日本血吸虫虫卵排泄分泌物中具有诊断价值的抗原分子的初步鉴定  

Screening of antigen molecules with the ability to identify the excretory-secretory proteins of Schistosoma japonicum eggs

作　　者：宋丽君[1] 殷旭仁[1] 王玠[1] 肖迪[2] 张建中[2] 高玒[3] 周伟[1] 许永良[1] 余传信[1] Li-jun Song;Xu-ren Yin;Jie Wang;Di Xiao;Jian-Zhong Zhang;Hong Gao;Yong-Liang Xu;Chuan-xin Yu(Jiangsu Instituteof Parasitic Diseases,Wuxi,Jiangsu Province 214064;National Institute for Commu-nicable DiseaseControl and Prevention,Chinese Center for Disease Control and Prevention;Nanjing Drum Tower Hospital.)

机构地区：[1]江苏省血吸虫病防治研究所,江苏无锡214064 [2]中国疾病预防控制中心传染病预防控制所 [3]南京鼓楼医院

出　　处：《中国病原生物学杂志》2020年第3期311-316,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金青年项目(No.81802035);江苏省卫生计生委面上项目(No.H201635);无锡市卫生计生委青年项目(No.Q201656);江南大学公共卫生研究中心面上项目(No.JUPH201812)。

摘　　要：目的鉴定日本血吸虫虫卵分泌排泄物(ESA)中有血吸虫病诊断价值蛋白分子,为建立新的高敏感性和特异性的日本血吸虫病诊断方法奠定基础。方法日本血吸虫尾蚴感染家兔后42d,解剖家兔,取肝脏,匀浆后收集虫卵,置于PBS中,4℃过夜,收集上清,制备虫卵排泄分泌物。以虫卵排泄分泌物包被酶标板,采用ELISA检测不同感染度、不同感染时间小鼠血清抗体滴度。同时以虫卵ESAELISA分别检测血吸虫病患者血清、健康人血清和弓形虫病患者血清,观察虫卵ESA用于血吸虫病诊断的敏感性和特异性及交叉反应性。通过一维电泳免疫印迹、二维电泳免疫印迹和飞行质谱分析,鉴定虫卵ESA中具有诊断潜能的蛋白分子。结果ELISA检测结果显示血吸虫感染小鼠血清中抗虫卵ESA抗体IgG水平随着感染时间的延长而升高,且与血吸虫感染度呈正相关,在感染早期即检出相应抗体。虫卵ESA检测血吸虫病患者血清的敏感性为97.2%,检测健康人血清的特异性为95.2%,与弓形虫病患者血清的交叉反应性为25.0%。一维电泳免疫印迹试验显示血吸虫感染兔血清可识别170×103、150×103、130×103、100×103、72×103、70×103、30×103ku左右的ESA蛋白;二维电泳免疫印迹试验显示血吸虫感染兔血清可识别8个蛋白斑点,质谱分析其中1个蛋白斑点为日本血吸虫主要的虫卵蛋白P40。结论日本血吸虫虫卵排泄分泌抗原具有一定的血吸虫病诊断与早期诊断价值,虫卵的主要蛋白P40可能是一个具有诊断潜能的抗原分子。Objective To determine the potential ability of proteins to identify the excretory and secretory antigens(ESA)fromSchistosoma japonicum eggs in order to establish a new method of diagnosing schistosomiasis with a high level of sensitivity and specificity.Methods Rabbits were sacrificed 42 days after infection with S.japonicumcercariae.The liver was homogenized,and the eggs were collected and placed in PBS overnight at 4℃.The supernatant consisted of the ESA proteins of eggs.Enzyme-labeled plates were coated with the ESA of S.japonicumeggs.An enzymelinked immunosorbent assay(ELISA)was used to detect antibody titers in serum of mice with different degrees of infection and different times of infection.The sensitivity and specificity with which ESA from eggs were able to identify schistosomiasis were determined by testing sera from patients with schistosomiasis,healthy persons,and patients with toxoplasmosis.A one-dimensional electrophoretic immunoblotting assay,two-dimensional electrophoresis,and mass spectrometry were used to analyze spots recognized by the ESA fromS.japonicumeggs in infected rabbit serum.ResultsELISA indicated that the level of ESA antibody IgG in the serum of mice infected with S.japonicumincreased with the infection time and was correlated with the degree of infection with S.japonicum.When infected with 5 or 10 cercariae,the anti-ESA antibody titer in 80.0%of the mice serum exceeded the positive threshold;when infected with 15 or more cercariae,the serum antibody titer in 100%of mice exceeded the positive threshold.In the second week after infection with 20 cercariae,the serum anti-ESA antibody titer reached the positive threshold in 16.7%of the mice;in the third and fourth weeks,it exceeded the positive threshold in 50.0%of the mice;and in the fifth week it exceeded the positive threshold in 100.0%of the mice.A positive reaction to the antibody began in the early stage of infection.ESA had a sensitivity of 97.2%in identifying patients with schistosomiasis.ESA had a specificity of 95.2%when di

关 键 词：虫卵排泄分泌抗原 诊断 蛋白组学分析 

分 类 号：R3[医药卫生—基础医学]