肝癌血清肿瘤标志物适配体的筛选及活性鉴定

目的:肝癌血清中含有已知和未知的标志物,可以作为SELEX筛选的靶标,获得一组已知和未知肝癌血清标志物的适配体,为肝癌早期诊断提供新的分子生物学检测方法。方法：收集50例经诊断证实为原发性肝癌患者的血清,以及50例体检无异常的正常人血清,并分别等比例混合制备成肝癌混合血清和正常人混合血清,作为筛选的靶标分子,磁珠作为分离载体。先将磁珠-正常人血清复合物与ssDNA文库结合,取上清再与磁珠-肝癌血清结合,经过洗脱、分离与肝癌血清结合的特异性ssDNA,并进行扩增,链霉亲和素磁珠法制备次级ssDNA,进行9轮筛选,将9轮筛选获得的饱和文库与PMD18-T载体连接进行转化、挑选单克隆团送上海生工进行测序,同时利用流式细胞术测定肝癌血清肿瘤标志物适配体的亲和力(Kd值)。结果：经9轮筛选,成功分离出200个核酸序列,其中序列不同的有10个。结论：特异性检测表明,筛选得到的肝癌血清肿瘤标志物适配体与肝癌血清的结合解离常数均在纳摩尔级水平,其中Seq-1、Seq-16、Seq-17、Seq-56、Seq-72号适配体能高特异性结合肝癌血清,与正常人血清不结合,再通过200例肝癌血清和200例正常人血清进一步鉴定5条肝癌血清肿瘤标志物适配体检出肝癌的阳性率,阳性检出率达91%以上,为肝癌的早期诊断提供了新方法。

Screening and identification of liver cancer serum tumor markers

Objective: The serum of liver cancer contains known and unknown markers, which can be used as SELEX screening target to obtain a set of known and unknown aptamers of liver cancer serum markers, for the early diagnosis of liver cancer to provide new molecular biological detection method. Methods: Serum samples from 50 patients with primary hepatocellular carcinoma and 50 normal serum samples without abnormal physical examination were collected and mixed in equal proportions to prepare mixed sera of hepatocellular carcinoma and normal subjects. The serum was used as the target molecule for screening. Magnetic beads-normal human serum complex was combined with ssDNA library. The supernatant was then combined with magnetic beads-liver cancer serum to elute and isolate the specific ssDNA binding to serum of liver cancer. Twelve rounds of ssDNA were screened by the method of streptavidin biotin method. Twelve rounds of the saturated library were transformed into PMD18-T vector. The single clones were selected and sequenced by Shanghai Biosystems. Affinity determination of serum tumor marker aptamers in patients with Flow Cytometry. RESULTS: After 12 rounds of screening, 200 nucleic acid sequences were successfully isolated, of which 10 sequences were different. Conclusion: Specificity test showed that the binding dissociation constants of serum tumor marker aptamers and hepatocarcinoma serum were all in nanomolar level. Seq-1, Seq-16, Seq-17, Seq-56, Seq-72 aptamers can be highly specific binding to serum of liver cancer, and normal serum does not combine. The positive rate of serum tumor marker aptamer was detected in 200 cases of liver cancer serum and 200 cases of normal human serum,positive detection rate of 91% or more, which provided a new method for the early diagnosis of liver cancer.