抗呋喃它酮代謝物的衍生物噬菌體單鏈抗體庫的構建

羅翠紅，王 弘,*，劉細霞，沈玉棟，孫遠明，黃佳佳，張宏斌

抗呋喃它酮代謝物的衍生物噬菌體單鏈抗體庫的構建

羅翠紅1，王 弘1,*，劉細霞1，沈玉棟1，孫遠明1，黃佳佳1，張宏斌2

(1.廣東省食品質量安全重點實驗室，華南農業大學食品質量與安全研究所，廣東 廣州 510642；2.廣州軍區廣州總醫院，廣東 廣州 510010)

目的：構建抗呋喃它酮代謝物(AMOZ)的衍生物噬菌體單鏈抗體庫。方法：從分泌抗AMOZ的單克隆抗體的雜交瘤細胞系(BC3-E8)中提取總RNA，經RT-PCR反轉錄成cDNA，設計通用簡并引物，PCR擴增抗體重鏈可變區基因(VH)和輕鏈可變區基因(VL)。經重疊延伸PCR (SOE-PCR)，將VH和VL基因用編碼(G1y4Ser)3的linker隨機拼接成單鏈抗體(scFv)基因，然后將其克隆到噬菌粒載體pCANTAB5E中，轉化大腸桿菌 (Escherichia coli) TG 1感受態細胞，經輔助噬菌體M13K07超感染，建立噬菌體單鏈抗體庫。隨機挑取10個陽性克隆，經PCR和雙酶切鑒定，并測序。登陸DNAMAN軟件對序列進行分析、比對。結果：成功擴增VH、VL及scFv基因，并得到庫容為1.2×106的噬菌體抗體庫，噬菌體的滴度為2.0×1010PFU，PCR鑒定及雙酶切鑒定文庫重組率較高，軟件對序列比對結果顯示，scFv基因全長序列之間差異為8.38%，VH序列差異為3.68%，VL序列差異為14.34%，且序列差異多集中在CDR抗原結合區域對應的核酸序列上。結論：已構建抗呋喃它酮代謝物的衍生物噬菌體單鏈抗體庫，為進一步富集篩選并表達抗AMOZ的衍生物的單鏈抗體提供參考。

抗呋喃它酮代謝物的衍生物；單鏈抗體；噬菌體展示；序列分析

呋喃它酮(furaltadone)屬于硝基呋喃類抗生素藥物，具有相當大的毒性，可導致畸胎突變和誘發癌癥。歐盟早在1995年已規定動物源性食品中硝基呋喃類藥物及其代謝物殘留為不得檢出[1-2]。但因其具有良好的抗菌效果和生長促進功能，非法使用現象依然存在。由于它對光敏感，會迅速被代謝和分解，從肉源性動物組織內很難檢出原藥殘留[3]。但呋喃它酮的代謝物5-甲基嗎啉-3-氨基-2-唑烷基酮(AMOZ)能以蛋白結合物的形式長期穩定存在組織內。故可通過檢測AMOZ達到檢測呋喃它酮的目的。

目前，AMOZ檢測方法主要有色譜法[4-5]、免疫分析法等[6]。但色譜法流程繁瑣、設備昂貴、檢測速度慢。而免疫分析法具有簡單、快速、靈敏度較高、特異性強等諸多優點。建立免疫法的關鍵是制備高特異性、高親和力的抗體。噬菌體表面呈現技術的出現，為抗體的制備提供了新途徑。同時，利用工程菌制備抗體周期短，成本低，有利于在實際中應用[7]。近年來，噬菌體表面展示技術已應用于新型疫苗的研制[8-9]、小分子有毒有害物抗體的制備[10]、藥物的開發[11-12]以及診斷試劑的研制[13-14]等領域。然而，應用噬菌體表面展示技術生產抗AMOZ的衍生物抗體的研究尚未見報道。本研究利用噬菌體表面展示技術，旨在構建抗AMOZ的衍生物單鏈抗體噬菌體表面展示文庫，為下一步篩選高特異性的抗AMOZ的衍生物抗體提供實驗基礎。

