TWI691598B - 以整體表現細胞作為抗原載體及其於製備疫苗或診斷試劑及篩選單株抗體之應用 - Google Patents

以整體表現細胞作為抗原載體及其於製備疫苗或診斷試劑及篩選單株抗體之應用 Download PDF

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TWI691598B
TWI691598B TW104133204A TW104133204A TWI691598B TW I691598 B TWI691598 B TW I691598B TW 104133204 A TW104133204 A TW 104133204A TW 104133204 A TW104133204 A TW 104133204A TW I691598 B TWI691598 B TW I691598B
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virus
animal cell
antigen
use according
surface protein
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TW201713771A (zh
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柯舜文
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台灣生物製劑股份有限公司
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Abstract

本發明係關於一種哺乳動物細胞經由共轉染方式製備，包含具有T7啟動子與目標抗原蛋白的開放閱讀框(ORF)之表現質體，及可表現T7聚合酶之vT7重組牛痘病毒，並以整體表現抗原的細胞，其製備方法及其作為抗原載體應用於製備疫苗，診斷試劑及篩選單株抗體的用途。

Description

以整體表現細胞作為抗原載體及其於製備疫苗 或診斷試劑及篩選單株抗體之應用
本發明係關於一種哺乳動物細胞，經由共轉染方式製備，包含具有T7啟動子與目標抗原蛋白的開放閱讀框(ORF)之表現質體，及可表現T7聚合酶之vT7重組牛痘病毒，並以整體表現抗原的細胞，其製備方法及其使用作為抗原載體應用於製備疫苗，診斷試劑及篩選單株抗體的用途。
疫苗的主要成分為抗原，最好的抗原當然是完整的病原，如口蹄疫病毒(Foot-and mouth disease virus，FMDV)或豬流行性下痢病毒(Porcine epidemic diarrhea virus，PEDV)等。有些病原難以培養或無法培養出具有商業生產價值的力價，或是需要在高生物安全規範之下生產抗原(如FMDV)時，可以引用生物技術生產此類抗原。
然而，病毒可依其結構是否具有封套(envelop)可分為封套(envelop)，無封套(non-envelop)病毒。封套病毒，如PEDV可以刺激免疫或作為疫苗的抗原，全為結合於細胞膜(封套)的表面蛋白。無封套病毒，如FMDV，則為在細胞內自行組合成完整病毒顆粒。能夠產生最佳免疫反應的病毒抗原當然是越接近於其原始型態的蛋白質或構型是最好的選擇。
傳統的病毒抗原製備是直接培養病毒本身，新一代的抗原製備是將具抗原性的蛋白質基因經分子選殖後進行表現及純化。由於病毒感染的對象如人類、經濟動物、寵物、家禽、魚、蝦等，均為具真核生物的高等生物。細胞的蛋白質合成機制與原核生物有極大的不同。對於是否能合成與病毒最近似的蛋白質，決定了抗原的品質以及其能否成功用於製備疫苗、診斷試劑及篩選單株抗體的先決條件。
因此，為生產具免疫原性的病毒抗原，嘗試將表面蛋白全長開放閱讀框(ORF，Open Reading Frame)(如PEDV表面蛋白)，或在N-端加上起始胺基酸碼ATG，在C-端加上結束胺基酸碼TAA/TAG/TGA)(如口蹄疫病毒之VP1，VP2，VP3)，直接在細胞進行表現，以生產能與原始病毒特性一致的抗原蛋白質。本發明目的即為達到最佳化(質與量)的抗原表現。
