劉媛,林曼曼,張霄,徐重新,焦凌霞,仲建鋒,武愛華,劉賢金(江蘇省食品質(zhì)量安全重點(diǎn)實(shí)驗(yàn)室和農(nóng)業(yè)部食用農(nóng)產(chǎn)品安全監(jiān)控重點(diǎn)開放實(shí)驗(yàn)室,南京210014)

基因突變技術(shù)在抗體親和力體外成熟中的應(yīng)用

劉媛,林曼曼,張霄,徐重新,焦凌霞,仲建鋒,武愛華,劉賢金*
(江蘇省食品質(zhì)量安全重點(diǎn)實(shí)驗(yàn)室和農(nóng)業(yè)部食用農(nóng)產(chǎn)品安全監(jiān)控重點(diǎn)開放實(shí)驗(yàn)室,南京210014)

摘要從天然抗體庫中篩選得到的抗體通常親和力較低,在微摩爾水平;同時(shí),經(jīng)過多次免疫獲得的天然抗體也存在100 pmol/L的親和力極限.然而,以抗體親和力體內(nèi)成熟為理論基礎(chǔ)的親和力體外成熟技術(shù),可以模擬體細(xì)胞高頻突變和克隆選擇過程.該技術(shù)可以解決庫來源抗體親和力不高、實(shí)際應(yīng)用難的問題,也可以幫助天然抗體突破其親和力極限.抗體基因體外突變技術(shù)可以模擬自體高頻突變過程,是抗體親和力體外成熟的重要手段,常見的抗體基因體外突變技術(shù)可以分為易錯(cuò)聚合酶鏈?zhǔn)椒磻?yīng)(polymerase chain reaction,PCR)、DNA改組、突變株、定點(diǎn)突變和鏈替換等方法.易錯(cuò)PCR可以在抗體基因全長或部分區(qū)域隨機(jī)引入突變,但隨著突變率的增加,陽性克隆數(shù)量呈指數(shù)性遞減.DNA改組包括抗體片段隨機(jī)化切割和重組步驟,可以加快抗體的體外進(jìn)化速度.突變株法易于構(gòu)建超大容量的抗體庫,但有害突變和突變率難以控制.定點(diǎn)突變的區(qū)域通常選擇與抗原直接接觸的互補(bǔ)決定區(qū)(complementary determination region,CDR)或自體突變熱點(diǎn)進(jìn)行操作.鏈替換通常保留母體抗體的一條重鏈或輕鏈,而對另一條鏈進(jìn)行隨機(jī)化組合.這些基因突變方法在提高抗體親和力中獲得了不同程度的應(yīng)用,但也存在效率不高,且方法選擇較為盲目等問題.可是,采用抗體X射線晶體衍射和計(jì)算機(jī)模擬等方法,卻可以幫助預(yù)測抗體結(jié)合部位,為突變位點(diǎn)的理性設(shè)計(jì)和方法選擇提供有效信息,因而成為親和力體外成熟技術(shù)未來發(fā)展的趨勢之一.

關(guān)鍵詞抗體;親和力成熟;易錯(cuò)PCR;DNA改組;突變株;定點(diǎn)突變;鏈替換

浙江大學(xué)學(xué)報(bào)(農(nóng)業(yè)與生命科學(xué)版) 42(1):1～7,2016

Journal of Zhejiang University(Agric.&Life Sci.)

http://www.journals.zju.edu.cn/agr

E-mail:zdxbnsb@zju.edu.cn

第一作者聯(lián)系方式:劉媛(http://orcid.org/0000-0003-3842-7571)，Tel:+86-025-84390401,E-mail:zeranol@163.com

URL:http://www.cnki.net/kcms/detail/33.1247.S.20151215.1739.002.html

Applications of mutagenesis methods on affinity maturation of antibodies in vitro.Journal of Zhejiang University(Agric.&Life Sci.),2016,42(1):1-7

LIU Yuan,LIN Manman,ZHANG Xiao,XU Chongxin,JIAO Linxia,ZHONG Jianfeng,WU Aihua,LIU Xianjin*(Key Laboratory of Food Safety of Jiangsu Province and Key Laboratory of Food Safety Monitoring and Management of Ministry of Agriculture,Nanjing 210014,China)

Summary Antibodies selected from naive antibody libraries usually have low affinity at the micromolar level.Meanwhile,the affinities of natural antibodies obtained by repeated immunization were also observed to have a limit around 100 picomole because of the inability to discriminate slower dissociation kinetics relative to intrinstric B cell receptor internalization rate.As an essential determinant for antibody function for therapeutic and diagnostics use,a lots of antibodies,either isolated from antibody libraries or cloned from monoclonal antibodies,have been targeted to generate high affinity by directed evolution.

