日本血吸虫天冬酰胺酰基内肽酶编码基因表达及诊断应用

Expression of gene encoding mature protease of Schistosoma japonicum asparaginyl endopeptidase (Sj32) and diagnostic application of recombinant protein

目的克隆和表达日本血吸虫天冬酰胺酰基内肽酶(Sj32),探索该重组抗原在动物血吸虫病诊断方面的应用潜能。方法用PCR法从Sj32前体编码基因中克隆编码成熟体的基因片段,以pET-28(a)为表达载体,用大肠埃希菌表达,制备重组抗原(rSj32);应用ELISA方法检测待检血清,并比较rSj32、日本血吸虫可溶性虫卵抗原(SEA)以及重组诊断抗原rSj23对人工感染兔、小鼠及水牛血吸虫病的检测效果。结果成功表达了Sj32成熟体,表达产物rSj32的分子量为41 kDa,可用H is柱纯化,得率约为68.8 mg/L培养物。用rSj32检测人工感染兔、小鼠和水牛血清以及对照兔、小鼠和水牛血清,特异性分别为100.0%、96.7%和96.9%,敏感性分别为88.9%、85.0%和71.8%,该抗原对牛血清的敏感性略低于SEA和rSj23,其余检测结果三者相比无显著差异。结论rSj32在诊断动物血吸虫病方面具有研究和应用价值。

Objective To express the gene encoding mature protease of Schistosoma japonicum asparaginyl endopeptidase（Sj32） and evaluate the potential of the recombinant protein rSj32 in diagnosis of domestic animal schistosomiasis.Methods The DNA fragment encoding mature protease of Schistosoma japonicum asparaginyl endopeptidase was cloned with PCR from pET-28（a）/Sj32,and a recombinant plasmid was previously constructed in the laboratory,which contained the ORF of the gene encoding the pro-enzyme Sj32.The amplified DNA fragment was subcloned into pET-28a（＋） and the recombinant plasmid was transformed into E.coli BL21（DE3） to express the mature protease of Sj32.Then the recombinant antigen（rSj32） was used in ELISA assay to diagnose schistosomiasis of mice,rabbits and water buffalo artificially infected.The detection effects of soluble Schistosoma japonicum egg antigen（SEA）,rSj32 and the recombinant 23 KDa membrane protein were compared.Results The recombinant antigen rSj32 with a molecular weight 41 KDa was successfully produced in E.coli BL21（DE3） and was purified with His Column with a yield of 25 mg/L E.coli culture.By using rSj32 as coating antigen in ELISA assay to detect the specific antibody in artificially infected mice,rabbits and buffalo,the sensitivities were 88.9%,85.0% and 71.8%,respectively,the specificities were 100%,96.7% and 96.9%,respectively.There were no significant differences among the detection results of rSj32,SEA and rSj23.Conclusion rSj32 is a promising antigen for serological diagnosis of domestic animal schistosomiasis.