甲状腺未分化癌人源单链抗体制备及初步鉴定  

Preparation and identification of human single chain variable fragments antibodies against anaplastic thyroid carcinoma

作　　者：刘倩[1] 刘琼[1] 李文波[1] 许璐[1] 周静[1] 刘影[1] 庞华[1] 

机构地区：[1]重庆医科大学附属第一医院核医学科,400016

出　　处：《免疫学杂志》2016年第11期981-985,共5页Immunological Journal

基　　金：国家自然科学基金(81071171);重庆市科技计划面上项目(CSCT2013JCYJA10072);国家临床重点专科建设项目(国卫办医函[2013]544号)

摘　　要：目的从大容量甲状腺未分化癌(anaplastic thyroid carcinoma,ATC)人源单链抗体(single chain variable fragments,sc Fv)库中筛选特异性抗ATC单链抗体并进行生物特性鉴定。方法对该抗体库进行4轮筛选后,挑取抗ATC阳性克隆感染E.coli HB2151;并用异丙基-β-D-硫代吡喃半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导该sc Fv抗体可溶性表达,ELISA法检测其可溶性表达;经HiTrap^(TM) Anti E-tag亲和柱层析纯化抗体;细胞ELISA鉴定可溶性抗体与细胞结合的特异性,分别设置TT细胞、ARO细胞、人肝癌Hep G2细胞及PBS空白对照组,用酶标仪检测各组在450 nm处的吸光度。SDS-PAGE法及Western blot检测抗体的表达和相对分子质量;流式细胞术观察细胞凋亡情况。结果经4轮筛选后抗体实现了显著富集,并得到了纯化抗体。ELISA结果表明筛选出的抗体与甲状腺未分化癌细胞能够特异性结合。SDS-PAGE和Western blot检测结果显示ATC抗体的相对分子质量约为2.9×10~4。流式细胞术结果显示ARO细胞经sc Fv特异性作用后细胞明显凋亡。结论成功获得抗ATC特异性人源单链抗体,鉴定结果显示抗体活性较好。This study was designed to prepare and identify the human single chain variable fragments antibodies（scFv） against anaplastic thyroid carcinoma（ATC） from human phage display library.Thyroid epithelial cell line（TEC cells） and ATC cell line（ARO cell） were used as antigen,and then positive clone against ATC was selected by four rounds of ＇ adsorption-elution-amplification＇ from the human phage display library.E.coli HB2151 was infected by the positive clone to induce the expression of soluble scFv by IPTG.ELISA was used to detect the expression of scFv,while HiTrap^TM Anti E-tag column was used for purification of soluble scFv.TEC cell,ARO cell,human liver cancer cell line（Hep G2 cell） and PBS blank control groups were set up to identify the specific of soluble scFv,and the absorbance value of A450nm was detected by microplate reader respectively.The expression and relative molecular mass of soluble scFv were tested by SDS-PAGE and Western blot.The ARO cells treated with scFv or PBS control group were set up to observe the apoptosis via flow cytometry.Data showed that the antibodies finished enrichment after four rounds screening,and the purification and expression of soluble antibody scFv was completed.Phage ELISA results showed that the screened antibody could specifically bind with ARO cells.SDS-PAGE and Western blot indicated that the relative molecular mass of soluble scFv was about 29 kD;obvious apoptosis was found in ARO cells treated with scFv by flow cytometry.Taken together,a human scFv against ATC,with well antibody activity,is prepared successfully in this study.

关 键 词：噬菌体 人源单链抗体 甲状腺未分化癌 鉴定 

分 类 号：R736.1[医药卫生—肿瘤]