1 材料與方法

1.1 菌株與載體

E. coli TG1菌株及分泌抗AMOZ的衍生物單克隆抗體的雜交瘤細胞株(BC3-E8)為本實驗室保存；噬菌粒載體pCANTAB5E購自Pharmacia公司。

1.2 試劑

cDNA第一鏈合成試劑盒 Promega公司；限制性內切酶、T4DNA連接酶和DNA聚合酶、膠回收試劑盒 大連TaKaRa公司；質粒提取試劑盒、M13K07 廣州佰路公司；蛋白胨、酵母提取物 Ameresco公司。

1.3 PCR引物

以文獻[15-16]報道的引物設計方法為基礎，參照GenBank數據庫以及所選linker(Gly4Ser)3的核苷酸編碼序列，設計鼠源單克隆抗體可變區基因引物，設計時既保證了引物的特異性，又適當增加了引物的簡并性。所設計引物均由上海生物工程技術服務有限公司合成。其中引物VH(back)、VH(for)為VH基因擴增引物，VL(back)、VL(for)為VL基因擴增引物，VHS(back)、VLS(for)分別為帶有SfiⅠ和NotⅠ酶切位點的擴增引物，VH-linker(for)、VL-linker(back)均為帶有部分linker序列的擴增引物，畫線部分為酶切位點(表1)。

1.4 總RNA的提取及逆轉錄

從分泌抗AMOZ的衍生物的單克隆抗體雜交瘤細胞系(BC3-E8)采用Trizol法提取總RNA，產物用Oligo (dT)18引物經RT-PCR逆轉錄合成cDNA。

1.5 VH和VL基因擴增

以cDNA第一鏈為模板，VH(back)、VH(for)為擴增VH的引物，反應條件為：94℃預變性2min；94℃、30s，56.7℃、1min，72℃、1min，共25個循環；72℃延伸10min。VL基因的擴增，以cDNA第一鏈為模板，VL(back)、VL(for)為擴增引物，反應條件同重鏈。反應產物經1%瓊脂糖凝膠電泳鑒定后，回收目的片段。

1.6 全長scFv基因的拼接及PCR擴增

以VH基因為模板，VHS(back)、VH-linker(for)為引物，擴增帶部分linker序列的VH基因片段，反應條件參照1.5節。以VL基因為模板，VL-linker(back)、VLS(for)為引物，擴增帶部分linker序列的VL基因片段，反應條件參照1.5節。反應產物經1%瓊脂糖凝膠電泳鑒定后，回收目的片段。然后以等體積的VH-linker和VL-linker片段互為模板，進行重疊延伸拼接PCR(SOE-PCR)反應，隨機拼接成scFv，如圖1所示，反應條件為：94℃預變性5min；94℃、45s，57℃、1min，72℃、1min，共20個循環；72℃延伸10min。然后以VHS(back)和VLS(for)為引物，二次PCR擴增scFv，反應條件為：94℃預變性5min，94℃、30s，57℃、1min，72℃、1min，共25個循環；72℃延伸10min。反應產物經1%瓊脂糖凝膠電泳鑒定后，回收目的片段。

表1 PCR擴增所用引物及其序列Table 1 Primer sequences used for the PCR amplification

圖1 scFv基因片段拼接示意圖Fig.1 Splicing diagram of scFv genes

1.7 噬菌體單鏈抗體表面展示文庫構建

回收后的scFv片段，先后用限制性內切酶SfiⅠ，NotⅠ雙酶切消化，產物純化后與同樣酶切處理的噬菌體載體pCANTAB5E連接。連接產物轉化感受態E. coli TG 1，轉化菌液100μL用以倍比稀釋，取稀釋105倍、106倍和107倍的菌液各100μL，涂布于LB AG(胰蛋白胨10g/L、酵母提取物5g/L、氯化鈉10g/L、氨芐青霉素100μg/mL、葡萄糖2g/100mL)瓊脂平板上，37℃溫箱培養過夜。觀察過夜培養的平板，計算生長的菌落數，推算庫容[17]。其余菌液用2×YT AG(胰蛋白胨16g/L、酵母提取物10g/L、氯化鈉5g/L、氨芐青霉素100μg/mL、葡萄糖終質量濃度2g/100mL)稀釋培養，加入輔助噬菌體M13K07，37℃振搖1h，4℃、5000r/min離心10min，沉淀用2×YT AK(K為卡那霉素，50μg/mL)重懸，振搖培養過夜。次日，4℃、5000r/min離心10min收集上清，加入1/5體積的PEG-NaCl溶液，冰浴45min，4℃、10000r/min離心20min，PBS重懸沉淀即為scFv噬菌體表面展示文庫。