本發明基於以上之目的，遂利用共轉染方式，獲得一包含具有T7啟動子與目標抗原蛋白的開放閱讀框(ORF)之表現質體，及一可表現T7聚合酶之vT7重組牛痘病毒的哺乳動物細胞。此一動物細胞/抗原系統可以表現出完整且符合原始結構的病毒性抗原，並用於作為製造疫苗，篩選單株抗體以及診斷用抗原的來源。
於是，本發明之一方面係關於，一種作為目標抗原載體之表現系統，其包含：(a)一含有T7啟動子與全長目標抗原蛋白的開放閱讀框(全長ORF)之表現質體；及(b)可表現T7聚合酶之vT7重組牛痘病毒；及(c)哺乳動物細胞。
於本發明之一些具體實施態樣，所述之抗原表現系統為一哺乳動物細胞，經過共轉染後可表現完整病毒或蛋白質。於本發明之一具體實施態樣，所述之動物細胞為Vero細胞系。於本發明之另一些具體實施態樣，所述之目標抗原蛋白的開放閱讀框係位於T7啟動子之下游。
於本發明之一些具體實施態樣，所述之目標抗原為一種病毒抗原。於本發明之一項具體實施態樣，所述之病毒抗原為封套病毒之表面蛋白。於本發明之一項具體實施態樣，所述之封套病毒為豬流行性下痢病毒(PEDV)。於本發明之另一項具體實施態樣，所述之病毒抗原為無封套病毒之類病毒顆粒。於本發明之一項具體實施態樣，所述之封套病毒 為豬環狀病毒二型(PCV2)。於本發明之另一項具體實施態樣，所述之目標抗原為口蹄疫(FMDV)病毒顆粒。
本發明之另一方面，係關於製備如前所述之抗原表現動物細胞的方法，其包含：將帶有T7聚合酶基因的重組牛痘病毒株感染動物細胞；將經感染的動物細胞以一包含一具有T7啟動子與編碼目標抗原蛋白的開放閱讀框(ORF)之表現質體進行共轉染，而獲得一表現目標抗原之抗原表現動物細胞。
於本發明之一些項具體實施態樣，所述之動物細胞為Vero細胞系。於本發明之另一些項具體實施態樣，所述之動物細胞為可感染牛痘病毒的哺乳動物細胞。
本發明之又一方面，係提供一種疫苗組成物，其包含如前所述之動物細胞作為抗原載體直接免疫動物。
本發明之又一方面，係提供一種用於篩選單株抗體之抗原組成物，其包含如前所述之動物細胞作為抗原載體。
本發明之又另一方面，係提供一種偵測動物是否受目標病毒感染之診斷套組，其包含如前所述之抗原表現動物細胞作為偵測血清樣本是否存在目標病毒抗體之抗原載體。
圖1為質體pT7CFE PEDv_TW_S之限制酶圖譜。
圖2係顯示以螢光免疫分析及螢光顯微鏡觀察，經由實施例二製備之Vero細胞表現PEDv表面蛋白的特異性螢光結果。
圖3係質體pT7CFE PCV2_ORF2之限制酶圖譜。
圖4係顯示於顯微鏡下觀察Vero細胞表現PCV2蛋白的特異性螢光結果標準的陽性如圖4A，陰性如圖4B。
本發明之其他特色及優點將於下列實施範例中被進一步舉例與說明，而該實施範例僅作為輔助說明，並非用於限制本發明之範圍。
實施例一、T7聚合酶基因之選殖
全長的T7聚合酶基因係由E.coli株BL21(DE3)經PCR而得，並以下列引子(前向引子：TTT GCGGCCG CTTTTTTTTT TTTTTTTTTT TGGCATATAA TAACATCGCT AAGAAC，SEQ ID No.1；反向引子：TTTGCGGCCG CTTACGCGAA CGCGAAGTCC，SEQ ID No.2)將牛痘的啟動子導入。所得到的DNA再以PCR方式送入pGS20質體的TK1，TKr中。最後以共轉染方式與野外株牛痘病毒(WR strain)感作Vero細胞。進行基因重組，並利用BrDu(Bromodeoxyuridine)篩選法，選殖出帶有T7聚合酶基因(2652核苷酸，883 a.a.，DNA序列如SEQ ID No.3所示)的重組牛痘病毒株。並經過三次溶病毒斑純化，得到純的vT7病毒株，命名為vT7FBI。
其特性經共轉染Co-transfect方式確認，可以表現含由T7啟動子控制之表現體系。方法為先將Vero細胞以vT7FBI病毒感染後(0.1MOI)，再加入pT7-GFP/turbofect的混和物。隔天後直接以螢光顯微鏡觀察，可見細胞充滿綠色螢光，而陰性控制組則無，可見經細胞、vT7FBI以及T7啟動子控制之ORF是可以操作表現目標蛋白質。
