旋毛虫肠道期虫体早期特异性诊断抗原的筛选与鉴定

The excretory-secretory (ES) antigens from Trichinella spiralis muscle larvae are widely used for the serodiagnosis of trichinellosis and they are recommended as diagnostic antigens by the International Commission on Trichinellosis (ICT), but anti-Trichinella antibodies could not be detected until 3-4 weeks after infection. There is the 2-3 week window period between Trichinella infection and anti-Trichinella antibody positivity. The patients with severe infection may died within 1-2 weeks after onset of the disease if they are not diagnosed and treated at early stage of infection. Our previous study showed that compared with muscle larval ES antigens, the detection time of anti-Trichinella antibodies could be advanced from 12 days after infection to 8-10 days after infection by using ES antigens from IIL and adult worms of T. spiralis , the specificity was increased from 89.31% to 96.86-98.11%. This project will change the traditional thinking of screening diagnostic antigens from muscle larvae. The early specific diagnostic antigens will be screened from the ES and surface proteins of IIL and adults by immunoproteomics, molecularly cloned, expressed and purified. The antigenicity, genus/species or stage specificity and tissue location of the early diagnostic antigens will be investigated by Western blot, q-PCR and immunofluorescent assay (IFA). The sensitivity and specificity of recombinant proteins are evaluated to detect anti-Trichinella antibodies in serum of mice infected with different dose of T. spiralis larvae. The project will lay the foundation for shortening the window period of serodiagnosis of trichinellosis, the early diagnosis and rapid screening of patients with trichinellosis while the public accident of outbreak of trichinellosis occurs.

目前国内外用于旋毛虫病血清学诊断的肌幼虫ES抗原在感染后3~4周才能检出抗旋毛虫抗体，在旋毛虫感染与抗体阳性之间具有2~3周的“窗口期”，而重症患者若未早期诊治可在发病后1~2周死亡。我们前期应用旋毛虫肠道感染性幼虫（IIL）和成虫ES抗原与肌幼虫ES抗原相比，可将检测抗旋毛虫抗体的时间从感染后12d提前至感染后8d~10d，特异性从89.31%提高到96.86%~98.11%。本项目将改变从肌幼虫筛选诊断抗原的传统思路，应用免疫蛋白组学从IIL和成虫 ES及表面蛋白中筛选早期特异性诊断抗原，进行克隆表达及纯化后，应用Western blot、q-PCR及IFA观察其抗原性、属/种和期特异性及虫体定位，观察重组蛋白检测旋毛虫感染小鼠后不同时间抗旋毛虫抗体的敏感性与特异性。该项目将为缩短旋毛虫病血清学诊断的“窗口期”、出现旋毛虫病暴发的突发公共事件时进行早期诊断与快速筛查等奠定基础。

目前，国内外用于旋毛虫病血清学诊断的肌幼虫ES抗原在感染后3~4周才能检出抗旋毛虫抗体，在旋毛虫感染与抗体阳性之间具有2~3周的“窗口期”，而重症患者若未早期诊治可在发病后1~2周死亡。本项目改变了从肌幼虫筛选诊断抗原的传统思路，应用免疫蛋白组学技术，从感染后6h 的肠道感染性幼虫（IIL）和3d成虫的表面蛋白与ES蛋白中筛选出了具有早期诊断潜能的抗原分子（丝氨酸蛋白酶TsSP与弹性蛋白酶TsEla等），克隆表达及纯化后，Western blot、IFA及ELISA等检测结果显示，TsSP与TsEla均为分泌蛋白，rTsSP与rTsEla具有良好的抗原性和旋毛虫属特异性，主要定位在虫体皮层与杆状体等部位。rTsSP-ELISA可检测旋毛虫感染不同动物宿主（小鼠、大鼠、兔、猪）血清中的抗旋毛虫IgG；检测100-500条旋毛虫感染小鼠后10~16天的血清抗体IgG阳性率达100%；对4种成囊型旋毛虫（旋毛虫、乡土旋毛虫、布氏旋毛虫、纳氏旋毛虫）感染小鼠血清特异性IgG检出率为80%~100%，对非成囊的伪旋毛虫感染小鼠特异性IgG检出率仅为41.67%；表明rTsSP可用于4种成囊型旋毛虫感染的诊断。rTsEla-ELISA检测小鼠感染旋毛虫后10-14天特异性IgM与IgG阳性率达100%，表明rTsEla检测特异性IgM与 IgG可用于旋毛虫感染的早期诊断。本项目建立的rTsSP-ELISA与rTsEla-ELISA检测感染后19天早期旋毛虫病人血清特异性IgG的敏感性分别为95.24%与92.31%，特异性分别为99.53%与99.10%。表明这两种重组抗原对早期旋毛虫病的血清学诊断具有良好的敏感性与特异性，可替代当前普遍应用的肌幼虫ES抗原用于旋毛虫病的诊断，可缩短旋毛虫病血清学诊断的“窗口期”，当出现旋毛虫病暴发的突发公共事件时可进行快速筛查与早期诊断。

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