1.8 噬菌體抗體庫滴度測定

取10μL scFv噬菌體表面展示文庫，用2×YT培養基連續10倍稀釋，每個稀釋度取100μL，加900μL新鮮制備的對數期E. coli TG 1，37℃放置15min，各取100μL涂布于LB AG平板。37℃溫箱培養過夜，計算菌落，估計噬菌體抗體庫滴度[17]。噬菌體抗體庫滴度(用噬菌體顆粒的菌落形成單位PFU表示)=10×集落數×稀釋倍數。

1.9 抗體庫鑒定

從平板上隨機挑取10個克隆，擴大培養后提取噬菌粒，以含有SfiⅠ和NotⅠ酶切位點的單鏈抗體引物VHS(back)和VLS(for)為引物進行PCR擴增，產物經1%瓊脂糖凝膠電泳檢測。PCR和雙酶切鑒定為陽性的克隆，送上海生物工程有限公司測序，所得序列用DNAMAN軟件進行比對分析。

2 結果與分析

2.1 總RNA的提取

圖2 總RNA 1%瓊脂糖凝膠電泳結果Fig.2 One percent agarose gel electrophoresis of the total RNA of BC3-E8

采用Trizol試劑從雜交瘤細胞株中提取總RNA，由圖2可知，瓊脂糖凝膠電泳結果可見明顯的3條帶，分別為28S、18S和5S，且條帶清晰，所提取的總RNA較完整。

2.2 VH、VL和scFv基因片段的擴增

圖3 VL、VH及scFv PCR擴增結果Fig.3 Results of PCR amplification of VL, VH and scFv

以cDNA第一鏈為模板，PCR擴增得到VH、VL基因，由圖3可知，VH和VL基因片段分別大約為340bp和330bp，與預期的片段大小相符。將純化后的VH和VL基因與編碼 (G1y4Ser)3的linker基因片段拼接成scFv并引入SfiⅠ、NotⅠ位點，結果顯示擴增產物約為750bp，與預期片段大小相符。

2.3 噬菌體抗體庫的構建與鑒定

圖4 重組噬菌體PCR鑒定Fig.4 Identification of recombinant phages by PCR amplification

圖5 重組噬菌體的酶切鑒定Fig.5 Identification of recombinant phages by double digestion

轉化菌液涂布于LB AG平板，經計算長出約1.2× 106個菌落。隨機挑取10個克隆進行PCR和雙酶切鑒定。結果顯示，10個克隆均擴增出750bp的片段，目的基因插入率較高(圖4)。以EcoRⅠ、Hind Ⅲ對克隆進行雙酶切鑒定，若克隆子DNA不含scFv插入片段，則大小約為1.4kb，若含scFv插入片段，則大小約為2.1kb，結果10個克隆均得到2.1kb和3.1kb兩個片段(圖5)，分別與含有scFv基因的片段及pCANTAB5E噬菌體的骨架片段吻合。結果表明，已成功將scFv基因克隆到pCANTAB5E噬菌粒載體，構建了單鏈抗體庫。經M13K07超感染后，噬菌體的滴度為2.0×1010PFU。

2.4 scFv基因序列分析

圖6 VH-CDR區對應核酸序列比對圖Fig.6 Alignment of VH-CDR corresponding nucleic acid sequences

圖7 VL-CDR區對應核酸序列比對圖Fig.7 Alignment of VL-CDR corresponding nucleic acid sequences

表2 VH-CDR區對應核酸序列比例差異表Table 2 Difference in the proportion of VH-CDR corresponding nucleic acid sequences

表3 VL-CDR區對應核酸序列比例差異表Table 3 Difference in the proportion of VL-CDR corresponding nucleic acid sequences

對10個陽性噬菌體克隆scFv序列進行分析，每種scFv序列均包含VH、VL和linker區，其中VH基因位于linker的上游，VL基因位于linker的下游，序列中包含有SfiⅠ和NotⅠ酶切位點，表明scFv基因片段成功插入載體pCANTEB5E。采用DNAMAN軟件對scFv序列進行比對，發現6條不同的scFv，且scFv基因全長序列之間差異為8.38%，VH序列差異為3.68%，VL序列差異為14.34%，且差異多集中在CDR區對應的核酸序列。從CDR區對應核酸序列位置分析，VH-CDR1差異為8.33%，VH-CDR2差異為2.54%，VH-CDR3差異為3.03%。VL-CDR1差異為16.67%，VL-CDR2差異為33.33%，VL-CDR3差異為26.00%，結果見圖6、7。6條不同scFv序列的VH基因CDR區對應的核酸序列差異構成見表2，VL基因CDR區對應的核酸序列差異構成見表3。

