羅翠紅，王 弘,*，劉細(xì)霞，沈玉棟，孫遠(yuǎn)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(gòu)建

羅翠紅1，王 弘1,*，劉細(xì)霞1，沈玉棟1，孫遠(yuǎn)明1，黃佳佳1，張宏斌2

(1.廣東省食品質(zhì)量安全重點(diǎn)實(shí)驗(yàn)室，華南農(nóng)業(yè)大學(xué)食品質(zhì)量與安全研究所，廣東 廣州 510642；2.廣州軍區(qū)廣州總醫(yī)院，廣東 廣州 510010)

目的：構(gòu)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(xì)胞系(BC3-E8)中提取總RNA，經(jīng)RT-PCR反轉(zhuǎn)錄成cDNA，設(shè)計(jì)通用簡并引物，PCR擴(kuò)增抗體重鏈可變區(qū)基因(VH)和輕鏈可變區(qū)基因(VL)。經(jīng)重疊延伸PCR (SOE-PCR)，將VH和VL基因用編碼(G1y4Ser)3的linker隨機(jī)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(zhuǎn)化大腸桿菌 (Escherichia coli) TG 1感受態(tài)細(xì)胞，經(jīng)輔助噬菌體M13K07超感染，建立噬菌體單鏈抗體庫。隨機(jī)挑取10個(gè)陽性克隆，經(jīng)PCR和雙酶切鑒定，并測序。登陸DNAMAN軟件對(duì)序列進(jìn)行分析、比對(duì)。結(jié)果：成功擴(kuò)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(duì)序列比對(duì)結(jié)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(jié)合區(qū)域?qū)?yīng)的核酸序列上。結(jié)論：已構(gòu)建抗呋喃它酮代謝物的衍生物噬菌體單鏈抗體庫，為進(jìn)一步富集篩選并表達(dá)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(dāng)大的毒性，可導(dǎo)致畸胎突變和誘發(fā)癌癥。歐盟早在1995年已規(guī)定動(dòng)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(jìn)功能，非法使用現(xiàn)象依然存在。由于它對(duì)光敏感，會(huì)迅速被代謝和分解，從肉源性動(dòng)物組織內(nèi)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(jié)合物的形式長期穩(wěn)定存在組織內(nèi)。故可通過檢測AMOZ達(dá)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(shè)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(qiáng)等諸多優(yōu)點(diǎn)。建立免疫法的關(guān)鍵是制備高特異性、高親和力的抗體。噬菌體表面呈現(xiàn)技術(shù)的出現(xiàn)，為抗體的制備提供了新途徑。同時(shí)，利用工程菌制備抗體周期短，成本低，有利于在實(shí)際中應(yīng)用[7]。近年來，噬菌體表面展示技術(shù)已應(yīng)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(fā)[11-12]以及診斷試劑的研制[13-14]等領(lǐng)域。然而，應(yīng)用噬菌體表面展示技術(shù)生產(chǎn)抗AMOZ的衍生物抗體的研究尚未見報(bào)道。本研究利用噬菌體表面展示技術(shù)，旨在構(gòu)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(shí)驗(yàn)基礎(chǔ)。

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(xì)胞株(BC3-E8)為本實(shí)驗(yàn)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(nèi)切酶、T4DNA連接酶和DNA聚合酶、膠回收試劑盒 大連TaKaRa公司；質(zhì)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(xiàn)[15-16]報(bào)道的引物設(shè)計(jì)方法為基礎(chǔ)，參照GenBank數(shù)據(jù)庫以及所選linker(Gly4Ser)3的核苷酸編碼序列，設(shè)計(jì)鼠源單克隆抗體可變區(qū)基因引物，設(shè)計(jì)時(shí)既保證了引物的特異性，又適當(dāng)增加了引物的簡并性。所設(shè)計(jì)引物均由上海生物工程技術(shù)服務(wù)有限公司合成。其中引物VH(back)、VH(for)為VH基因擴(kuò)增引物，VL(back)、VL(for)為VL基因擴(kuò)增引物，VHS(back)、VLS(for)分別為帶有SfiⅠ和NotⅠ酶切位點(diǎn)的擴(kuò)增引物，VH-linker(for)、VL-linker(back)均為帶有部分linker序列的擴(kuò)增引物，畫線部分為酶切位點(diǎn)(表1)。

