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目的寻找曼氏迭宫绦虫裂头蚴感染早期诊断抗原。方法从野蛙体内收集曼氏迭宫绦虫裂头蚴,经口接种感染20只昆明小鼠,每只小鼠接种2条裂头蚴,感染后6~28d隔天尾静脉采血,采用ELISA检测抗裂头蚴抗体;应用双向电泳(two-dimensional electrophoresis,2-DE)与Western blot对裂头蚴可溶性抗原进行分析。结果裂头蚴感染后8d,小鼠血清特异抗体阳性率为10%(2/20),感染后14d特异抗体阳性率达100%。2-DE显示,裂头蚴可溶性抗原有255±6个蛋白点,分子质量主要集中在25~117ku,其中>44ku的蛋白点有171±9个,占总蛋白点数的67.1%;等电点(pI)范围为4~7,其中227个蛋白点集中在pI 5~6.5,占蛋白点总数的89.1%。Western blot检测有14个蛋白点可被感染曼氏迭宫绦虫裂头蚴14d的小鼠血清识别,其分子质量集中在82、45、36、34及25ku;1~14号蛋白点所对应的pI值分别为5.93、6.06、6.17、6.30、6.39、5.82、6.01、6.17、5.69、5.46、5.63、6.1、5.71、6.19。结论曼氏迭宫绦虫裂头蚴可溶性抗原分子质量主要集中在25~117ku,pI值集中在5.0~6.5。感染14d的小鼠血清识别的裂头蚴蛋白可作为裂头蚴感染早期诊断候选抗原。
Objective To search for the early diagnostic antigen of metacercariae infection of Metastasis mansoni. Methods Tissue of M. rutheni were collected from field frogs and 20 Kunming mice were inoculated by oral inoculation. Two mice were inoculated with 2 sporozoites. Blood samples were taken from the tail veins 6 to 28 days after infection. The anti-sporozoite antibodies were detected by ELISA. Two-dimensional electrophoresis (two-dimensional electrophoresis, 2-DE) and Western blot analysis of soluble antigen of cercariae. Results The positive rate of serum specific antibody was 10% (2/20) on day 8 post infection, and the positive rate of specific antibody was 100% on the 14th day after infection. 2-DE showed that there were 255 ± 6 protein spots in the soluble antigen of the spider laevae and the molecular mass was mainly concentrated in the range of 25 to 117 ku, with 171 ± 9 protein spots of> 44 ku and 67.1% of the total protein spots. The isoelectric point ) Ranged from 4 to 7, of which 227 protein spots concentrated in the pI5 ~ 6.5, accounting for 89.1% of the total number of spots. Western blot detection of 14 protein spots can be infected with Mann’s E.coli Metapharyptococcosis 14d mouse serum identification, the molecular mass concentrated in the 82,45,36,34 and 25ku; 1 ~ 14 protein spots corresponding to the pI value Respectively, 5.93,6.06,6.17,6.30,6.39,5.82,6.01,6.17,5.69,5.46,5.63,6.1,5.71,6.19. Conclusion The molecular mass of soluble antigen in the larvae of M. mansoni is mainly concentrated in the range of 25 ~ 117ku, with the pI value of 5.0 ~ 6.5. Filaggiolae identified by sera from mice infected for 14 days could be used as an early candidate antigen for diagnosis of sporozoite infection.