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目的制备重组日本血吸虫中国大陆株热休克蛋白[rSj HSP70(Grp78)],检测其抗体反应特征及用于血吸虫病免疫诊断的价值。方法以日本血吸虫中国大陆株成虫mRNA为模板逆转录合成cDNA,采用基因特异性引物,通过PCR技术扩增编码Sj HSP70(Grp78)成熟肽的基因片段,并将该片段插入到表达质粒pET28a(+)中,构建表达质粒HSP70(Grp78)-pET28a。将重组质粒转化大肠埃希菌BL21(DE3)感受态细胞,经IPTG诱导表达后采用镍亲和层析法制备纯化的可溶性rSj HSP70(Grp78)。采用酶联免疫吸附试验(ELISA)及Western blot检测rSj HSP70(Grp78)的抗体反应特异性及抗rSj HSP70(Grp78)IgG在血吸虫感染小鼠血清中动态变化,以分析其早期诊断与疗效考核价值,并比较rSj HSP70(Grp78)-ELISA与SEA-ELISA检测血吸虫感染者血清抗体IgG的敏感性与特异性。结果编码Sj SP70(Grp78)成熟肽基因片段克隆成功,并获得纯化的分子质量单位约69ku的重组表达Sj HSP70(Grp78)蛋白。Western blot显示Sj HSP70(Grp78)蛋白能被日本血吸虫感染的小鼠及人血清识别,但不与华支睾吸虫病和卫氏并殖吸虫病患者血清及健康人、鼠血清反应。感染小鼠血清抗Sj HSP70(Grp78)IgG水平与血吸虫感染度呈正相关,并随着感染时间延长呈增强趋势,在感染后16~18周达到峰值,随后开始回调,但始终维持在较高水平。治疗组小鼠经药物治疗后第2周(感染后第8周),体内抗体水平迅速上升并达到高峰,经过短暂下降后再次上升,出现第二个高峰,随后逐渐回调,至治疗16周(感染后第22周),部分小鼠抗体水平已接近阴性阈值。部分个体在血吸虫感染后1周即可检测到血清抗rSj HSP70(Grp78)IgG。分别以rSj HSP70(Grp78)和SEA血吸虫、华支睾吸虫及卫氏并殖吸虫感染者血清及健康人血清,敏感性分别为82.9%和95.7%,特异性分别为94.3%和85.7%,与健康人血清的交叉反应率分别为0和16.67%,与华支睾吸虫感染者血清的交叉反应率分别为5.7%和14.29%;与卫氏并殖吸虫感染者血清的交叉反应率分别为5%和65%。结论 Sj HSP70(Grp78)具有高度的抗原特异性,以此为抗原采用ELISA检测抗Sj HSP70(Grp78)IgG具有日本血吸虫病辅助诊断价值,并具有一定的早期诊断与疗效考核潜能。
Objective To prepare heat shock protein [rSj HSP70 (Grp78)] of Chinese Schistosoma japonicum from Chinese mainland and test its antibody response and its value in immunodiagnosis of schistosomiasis. Methods The cDNA of Schistosoma japonicum adult strain mRNA was used as a template to reverse transcribe cDNA. Gene-specific primers were used to amplify the gene fragment encoding the mature peptide of Sj HSP70 (Grp78) by PCR. The fragment was inserted into expression vector pET28a (+ ), The expression plasmid HSP70 (Grp78) -pET28a was constructed. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells. After induced by IPTG, purified soluble rSj HSP70 (Grp78) was prepared by nickel affinity chromatography. The antibody specificity of rSj HSP70 (Grp78) and the anti-rSj HSP70 (Grp78) IgG in sera of mice infected with Schistosoma japonicum were detected by enzyme linked immunosorbent assay (ELISA) and Western blot. The results of early diagnosis and therapeutic evaluation , And compared the sensitivity and specificity of rSj HSP70 (Grp78) -ELISA and SEA-ELISA to detect serum antibody IgG in schistosomiasis-infected persons. Results The fragment encoding the mature peptide of SjSP70 (Grp78) was successfully cloned and the purified recombinant Sj HSP70 (Grp78) protein of about 69ku was obtained. Western blot showed that the Sj HSP70 (Grp78) protein was recognized by both Schistosoma japonicum-infected mice and human sera, but not with serum from clonorchiasis and Paragonimiasis patients and healthy human and mouse sera. The level of anti-Sj HSP70 (Grp78) IgG in sera of infected mice was positively correlated with the infection rate of Schistosoma japonicum, and increased with the prolongation of infection time. It peaked at 16-18 weeks after infection and then started to call back, but remained at a relatively high level . After treatment for 2 weeks (8 weeks after infection), the antibody level of the mice in the treatment group rose rapidly and reached a peak, and then rose again after a brief decrease. The second peak appeared and then gradually returned to 16 weeks after the treatment 22 weeks after infection), some mouse antibody levels are close to the negative threshold. Some individuals detect serum anti-rSj HSP70 (Grp78) IgG one week after infection with schistosomiasis. The sensitivity and specificity of rSj HSP70 (Grp78), SEA, Clonorchis sinensis and Paragonimus westermani were 82.9% and 95.7%, respectively, with specificity of 94.3% and 85.7% respectively The cross-reaction rates of serum from healthy volunteers were 0 and 16.67%, respectively. The rates of cross-reaction with sera of Clonorchiasis sinensis were 5.7% and 14.29%, respectively. The cross-reactivity with Serum of Paragonimus westermani infection was 5 % And 65%. Conclusion Sj HSP70 (Grp78) is highly antigen specific, which is an antigen for the detection of anti-Sj HSP70 (Grp78) IgG. It has the diagnostic value of schistosomiasis japonica and has certain potential for early diagnosis and therapeutic evaluation.