1.4 總RNA的提取及逆轉(zhuǎn)錄

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

1.5 VH和VL基因擴(kuò)增

以cDNA第一鏈為模板，VH(back)、VH(for)為擴(kuò)增VH的引物，反應(yīng)條件為：94℃預(yù)變性2min；94℃、30s，56.7℃、1min，72℃、1min，共25個(gè)循環(huán)；72℃延伸10min。VL基因的擴(kuò)增，以cDNA第一鏈為模板，VL(back)、VL(for)為擴(kuò)增引物，反應(yīng)條件同重鏈。反應(yīng)產(chǎn)物經(jīng)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(kuò)增

以VH基因?yàn)槟０?，VHS(back)、VH-linker(for)為引物，擴(kuò)增帶部分linker序列的VH基因片段，反應(yīng)條件參照1.5節(jié)。以VL基因?yàn)槟０?，VL-linker(back)、VLS(for)為引物，擴(kuò)增帶部分linker序列的VL基因片段，反應(yīng)條件參照1.5節(jié)。反應(yīng)產(chǎn)物經(jīng)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(jìn)行重疊延伸拼接PCR(SOE-PCR)反應(yīng)，隨機(jī)拼接成scFv，如圖1所示，反應(yīng)條件為：94℃預(yù)變性5min；94℃、45s，57℃、1min，72℃、1min，共20個(gè)循環(huán)；72℃延伸10min。然后以VHS(back)和VLS(for)為引物，二次PCR擴(kuò)增scFv，反應(yīng)條件為：94℃預(yù)變性5min，94℃、30s，57℃、1min，72℃、1min，共25個(gè)循環(huán)；72℃延伸10min。反應(yīng)產(chǎn)物經(jīng)1%瓊脂糖凝膠電泳鑒定后，回收目的片段。

表1 PCR擴(kuò)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(gòu)建

回收后的scFv片段，先后用限制性內(nèi)切酶SfiⅠ，NotⅠ雙酶切消化，產(chǎn)物純化后與同樣酶切處理的噬菌體載體pCANTAB5E連接。連接產(chǎn)物轉(zhuǎn)化感受態(tài)E. coli TG 1，轉(zhuǎn)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(yǎng)過夜。觀察過夜培養(yǎng)的平板，計(jì)算生長的菌落數(shù)，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(zhì)量濃度2g/100mL)稀釋培養(yǎng)，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(yǎng)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(yǎng)基連續(xù)10倍稀釋，每個(gè)稀釋度取100μL，加900μL新鮮制備的對(duì)數(shù)期E. coli TG 1，37℃放置15min，各取100μL涂布于LB AG平板。37℃溫箱培養(yǎng)過夜，計(jì)算菌落，估計(jì)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(shù)×稀釋倍數(shù)。

1.9 抗體庫鑒定

從平板上隨機(jī)挑取10個(gè)克隆，擴(kuò)大培養(yǎng)后提取噬菌粒，以含有SfiⅠ和NotⅠ酶切位點(diǎn)的單鏈抗體引物VHS(back)和VLS(for)為引物進(jìn)行PCR擴(kuò)增，產(chǎn)物經(jīng)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(jìn)行比對(duì)分析。

2 結(jié)果與分析

2.1 總RNA的提取

圖2 總RNA 1%瓊脂糖凝膠電泳結(jié)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(xì)胞株中提取總RNA，由圖2可知，瓊脂糖凝膠電泳結(jié)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(kuò)增

圖3 VL、VH及scFv PCR擴(kuò)增結(jié)果Fig.3 Results of PCR amplification of VL, VH and scFv

以cDNA第一鏈為模板，PCR擴(kuò)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(yù)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(diǎn)，結(jié)果顯示擴(kuò)增產(chǎn)物約為750bp，與預(yù)期片段大小相符。