Affinity maturation in vitro could mimic the mutation and selection process of affinity maturation in vivo,it couldhelp to improve the affinity of the antibodies derived from antibody libraries and make the natural antibodies to break through affinity ceiling.At the mutagenesis level,strategies to affinity maturation in vitro can be grouped in five categories:error prone PCR,DNA shuffling,mutator strains,site directed mutagenesis and chain shuffling.Error PCR could generate random mutations into antibodies throughout the whole antibodies gene.But it is notable that when the mutation rate increases,the active clones decreases exponentially.DNA shuffling includes random fragmentation and reassembly steps;it can be used with longer DNA sequences,and also allows for the selection of clones with mutations outside the binding or active site of the antibodies.By mutator strains,the mutations are produced after the transformation,which allows the randomly mutated libraries of even 1012_1014clones be produced.However,by mutator strains,mutations are also produced in the vector part to cause deleterious effects.In site-directed mutagenesis,selected residues are targeted to be mutated.The most common sites for mutation are the CDR regions which directly contact with antigen.However,mutations that do not directly contact antigen can also result in enhanced affinity.And the mutation hotspots in affinity maturation in vivo can also be targeted to the mutation sites.In chain shuffling,a variable heavy or light chain of a specific antibody is recombined with a complementary variable domain library.In a lot of experiments,light chains were used to be shuffling,because heavy chain is crucial for the antigen binding.But there are also some reports on producing the affinity-matured antibodies by heavy chain shuffling.

These mutagenesis methods have been used with various degrees of success.However,the efficiency of these methods was relatively low and there was a lack of a rational guidance to choose these methods.Knowledge gained on antibody structure through X-ray crystallography or computer-aided design could help to predict the paratope and mutation sites,which is one of the development trend of affinity maturation in vitro.

Key words antibodies;affinity maturation;error PCR;DNA shuffling;mutator strains;site-directed mutagenesis;chain shuffling

抗體制備技術(shù)的發(fā)展,已經(jīng)歷了多克隆抗體技術(shù)、單克隆抗體技術(shù)和抗體庫技術(shù)3個(gè)發(fā)展階段[1].單克隆抗體技術(shù)的出現(xiàn),解決了多克隆抗體親和力、特異性相對較低和難以重復(fù)制備的問題.而抗體庫技術(shù)的出現(xiàn),則能以體外建庫和篩選代替單克隆抗體復(fù)雜的免疫和雜交瘤篩選過程,被譽(yù)抗體制備創(chuàng)新性的換代技術(shù),隨著相關(guān)行業(yè)的發(fā)展迅猛,國內(nèi)外已有大量抗體庫篩選的報(bào)道[2 5].

然而,抗體庫來源的抗體與傳統(tǒng)的單、多克隆抗體相比,實(shí)際應(yīng)用非常少見,究其原因是由于庫來源抗體與天然抗體相比,親和力偏低,難以滿足多種實(shí)際應(yīng)用需求.特別是來自于未經(jīng)免疫的天然抗體庫中的抗體,抗體的親和力通常在微摩爾水平,這僅與動(dòng)物在初次免疫應(yīng)答后獲得的抗體親和力水平相當(dāng)[6].與此同時(shí),天然抗體的親和力通常存在一個(gè)“天花板效應(yīng)”,通過免疫獲得的天然抗體親和力通常低于100 pmol/L,這是由于B細(xì)胞受體難以區(qū)分更低的解離速率造成的.可是,抗體的親和力體外成熟技術(shù)卻可以幫助解決這些問題.