3 討 論

噬菌體抗體庫技術作為第三代抗體制備技術，具有基因型與表型相統一、選擇與擴增相統一的優點，已被廣泛用于抗體高通量篩選[18-21]。目前針對小分子有害物所構建噬菌體中，大部分是以雜交瘤細胞系為基因來源。這主要是因為，一方面，免疫過程在體內經過了抗原選擇和親和力成熟，富集了編碼可變區靶特異性抗體基因，增加了獲得針對靶抗原較高親和力的抗體的可能性[22]；另一方面，經過篩選獲得的雜交瘤細胞中抗體基因單一，干擾少，易于從RNA中采用RT-PCR方法克隆抗體可變區基因，甚至可在表達載體中直接表達出功能性單鏈抗體[23]。本研究以分泌抗AMOZ的衍生物單克隆抗體的雜交瘤細胞(BC3-E8)為源，所建抗體庫庫容可達1.2×106。同時，通過DNAMAN軟件對scFv基因序列的分析顯示，在重鏈和輕鏈的互補決定區(CDRs)，6條序列均顯示較為顯著差異。因CDR區是識別抗原的主要區域，可以看出，本研究所構建抗體庫具有多種抗原識別區。該結果為后期篩選特異性抗體提供參考。

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Construction of a Phage Single Chain Fv Library against Derivatives of Furaltadone Metabolites

LUO Cui-hong1，WANG Hong1,*，LIU Xi-xia1，SHEN Yu-dong1，SUN Yuan-ming1，Huang Jia-jia1，ZHANG Hong-bin2
(1. Key Laboratory of Food Quality and Safety of Guangdong Province, Research Institute of Food Quality and Safety, South China Agricultural University, Guangzhou 510642, China；2. Guangzhou Army General Hospital, Guangzhou 510010, China)

Objective: To construct a phage single chain Fv (scFv) library against derivatives of furaltadone metabolites (AMOZ). Methods: The total RNA extracted from a hybridoma cell line (BC3-E8) secreting monoclonal antibodies against AMOZ was reverse-transcribed into cDNA by RT-PCR. The heavy chain (VH) and the light chain (VL) variable region genes were amplified respectively by PCR with the previously designed degenerate primer. The VH and VL genes were spliced into scFv fragment with a DNA linker encoding (G1y4Ser)3 by splicing overlap extension (SOE). Then the scFv fragment was cloned into the phagemid pCANTAB5E and after the cloning, the phagemid was transformed into the competent Escherichia coli TG1. With the rescue of helper phage M13K07, a phage scFv library was constructed. Ten positive clones were randomly selected and identified by PCR and double enzymatic digestion. Furthermore, the sequences of these positive clones were sent for sequencing and analyzed by the DNAMAN software. Results: VH, VL and scFv DNA fragments were amplified successfully. The constructed phage scFv library had a capacity of 1.2 × 106and the titre was about 2.0 × 1010PFU. PCR and double enzymatic digestion identification showed that the ratio of positive insert was high. Multiple sequence alignment showed that the difference of scFv sequences was 8.38%, and those of VH and VL sequences were 3.68% and 14.34%, respectively, and the discrepancy were mostly concentrated in corresponding nucleic acid sequences of the CDR antigen region. Conclusion: a scFv phage antibody library against derivaties of furaltadone metabolites has been constructed successfully. This will lay a foundation for the further enrichment and expression of scFv.

derivatives of furaltadone metabolites；single chain Fv (scFv)；phage display；sequencing

Q78

A

1002-6630(2010)19-0283-05

2010-02-04

國家“863”計劃項目(2007AA10Z437)；國家自然科學基金項目(30871755)；廣東省科技廳科技計劃項目(2009A020101004)；廣東省-教育部產學研結合項目(2009B090300409)

羅翠紅(1984—)，女，碩士研究生，研究方向為食品質量與安全。E-mail：yhcuihluo@yahoo.cn

*通信作者：王弘(1973—)，女，副教授，博士，研究方向為食品質量安全。E-mail：gzwhongd@163.com