双抗原夹心法基因工程Em18泡状棘球蚴快速诊断试剂盒的研制

发布时间：2018-05-17 17:45

本文选题：泡状棘球蚴病 + 基因克隆　；参考：《新疆医科大学》2014年硕士论文

【摘要】：目的：(1)通过基因工程技术克隆多房棘球绦虫Em18抗原。(2)将HRP标记在基因工程制备的Em18抗原上,利用双抗原夹心法,制备泡状棘球蚴快速诊断试剂盒。方法：(1)从多房棘球蚴中使用RNA提取试剂盒提取RNA,反转录成cDNA,通过克隆、构建、测序,利用DNAman软件对Em18基因特点进行分析并构建Em18核酸序列。用DNAman软件设计引物,分别在5’端3’端添加EcoRI和Xhol酶切位点,以pET28a/Em18原核表达质粒为模板,PCR扩增Em18基因片断,经酶切,转化入大肠埃希菌表达载体BL21,酶切、PCR及测序鉴定其插入序列的正确性。扩增出Em18抗原,测序鉴定序列正确。(2)经IPTG诱导表达rEm18,洗脱纯化,蔗糖浓缩,通过SDS-PAGE电泳和Western blot试验鉴定重组蛋白的表达水平。(3)采用HRP标记基因重组抗原。(4)棋盘法筛选抗原包被浓度,酶标抗原稀释倍数,待检抗体稀释倍数,利用双抗原夹心法制备Em18泡状棘球蚴快速诊断试剂盒。结果：(1)成功克隆并构建了pET28a/Em18原核表达质粒,经IPTG诱导,SDS-PAGE电泳检测表明pET28a/Em18重组蛋白得到成功表达,在相对分子量为50kD处有表达条带。(2) Western Blot分析显示rEm18蛋白能被多房棘球蚴病人阳性血清识别,具有良好的抗原性。(3)通过酶结合物的评价指标判断HRP成功标记抗原。将重组蛋白包被在酶联板上,按照已筛选好的条件进行组装试剂盒,选择待测样本,检测出试剂盒具有较高的敏感性和特异性。(4)通过单盲法利用配对χ2检验比较双抗原夹心法和斑点免疫胶体金法同金标准(手术)的敏感性和特异性。P0.05,差异有统计学意义。结论：运用基因克隆技术对Em18抗原进行克隆,并成功表达、鉴定、浓缩和纯化,成功使用HRP标记重组抗原,利用棋盘滴定法筛选出试剂盒各因素最适条件,通过ELISA双抗原夹心法原理组装试剂盒,采用配对χ2检验比较双抗原夹心法和斑点免疫胶体金法同金标准的敏感性和特异性。χ2=5.1,P0.05,差异有统计学意义。
[Abstract]:Objective: to clone Em18 antigen of Echinococcus multilocularis by genetic engineering. To label HRP on Em18 antigen prepared by genetic engineering, and to prepare a rapid diagnostic kit for hydatid alveolar echinococcus by double antigen sandwich method. Methods RNA extraction kit was used to extract RNAs from echinococcus multilocularis and reverse transcribed into cDNA.Through cloning, construction and sequencing, the characteristics of Em18 gene were analyzed by DNAman software and the nucleic acid sequence of Em18 was constructed. DNAman software was used to design primers, EcoRI and Xhol sites were added at the 3 'end of 5' end respectively. The pET28a/Em18 prokaryotic expression plasmid was used as template to amplify the Em18 gene fragment, and was digested by enzyme. The recombinant plasmid BL21 was transformed into Escherichia coli expression vector BL21, and its insertion sequence was confirmed by restriction endonuclease polymerase chain reaction (PCR) and sequencing. Em18 antigen was amplified and sequenced to identify the correct sequence. It was induced by IPTG to express rEm18, eluted and purified, and sucrose was concentrated. The expression level of recombinant protein was identified by SDS-PAGE electrophoresis and Western blot test. The antigen coating concentration, dilution multiple of enzyme labeled antigen, dilution multiple of antibody to be detected were screened by using HRP labeled gene recombinant antigen. 4) chessboard method was used to screen the concentration of antigen, the dilution multiple of enzyme labeled antigen, and the dilution multiple of antibody to be detected. The rapid diagnostic kit of Em18 alveolar echinococcus was prepared by double antigen sandwich method. Results the prokaryotic expression plasmid of pET28a/Em18 was cloned and constructed successfully. IPTG induced SDS-PAGE electrophoresis showed that the recombinant pET28a/Em18 protein was successfully expressed. Western Blot analysis showed that the rEm18 protein could be recognized by the positive sera of patients with multilocularis echinococcosis, and had good antigenicity. The recombinant protein was coated on the enzyme linked plate, and the kit was assembled according to the selected conditions, and the samples to be tested were selected. The sensitivity and specificity of double antigen sandwich assay and dot immune colloidal gold standard (P0.05) were compared by paired 蠂 2 test with single blind method. The difference was statistically significant. Conclusion: the Em18 antigen was cloned by gene cloning technique and successfully expressed, identified, concentrated and purified. The recombinant antigen labeled with HRP was successfully used. The optimum conditions of each factor of the kit were screened by chessboard titration. The kit was assembled by ELISA double antigen sandwich method, and the sensitivity and specificity of double antigen sandwich method and dot immunocolloid gold method were compared by paired 蠂 2 test. The difference was statistically significant.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R446.6;R532.32

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