1　抗體的親和力體外成熟的理論基礎(chǔ)

要想實(shí)現(xiàn)抗體的親和力體外成熟,就必須充分地了解天然抗體的親和力體內(nèi)成熟原理;設(shè)計(jì)模擬體內(nèi)可能出現(xiàn)和存在的變化,從而促進(jìn)抗體的體外進(jìn)化[7].

天然抗體的親和力成熟可以分為體細(xì)胞高頻突變和克隆選擇2個(gè)過程.機(jī)體接受抗原刺激后,體細(xì)胞發(fā)生高頻突變,突變產(chǎn)生的各種親和力不同的B細(xì)胞克隆,經(jīng)過濾泡樹突細(xì)胞捕獲,使得后代B細(xì)胞及其產(chǎn)生的抗體對抗原的平均親和力得到了提升,天然抗體從而實(shí)現(xiàn)了親和力的體內(nèi)成熟.

在天然抗體親和力成熟的過程中,抗原刺激下的體細(xì)胞高頻突變有著舉足輕重的作用,因此親和力體外成熟的策略也多在抗體基因突變水平上,即采用各種突變方法來模擬體內(nèi)的高頻突變.本文將對抗體親和力體外成熟技術(shù)中常用的突變方法加以介紹.

2　抗體基因的體外突變技術(shù)

2.1易錯(cuò)PCR

易錯(cuò)PCR技術(shù)是最常用的抗體基因突變技術(shù),可以在抗體基因的全長或部分區(qū)域隨機(jī)引入突變.該技術(shù)可以通過提高鎂離子濃度、加入錳離子、失衡4種脫氧核苷三磷酸(deoxyribonucleoside triphosphate,d NTPs)濃度、使用低保真DNA聚合酶等方法,來提高抗體基因的突變率[8].

除此之外,突變率的高低也可以采用改變模板DNA的復(fù)制次數(shù)進(jìn)行控制.復(fù)制次數(shù)越多,突變率將累積得越高.因此,要想獲得高突變率,可以采用低濃度的DNA模板同時(shí)增加PCR循環(huán)數(shù)來實(shí)現(xiàn).但是,隨著突變率的提高也將造成大量突變抗體失活,從而影響突變庫庫容.表1中列出了一些易錯(cuò)PCR的應(yīng)用實(shí)例,DAUGHERTY等[9]對易錯(cuò)PCR的突變率與抗體活性保留關(guān)系進(jìn)行了研究.通過對地高辛單鏈抗體(single-chain variable fragment,scFv)進(jìn)行低(0.22%)、中(0.5%)和高(3%)3個(gè)水平的隨機(jī)突變研究,表明隨著突變率的提高,突變庫中活性克隆所占比例大幅下降,在3種突變率下對應(yīng)的活性克隆比例分別為40%、6.7%和0.17%.但是,親和力提高最多的克隆(提高1.9倍)仍然來自于高頻突變庫.

GRAM等[10]從非免疫鼠組合抗體庫中獲得的Fab抗體,對黃體酮-牛血清白蛋白(bovine serum albumin,BSA)的親和力僅為10-4～10-5mol/L,作者隨后采用易錯(cuò)PCR法對4個(gè)抗體基因模板重、輕鏈可變區(qū)隨機(jī)突變,PCR體系采用了系列濃度的模板DNA和優(yōu)化的MgCl2、MnCl2以及d NTP濃度,篩選到的最佳克隆較母體抗體親和力提高了30 倍,突變率為1.5%.作者觀察到的最主要突變形式是A/G和T/C的替換,同時(shí)發(fā)現(xiàn)突變熱點(diǎn)的存在.PERSSON等[11]采用Ampli Taq聚合酶,對將睪丸激素抗體結(jié)合片段(fragment antibody-binding,Fab)進(jìn)行隨機(jī)突變,結(jié)果表明構(gòu)建的2個(gè)突變庫CTep1和CTep2的突變率分別是0.77%和0.87%,其中89%的突變含有插入堿基,而80%突變存在于抗體的框架結(jié)構(gòu).作者隨后采用了逐輪減少抗原濃度、改變和洗脫捕獲條件等方法,獲得的最佳克隆親和力為2 nmol/L,親和力提高了200倍.

