甲状腺髓样癌人源噬菌体单链抗体的筛选及初步鉴定  

Screening and preliminary identification of human single chain variable fragments antib Aies against medullary thyroid cancer

作　　者：刘琼[1] 庞华[1] 奚级梅 李文波[1] 许璐[1] 

机构地区：[1]重庆医科大学附属第一医院核医学科,重庆400016

出　　处：《重庆医科大学学报》2015年第9期1199-1203,共5页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81071171)

摘　　要：目的:从大容量甲状腺髓样癌(medullary thyroid carcinoma,MTC)人源噬菌体单链抗体(single chain variable fragments,sc Fv)库中筛选出特异的抗MTC单链抗体,并对其生物学特性进行鉴定。方法:分别以甲状腺上皮细胞TEC细胞株和MTC细胞TT细胞株为抗原进行5轮"吸附-洗脱-扩增"的富集循环,筛选出抗MTC抗体,将筛选出的阳性克隆感染E.coli HB2151;异丙基-β-D-硫代吡喃半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导抗体可溶性表达,HiTrapTM Anti E-tag柱纯化表达抗体;SDS-PAGE和Western blot检测抗体表达和相对分子质量;ELISA检测可溶性sc Fv的特异性和免疫活性,分为TT细胞组、SW480细胞组、TEC细胞组和PBS空白对照组,分别检测各组在酶标仪A450 nm处的吸光度;四甲基偶氮唑盐比色分析法(methyl thiazolyl tetrazolium,MTT)分别检测TT细胞组、SW 480细胞组与不同浓度sv Fv反应并读取A490 nm处的吸光度,设置空白对照PBS组,分别计算细胞抑制率。结果:5轮筛选后,抗体得到了明显富集。随机挑取单个细菌克隆,phage ELISA和sc Fv ELISA阳性率分别为65%和50%。SDS-PAGE、Western blot结果显示MTC特异性抗体分子量为28 k D左右。ELISA结果显示,TT细胞组的吸光度(0.41±0.12)较SW 480细胞组(0.20±0.03)、TEC细胞组(0.13±0.01)、PBS空白对照组(0.07±0.01)明显增加(P=0.000)。MTT法结果显示,TT细胞组和SW 480细胞组在sc Fv浓度为0.1、1、10μmol/L时,2细胞株间抑制率有统计学意义,统计量分别是t=2.881,P=0.02;t=5.073,P=0.006;t=7.324,P=0.000,且在sc Fv浓度为10μmol/L时,对TT细胞的抑制率最高(0.59±0.15)%。结论:利用噬菌体抗体库技术成功获得了特异性的抗MTC人源单链抗体。Objective：To isolate single chain variable fragments （scFv） antibAies, against medullary thyroid carcinoma （MTC） from a large human phage display library and to identify the biological activity of positive clones. Methods：Thyroid epithelial cell line（TEC cell） and MTC cell line（TF cell） were used as negative and positive antigen respectively and then five rounds of ＇adsorption elution- amplification＇from the large human phage display library against MTC was conducted. Specific antibAy was tested by ELISA. E.coli HB2151 was infected to induce the expression of soluble scFv. Soluble scFv was purified by HiTrapTM Anti E-tag column. The ex- pression and relative molecular weight of the soluble scFv were detected by SDS-PAGE and Western blot. Immune activity of specific soluble scFv was detected by cell ELISA. They were divided into four groups：Tr cells group,SW 480 cells group,TEC cells group and control group with PBS;A450nm was detected by microplate reader respectively. The effects of proliferation of TT cell at different concen- trations were explored by methyl thiazolyl tetrazolium（MTT） assay. They were divided into：Tr cells group,SW 480 cells group and control group with PBS, A450nm absorbanee was detected by microplate reader and inhibition rates were calculated. Results：After five panning, antibAies obtained obvious enrichment. Phage ELISA and scFv ELISA results showed that positive reactions to TT ceils were 65% and 50% respectively. SDS-PAGE and Western blot showed that the relative molecular weight of the soluble scFv was about 28 kD. Cell ELISA results showed that the absorbance of TT cell group（0.41 ± 0.12） was significantly increased compared with that of SW 480 cells group （0.20 ± 0.03 ）, TEC cell group （ 0.13 ± 0.01 ）, PBS control group （0.07 ± 0.01）（P= 0.000）. MTF assay showed that when the concentration of scFv was 0.1,1,10 μmol/L, the inhibition rate between the two cell lines was statis-tically significant different （t=2.881, P=0.02; t=5

关 键 词：噬菌体抗体库 人源单链抗体 甲状腺髓样癌 筛选 

分 类 号：R392.11[医药卫生—免疫学] R446.62[医药卫生—基础医学]