关键词: 巨片形吸虫, 硫氧还蛋白过氧化物酶, 免疫学筛选, 原核表达, ELISA

Abstract:

Objective  To immunoscreen the gene encoding thioredoxin peroxidase（TPx） from a cDNA library made from adult Fasciola gigantica worms， clone and express the gene， and evaluate the immunodiagnostic value of TPx recombinant protein.  Methods  The λ ZAP cDNA library was immunoscreened with pooled serum of fascioliasis gigantica patients. The obtained positive clones were sequenced and analyzed by multiple sequence alignment. The full-length （rFgTPx） and N-termianal truncated（rFgTPx_nt） sequence of FgTPx was subcloned into prokaryotic plasmid pET28a（+） with a non-fusion expression technique， respectively. The recombinant proteins of rFgTPx and rFgTPx_nt were purified by His-bind affinity column（Ni-NTA）. rFgTPx and rFgTPx_nt were used in indirect ELISA to test the antibody response of the serum samples. Sera of 27 fascioliasis gigantica patients， 15 patients with schistosomaisis japonica， 15 clonorchiasis sinensis patients， and 32 healthy donors were tested by using the recombinant protein based ELISA.  Results  The TPx recombinant proteins were obtained through expression， purification and renaturation， the relative molecular mass of rFgTPx and rFgTPx_nt were Mr 30 000 and Mr 26 000， respectively. The total diagnostic coincidence rate， sensitivity and specificity of rFgTPx_nt-based ELISA was 87.6%（78/89）， 66.7%（18/27）， and 96.8%（60/62）， respectively. The cross reaction with Schistosoma japonicum and Clonorchis sinensis was 0 and 1/15 for rFgTPx_nt， respectively. Before and after treatment， A450 value of the serum samples from fascioliasis patients was 0.233±0.088 and 0.129±0.072， respectively（t=4.27， P<0.01）.  Conclusion  The gene encoding TPx is expressed in the prokaryotic expression system. The recombinant protein shows proper sensitivity and high specificity for the serodiagnosis of Fasciola gigantica infection.

Key words: Fasciola gigantica, Thioredoxin peroxidase, Immunoscreen, Expression, Immunodiagnosis