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目的免疫筛选巨片形吸虫成虫c DNA表达文库,并克隆和重组表达巨片形吸虫硫氧还蛋白过氧化物酶(TPx),初步评价其免疫诊断价值。方法用巨片形吸虫病患者的混合血清免疫学筛选巨片形吸虫成虫λZAP c DNA表达文库,取阳性噬菌体进行克隆、测序及序列比对分析。将TPx基因全长片段和N段截短片段分别克隆至原核表达质粒p ET28a(+)中,用组氨酸标签亲和纯化柱(Ni-NTA树脂)纯化2种重组蛋白r Fg TPx和r Fg TPx_nt(N端截短型)。以间接ELISA法,检测27例巨片形吸虫病患者治疗前后的血清,15例日本血吸虫病和15例华支睾吸虫病的患者血清,32位健康人血清,评价重组蛋白的免疫诊断价值。结果克隆的巨片形吸虫TPx基因,经原核表达、纯化并复性,获得其可溶性重组蛋白r Fg TPx和r Fg TPx_nt,相对分子质量(Mr)分别为30 000和26 000。以重组蛋白r Fg TPx_nt为检测抗原的ELISA检测的敏感性为66.7%(18/27),特异性为96.8%(60/62),总符合率为87.6%(78/89),与日本血吸虫病和华支睾吸虫病患者血清的交叉反应率分别为0和1/15。巨片形吸虫病患者治疗前后血清的A450值分别为0.233±0.088和0.129±0.072,差异有统计学意义(t=4.27,P<0.01)。结论筛选并克隆了巨片形吸虫TPx抗原基因,实现原核表达,并证明该重组蛋白作为人体巨片形吸虫病的免疫诊断抗原具有较好的诊断意义,亦有可能具有早期诊断和疗效考核价值。
OBJECTIVE: To immunopurify the c DNA expression library of adult giant formidoglus, and clone and recombinantly express the thioredoxin peroxidase (TPx) of the giant form schistosome, so as to evaluate its immunological diagnostic value. Methods The cDNA library of A. zhabaciens λZAP c was immunologically screened by mixed serum of patients with giant fistula. The positive phage were cloned, sequenced and sequenced. The full-length and the truncated fragments of TPx gene were cloned into the prokaryotic expression plasmid pET28a (+) respectively. Two kinds of recombinant protein r Fg TPx and r were purified by using histidine tag affinity purification column (Ni-NTA resin) Fg TPx_nt (N-terminal truncation). Serum samples of 27 patients with giant fasciola asiatica, 15 patients with Schistosoma japonicum and 15 cases of Clonorchis sinensis, and 32 healthy volunteers were detected by indirect ELISA. The immunological diagnostic value of the recombinant protein was evaluated. Results The cloned Toxoplasma gondii TPx gene was prokaryotic expressed, purified and renatured. The soluble recombinant proteins rFg TPx and rFg TPx_nt were obtained. The relative molecular masses (Mr) were 30 000 and 26 000, respectively. The ELISA detection of recombinant protein r Fg TPx_nt was 66.7% (18/27), 96.8% (60/62) and 87.6% (78/89), respectively, The cross-reactivity rates of patients with Clonorchiasis sinensis were 0 and 1/15, respectively. The A450 values of serum of patients with giant fasciolae were 0.233 ± 0.088 and 0.129 ± 0.072 respectively before treatment, the difference was statistically significant (t = 4.27, P <0.01). Conclusion The gene of TPx antigen of giant toxoplasma gondii was screened and cloned. The prokaryotic expression of the gene was confirmed and it was proved that the recombinant protein could be used as an immunodiagnostic antigen for human giant-belliculosis. It may also have the value of early diagnosis and therapeutic evaluation .