2.2DNA改組

DNA改組技術(shù)是對同源的抗體基因,采用脫氧核糖核酸酶Ⅰ將其切割成不超過50 bp的片段,再隨機(jī)組合后進(jìn)行PCR擴(kuò)增成完整的抗體基因.它包含了抗體片段隨機(jī)化切割、重組和篩選的過程,一定程度上模擬了天然抗體的親和力成熟過程,并加快了體外定向進(jìn)化速度[18 19].采用連續(xù)多輪的DNA改組具有去除有害突變優(yōu)勢[20].另外,DNA改組獲得的突變體,突變位點(diǎn)可以位于非抗原直接接觸的區(qū)域,有擴(kuò)大庫容的作用[7].

為了獲得足夠多的點(diǎn)突變,DNA改組技術(shù)還常與易錯(cuò)PCR技術(shù)聯(lián)用.BODER等[15]以熒光素單鏈抗體4-4-20為研究對象,其親和力為0.3 nmol/L,已接近于天然抗體所能獲得的最大親和力,但是通過DNA改組和易錯(cuò)PCR的組合應(yīng)用對其進(jìn)行了四輪突變及篩選后,獲得的最佳克隆4M5.3在PBS的半數(shù)解離常數(shù)達(dá)到了5 d,比母體抗體延長了4個(gè)數(shù)量級,解離時(shí)間超過了生物素與鏈霉親和素的解離時(shí)間.在LSB溶液中與生物素化熒光素的親和力可達(dá)48 fmol/L,比4-4-20提高了208倍,是目前公開報(bào)道的親和力最高的非共價(jià)結(jié)合物.

2.3突變株

Escherichia coli MutD5是最常用的大腸埃希菌突變株,該菌株具有校對和復(fù)制后錯(cuò)配修復(fù)缺陷,可以產(chǎn)生高頻率的單個(gè)堿基替換,突變率高于普通菌株105倍.突變株法的優(yōu)勢是突變發(fā)生在轉(zhuǎn)化宿主菌后,而其他方法的突變均發(fā)生于轉(zhuǎn)化之前,而轉(zhuǎn)化效率是限制大容量突變庫構(gòu)建的關(guān)鍵因素之一.使用突變株法可以構(gòu)建庫容為1012～1014個(gè)克隆的超大容量抗體庫,但是該方法較難對突變率進(jìn)行控制,且獲得的隨機(jī)突變也可能發(fā)生于載體部分,造成有害突變.

LOW等[17]采用E.coli MutD5對半抗原3-苯基-噁唑酮抗體進(jìn)行突變,獲得的突變體親和力可達(dá)3.2 nmol/L,親和力提高了100倍.MUTEEB等[21]也報(bào)道了采用E.coli XL1-red作為突變菌株的研究.

2.4定點(diǎn)突變

由于天然抗體在親和力成熟過程中,體細(xì)胞高頻突變發(fā)生區(qū)域并非均勻分布,而是主要集中在與抗原直接接觸的CDR區(qū).這樣既可以獲得足夠的序列多樣性,又不會(huì)破壞蛋白質(zhì)結(jié)構(gòu).因此在抗體的親和力體外成熟中,CDR區(qū)是最常選用的定點(diǎn)突變區(qū)域.

在表2所列出的一系列定點(diǎn)突變中,有平行對多個(gè)CDR進(jìn)行突變或?qū)Χ鄠€(gè)CDR進(jìn)行逐步優(yōu)化,即CDR步行法(CDR walking).STEIDL等[22]報(bào)道了對CDR-H2區(qū)的突變,將母體抗體親和力提高近300 倍.除了CDR區(qū)的點(diǎn)突變外,LAMMINMAKI[23]和PARHAMI-SEREN[24]通過增加CDR-H2 loop環(huán)長度的方法提高半抗原特異性抗體的親和力.KOBAYASHI[25]則對半抗原17β-雌二醇母體抗體進(jìn)行了2步法突變,首先是在抗體的CDR H2,H3,L1 和L3進(jìn)行了CDR改組,對該突變庫中篩選出親和力提高的克隆,再進(jìn)行重、輕鏈可變區(qū)的易錯(cuò)PCR,多輪篩選獲得的最佳克隆親和力提高了10倍.