實施例二、製備表現PEDV表面蛋白之細胞
利用RT-PCR方式，選殖得到由彰化地區所分離之PEDV病毒株的全長Spike蛋白質(a.a.：1386)基因(4161bp)，進行，其cDNA序列如SEQ ID No.4所示。利用PCR方式，將該cDNA安置於T7啟動子的下游。並建構可以使用T7啟動表現的質體pT7CFE PEDv_TW_S(其質體圖譜如圖1所示)。
細胞的表現方式為利用Vero細胞為宿主細胞，T7重組牛痘病毒(vT7FBI，1MOI)，以及質體pT7CFE PEDV_TW_S。Vero細胞先消化後生長於培養瓶過夜後，約 70-80%滿。棄去培養液。以1MOI的比例，加入如實施例一製備之vT7重組牛痘病毒，並於37℃下先感作約3小時。
製備質體pT7CFE PEDV_TW_S與PEI的混和液。於150cm培養瓶中置入10mL不含血清抗生素phenol red的DMEM，及20μg質體pT7CFE PEDV_TW_S DNA，將PEI(Polysciences,Inc.Cat#23966 1μg/μl)20ul加入，混合均勻。將所得之混合液慢慢加入上述已感染vT7重組牛痘病毒的Vero細胞中。混合均勻後，加入含2%血清的DMEM，於37℃、5%二氧化碳培養箱中培養24-36小時。之後，立刻以0.02%甲醛固定細胞，並測定於該重組Vero細胞之PEDV蛋白的表現量。
採取部分細胞進行免疫螢光染色。待測樣品，以PBS先進行清洗後加入抗-PEDV單株抗體(Anti-PEDV S-M1-5，自行製備，製備方式見實施例七)，於室溫作用一小時後，以PBS清洗三次。再加入FITC結合兔子抗-豬IgG抗血清(Biomedicals,FITC-conjugated rabbit fraction to swine IgG Cat# 55824)，以PBS 500倍稀釋)，室溫作用一小時後。以PBS清洗三次。即可觀察抗PEDv表面蛋白的特異性螢光。結果如圖2所示。多數(大於95%)細胞的表面均呈現螢光。
PEDV中和抗體檢測
血液樣本先經56℃加熱感作三十分鐘，以去除血清中之補體。進行2X連續樣品稀釋：先於96孔盤內加入2% FBS DMEM細胞培養液，每孔均加入100μL。將未稀釋之待測血清置於96孔盤最下層之H列，每個樣品加入鄰近兩孔，每孔均加入100μL。以十二爪微量吸管在H列將血清與2% FBS DMEM細胞培養液充分混合，吸出100μL移注至G列，再以12爪微量吸管與2% FBS DMEM細胞培養液充分混合，再吸出100μL移注至F列，重複上述步驟將血清自H列以連續兩倍稀釋至A列，最後從A列吸取100μL混合稀釋液丟棄。
將PEDV病毒以2% FBS DMEM細胞培養液作適當稀釋，使病毒濃度為100 TCID50/50μL(即2×103 TCID50/mL)，於含有已經稀釋好之血清樣品的96孔盤，每孔加入100μL稀釋標準病毒液，置於37℃、5%二氧化碳培養箱中感作1小時。每次試驗均需設置對照組，包括細胞對照組、陽性血清對照組、血清對照組(為測試該批血清是否具細胞毒性)、陰性血清對照組等。其中，細胞對照組：對照細胞的位置，加入200μL 2% FBS DMEM細胞培養液；陽性血清對照組：取已知中和抗體力價的血清作為陽性對照。其操作方法與前述檢測血清的操作方法相同。在作用5-7天後於顯微鏡下觀察其CPE(Cytopathic effect)。沒有CPE者代表有中和抗體反應。
實施例三、製備表現PCV2 ORF2之重組載體細胞
