基因组DNA羟甲基化的光电化学检测及其对禽白血病指示作用研究

The early diagnosis of avian leukosis is one of the important challenges for poultry farming. Through the search and detection of avian leukosis biomarkers in peripheral blood of chicken serum, we can achieve the early diagnosis of avian leukosis with the advantages of non-invasion and non-vulnus. In this project, a series of photoelectrochemical biosensors with signal-on model will be fabricated for detecting the expression level of 5-hydroxymethylcytosine (5hmC) in genomic DNA of chick serum with high sensitivity and selectivity. The strategy for the fabrication of these biosensors are mainly based on MoS2 nanosheets as photoactive materials, and the specific recognition and capture of 5hmC can be achieved according to the covalent chemical reaction of hydroxymethyl group of 5hmC, and the signal amplification can be attained based on enzymatic in situ generation of electron donor technique and nanomaterial amplification technique. The expression level of 5hmC in genomic DNA of chicken fibroblast, tissue and serum infected with avian leukosis virus will be detected with high sensitivity and selectivity. According to the detection results, the application feasibility of the expression level change of 5hmC in genomic DNA of chicken serum on the early diagnose of avian leukosis will be discussed and assayed. Moreover, the effect of anti-virus on 5hmC expression level will be further investigated in chicken fibroblast, various tissues, and serum infected with avian leukosis virus. Through the implementation of this project, we can not only provide new detection method for 5hmC, understand the indicative effect of the expression level change of 5hmC on avian leukosis, but also propose new strategy and convenient detection technique for the early diagnosis of avian leukosis.

禽白血病的早期检测是养禽业面临的主要难题之一。通过筛选合适的禽白血病生物标志物，可实现该病的早期检测。本项目拟以硫化钼纳米片层复合材料作为光活性材料，利用5-羟甲基胞嘧啶（5hmC）上羟甲基的共价反应实现对5hmC的特异识别，结合酶催化原位生成电子供体和纳米粒子信号扩增技术，构建信号开模式的光电化学生物传感器，实现对5hmC的灵敏和特异检测。以感染J亚型禽白血病病毒的鸡胚成纤维细胞和雏鸡为研究对象，运用光电化学技术检测感染与未感染病毒的鸡胚成纤维细胞、雏鸡多种病变组织与对应的正常组织、感染与未感染病毒雏鸡的血清基因组DNA中5hmC表达水平变化，明确基因组DNA中5hmC表达量变化对禽白血病的指示作用，以及所构建方法在禽白血病早期诊断中的应用性。通过本项目的研究，不仅为5hmC检测提供新的方法，了解5hmC表达变化对禽白血病的指示作用，还为禽白血病的早期诊断提供新的思路和策略。

通过筛选合适的禽白血病生物标志物，可实现该病的早期检测。本项目以硫化钼、硫化钨、MXene等材料为基础制备复合纳米材料，改进材料的光电活性，如MoS2-g-C3N4/ZnO、MoS2/g-C3N4/TiO2、WS2/PDA、CdS/MXene/ZnSnO3、Bi2S3/MXene、Bi4TaO8Cl/PTCA。运用扫描电子显微镜、透射电子显微镜、原子力显微镜、紫外可见漫反射、X射线光电子能谱、紫外-可见光谱、红外光谱、拉曼光谱等技术对材料进行了表征。运用电化学阻抗技术、荧光技术、紫外可见漫反射技术、电化学莫特肖特基技术、价带-X射线光电子能谱技术等研究了复合材料光电活性提高的机理，计算了纳米材料的能带宽度、导电电位和价带电位，构建了复合材料中电子迁移路径。. 基于制备的纳米复合材料，结合5hmC上羟甲基在酶催化作用下与糖基、巯基化合物的共价反应，以及酶催化原位生成电子供体、纳米材料负载光电信号扩增分子、纳米材料能带匹配扩增技术等信号扩增技术，构建了多种光电化学生物传感器，实现对5hmC的各种衍生物的灵敏性和特异性检测，如5hmdC、5hmdCTP。构建的传感器具有稳定性和重现性。. 研究了5hmC对J亚型禽白血病的指示作用，具体探究了感染时间、病毒浓度对雏鸡成纤维细胞、雏鸡肝组织和雏鸡血清基因组DNA中5hmC含量的影响。结果表明，随着感染时间延长、病毒浓度增大，雏鸡成纤维细胞、雏鸡肝组织和雏鸡血清基因组DNA中5hmC含量均降低，说明5hmC对感染J亚型禽白血病病毒具有一定的指示作用。研究了马立克氏病毒（CVI998、RMD5）对鸡胚成纤维细胞基因组DNA中5hmC的影响，结果表明，马立克氏病毒也抑制雏鸡成纤维细胞基因组DNA中5hmC的含量。此外，研究了重金属、抗生素、外源植物激素、全氟化合物对水稻、小麦和玉米组织中5hmC表达的影响。