然而,由于單鏈抗體或Fab抗體具有6個(gè)CDR 區(qū),抗體與抗原結(jié)合過程中又涉及多個(gè)CDR.因此在確定對抗原結(jié)合起作用氨基酸殘基,以及CDR邊緣氨基酸殘基對親和力起作用方面相當(dāng)復(fù)雜.為了解決這一問題,近年來最突出的進(jìn)展是利用抗體晶體結(jié)構(gòu)信息或計(jì)算機(jī)輔助設(shè)計(jì),來分析與抗原直接接觸的氨基酸位點(diǎn).BARDERAS等[26]采用了胃泌激素抗體可變區(qū)的3D結(jié)構(gòu)模型來輔助預(yù)測抗原結(jié)合部位的氨基酸位點(diǎn),設(shè)計(jì)采用的CDR步行法將胃泌激素單鏈抗體的親和力提高了454倍.

除了CDR區(qū)突變外,天然抗體親和力成熟中的突變熱點(diǎn)(hotspot)也是常見的定點(diǎn)突變位點(diǎn).這些熱點(diǎn)通常包含或接近AGY和RGYW(R=A or G;Y=C or T;W=A or T)序列[32].

2.5鏈替換

鏈替換是保留某個(gè)特定抗體的重鏈或輕鏈,并與一個(gè)隨機(jī)化的互補(bǔ)鏈進(jìn)行組合,從中篩選更高活性的突變株(表3).由于通常認(rèn)為抗體的重鏈CDR3區(qū)對抗體的結(jié)合能力至關(guān)重要,因此輕鏈的替換較為常見.FITZGERALD等[33]將提取第5輪篩選后的溴氯哌喹酮scFv重鏈可變區(qū)基因與原始抗體庫的輕鏈基因隨機(jī)組合,構(gòu)建了庫容1.25×107的輕鏈替換庫,從中篩選到最佳克隆建立的ELISA檢測方法,靈敏度可達(dá)80 pg/m L,比母體抗體提高了185倍.

鏈替換技術(shù)除了可以用于提高抗體親和力外,也有對抗體特異性、穩(wěn)定性提高方面的報(bào)道.該技術(shù)可以用于鼠源抗體的人源化改造[40],幫助解決雜交瘤來源的抗體轉(zhuǎn)變?yōu)閱捂溈贵w形式后,在原核表達(dá)中出現(xiàn)失活的問題[34].

除了輕鏈替換外,也有重鏈替換或兩者組合應(yīng)用的成功報(bào)道.PARKER等[37]通過對母體抗體結(jié)構(gòu)分析,認(rèn)為輕鏈在抗原識(shí)別中起到?jīng)Q定性作用,因此采用了重鏈替換的方法對肝炎B病毒scFv進(jìn)行改造,獲得的最佳克隆比母體抗體親和力提高了6.5倍.KRAMER[38]則對阿特拉津單鏈抗體的重鏈和輕鏈依次進(jìn)行置換,親和力成熟抗體的檢測靈敏度由最初的5.1μg/L提高到0.2μg/L,抗體的特異性也有所改善.

3　問題與展望

近年來學(xué)術(shù)界雖然在應(yīng)用基因突變技術(shù)實(shí)現(xiàn)抗體親和力體外成熟中取得了一些進(jìn)展,但由于人們對親和力體外成熟的分子基礎(chǔ)方面的認(rèn)知不夠明確,抗體的突變策略選擇仍然較為盲目,獲得成熟抗體的效率不高.

在現(xiàn)有的抗體親和力成熟分子基礎(chǔ)研究中,通過對成熟抗體與母體抗體序列比對分析,可以證明抗體的CDR3氨基酸對于親和力成熟演化過程非常重要,然而主要用于維持抗體結(jié)構(gòu)保守性、折疊功能和穩(wěn)定性的框架結(jié)構(gòu)序列,對抗原結(jié)合作用也存在影響[41-42].氨基酸序列的差異對抗體功能性影響,在不同區(qū)域呈現(xiàn)的相關(guān)性又會(huì)不同,因此僅采用氨基酸序列分析的方法,難以精確預(yù)測出哪些位點(diǎn)的突變會(huì)對抗體親和力的改變產(chǎn)生影響[43].

