乙基）氧化苯續酰（4-(2-aminoethyl)benzenesulfonyl fluoride, AEBSF) ' 胃蛋白酶抑制劑（pepstatinA)、絲胺酸蛋白酶抑制劑（E-64)、胺肽酶抑製劑（bestatin)、亮抑酶肽（leupeptin)、以及抑胰肽酶 (aprotinin)。一蛋白酶抑制劑混合物可不含金屬螯合物 (metal chelator)，亦得以自商業公司獲得。細胞萃取物（cell extracts)係在含有蛋白酶抑制劑混合物之溶解缓衝液（lysis buffer)(含 50 mM HEPES pH 7.6、 21 201035312 0.5% SDS、1% 脫氧膽酸納（sodium deoxycholate)、以及 5 mM EDTA)(Sigma)中製備而成。部分蛋白質樣本係於12% SDS-PAGE瓊膠上分離’並以電轉潰（electroblot)至聚偏二氣乙稀（polyvinylidene fluoride)膜（Amersham)上。西方墨點法係採用一 GFP抗體（Chemicon International)及一肌動蛋白（actin)抗體（1-19; Santa Cruz Biotechnology)進行。接合有辣根過氧化酶（horseradish peroxidase)的二次抗體 (Secondary antibodies)(Sigma)與加強化學發光測定系統 O (enhanced chemiluminescence)(ECL; Amersham)則被用以進行偵測。如第4B圖所示，在50 " g/m卜400 " g/ml或1〇〇〇 " g/ml濕黴素B的條件下培養的細胞以西方墨點法觀察到增加的綠螢光蛋白產出量。又’關於其於細胞萃取物之凝膠中施用抗肌動蛋白之單株抗體以作為蛋白質載入量（1〇ading level)之控制組（contr〇i)則未示於本圖。〇結果上述實施例清楚地表示出具有核錨定元件與反向重覆元件的表現載體，無論在錯配修復系統缺陷與錯配修復系統正常細胞_都加強了轉染基因（transgene)的基因表現。本發明所提及的實驗是將具有核錨定元件、一或二個反向重覆70件、二抗藥基因（例如濕黴素B抗藥性基因與安比西林抗藥性基因）、卩及一報導基因（例如強化綠榮光蛋基口）的表現載體轉染至人類細胞中。如第3圖所示， 22 201035312 ο 由於更多殖系在1000 Mg濕黴素Β存活，顯示轉染細胞之抗藥性（drug-resistance)大幅地改善（例如對於濕黴素Β抗性）。又如第4圖所示’由提昇的螢光強度（intensity 〇f fluorescence)與 SDS-polyacrylamide 瓊膠上較高的綠螢光 =白染色，可知在高濃度濕黴素B存活的殖系明顯地比低濃度濕黴素B存活的對照組殖系（其未經表現載體轉染）具有較高濃度的螢光蛋白。此結果確認了在轉染細胞中基因擴增與基因表現均受到加強。最為明顯的是這些改善非僅見於錯配修復系統缺陷細胞（其無法壓抑基因擴增與基因表現）中，在錯配修復系統正常細胞（其通常具有壓抑基因擴增與基因表現之效果）中亦有這些改善。 '在本發明中構築的各載體中，pGTQ4/id反向雙聚體作為表現載體係構成為環狀反向雙聚體（Ocular ’ Circular id) ’其在宿主細胞（h〇st Μ")中提供最高的抗樂性效果與綠螢光蛋白濃度。〇藥基領域的技術人員將可以理解所使用的前述抗換土—螢光基因可以採用編碼其他蛋白質的基因代 7例如生長因子、激素、細胞介素、趨化因子管生出·^ f仏因子、抗血管生成因子、血 :生成别趨因子、運送蛋白、受體、配位子、調控蛋白、、、’°構蛋白、轉錄因子、核糖酵f 工雖然本發明的許多特性與、、明詳細的結構與性質 ’ ：5己述’關於本發對於本'达的揭露用思在於說明本發明。下，依昭申枝直&〜尤其疋在本發明揭橥原則專利乾圍用語最廣泛的解釋對於形狀、大小、 23 201035312 以及部件配置進行修改。【圖式簡單說明】第1A圖係關於本發明之表現載體PGT01-GFP之圖譜 (map) ° 第1B圖係關於本發明之表現載體PGT02-GFP之圖譜。第1C圖係關於本發明之表現載體PGT03-GFP之圖Bodnar and Schaffer, 1992, Virology 187, 591-603; Yates et al., 1984, Proc. Natl, Acad. Sci. USA 81, 3806-3810). Please refer to FIG. 1A, FIG. 1B and FIG. 1C. 'The foregoing structure including the ebv element and the at least one reverse-repetitive element is used to confirm whether it can be controlled by at least one reverse-repetitive element. The DNA rearrangement of the mediate enhances gene expression by increased drug selection and increased gene copies. The enhanced green glory gene encoding the enhanced green fluorescent protein and driven by the cellular giant viral promoter is selected as a gene into the expression vectors of A. Please refer to Fig. 2 for the pGT04/ID reverse dimerization system obtained from the pGT03-GFP system by means of reverse repetitive element regulation. Example 2 18 201035312 Complete cell HCT116 (hMLH1-/- human colorectal tumor) cell line from Dr. CR Boland (UCSD, CA) (Koi et al., 1994, Cancer Res. 54, 4308-43 12; ATCC# CCL-247). The pC9MLHWT plastid contains wild-type hMLH1 complement DNA (cDNA), which is transfected with HCT1 16 cells by electroporation to restore mismatch repair function. The aforementioned PC9MLHWT system is from Dr. B. Vogelstein (Lin et al., Nucleic Acids Res. 29, 3304-3310). Its G418-resistant clone of hMLH1 protein is called HCT116+hMLH1. The HCT116 + hMLH1 cell line was cultured in an environment containing 100 // g/ml G41 8 . Another human colon cancer cell DLD-1 (hMSH6-) (ATCC #CCL-221) was from Dr. Thomas A. Kunkel (NIEHS, NC). DLD-1 cells were cultured in complete RPMI medium supplemented with 10% fetal bovine serum. Example 3 High Resistance Tests [About 1X104 of unconfluent cells were seeded with intact RPMI-1640 