筛选日本血吸虫童虫早期诊断抗原的研究

Study on early diagnostic antigens of Schistosoma japonicum schistosomula

目的筛选日本血吸虫童虫早期诊断抗原及其表位。方法制备日本血吸虫可溶性童虫抗原(SSA),经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及Westernblot后分别与日本血吸虫感染后10、21、42天兔血清进行酶联免疫印迹试验(EITB),筛选出能被早期感染血清所识别的抗原组分。用日本血吸虫感染后21天血清从噬菌体12随机肽库中筛选、富集、纯化、扩增与早期感染兔血清有高亲和力的噬菌体克隆,经动物实验初步评估其对日本血吸虫感染的早期诊断效果。结果SDS-PAGE显示SSA的总条带数多于可溶性成虫抗原(SAWA),但大多为共同抗原。EITB结果显示SSA共出现12条早期反应抗原带,其中97kDa和47kDa抗原分子只能被感染后10天兔血清特异性识别;SAWA仅出现7条反应带,未显示只能被感染后10天兔血清识别的期特异性抗原分子。挑选出3个与感染后21天兔血清具有高亲和力的单克隆噬菌体,混合后分别用于检测感染日本血吸虫10、21d鼠血清中IgG特异性抗体,阳性检出率分别为48.8%和64.4%。结论SSA中的97kDa和44kDa抗原分子及3个噬菌体随机12肽抗原模拟表位可能具有日本血吸虫感染早期诊断价值。

Objective To screen the early diagnostic antigens of Schistosoma japonicum and their epitopes. Methods Schistosomulum and adult worm antigens were prepared and separated by SDS- PAGE. Then Western blotting（s） with the rabbit sera of 10, 21, and 42 days post-infection were performed, respectively, to detect some antigen bands recognized by early infected sera. Phage clones with high affinity to the specifical IgG antibobies were screened, enriched, purified and amplified from a random 12-peptides phage library by using the rabbit sera of 21 days post-infection. Positive phage clones were compared with SEA in early diagnostic value of infected mice by ELISA. Results SDS-PAGE showed that the protein bands of SSA were more than that of SAWA, and there were many similar protein bands between SSA and SAWA. SSA had 12 bands recognized by the early infected sera, including 97 kDa and 47 kDa antigens recognized only by the rabit sera of 10 days post-infection. SAWA only showed 7 bands, without any one recognized merely by the rabbit sera of 10 days post-infection. The mixture of 3 positive phage clones showed 48.8% and 64.4% of sensitivity in diagnosing mice of 10 days and 21 days post-infection, without any statistic differences compared with SEA. However, in healthy mice, these phage clones did not show any false positive result, but SEA showed 11 false positive results （11/45, 24.4%）. Conclusions Many common antigen molecules between SSA and SAWA have been found, and 97 kDa and 44 kDa antigen molecules of schistosomulum might be the potential early diagnostic antigens. Three random 12-peptides from positive phage clones recognized by the rabbit sera of 21 days post-infection might have early diagnositic value of schistosomiasis.