利用PCR方法，以台南地區所分離之豬環狀病毒二型病毒株(FBI_PCV2)之全長ORF2(702核苷酸，233 a.a.)為模板，進行基因選殖，其cDNA序列如SEQ ID No.5所示。利用PCR方式將該cDNA置於T7啟動子的下游。並建構可以使用T7啟動表現的質體pT7CFE PCV2_ORF2(其質體圖譜如圖3所示)。細胞的表現方式為利用Vero細胞為宿主細胞、T7重組牛痘病毒(vT7FBI，1MOI)，以及質體pT7CFE PCV2_ORF2。方法如下述：Vero細胞先消化後生長於培養瓶過夜，約達70-80%滿。棄去培養液。以1MOI的比例，將如實施例一製備之vT7FBI毒加入該Vero細胞，並於37℃下先感作約3小時進行感染。
製備包含質體pT7CFE PCV2_ORF2與PEI(Polysciences,Inc.Cat#23966 1ug/ul)的混和液。於150cm培養瓶中包含：10mL不含血清抗生素phenol red的DMEM、20μg質體pT7CFE PCV2_ORF2 DNA及20ul PEI(Polysciences,Inc.Cat#23966 1μg/μl)。混合均勻後之後，將所得之混合液 慢慢加入上述已經vT7FBI重組牛痘病毒感染的Vero細胞中。混和均勻後，加入含2%血清的DMEM培養液，置於37℃、5%二氧化碳培養箱中進行培養24-36小時。之後立刻以0.02%甲醛固定細胞。並測定該重組Vero細胞之PCV2 ORF2表現量。
採取部分細胞進行免疫螢光染色。待測樣品(重組Vero細胞，2X104/cell/well)以PBS先進行清洗後，加入豬抗-PCV2抗體L36(SPF豬感染PCV2之抗血清，自行製備)，於室溫作用一小時後，以PBS清洗三次。再加入FITC結合山羊抗-豬IgG抗血清(MP Biomedicals,FITC-conjugated goat IgG fraction to mouse immunoglobulins IgG,IgA,IgM,Cat number 55499)，以PBS 500倍稀釋)，於室溫作用一小時。以PBS清洗三次。之後以顯微鏡觀察抗-PCV2 ORF2的特異性螢光。細胞的表現量一般都大於95%。
將製得之完整的重組Vero細胞作為抗原進行免疫注射，約106/細胞可作成一劑。採用油質佐劑，或鋁膠佐劑進行免疫注射SPF豬隻。經過三次免疫注射後，取血清以下列PCV2 IFA抗體法檢測其抗體力價。
實施例四、PCV2 ORF2抗原盤之製備
準備達80%佈滿之Vero細胞置於96-孔細胞平盤之各孔(2X104/cell)，並加入0.1MOI如實施例一製備之重組vT7病毒(vT7FBI)進行感染。以maxi-prep抽取質體pT7CFE PCV2_ORF2 DNA並定量。將質體pT7CFE PCV2_ORF2 DNA與PEI混和均勻。將DNA/Turbofect®加入細胞盤。進行轉染24小時後棄去上清液，並加入80%丙酮固定。棄去丙酮，乾燥，此即為以重組細胞為抗原表現載體之PCV2 ORF2抗原盤。包上護膜。保存於-80℃。
將待測血清加入96孔平盤之各孔內，以PBS先進行連續2X稀釋(初始稀釋倍數initial dilution為50X)後，再轉移至上述製備之PCV2 ORF2抗原盤，於室溫作用一小時後，以PBS清洗三次。再加入FITC結合山羊抗豬IgG抗血清 ((MP Biomedicals,FITC-conjugated goat IgG fraction to mouse immunoglobulins IgG,IgA,IgM,Cat number 55499)，以PBS 500倍稀釋)，於室溫作用一小時。之後以PBS清洗三次。於顯微鏡下觀察螢光抗體力價。標準的陽性如圖4A，陰性如圖4B。上圖4(A)為標準的PCV2陽性螢光，多數細胞的螢光均出現在細胞核，與此一蛋白質的特性相符合。下圖4(B)為同一區域之可見光。
實施例五、表現PEDV表面蛋白質全長細胞作為抗原的免疫試驗
Vero細胞先消化後生長於培養瓶過夜後，約70-80%滿。棄去培養液。以1MOI的比例加入vT7重組牛痘病毒。並於37℃先感作約3小時。之後加入質體pT7CFE PEDV_TW_S與PEI的混和液。150cm培養瓶：10mL(不含血清抗生素phenol red的DMEM)20ug質體pT7CFE PEDV_TW_S DNA加入PEI(Poysciences,Inc.Cat#23966 1μg/μl)20μl。混合均勻後慢慢加入以感染vT7重組牛痘病毒vT7FBI的Vero細胞中。混和均勻後，加入含2%血清的DMEM培養24-36小時。之後立刻以0.02%甲醛固定細胞。並測定表現量。細胞的表現量一般都大於95%(如圖2)。