medium or added with 10% fetal bovine serum 24 hours prior to drug selection (available from Gibco Business) Obtained in a 100x20 mm culture dish of DMEM medium. With the nutrient depletion of the medium 19 201035312 and the accumulation of ceU waste, the aged lip base is renewed with fresh medium over a certain period of time. After the medium is refreshed, a specified concentration of thirst ^^ method, and a factor B are added to each plate. The culture dish is cultured at 37 ° C and added to a 5% dioxin, incubator (incubator). Cytotoxicity caused by ik's hygromycin B' After treatment for 2 to 4 ,, fresh medium was replenished to the cells. The group in the group (0〇1〇117) observed its growth status after the next day. The method of measuring the growth 细胞 efficiency (8) aUng ef of the cells in the culture nucleus is carried out by colonizing the cells into a 100×20 mm culture dish, and the sample is then grown for 10 曰 and then the community is calculated. Number ° Example 4 (1) Measurement of green fluorescent protein production The cells were washed with PBS and subjected to trypsin treatment to suspend the cells in PBS. The green fluorescent protein green was then determined using a FACS Vantage cell sorter (Becton Dickinson, Mountain View, CA) with an excitation wavelength of 480 nm and an emission wavelength of 525 nm. Fluorescent. (2) Enhancing recombinant gene expression As human colon cancer cells, OLD-1 cells, HCT1 16 cells, and HCT116 + hMLH1 cell lines were transfected with the above various expression vectors and selected for stable colonization with 50 # g/ml wet mycin b. . Multiple stable cell lines were cultured in each stable lineage. Various human colon cancer cells were cultured in a culture medium containing 1 X1 〇4 stable colonies and cultured in a medium containing 4 〇〇 "g/ml or 1 湿 wet 20 201035312 mycin B. As shown in Fig. 3A, Fig. 3B, Fig. 3C and Fig. 3D, colon cancer cell line DLD-transfected with pGT〇4/ID reversed dimer under the selection conditions of 1000 hygromycin B l (MMR-) cells, HCT116 (MMR-) and HCT1 16+hMLH1 (MMR+) cells were 50 times more colony than other cells with at least one inverted repetitive element. Please refer to Figure 4A 'Under 400 #g/ml or 10〇〇#g/ml wetmycin b under the selection conditions' to increase the green chelate light by flow cytometry (fl〇w cytometry) Protein yield (yield). Example 5 Western blotting method for cocktails of protease inhibitors such as serine, cysteine and aspartic pro teases and amine peptidase ( Aminopeptidases) and the like have broad specificity. A protease inhibitor mixture may include 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) 'pepsstatin A, a serine protease inhibitor ( E-64), bestatin inhibitor, leupeptin, and aprotinin. A protease inhibitor mixture can be free of metal chelator and is also available from commercial companies. Cell extracts are in a lysis buffer containing a mixture of protease inhibitors (containing 50 mM HEPES pH 7.6, 21 201035312 0.5% SDS, 1% sodium deoxycholate, and 5 mM) Prepared in EDTA) (Sigma). A portion of the protein sample was separated on a 12% SDS-PAGE agar and electroblotted onto a polyvinylidene fluoride membrane (Amersham). Western blotting was performed using a GFP antibody (Chemicon International) and an actin antibody (1-19; Santa Cruz Biotechnology). Secondary antibodies (Sigma) and enhanced chemiluminescence (ECL; Amersham) with horseradish peroxidase were used for detection. As shown in Figure 4B, cells cultured under conditions of 50 " g/m Bu 400 " g/ml or 1〇〇〇" g/ml hygromycin B were observed by Western blotting. Green fluorescent protein production. Further, the control group (contr〇i) which is a monoclonal antibody against actin in the gel of the cell extract as a protein loading level (contr〇i) is not shown in the figure. 