【摘要】 目的利用噬菌体肽库技术筛选华支睾吸虫童虫模拟抗原表位,观察影响筛选效率和噬菌体滴度的关键因素。方法收集华支睾吸虫感染童虫期大鼠血清,用正辛酸硫酸铵法提纯多克隆抗体,用提纯的多克隆抗体对噬菌体十二肽库进行3轮筛选。随机挑取噬菌体克隆,用ELISA法检测其特异性。结果经3轮免疫学筛选获得19个噬菌体克隆,ELISA测定有17个能被纯化的华支睾吸虫(童虫期)感染大鼠血清抗体识别。结论利用噬菌体肽库技术可以筛选出华支睾吸虫童虫模拟抗原表位,靶分子浓度以及噬菌体液的稀释度等因素是技术关键。更多还原

【Abstract】 Objective Clonorchis sinensis DNA was detected in gallbladder stones from patients with cholecystolithiasis using TaqMan-based real-time PCR.Methods Gallbladder stones from 40 patients with cholecystolithiasis who underwent endoscopic gallbladder-preserving cholelithotomy from March to April 2012 were studied.The stones were first analyzed using Fourier infrared spectrometry and were then ground for microscopic examination.Specific primers and TaqMan probes targeting the cytochrome c oxidase gene of C.sinensis were designed and synthesized,and then the DNA of gallbladder stones was extracted and the C.sinensis gene was detected with real-time PCR.Results The 40 gallbladder stones were divided into three types based on Fourier infrared spectrometry: bile pigment stones(25 patients),cholesterol stones(6 patients) and mixed stones(9 patients).Parasite eggs were found in 23 stones,including 19 bile pigment stones and 4 mixed stones(positivity: 57.5%).No eggs were found in cholesterol stones.Positivity for infection according to real-time PCR was 65.0%(26/40).C.sinensis DNA was detected in all 23 stones that were positive for eggs and 3 stones that were negative for eggs.Conclusion C.sinensis DNA was detected in gallbladder stones using real-time PCR,which was superior to microscopic examination and appears to be the most effective means of assessing C.sinensis infection status.This research presents the molecular biological evidence for the formation of gallbladder stones induced by C.sinensis infection and it also provides a new approach to detecting C.sinensis infection.This has a great significance in diagnosing patients with cholecystolithiasis and in facilitating treatment of the condition and prevention of its recurrence following surgery.更多还原