2.3 噬菌體抗體庫的構(gòu)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(zhuǎn)化菌液涂布于LB AG平板，經(jīng)計(jì)算長出約1.2× 106個(gè)菌落。隨機(jī)挑取10個(gè)克隆進(jìn)行PCR和雙酶切鑒定。結(jié)果顯示，10個(gè)克隆均擴(kuò)增出750bp的片段，目的基因插入率較高(圖4)。以EcoRⅠ、Hind Ⅲ對(duì)克隆進(jìn)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(jié)果10個(gè)克隆均得到2.1kb和3.1kb兩個(gè)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(jié)果表明，已成功將scFv基因克隆到pCANTAB5E噬菌粒載體，構(gòu)建了單鏈抗體庫。經(jīng)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(qū)對(duì)應(yīng)核酸序列比對(duì)圖Fig.6 Alignment of VH-CDR corresponding nucleic acid sequences

圖7 VL-CDR區(qū)對(duì)應(yīng)核酸序列比對(duì)圖Fig.7 Alignment of VL-CDR corresponding nucleic acid sequences

表2 VH-CDR區(qū)對(duì)應(yīng)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(qū)對(duì)應(yīng)核酸序列比例差異表Table 3 Difference in the proportion of VL-CDR corresponding nucleic acid sequences

對(duì)10個(gè)陽性噬菌體克隆scFv序列進(jìn)行分析，每種scFv序列均包含VH、VL和linker區(qū)，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(diǎn)，表明scFv基因片段成功插入載體pCANTEB5E。采用DNAMAN軟件對(duì)scFv序列進(jìn)行比對(duì)，發(fā)現(xiàn)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(qū)對(duì)應(yīng)的核酸序列。從CDR區(qū)對(duì)應(yīng)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(jié)果見圖6、7。6條不同scFv序列的VH基因CDR區(qū)對(duì)應(yīng)的核酸序列差異構(gòu)成見表2，VL基因CDR區(qū)對(duì)應(yīng)的核酸序列差異構(gòu)成見表3。

3 討 論

噬菌體抗體庫技術(shù)作為第三代抗體制備技術(shù)，具有基因型與表型相統(tǒng)一、選擇與擴(kuò)增相統(tǒng)一的優(yōu)點(diǎn)，已被廣泛用于抗體高通量篩選[18-21]。目前針對(duì)小分子有害物所構(gòu)建噬菌體中，大部分是以雜交瘤細(xì)胞系為基因來源。這主要是因?yàn)?，一方面，免疫過程在體內(nèi)經(jīng)過了抗原選擇和親和力成熟，富集了編碼可變區(qū)靶特異性抗體基因，增加了獲得針對(duì)靶抗原較高親和力的抗體的可能性[22]；另一方面，經(jīng)過篩選獲得的雜交瘤細(xì)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(qū)基因，甚至可在表達(dá)載體中直接表達(dá)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(xì)胞(BC3-E8)為源，所建抗體庫庫容可達(dá)1.2×106。同時(shí)，通過DNAMAN軟件對(duì)scFv基因序列的分析顯示，在重鏈和輕鏈的互補(bǔ)決定區(qū)(CDRs)，6條序列均顯示較為顯著差異。因CDR區(qū)是識(shí)別抗原的主要區(qū)域，可以看出，本研究所構(gòu)建抗體庫具有多種抗原識(shí)別區(qū)。該結(jié)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(jì)劃項(xiàng)目(2007AA10Z437)；國家自然科學(xué)基金項(xiàng)目(30871755)；廣東省科技廳科技計(jì)劃項(xiàng)目(2009A020101004)；廣東省-教育部產(chǎn)學(xué)研結(jié)合項(xiàng)目(2009B090300409)

羅翠紅(1984—)，女，碩士研究生，研究方向?yàn)槭称焚|(zhì)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?yàn)槭称焚|(zhì)量安全。E-mail：gzwhongd@163.com