〇Results The above examples clearly show that the expression vector having the nuclear anchoring element and the reversely repetitive element enhances the gene of the transgene regardless of the defect in the mismatch repair system and the normal cell of the mismatch repair system. which performed. The experiments mentioned in the present invention are to have a nuclear anchoring element, one or two inverted repeats of 70 pieces, a secondary drug resistance gene (such as a hygromycin B drug resistance gene and an ampicillin resistance gene), and a The expression vector of the reporter gene (eg, the enhanced green glory base) is transfected into human cells. As shown in Fig. 3, 22 201035312 ο As more colonies survived in 1000 Mg damphomycin, the drug-resistance of the transfected cells was significantly improved (e.g., for hygromycin resistance). As shown in Fig. 4, 'increased fluorescence intensity (intensity 〇f fluorescence) and higher green fluorescence on SDS-polyacrylamide agar = white staining, it is known that the growth of high concentration of wet mycin B is obvious. The control line that survived lower than the low concentration of wet mycin B (which was not transfected with the expression vector) had a higher concentration of fluorescent protein. This result confirmed that both gene amplification and gene expression were enhanced in transfected cells. Most notably, these improvements are not only found in defective cells of the mismatch repair system (which cannot suppress gene amplification and gene expression), but also in normal cells of the mismatch repair system (which usually have the effect of suppressing gene amplification and gene expression). There are also these improvements. 'In each of the vectors constructed in the present invention, the pGTQ4/id reverse dimer is configured as a expression vector to form a cyclic reversed dimer (Ocular 'Circular id'' in a host cell (h〇st Μ") Provides the highest anti-music effect and green fluorescent protein concentration. It will be understood by those skilled in the art of bismuth-based medicine that the aforementioned anti-replacement-fluorescent gene can be encoded by genes encoding other proteins such as growth factors, hormones, interleukins, chemokines, and factors. , anti-angiogenic factors, blood: production of alternative factors, transport proteins, receptors, ligands, regulatory proteins, , 'construction proteins, transcription factors, ribolysis, although many of the characteristics and Detailed Structure and Properties ': 5 Having described the present invention for the disclosure of the present invention. Next, Yizhao Shenzhizhi &~ Especially in the invention's principle of disclosure The most extensive interpretation of the patented terms is to modify the shape, size, 23 201035312 and component configuration. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1A is a map of the expression vector PGT01-GFP of the present invention. Fig. 1B is a map showing the expression vector PGT02-GFP of the present invention. Figure 1C is a diagram of the expression vector PGT03-GFP of the present invention.

