巨片形吸虫硫氧还蛋白过氧化物酶的克

发布时间：2018-06-21 18:43

本文选题：巨片形吸虫 + 硫氧还蛋白过氧化物酶　；参考：《中国寄生虫学与寄生虫病杂志》2015年02期

【摘要】：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,P0.01)。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。
[Abstract]:Objective to screen the c DNA expression library of adults of paragonimiasis monocytogenes and to clone and recombine the expression of thioredoxin peroxidase (TPxX) of paragonimiasis megacephala, and to evaluate its immunological diagnostic value. Methods Immunology was used to screen 位 ZAP c DNA expression library of adult adults of paragonimiasis macrophylla. The positive phage was cloned, sequenced and sequenced. The full length and N fragment of TPX gene were cloned into prokaryotic expression plasmid pET28a (), and two recombinant proteins, rFg TPx and rFg TPxntttttttttir, were purified by affinity purification column (Ni-NTA resin) with histidine label. The sera of 27 patients with paragonimiasis japonicum and 15 patients with clonorchiasis sinensis before and after treatment were detected by indirect Elisa. The serum samples of 32 healthy people were collected from 15 patients with clonorchiasis sinensis and 15 patients with clonorchiasis sinensis. The immunological diagnostic value of recombinant protein was evaluated. Results the recombinant protein rFg TPx and rFg TPxntwere obtained by prokaryotic expression, purification and renaturation. The relative molecular weight of TPx gene was 30 000 and 26 000, respectively. The sensitivity of Elisa using recombinant protein rFg TPxNT as antigen was 66.7 / 27, the specificity was 96.860 / 62 / 62, the total coincidence rate was 87.6% / 89 / 89, and the cross reaction rates with serum of schistosomiasis japonicum and clonorchiasis sinensis were 0 and 1 / 15, respectively. The serum A450 levels were 0.233 卤0.088 and 0.129 卤0.072 in patients with paragonimiasis before and after treatment, respectively. The difference was statistically significant. Conclusion the TPx antigen gene was screened and cloned to realize prokaryotic expression, and the recombinant protein was proved to be a good diagnostic antigen for human paragonimiasis macrophylla. May also have the early diagnosis and the curative effect appraisal value.
【作者单位】：中国疾病预防控制中心寄生虫病预防控制所卫生部寄生虫病原与媒介生物学重点实验室世界卫生组织疟疾血吸虫病和丝虫病合作中心;大理州血吸虫病防治研究所;宾川县血吸虫病防治站;
【基金】：国家科技重大专项(No.2012ZX10004-220)~~
【分类号】：R532.2

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