人源结缔组织生长因子单链可变区抗体片段的筛选  

Screening of humanized single-chain variable fragments against connective tissue growth factor

作　　者：吴国球[1] 刘乃丰[2] 赵桂琴[2] 范小波[3] 张臣[1] 王明虹[2] 

机构地区：[1]东南大学附属中大医院临床检验中心 [2]东南大学临床医学院心血管研究所 [3]中国药科大学生命科学与技术学院,南京210009

出　　处：《中国药科大学学报》2007年第5期464-468,共5页Journal of China Pharmaceutical University

基　　金：国家自然科学基金资助项目(No.30070300)~~

摘　　要：目的:从人源单链可变区片段(ScFv)文库中筛选与结缔组织生长因子(CTGF)特异结合的噬菌体克隆,获得ScFv氨基酸序列。方法:以重组硫氧化还原蛋白-结缔组织生长因子(TrxA-CTGF)融合蛋白为靶目标,用差异吸附法,逐步增加选择压力,经过4轮亲和筛选后随机挑取8个克隆,提取DNA后测序;测序阳性的DNA转化E.coliHB2151,IPTG诱导后用间接ELISA方法检测培养上清单链抗体的活性。MTT法测定单链抗体对CTGF促人近曲肾小管上皮HK-2细胞增殖作用的影响。结果:从8个克隆中获得7个完全一致的DNA序列(W0616);另有1个克隆丢失了重链,且轻链框架区(FR)和互补决定区(CDR)序列与W0616不一致;阳性克隆经诱导后,培养上清用间接ELISA试验证实能与CTGF发生特异性结合,与TrxA及阴性对照比较差异显著(P<0.001)。同时,该单链抗体片段能明显抑制CTGF引起的HK-2细胞的增殖。结论:成功获得与CTGF特异结合的人源单链抗体可变区序列,为CTGF抑制剂的研究奠定基础。Aim： To screen phage clones specifically combined with connective tissue growth factor（CTGF） and to obtain the amino acid sequence of humanized single-chain variable fragment（ScFv）. Methods： TrxA-CTGF as a target, a ScFv library was screened by the differential absorption method. Phage clones were obtained after 4 cycles of biopanning through a gradual increase in the selective pressure. The DNAs of eight clones randomly picked up were abstracted and sequenced, and the DNAs of positive clone were transferred into E. coli HB2151. The specificity of ScFv in the medium supematant after inductign with IPTG combined with CTGF was determined by indirect-ELISA. The influence of ScFv on the proliferation of human proximal tubular cells （HK-2） caused by CTGF was investigated by MTT method. Results： Seven clones shared a consensus sequence（W0616）. Another clone lost its heavy-chain variable domain and the sequences of frame regions（FR） as well as complementarity-determining regions（CDR） of lightchain variable domain were not in accordance with those of W0616. ScFv in the medium supematant after being induction can be specially combined with recombinant TrxA-CTGF. The reaction with TrxA-CTGF were significantly increased compared with those of TrxA and negative control（ P 〈 0.001 ）. ScFv could significantly inhibit cell pmliferation of HK-2 cells caused by CTGF. Conclusion： The amino acid sequence of humanized ScFv specially combined with CTGF was obtained successfully, and the findings could lay foundations in the study of CTGF inhibitor.

关 键 词：结缔组织生长因子 噬菌体展示 人源单链可变区片段 

分 类 号：R392[医药卫生—免疫学]