Daudi细胞系特异性Fab噬菌体抗体库的构建_筛选与初步鉴定

#论著#

文章编号:1007-8738(2009)02-0150-05

Daudi 细胞系特异性Fab 噬菌体抗体库的构建、筛选与初步鉴定

申咏梅1,2*,杨晓春3,董宁征4,白 霞4

(苏州大学附属第二医院:1放免中心,2检验教研室,3磁共振室,江苏苏州

215004;

4

江苏省血液研究所,苏州大学附属第一医院,江苏苏州215006)

收稿日期:2008-06-30; 接受日期:2008-09-08

基金项目:国家自然科学基金资助项目(30400111);江苏省自然科学

基金资助项目(BK2004041)

作者简介:申咏梅(1969-),女,重庆人,副教授,医学博士

Te:l 0512-********;E-m ai:l szs y m@yahoo .co

*Corres pond i ng author

G enerati on ,screeni ng and prelim i nary

i dentification of specific Fab phage an -ti body li brary agai nst D audi cell strai n

SHE N Yong-m ei 1,2*,YANG X iao -chun 3,DONG N ing-

zheng 4,BAI X ia

4

1R adi o i m munoassay Cente r ,2

D epart m ent of C linical L abo rato ry ,

3

D epart m ent o f M agnetic R esonance ,Second A ffiliated H osp ita,l

Soocho w U niversity ,Suzhou 215004;4

Ji ang su Institutie o fH em a -to l ogy ,F irst A ffiliated H ospita ,l

Soochow U n i ve rsity ,

Suzhou

215006,Ch i na

[Abstract] A I M:To gene ra t e and screen the specific

Fab p hage an ti b ody library aga inst human Daud i ce ll stra in in B-ly mphoma and i d en tify t he positive c l o nes .M ETH-OD S :BA LB /c m ice we re m i mun ized w it h Daud i ce lls ,and an tisera we re titra ted by EL I SA .Fo llo w ing t he demonstra -tion o f su fficien t an tibody tit e r ,to t a l RNA was extract ed from sp l e n ic ly mphocytes o f the m i m unized m i c e and RT -PCR w as used to am p lify J ligh t cha in and Fd fragmen ts o f heavy chain .A ft e r re strictive d igestion w ith S a c I/Xba I and X ho I/S pe ,I the J ligh t cha in and the Fd fragm ents were succes -sive ly inse rt ed in t o the p hagem id vector pComb 3H -SS and then e l e ctropo ra t ed in t o E.co li XL 1-B l u e .The spe cific Fab phage antibody li b ra ry aga inst Daud i ce ll stra i n in hu man B-l y m pho m a was constructed by in f ec tion o f he l p e r phage VC -SM 13.Fo llo w ing six rounds of b iopanning w ith Daud i ce lls ,the antigen b inding acti v ities o f rando m clone s we re t ested by EL I S A to se lect the positive c l o nes ,wh ich we re further DNA sequenced ,expre ssed i n E.co li X L 1-B lue and i d en t -i fi e d byW estern b lo.t RE SULTS :The Fab phage an tibody l -i b ra ry w it h 3.13@107

size was construct ed and f our positive c lones wh ich spec ifica lly recogn ized Daudi ce ll stra in w ere -i so l a t ed .In am i n o ac i d seq uences ,the var i a b le heavy do -m ains (V H )we re found to be 80%-94%and va riab l e ligh t

dom a i n s (V L )88%-95%homo logous w ith respective m u -r i n e ge r m line genes in GenBank .Fu rthe r mo re ,so lub l e Fab antibod ies o f t he po sitive clone s we re successfu ll y ex -p ressed in E.c o l i XL 1-B l u e and t he reactivity w ith t he memb rane p ro t e ins o f Daud i ce lls wa s demonstra t ed by W estern b l o .t CONCLUSI ON :Fab phage an ti b ody library is succe ssfu lly constructed and specific an tibod ies aga inst

memb rane antigens in Daud i ce lls are ob ta ined ,wh ich pro -vides an expe rm i en t a l f ounda tion for the further i n ve stiga tion o f B -ly m phoma m i munotherapy .[K ey w ords]

B-ly mphoma ;Fab ;Phage anti b ody li b ra ry ;Daud i ce ll stra i n ;pComb 3H -SS vector

[摘要] 目的:构建针对B 细胞淋巴瘤D aud i 细胞系噬菌体抗体库,从中筛选特异性抗体,并对所筛选出的阳性克隆进行鉴定。方法:以D aud i 细胞免疫B A LB /c 小鼠,EL IS A 检测抗血清滴度后,分离抗体阳性的小鼠脾淋巴细胞,提取RNA 。利用RT-PCR 扩增抗体¼轻链和重链Fd 片段,经Sac I /X ba I 和Xho I /Sp e I 双酶切,依次克隆入噬菌体载体p Comb3H-SS 的相应酶切位点,并电转化大肠杆菌XL1-B lue ,以辅助噬菌体VCS M 13进行超感染,构建B 细胞淋巴瘤D audi 细胞系特异性F ab 噬菌体抗体库。以D audi 细胞为抗原对抗体库进行6轮/吸附-洗脱-扩增0筛选,并通过EL ISA 法对随机挑选的克隆进行抗原结合活性测定,获得的阳性克隆进一步做DNA 序列测定、在大肠杆菌XL1-B l ue 中进行可溶性表达,并用W estern b l o t 对表达产物的特异性作了鉴定。结果:构建了容量为3.13@107的抗人B 细胞淋巴瘤Daud i 细胞系的F ab 噬菌体抗体库,并筛选获得了与Daud i 细胞系特异性识别结合的4株阳性克隆。氨基酸序列分析结果显示:阳性克隆的重链、轻链可变区序列分别与基因库中已注册的鼠源性免疫球蛋白重链、轻链可变区序列有80%~94%和88%~95%同源性。阳性克隆成功在大肠杆菌细胞中可溶性表达,W estern b l o t 结果表明所获得的可溶性表达产物可与D audi 细胞膜抗原特异性结合。结论:成功地构建F ab 噬菌体抗体库并筛选出针对D audi 细胞系膜抗原的抗体,为进一步研究B 细胞淋巴瘤的免疫治疗提供了实验基础。

[关键词]B 细胞淋巴瘤;Fab ;噬菌体抗体库;D aud i 细胞

系;pCo m b3H-SS 载体

[中图分类号]Q 782 [文献标识码]A

恶性淋巴瘤是血液系统常见的恶性肿瘤,传统的治疗主要包括放疗、化疗及施行骨髓移植或外周血干细胞移植,但效果都不十分理想。目前,未标记