十二肽库中与结缔组织生长因子特异结合噬菌体的筛选

目的:从随机十二肽库中筛选与结缔组织生长因子(CTGF)特异结合的噬菌体克隆。方法:用基因重组方法制备硫氧化还原蛋白(TrxA)以及硫氧化还原蛋白-结缔组织生长因子(TrxA-CTGF)融合蛋白;噬菌体肽库经TrxA预吸附后,与TrxA-CTGF结合,逐步增加选择压力,经过4轮亲和筛选后随机挑取10个克隆,提取单链DNA后测序;用直接和间接ELISA方法检测噬菌体阳性克隆与CTGF结合的特异性。结果:10个克隆中8个DNA序列完全一致(810A),另有2个有不同序列(810B,810C);3个阳性克隆用直接和间接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<0.05)。结论:成功获得与CTGF特异结合的噬菌体克隆,为CTGF小分子抑制剂的研究打下基础。

Screening of phages specifically combined with connective tissue growth factor from a random 12-peptide library

Aim:To screen phage clones specifically combined with connective tissue growth factor(CTGF)from a random 12-peptide library.Methods:Thioredoxin(TrxA)and TrxA-CTGF were prepared by gene recombination.A random 12-peptide phage display library was pre-absorbed with TrxA and then screened with TrxA-CTGF.Phage clones were obtained after 4 cycles biopanning by gradually increased selective pressure.The single strand DNAs of ten randomly selected clones were abstracted and sequenced.The specificity of phages combined with CTGF was identified by direct and indirect-ELISA.Results:Eight clones shared a consensus sequence(810A),and two different sequences(810B,810C)were also obtained.Three positive clones can specifically combine with recombinant CTGF.The reaction of positive clones with CTGF was significantly increased compared with those of TrxA and negative control(P<0.05).Conclusion:Phage clones specifically combined with CTGF have been obtained successfully,and it is valuable for the study of CTGF inhibitor of small molecule.