第2圖係由第1C圖所示表現載體pGT03-GFP製作 (generating)表現載體pGT04/ID之流程圖。第3A圖係以pGT03-GFP或pGT04/ID轉染之DLD-l(hMSH6·)細胞，且該轉染細胞係施以400私g/ml或1000 // g/ml 之濕黴素 B(hygromycin B)處理。第 3B 圖係以 pGT02-GFP、pGT03-GFP 或 pGT04/ID 轉染之HCT116(hMLHl-)細胞或HCT116+hMLHl細胞，且該轉染細胞係施以1 〇〇〇 // g/ml之濕黴素B處理。第3C圖係呈現第3A圖轉染細胞存活群落（colony)數量之柱狀圖。第3D圖係呈現第3B圖pGT03-GFP或pGT04/ID轉染細胞存活群落（colony)數量之柱狀圖。第4A圖係細胞數對綠螢光強度（intensity)之圖表，其所示者係以PGT04/ID轉染並施以50 //g/m卜400 /zg/ml 或1000 // g/ml濕黴素B處理之DLD-l(hMSH6-)細胞。第4B圖係第4A圖所示細胞之西方點墨法（Western- 24 201035312Fig. 2 is a flow chart showing the expression vector pGT04/ID generated by the expression vector pGT03-GFP shown in Fig. 1C. Figure 3A is a DLD-1 (hMSH6·) cell transfected with pGT03-GFP or pGT04/ID, and the transfected cell line is administered 400 g/ml or 1000 // g/ml of damptomycin B ( Hygromycin B) treatment. Figure 3B is HCT116 (hMLH1-) cells or HCT116+hMLH1 cells transfected with pGT02-GFP, pGT03-GFP or pGT04/ID, and the transfected cell line is applied at 1 〇〇〇//g/ml wet Treatment withmycin B. Figure 3C is a bar graph showing the number of colonies of transfected cells in Figure 3A. Figure 3D is a bar graph showing the number of colonies of pGT03-GFP or pGT04/ID transfected cells in Figure 3B. Figure 4A is a graph of the number of cells versus green fluorescence intensity, which is shown transfected with PGT04/ID and applied at 50 //g/m b 400 /zg/ml or 1000 // g/ml DDT-1 (hMSH6-) cells treated with wetmycin B. Figure 4B is a Western blotting method for cells shown in Figure 4A (Western- 24 201035312)

blotting)分析圖；其以未受轉染之DLD-1細胞作為負控制組（negative control) ° 【主要元件符號說明】無 25Blotting) analysis; the untransfected DLD-1 cells were used as a negative control group. [Main component symbol description] None 25

Claims

201035312 VII. Patent application scope: 1. An expression vector comprising: at least one gene; a nuclear anchoring element; and at least one inverted repeat (IR), Having a lateral nucleic acid sequence, each side nucleic acid sequence is complementary to the other side nucleic acid sequence; wherein 'month ί 述 expression vector is used to transfect a mammalian cell; An extrachromosomal vector (episomal vector) located in the aforementioned mammalian cells. 2. The expression vector according to claim 1, wherein the gene encodes a protein selected from the group consisting of a signal peptide, a growth factor, a hormone, a cytokine. (cytokine), chemokine, neuropeptide, antigen, antibody, enzyme, clotting factor, anti-angiogenic factor Factor), pro-angiogenic factor, transport protein, receptor, ligand, regulatory protein, structural protein, report A group consisting of a reporter protein, a transcription factor, a ribozyme 'fusion protein, and a drug-resistance protein. 3. The expression vector of claim 2, wherein the protein 26 201035312 is a protein of natural origin or an artificially modified protein. 4. The expression vector according to claim 3, wherein the reported protein is an enhanced green fluorescent protein (EGFP). 5. The expression vector of claim 1, wherein the gene is transcribed into an RNA selected from the group consisting of tRNA, rRNA, antisense RNA, micro-RNA, and double-stranded O. A group of RNA (double stranded RNA). 6. The expression vector according to claim 1, wherein the mammalian cell line is a human cell. 7. The performance vector of claim 6, wherein the human cell line is a mismatch repair-deficient (MMR-) cell. 8. The expression vector according to claim 7, wherein the defective cell repair system is a defective cell line of HCT116 (hMLH1-) cells or a LD_® l (hMSH6-) cell. 9. The expression vector of claim 6, wherein the human cell line is a mismatch repair-proficient (MMR+) cell. 10. The performance vector according to claim 1, wherein the nuclear-anchoring element comprises an EB virus gene encoding Epstein-Barr Virus (EBV) nuclear protein EBNA-1 and a Epstein-Barr virus replicon oriP. 11. The performance vector of claim 1, wherein the reverse 27 201035312 to the repetitive element further comprises a mediation sequence (central seqUenee) located between the two lateral nucleic acid sequences and different from the foregoing two A nucleic acid sequence of any of the flanking nucleic acid sequences. 12. The performance carrier of claim 1, wherein the at least one reverse overlapping element comprises a reverse overlapping element. 1 3. The expression vector of claim 12, wherein the expression vector is PGT02-GFP. 14. The performance carrier of claim 1, wherein the at least one reverse overlapping element comprises two reverse overlapping elements; wherein the two reversed overlapping elements are identical or different from one another. 