噬菌体随机肽库中乳腺癌肿瘤标志物的筛选

Screening of breast cancer antigen from a phage display random peptide library

目的:利用噬菌体随机12肽库对乳腺癌患者血清进行差异性筛选,以获得乳腺癌特异性的肿瘤标志物。方法:以混合乳腺癌患者血清IgG作为筛选分子,对噬菌体随机12肽库进行亲和筛选;3轮淘选后,应用常规ELISA鉴定噬菌体单克隆;阳性克隆经测序后,选择与乳腺癌血清结合活性最高的多个噬菌体的共有小肽序列来人工合成其相应的寡核苷酸序列,并将其克隆至原核表达载体pGEX-4T-1中,经诱导表达及纯化GST-小肽融合蛋白后,应用常规ELISA检测20例乳腺癌患者和20例正常人与GST-小肽的结合反应。结果:3轮筛选没有明显的富集现象,但获得38个可与乳腺癌患者血清反应,而不与正常人血清反应的阳性克隆。经扩大乳腺癌患者血清样本例数进一步检测后对8个结合活性始终较高的阳性克隆进行测序发现部分克隆序列相同;将其中结合活性最高的小肽序列P15与GST蛋白进行融合表达,并分别检测其与20例乳腺癌患者和正常人血清的结合反应,结果提示患者血清与GST-P15的反应明显高于正常人,差异有统计学意义(P<0.01)。结论:筛选到的小肽P15可以特异结合乳腺癌患者血清中的抗体。该小肽为进一步寻找乳腺癌患者体内相应的肿瘤抗原奠定了一定的基础。

AIM：To isolate novel breast cancer antigen for developing cancer marker and vaccine,a phage display peptide library was screened with serum antibody from breast cancer patients.METHODS：A phage display peptide library was screened with serum IgG from six breast cancer patients,and then identified the positive clones by ELISA.According to the sequences of the positive clones,the synthesized oligonucleotides of one small peptide （P15） was cloned into PGEX-4T-1.Finally the fusion protein （GST-P15）,which was purified from E.coli.BL21,was checked its binding activity with two kinds of serum antibodies （20 breast cancer patients and 20 normal people） by ELISA.RESULTS：38 positive clones which specifically bound with serum IgG from breast cancer patients were screened.Some of these clones had the same DNA sequence.A small peptide （P15） and purified its fusion protein （GST-P15） was obtained successfully.ELISA assay showed GST-P15 had higher affinity with the serum antibody from breast cancer patients than with the normal serum and there was a significant difference （P〈0.01）.CONCLUSION：The peptide P15 can be recognized specifically by serum antibody from the breast cancer patients and they will be helpful for isolate new cancer antigen from breast cancer.