為了解決這些問題,可以采用X射線晶體衍射或是核磁共振的方法對親和力成熟抗體及其抗原復(fù)合物的三維結(jié)構(gòu)進(jìn)行分析[44 45].或采用計(jì)算機(jī)模型來模擬“抗原-抗體”的互作過程,用于預(yù)測親和力成熟的關(guān)鍵位點(diǎn).對范德華力、氫鍵、親水性、構(gòu)型、電荷互補(bǔ)、變構(gòu)效應(yīng)、可塑性和協(xié)調(diào)性等物化因子進(jìn)行測定,也可對“抗原-抗體”復(fù)合物的形成做定量分析,以增加對親和力成熟過程的理解,并為突變位點(diǎn)的理性設(shè)計(jì)提供有效的信息[46].

隨著人們對天然抗體親和力成熟分子基礎(chǔ)認(rèn)知的不斷加深,以及突變技術(shù)應(yīng)用于抗體親和力體外成熟實(shí)踐的積累,抗體基因體外突變技術(shù)將日趨發(fā)展成熟,成為更為有效的抗體體外定向進(jìn)化手段.

參考文獻(xiàn)(References):

[1]WEISSER N E,HALL JC.Applications of single-chain variable fragment antibodies in therapeutics and diagnostis.Biotechnology Advances,2009,27:502-520.

[2]LI T,ZHANG Q,LIU Y,et al.Production of recombinant ScFv antibodies against methamidophos from a phage-display library of a hyperimmunized mouse.Journal of Agricultural and Food Chemistry,2006,54:9085-9091.

[3]WEN S,ZHANG X,LIU Y,et al.Selection of a single chain variable fragment antibody against ivermectin from a phage displayed library.Journal of Agricultural and Food Chemistry,2010,58(9):5387-5391.

[4]NIEMIM H,TAKKINEN K,AMUNDSEN L K,et al.The testosterone binding mechanism of an antibody derived from a na?ve human scFv library.Journal of Molecular Recognition,2011,24:209-219.

[5]MCELHINEY J,DREVER M,LAWTON L A,et al.Rapid isolation of a single-chain antibody against the cyanobacterial toxin microcystin-LR by phage display and its use in the immunoaffinity concentration of microcystins from water.Applied and Environmental Microbiology,2002,68(11):5288-5295.

[6]SHEEDY C,MACKENZIE C R,HALL J C.Isolation and affinity maturation of hapten-specific antibodies.Biotechnology Advances,2007,25:333-352.

[7]胡曉林,張春.基因工程抗體親和力成熟的策略.免疫學(xué)雜志,2006,22(6):702-704.HU X L,ZHANG C.Strategies for affinity maturation of engineering antibodies.Immunological Journal,2006,22(6):702-704.(in Chinese with English abstract)

[8]BROCKMANN E C.Evolution of bioaffinity reagents by phage display.Turku:University of Turku,2010.

[9]DAUGHERTY P S,CHEN G,IVERSON L,et al.Quantitative analysis of the effect of the mutation frequency on the affinity maturation of single chain Fv antibodies.Proceedings of the National Academy of Sciences of the United States of America,2000,97(5):2029-2034.

[10]GRAM H,MARCONI L A,BARBAS C F,et al.In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library.Proceedings of the National Academy of Sciences of the United States of America,1992,89(8):3576-3580.

[11]PERSSON H,WALLMARK H,LJUNGARS A,et al.In vitro evolution of an antibody fragment population to find high-affinity hapten binders.Protein Engineering Design and Selection,2008,21:485-493.

[12]VAN DEN BEUCKEN T,PIETERS H,STEUKERS M,et al.Affinity maturation of Fab antibody fragments by fluorescentactivated cell sorting of yeast-displayed libraries.FEBS Letters,2003,546(2/3):288-294.

[13]CHIN S E,FERRARO F,GROVES M,et al.Isolation of highaffinity,neutralizing anti-idiotype antibodies by phage and ribosome display for application in immunogenicity and pharmacokinetic analyses.Journal of Immunological Methods,2015,416:49-58.