1 5. The expression vector of claim 14, wherein the expression vector is pGT03-GFP or pGT04-ID. 16. The expression vector of claim 1, wherein the expression vector is used to enhance expression of the aforementioned gene in mammalian cells. 17. A human cell comprising a performance vector as described in claim 1 of the patent application. 〇 18. The human cell according to claim 17, wherein the human cell line is a mismatch repair system defective cell. The human cell described in claim 17 of the patent application, wherein the human cell line is a mismatch repair system normal cell. 20. An organized carrier of inverte (j dimer ' ID) comprising a circular nucieic aeid m〇lecule having a two-length sequence 〇〇ng sequence, The long sequence is separated by two inverted repeating elements; wherein 'the aforementioned two long sequences are complementary to each other, and each long sequence includes 28 201035312 to > a gene and a nuclear anchoring element; Each of the reverse overlapping elements has a face nucleic acid sequence complementary to the other side nucleic acid sequence; and the 二 sequence of the two inverted repeat elements are identical or different from each other. The aforementioned expression vector is used to transfect a mammalian cell. And the vector expression vector is located outside the chromosome in the aforementioned mammalian cells.

The performance vector described in the 2G item of the Shishen 4 patent, the protein encoded by the aforementioned gene, which is selected from the group consisting of a message peptide, a growth factor, a hormone, an interleukin, a chemokine, a neuropeptide, an antigen, Antibodies, enzymes, coagulation factors, anti-angiogenic factors, pre-angiogenic factors +, transport proteins, receptors, ligands, regulatory proteins, structural proteins, reporter proteins, transcription factors, ribozymes, fusion proteins, and drug-resistant proteins The group formed. 22. The expression vector of claim 2, wherein the mammalian cell line is a human cell.

23. The expression vector of claim 22, wherein the human cell line is a mismatch repair system defective cell. 24. The expression vector of claim 22, wherein the human cell line is a mismatch repair system normal cell. 25. A human cell comprising a performance vector as described in claim 20 of the patent application. 26. The human cell of claim 25, wherein the human cell line is a mismatch repair system defective cell. 27. The human cell of claim 25, wherein the human cell line is a mismatch repair system normal cell. 29 201035312 28. A method for expressing a gene in a mammalian cell, comprising: transfecting the mammalian cell with the expression vector described in claim 4 to prepare a transfected cell; The aforementioned transfected cells are incubated to produce the protein encoded by the above-mentioned transfected cells; and the aforementioned proteins are obtained. 29. The method of claim 28, wherein the protein is selected from the group consisting of a message peptide, a growth factor, a hormone, an interleukin, a chemokine, a neuropeptide, an antigen, and an antibody. , enzymes, coagulation factors, anti-angiogenic factors, angiogenesis predisposing factors, transport proteins, receptors, ligands, regulatory proteins, structural proteins, reporter proteins, transcription factors, ribozymes, fusion proteins, and drug-resistant proteins Group of. 30. The method of claim 28, wherein the gene is transcribed into an RNA selected from the group consisting of tRNA, rRNA, antisense RNA, microRNA, and double-stranded rna. Q 3 1 . The expression vector according to claim 28, wherein the mammalian cell line is a human cell. 32. The expression vector of claim 31, wherein the human cell line is a mismatch repair system defective cell. 33. The expression vector of claim 32, wherein the defective cell repair system is a defective cell line of HCT116 (hMLH1-) cells or a DLD-1 (hMSH6-) cell. 34. The expression vector of claim 31, wherein the human cell line is a mismatch repair system normal cell. 30 201035312 35. A method for expressing a gene in a mammalian cell, comprising: transfecting the mammalian cell with the expression vector described in claim 20 to prepare a transfected cell; and cultivating the transfected cell The aforementioned transfected cells are made to produce the protein encoded by the aforementioned gene; and the aforementioned protein is obtained.

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