摘要
目的：纳米金生物传感器是一种基于局域表面等离子体共振(Localized surface plasmon resonance, LSPR)的非标记生物传感器。大量研究证实LSPR生物传感器能辅助疾病的诊断。本文应用噬菌体随机12肽库筛选结核分枝杆菌抗原模拟肽,构建基于结核分枝杆菌抗原模拟肽的纳米金生物传感器,评价其诊断结核病的能力,为结核病(Tuberculosis, TB)的诊断提供新的理论依据和技术支持。
方法：①以结核患者血清IgG为靶分子,3轮淘选噬菌体随机12肽库,筛选与结核患者血清IgG具有亲和性的噬菌体克隆,经酶联免疫吸附试验(Enzyme linked immunosorbent assay, ELISA)鉴定得到阳性克隆；提取阳性噬菌体克隆ssDNA,进行测序分析,获得结核分枝杆菌抗原模拟肽序列；②选择高频出现的2种模拟短肽,进行人工合成,ELISA分析合成短肽及对应的阳性噬菌体克隆诊断结核病的灵敏度和特异性；③种子生长法制备金纳米棒,经十一巯基十一烷酸(MUA)、1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)的化学修饰,将合成短肽包被金纳米棒,构建纳米金生物传感器,评价其诊断结核病的能力。
结果：(1)经3轮淘筛噬菌体得到有效富集,12个阳性噬菌体克隆经测序分析获得6种短肽序列,其中有8个克隆中的每4个各编码同一条肽段；蛋白质BLAST比对未发现有同源性一致的序列。(2)人工合成高频出现的2条12肽(T1和T2),经ELISA初步鉴定,其敏感性和特异性分别为66.1%,35.7%和100.0%,100.0%。(3)种子生长法制备得到分散度良好的金纳米棒,将T1包被到金纳米棒,对112份血清样品进行血清学检测,敏感性和特异性分别为57.1%和83.9%,成功构建了有诊断价值的纳米金生物传感器。
结论：(1)结核患者血清IgG筛选噬菌体随机12肽库获得了对抗结核抗体高度亲和的噬菌体展示短肽；(2)种子生长法制备获得分散度良好的金纳米棒；(3)基于合成短肽的纳米金生物传感器对结核病有着较好的诊断价值。
Objective:Au NRs biosensor is a kind of biosensor based on Localized surface plasmon resonance (LSPR) technology of gold nanorods. It has been proved that the LSPR biosensor can be used to aid disease diagnosis without using any labels. The mimic peptides of Mycobacterium tuberculosis antigen were immunoscreened from phage displayed 12-mer peptide library, and the diagnostic potential of the Au NRs biosensor based on the mimic peptide of Mycobacterium tuberculosis antigen was explored. Study on the novel diagnosis methods will provide theoretical basis and technical support for diagnosis of TB.
Methods:①Specific IgG was isolated from sera of patients with tuberculosis and used as the target to immunoscreen its short affinity peptide from a phage random peptide library of 12 amino-acid residues displayed as a fusion to proteinⅢof filamentous phage M13. The positive phage clones were obtained after three rounds of biopanning, their specificity were detected by enzyme linked immunosorbent assay (ELISA), followed by single stranded DNA extraction and sequence analysis.②The high frequent mimic peptides were synthesized based on their amino acid sequences. Sensitivity and specificity of the synthetic short peptides and corresponding positive phage clones were detected by ELISA.③The gold nanorods (Au NRs) were synthesized with seed- mediated method. After chemical modification with 11-mercaptoundecanoic acid (MUA), l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), these Au NRs were conjugated with the synthetic short peptides so as to develop a Au NRs biosensor, then the diagnostic potential of the biosensor for TB was detected by seroanalysis.
Results:(1) After 3 rounds of immunoscreening, the radio of output to input increase to 5.18×10-4, which means that the enrichment was effective. Six kinds of animo acid sequence were obtained from twelve positive phage clones, there were not homologous sequence in NCBI when compared with protein BLAST. (2) The sensitivity and specificity of the synthetic short peptides (T1 and T2) were 66.1%,35.7%and 100.0%,100.0%respectively. (3) Au NRs with good dispersity were prepared with seed-mediated method. T1 was conjugated to Au NRs successfully to develop the biosensor. Seroanalysis of 112 sera tests showed that the sensitivity and specificity of the biosensor were 57.1% and 83.9%respectively.
Conclusions:(1) The antigen-mimic peptide was successfully obtained from 12 random phage peptide library and the peptides can be recognized by tuberculosis patients'polyclonal antibodies. (2) Au NRs with good dispersity were prepared with seed-mediated method. (3) Au NRs biosensor based on the mimic peptide can be used in the diagnosis ofTB.
引文
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