[14]FERMER C,ANDERSSON I,NILSSON K,et al.Specificity rescue and affinity maturation of a low-affinity IgM antibody against pro-gastrin-releasing peptide using phage display and DNA shuffling.Tumour Biology,2004,25(1/2):7-13.

[15]BODER E T,MIDELFORT K S,WITTRUP K D.Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity.Proceedings of the National Academy of Sciences of the United States of America,2000,97(20):10701-10705.

[16]ZAHND C,SPINELLI S,LUGINBUHL B,et al.Directed in vitro evolution and crystallographic analysis of a peptide-binding single chain antibody fragment(scFv) with low picomolar affinity.The Journal of Biological Chemistry,2004,279(18):18870-18877.

[17]LOW N M,HOLLIGER P H,WINTER G.Mimicking somatic hypermutation:affinity maturation of antibodies displayed on bacteriophage using a bacterial mutator strain.Journal of Molecular Biology,1996,26(3):359-368.

[18]STEMMER W P.DNA shuffling by random fragmentation and reassembly:in vitro recombination for molecular evolution.Proceedings of the National Academy of Sciences of the United States of America,1994,91(22):10747-10751.

[19]汪保安,王琰.基因工程抗體的體外親和力成熟.海軍總醫(yī)院學(xué)報(bào),2003,16(4):222-225.WANG B A,WANG Y.In vitro affinity maturation of engineering antibodies.Journal of Navy General Hospital,2003,16(4):222-225.(in Chinese with English abstract)

[20]VAN DERLINDEN R H,DE GEUS B,FRENKEN G J,et al.Improved production and function of llama heavy chain antibody fragments by molecular evolution.Journal of Biotechnology,2000,80(3):261-270.

[21]MUTEEB G,SEN R.Random mutagenesis using a mutator strain.Methods in Moecular Biology,2010,634:411-419.

[22]STEIDL S,RATSCH O,BROCKS B,et al.In vitro affinity maturation of human GMCSF antibodies by targeted CDR-diversification.Molecular Immunology,2008,46:135-144.

[23]LAMMINMAKI U,PAUPERIO S,WESTERLUNDKARLSSON A,et al.Expanding the conformational diversity by random insertions to CDRH2 results in improved anti-estradiol antibodies.Journal of Molecular Biology,1999,291(3):589-602.

[24]PARHAMI-SEREN B,VISWANATHAN M,MARGOLIES M N.Selection of high affinity p-azophenyl arsonate Fabs from heavy-chain CDR2 insertion libraries.Journal of Immunological Methods,2002,259:43-53.

[25]KOBAYASHI N,OYAMA H,KATO Y,et al.Two-step in vitro antibody affinity maturation enables estradiol-17beta assays with more than 10-fold higher sensitivity.Analytical Chemistry,2010,82:1027-1038.

[26]BARDERAS R,DESMET J,TIMMERMAN P,et al.Affinity maturation of antibodies assisted by in silico modeling.Proceedings of the National Academy of Sciences,2008,105 (26):9029-9034.

[27]CHEN G,DUBRAWSKY I,MENDEZ P,et al.In vitro scanning saturation mutagenesis of all the specificity determining residues in an antibody binding site.Protein Engineering,1999,12(4):349-356.

[28]BROCKS B,KRAFT S,ZAHN S,et al.Generation and optimization of human antagonistic antibodies against TIMP-1 as potential therapeutic agents in fibrotic diseases.Human Antibodies,2006,15(4):115-124.

[29]NAGY Z A,HUBNER B,LOHNING C,et al.Fully human,HLA-DR-specific monoclonal antibodies efficiently induce programmed death of malignant lymphoid cells.Natural Medicine,2002,8:801-807.

[30]SCHIER R,MCCALL A,ADAMS G P,et al.Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site.Journal of Molecular Biology,1996,263(4):551-567.

[31]YANG W P,GREEN K,PINZ-SWEENEY S,et al.CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range.Journal of Molecular Biology,1995,254(3):392-403.

[32]YAU K Y F,DUBUC G,LI S,et al.Affinity maturation of a VHH by mutational hotspot randomization.Journal of Immunological Methods,2005,297(1/2):213-224.

