日本血吸虫琥珀酸脱氢酶铁硫蛋白重组抗原的制备及其诊断价值的研究

目的 探讨重组日本血吸虫琥珀酸脱氢酶铁硫蛋白(rSjSDISP)用于血吸虫病的诊断价值。方法 利用经原核表达并纯化的rSjSDISP, 建立基于重组抗原的快速酶联免疫吸附试验(F-ELISA),检测血吸虫感染不同时期的兔血清、健康人血清、寄生虫病患者血清(其中包括血吸虫病患者血清、肝吸虫病患者血清、肺吸虫病患者血清),比较rSjSDISP F-ELISA与SEA-IHA两者的早期诊断价值及对rSjSDISP F-ELISA诊断方法的诊断效能做出评价;并与SEA-IHA法和SEA F-ELISA法平行检测血吸虫病疫区335份待测人血清比较3者的敏感性和特异性。结果 成功构建pET32a/rSjSDISP重组质粒,当IPTG终浓度为0.1 mmol/L,28 ℃诱导6 h时SjSDISP重组蛋白表达量达到最大。rSjSDISP F-ELISA与SEA-IHA可区别出血吸虫感染28 d后的兔血清,两者检测结果差异无统计学意义;rSjSDISP F-ELISA敏感度为94.4,特异度为93.0,Youden指数为0.88。在诊断335份血吸虫病疫区现场血清中,该方法与SEA-IHA 、SEA F-ELISA具有相似的敏感性和特异性(P>0.05)。结论 rSjSDISP重组抗原可作为一种成分均一的优势诊断抗原,替代成分复杂的虫卵可溶性抗原(SEA)用于血吸虫病的诊断。rSjSDISP F-ELISA方法操作简便、快速,抗原制备易于标化,稳定性强,在血吸虫病诊断上具有较大的应用潜能。

Prokaryotic expression of succinate dehydrogenase iron-sulfur protein and evaluation of it on immunodiagnosis of schistosomiasis japonica

To evaluate the value of rSjSDISP on the diagnosis of schistosomiasis japonica by fast ELISA, the recombi- nant plasmid pET32a/rSjSDISP was constructed, induced and purified. For comparing the value of early diagnosis on schisto- somiasis japonica, we used methods of rSjSDISP F-ELISA and SEA-IHA to detect serum of rabbit in different period of infec- tion with Schistosorna japonicum. By detecting the serum from 31 patients of parasitic diseases （18 with schistosomiasis, 9 with lung flukes, and 18 with liver fluke）, we preliminary evaluated the diagnostic efficiency of the F-ELISA based on rSjS- DISP antigen. By detecting 335 samples of human serum from the epidemic field of schistosomiasis japonica, we compared the sensitivity and specificity among methods of IHA, SEA F-ELISA and rSjSDISP F-ELISA. It was found that F-ELISA using rSjSDISP as coating antigen could distinguish the sera of rabbits infected with schistosomiasis after 28 days and there was no significantly statistical difference in the test results between rSjSDISP F-ELISA and SEA-IHA. And the sensitivity, specifici- ty, and Youden ration of rSjSDISP F-ELISA method was 94.4, 93.0, and 0.88, respectively. There was no significantly sta- tistical difference in the results among rSjSDISP in F-ELISA, SEA in F-ELISA, and SEA in IHA （P~0.05）. It is evident that recombinant SjSDISP protein is a substitute for soluble egg antigen （SEA） as coating antigen in serodiagnosis of schistoso- miasis. The rSjSDISP constructed by F-ELISA would have great potential in schistosomiasis diagnosis for its simplicity, stabil-Funded by the National Scientific and Technological Support Projects （No. 2009BAI78B05）, the National Natural Science Foundation of China （No. 81271862）, and the Natural Science Foundation of Hunan Province （No. 12JJ3107）