以如上所述製備之完整的Vero細胞作為抗原進行免疫注射，約106/細胞可作成一劑。採用油質佐劑，或鋁膠佐劑進行免疫注射SPF豬隻。經過三次免疫注射後(免疫行程如表1)，以PEDV中和抗體法檢測血清中的PEDV抗體力價。結果如下表1所示。
Figure 104133204-A0305-02-0009-1
Figure 104133204-A0305-02-0010-2
結果顯示，利用本發明之Vero細胞表現PEDv表面蛋白作為抗原免疫之SPF豬隻血清以中和抗體力價以注射2ml/劑量的抗體力價最佳，且最少需要兩次注射。
實施例六、特異性病毒病原抗原盤的製備
依照實施例四中所述製備用於檢測PCV2 IFA抗體力價之PCV2 ORF2抗原盤的方法，利用動物細胞為宿主細胞，以vT7重組牛痘病毒(1MOI)，與包含置於T7啟動子下游之編碼所希望抗原的DNA片段之表現質體進行共轉染，即可直接在習知用於抗原偵測的物品如96孔細胞平盤，製作用於檢測該所希望抗原之抗體的抗原盤。
實施例七、抗-PEDV單株抗體之篩選
將根據上述實施例二之方法製備得之表現PEDV表面蛋白的細胞(106細胞/隻)，直接以腹腔注射方式免疫小鼠(Balb/C)，待中和抗體達100X以上之後，犧牲小鼠取脾臟細胞進與SP2細胞行融合。抗原的篩選係利用實施例四相同的方法，將表現PEDV表面蛋白的細胞培養於96孔盤，作為篩選單株抗體用之抗原盤。結果，獲得一株陽性對PEDV表面蛋白有反應的單株抗體命名為S-M1-5。
實施例八、製備表現口蹄疫FMDV病毒顆粒之重組載體細胞
口蹄疫病毒外殼蛋白可以自組裝成處理形成空外殼顆粒。而且，口蹄疫病毒感染的細胞產生的空外殼顆粒，不具傳染性，與完整病毒具有相同的抗原性和免疫原性。本實例係依照前述之方法，將口蹄疫病毒的多肽，先經由切割而形成小的次單位蛋白，在其N-端加上起始胺基酸碼ATG， 在C-端加上結束胺基酸碼TAA/TAG/TGA，於T7啟動子之調控下由本發明之重組細胞表現出對應的蛋白質片段，例如口蹄疫病毒之3A、3B、3C、3AB或3ABC等次單位蛋白。
以FMDV O/97的VP4(1A)，VP2(1B)，VP3(1C)進行表現並以11種來自台灣大學獸醫學系的單株抗體進行染色，發現只有一株有反應其他均為陰性。而於P1全長(VP4231)的表現系統，僅單株抗體Q10E-3、K12F-1、及N10E-1出現陽性，但是其他均為陰性，顯示只有P1無法組合出完整病毒顆粒。具有P1的主要抗原VP231全長的DNA無論同時加入3ABC，或3C共同轉染細胞後均可與多數單株抗體產生反應結果與P1同時加入3ABC，或3C相似，但是螢光反應更強，顯示可形成更多的對應抗原(Viroid)(參見下表2)。
Figure 104133204-A0305-02-0011-3
結果顯示，P1_2A將P12A再接上3ABC組合成P1_2A_3ABC或VP231_2A將VP2312A再接上3ABC組合成P1_2A_3ABC均可表現出與兩種DNA共同轉染細胞所得的螢光結果，顯示兩種蛋白質均可在細胞內合成並進行其原有之功能。完整的FMDV病毒結構除了需要P1全長(或僅需要VP231)之外，還需要2A以及3ABC，或3C的協同作用。最佳的DNA組合必須連結P1_2A_3ABC或VP231_2A_3ABC，而且後者又比前者反應更佳，顯示VP4對於組合完整病毒顆粒沒有決定性的影響。顯然，本發明之表現系統可以在沒有感染性FMDV的前提下，合成具完整抗原性的病毒顆粒，因此經過超過濾後，即可製作口蹄疫疫苗的原料。而且，本實例之結果同時也證明，本發明之表現系統可直接用於分析一單株抗體所對應的抗原。
【生物材料寄存】
國內寄存資訊【請依寄存機構、日期、號碼順序註記】
