日本血吸虫核糖体蛋白S4基因的克隆、表达、纯化及免疫诊断价值的初步研究  

Cloning, expression and purification of Schistosoma japonicum ribosomal protein S4 as well as the preliminary study of the diagnostic value of the recombinant protein

作　　者：高冬梅[1] 汪世平[1] 余路新[1] 何卓[1] 陈秀春[1] 

机构地区：[1]中南大学湘雅医学院血吸虫病免疫与传播控制重点实验室,长沙410078

出　　处：《中华预防医学杂志》2010年第6期500-504,共5页Chinese Journal of Preventive Medicine

基　　金：基金项目：国家科技支撑计划（2009BA178805）;973计划（2007CB513108）;中南大学研究生学位论文创新基金（2009bsxt016）

摘　　要：目的 体外重组日本血吸虫核糖体蛋白S4（SjRPS4）,并评价其在日本血吸虫病免疫诊断中的应用价值.方法 从日本血吸虫尾蚴cDNA文库获得了SjRPS4蛋白相关基因,将该段基因克隆入pQE30表达载体并转化到大肠杆菌M15中,异丙基-β-D-硫代半乳糖苷（IPTG）诱导融合蛋白表达.融合蛋白经Ni2＋-NTA树脂柱进行亲和层析纯化,并通过十二烷基磺酸钠一聚丙烯酰胺凝胶电泳（SDS-PAGE）鉴定蛋白反应,通过Western blot和ELISA对其免疫原性进行鉴定.结果 成功构建了pQE30/SjRPS4重组菌.重组菌诱导培养后,SDS-PAGE电泳分析可见在相对分子质量约30×103处有SjRPS4融合蛋白的表达,经纯化获得纯度达90%以上的表达蛋白.Western blot检测结果表明该重组蛋白能被日本血吸虫病患者血清识别.用ELISA法检测疑似血吸虫病患者血清,敏感性为90.91%（70/77）,特异性为92.59%（25/27）,SjRPS4重组表达蛋白阳性率为67.30%（70/104）,与肺吸虫病患者血清无交叉反应.结论 成功克隆表达了SjRPS4蛋白,初步证实了SjRPS4在诊断血吸虫病中具有良好的应用价值.Objective To express and purify Schistosomajaponicum ribosomal protein S4（SjRPS4） in Escherichia coli,and asses its value in immunodiagnosis of Schistosomiasis iapanica.Methods Gene fragment of sjBRS4 was amplified by screening the cercaria cDNA library of Schistosoma iaponicum.The target gene was cloned into the expressive vector pQE30 and transformated into E coli M15.The recombinant protein expression was induced by isopropyhhio-β-D-galactoside（IPTG）.This fusion protein was purified by Ni2＋-NTA chromatography and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDSPAGE）,Western blot and ELISA.Results The plasmid pQE30/sjRPS4 was constructed successfully and expressed a sjRPS4 fusion protein in E coli as showing a single special band on SDS-PAGE gel at Mr 30×103 position.It reached a purity of above 90% after purification.The Western blot result confirmed that the recombinant protein could specifically react with the serum samples from patients of schistosomiasis.Detecting the serum of Schistosomiasis japonica patients by ELISA,the sensitivity and specificity of the ELISA method were 90.91%（70/77） and 92.59%（25/27）,the positive rate of recombinant protein expression was 67.30%（70/104）.There was no cross-reaction with paragonimiaais patients＇serum.Conclusion Protein SjRPS4 was successfully cloned and expressed,and it was confirmed that SjRPS4 antibodies were valuable in the diagnosis of Schistosomiasis iaponica.

关 键 词：血吸虫 日本 重组蛋白质类 核糖体蛋白质类 免疫学试验 

分 类 号：R532.21[医药卫生—内科学]