[33]FITZGERALD J,LEONARD P,DARCY E,et al.Light-chain shuffling from an antigen-biased phage pool allows 185-fold improvement of an anti-halofuginone single-chain variable fragment.Analytical Biochemistry,2011,410(1):27-33.

[34]ROJAS G,TALAVERA A,MUNOZ Y,et al.Light-chain shuffling results in successful phage display selection of functional prokaryotic-expressed antibody fragments to N-glycolyl GM3 ganglioside.Journal of Immunological Methods,2004,293(1/2):71-83.

[35]SHI L,WHEELER JC,SWEET R W,et al.De novo selection of high-affinity antibodies from synthetic fab libraries displayed on phage as pIX fusion proteins.Journal of Molecular Biology,2010,397(2):385-396.

[36]HUR B U,CHOI H J,SONG S Y,et al.Development of the dual-vector system-Ⅲ(DVS-Ⅲ),which facilitates affinity maturation of a Fab antibody via light chain shuffling.Immunology Letters,2010,132(1/2):24-30.

[37]PARK S G,LEE J S,JE E Y,et al.Affinity maturation of natural antibody using a chain shuffling technique and the expression of recombinant antibodies in Escherichia coli.Biochemical and Biophysical Research Communications,2000,275(2):553-557.

[38]KRAMER K.Evolutionary affinity and selectivity optimization of a pesticide-selective antibody utilizing a hapten-selective immunoglobulin repertoire.Environmental Science &Technology,2002,36(22):4892-4898.

[39]WANG N,YUAN A,DENG Z,et al.Engineering the malespecificity of Fab against SDM antigen by chain shuffling.Theriogenology,2013,79(8):1162-1170.

[40]DAMSCHRODER M M,WIDJAJA L,GILL P S,et al.Framework shuffling of antibodies to reduce immunogenicity and manipulate functional and biophysical properties.Molecular Immunology,2007,44(11):3049-3060.

[41]DEGENST E,HANDELBERG F,VAN MEIRHAEGHE A,et al.Chemical basis for the affinity maturation of a camel single domain antibody.The Journal of Biological Chemistry,2004,279(51):53593-53601.

[42]ACIERNO J P,BRADEN B C,KLINKE S,et al.Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies.Journal of Molecular Biology,2007,374(1):130-146.

[43]PAMELA M C,DAVID C,JAMES J,et al.A study of the structural correlates of affinity maturation:antibody affinity as a function of chemical interactions,structural plasticity and stability.Molecular Immunology,2007,44(6):1342-1351.

[44]鄧龍,周思,郭新東.基于基因突變的基因工程抗體親和力成熟研究.生物技術(shù)進(jìn)展,2014,4(6):400-404.DENG L,ZHOU S,GUO X D.Affinity maturation of genetically engineered antibody by gene mutation.Current Biotechnology,2014,4(6):400-404.(in Chinese with English abstract)

[45]于禮,梁米芳.基因工程抗體親和力成熟研究進(jìn)展.中國醫(yī)藥生物技術(shù),2012,7(4):286-289.YU L,LIANG M F.Reviews on the affinity maturation of genetically engineered antibody.Chinese Medicine Biotechnology,2012,7(4):286-289.(in Chinese)

[46]YIN J,BEUSCHER A E,ANDRYSKI S E,et al.Structural plasticity and the evolution of antibody affinity and specificity.Journal of Molecular Biology,2003,330(4):651-656.

收稿日期(Received):2015-03-25;接受日期(Accepted):2015-06-26;網(wǎng)絡(luò)出版日期(Published online):2015-12-15

*通信作者(

Corresponding author):劉賢金(http://orcid.org/0000-0003-0184-7978),E-mail:jaasliu@163.com

基金項(xiàng)目:國家自然科學(xué)基金(31201535);江蘇省社會(huì)發(fā)展項(xiàng)目(BE2012750);江蘇省農(nóng)業(yè)自主創(chuàng)新基金(CX(12)5042).

DOI:10.3785/j.issn.1008-9209.2015.03.251

中圖分類號Q789

文獻(xiàn)標(biāo)志碼A