財團法人食品工業發展研究所、2015年09月02日、BCRC970066
<110> 臺灣生物製劑股份有限公司
<120> 以整體表現細胞作為抗原載體及其於製備疫苗或診斷試劑及篩選單株抗體之應用
<160> 5
<170> PatentIn version 3.2
<210> 1
<211> 56
<212> DNA
<213> 人造序列
<220>
<223> 正向引子
<400> 1
Figure 104133204-A0305-02-0013-11
<210> 2
<211> 30
<212> DNA
<213> 人造序列
<220>
<223> 反向引子
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Figure 104133204-A0305-02-0013-13
<210> 3
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<213> T7聚合酶
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Figure 104133204-A0305-02-0013-16
Figure 104133204-A0305-02-0014-4
<210> 4
<211> 4161
<212> DNA
<213> 豬流行性下痢病毒(PEDV)
<400> 4
Figure 104133204-A0305-02-0015-17
Figure 104133204-A0305-02-0016-5
<210> 5
<211> 702
<212> DNA
<213> 豬環狀病毒二型(PCV2)ORF2
<400> 5
Figure 104133204-A0305-02-0016-18
Figure 104133204-A0305-02-0017-6

Claims (14)

  1. 一種動物細胞作為整體表現目標病毒表面蛋白抗原載體之用途，其中該動物細胞包含：(a)一含有T7啟動子與全長目標病毒表面蛋白抗原的開放閱讀框(ORF)或其片段之表現質體；(b)一表現T7聚合酶之vT7重組牛痘病毒(vT7FBI，寄存編號BCRC970066)，其中該T7聚合酶基因具有如SEQ ID No.3所示的核苷酸序列；及(c)表現之該目標病毒表面蛋白抗原位於該動物細胞表面。
  2. 如請求項1所述之用途，其中該病毒表面蛋白抗原為一封套病毒之表面蛋白。
  3. 如請求項2所述之用途，其中該封套病毒為豬流行性下痢病毒(PEDV)。
  4. 如請求項2所述之用途，其中該封套病毒為豬環狀病毒二型(PCV2)。
  5. 如請求項1所述之用途，其中該病毒表面蛋白抗原為無封套病毒之類病毒顆粒。
  6. 如請求項5所述之用途，其中該病毒表面蛋白抗原為口蹄疫病毒(FMDV)之外殼顆粒。
  7. 如請求項1所述之用途，其中該目標病毒表面蛋白抗原的開放閱讀框或其片段係位於該T7啟動子的下游。
  8. 如請求項1所述之用途，其中該動物細胞為Vero細胞系。
  9. 如請求項1所述之用途，其中該動物細胞為用於感染牛痘病毒的哺乳動物細胞。
  10. 一種動物細胞作為製備疫苗組成物之用途，包含如請求項8所述之動物細胞及一免疫上可接受的載劑或佐劑，其中該動物細胞係作為抗原載體直接免疫動物。
  11. 一種動物細胞作為製備用於篩選單株抗體的組成物之用途，包含如請求項1所述之動物細胞，其中該動物細胞係作為篩選單株抗體之抗原。
  12. 一種動物細胞作為製備偵測動物是否受目標病毒感染的診斷套組之用途，包含如請求項1所述之動物細胞，其中該動物細胞係作為偵測血清樣本是否存在目標病毒抗體之抗原載體。
  13. 如請求項12所述之用途，其中該套組為一種免疫螢光分析套組。
  14. 如請求項12或13所述之用途，其中該動物細胞係貼附及固定於分析平盤。
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