报道一条具有pH敏感解吸附特征的羟基磷灰石结合肽序列

Hydroxyapatite-binding Peptide Sequence with pH-sensitive Desorption Characteristics:A Report

目的评价基于噬菌体展示技术的羟基磷灰石结合肽的解吸附特征。方法采用成品羟基磷灰石(hydroxyapatite,HAP)作为结合底物,通过X射线衍射(X-ray diffraction,XRD)对其进行物相鉴定,提取噬菌体DNA测序读取展示肽的序列,扩增制备目标序列YSLHWGE(YSL)的单克隆储液,以肽库中随机挑选的序列VTDSKPK(VTD)为阴性对照;滴度测试测定投入回收比,免疫荧光法验证目标序列的标靶亲和力。参照标准淘选方案,在不同pH值(7.5、6.5、5.5、4.5)缓冲洗脱液中分析目标序列的解吸附特征。结果YSL和VTD的单克隆储液扩增后滴度均达到10;pfu/10μL,符合后续实验要求。测序结果得到目标序列和随机序列分别为YSL和VTD。通过重复3次滴度测试计算投入回收比发现,目标序列YSL与HAP的亲和度较高,投入回收比为随机肽VTD投入回收比的17.5倍。这种结合亲和力也通过免疫荧光实验得以验证,结果显示YSL组羟基磷灰石粉末被荧光点亮的面积比例和平均吸光度分别为(71.98±2.30)%和(0.10090±0.00544),均显著高于VTD组的(49.95±2.28)%(P<0.01)和(0.04637±0.00117)(P<0.01);发现在不同pH值环境中,YSL解吸附释放的噬菌体数量占原吸附总量的比例均高于随机肽VTD解吸附的比例(P<0.01)。结论羟基磷灰石结合肽序列YSLHWGE对结合底物羟基磷灰石有较好的亲和力且具有一定的随环境pH值变化的缓释能力。

Objective To evaluate the desorption characteristics of hydroxyapatite binding peptides(HABPs)based on phage display technology.Methods The commercial hydroxyapatite(HAP)was used as the binding substrate,and X-ray diffraction(XRD)was performed for phase identification.Phage DNA was extracted and sequenced to read the display peptide,and the monoclonal stock solution of the target sequence was prepared by amplification.The randomly selected sequence in the peptide library was used as a negative control.The input-recovery ratio of phage was analyzed through titer test and immunofluorescence method was used to verify the target-binding affinity of HABPs.With reference to the standard panning scheme,the desorption characteristics of the target sequence was analyzed in buffer eluents with different pH value(7.5,6.5,5.5,4.5).Results Both YSLHWGE(YSL)and VTDSKPK(VTD)of the monoclonal phage stock solutions were amplified to a titer of 10;pfu/10μL,which met the requirements of subsequent experiments.The sequencing results showed that the target sequence and the random sequence were YSL and VTD,respectively.The tier test was repeated three times.The target sequence YSL showed a high affinity with HAP,and input-recovery ratio was 17.5 times higher than that of random peptide(VTD).This binding affinity was also verified by immunofluorescence experiments.The results showed that the ratio of fluorescence area to hydroxyapatite powder area[(71.98±2.30)%]and the mean optical density(0.10090±0.00544)in the YSL group were significantly higher than those of the random sequence group(VTD),which were(49.95±2.28)%and(0.04637±0.00117)respectively(both P<0.01);the amounts of desorption phage released through YSL in buffers with different pH were higher than that released through random peptide(VTD)(P<0.01).Conclusion The hydroxyapatite binding peptide sequence YSLHWGE has a satisfying affinity for the binding substrate hydroxyapatite and has a sustained release ability that varies with the environmental pH.