摘要
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。
Objectives To prepare a recombinant form of the heat shock protein of Schistosoma japonicum [rSj HSP70（Grp 78）]and to characterize the antibody response to that protein in hosts and its immunological value in diagnosing schistosomiasis. Methods mRNA from adult S.japonicumworms was used to synthesize cDNA by reverse transcription.A DNA fragment encoding Sj HSP70（Grp 78）was amplified fromS.japonicumcDNA by PCR using apair of gene specific primers.The fragment was directionally subcloned into the expression plasmid pET28a（＋）to construct the recombinant plasmid Sj HSP70（Grp78）-pET28 a.The recombinant plasmid was transferred into E.coli BL21（DE3）competent cells by electric pulse,and rSj HSP70（Grp78）was expressed via induction with IPTG.Purified rSj HSP70（Grp78）protein was prepared using nickel affinity chromatography.An enzyme-linked immunosorbent assay（ELISA）and Western blotting were used to observe dynamic changes in the levels of IgG antibodies against rSj HSP70（Grp78）in hosts and the specificity of the antibody response to rSj HSP70（Grp78）in order to determine the value of the recombinant protein in early diagnosis and monitoring of therapeutic effectiveness.The clinical immunological diagnostic value of detecting IgG antibodies against rSj HSP70（Grp 78）was also investigated by comparing the sensitivity and specificity of ELISA with rSj HSP70（Grp 78）and ELISA with SEA. Results The gene encoding the mature peptide of S.japonicumheat shock protein HSP70（Grp 78）was successfully cloned and a purified recombinant Sj HSP70（Grp78）with a molecular weight of about 69 ku was prepared.The results of Western blotting indicated that the rSj HSP70（Grp 78）protein was recognized by sera from humans and mice with schistosomiasis but was not recognized by sera from humans or mice with clonorchiasis or paragonimiasis or by sera from healthy humans.This indicated that antibody immunoreaction is highly specific.There was a correlation between the extent of infection and the level of anti-rSj HSP70（Grp 78）IgG antibodies in animals infected with S.japonicum.IgG antibody levels rose along with the duration of infection.Those levels peaked 16-18 weeks post-infection and then declined slowly but still remaining high.In treatment groups,the level of anti-rSj HSP70（Grp 78）IgG antibodies initially peaked 2weeks post-treatment（8weeks after infection）and then temporarily declined.The level peaked a second time and then rapidly declined again.Anti-rSj HSP70（Grp 78）IgG antibodies were not detected in some animals 16 weeks after treatment（22weeks after infection）.In addition,anti-rSj HSP70（Grp78）IgG antibodies were detected in the sera of some infected animals 1week after infection.Sera from individuals with schistosomiasis,clonorchiasis,or paragonimiasis and sera from healthy individuals recognized rSj HSP70（Grp 78）with a sensitivity of 82.9%and SEA with a sensitivity of 95.7%according to ELISA.Sera recognized rSj HSP70（Grp 78）with a specificity of 94.3%and SEA with a specificity of 85.7%according to ELISA.Cross-reactivity with sera from healthy individuals was 0% for rSj HSP70（Grp 78）and 16.67% for SEA.Cross-reactivity with sera from individuals with clonorchiasis was with 5.7%for rSj HSP70（Grp78）and 14.29%for SEA.Cross-reactivity with sera from individuals with paragonimiasis was 5%for rSj HSP70（Grp78）and 65%for SEA. Conclusion Sj HSP70（Grp 78）is a highly specific antigen,so detection of specific IgG antibodies against rSj HSP70（Grp 78）can be used for supplemental diagnosis.This recombinant protein has potential for early diagnosis of schistosomiasis and evaluation of the effectiveness of treatments.
出处
《中国病原生物学杂志》
CSCD
北大核心
2014年第8期699-705,共7页
Journal of Pathogen Biology
基金
国家重大科技专项(No.2012ZX10004220)
国家自然科学基金项目(No.81201316,30972581,30471515)
江苏省自然科学基金项目(No.BK2012544,BK2008110)
关键词
血吸虫
日本
热休克蛋白70
表达
抗体反应
免疫诊断
Schistosoma japonicum
heat shock protein 70
expression
antibody response
immunological diagnosis
