摘要
目的利用基因工程方法表达细粒棘球绦虫抗原B重组蛋白(rAgB),并分析其免疫反应性。方法将rAgB基因片段插入原核表达载体pET41a(+)中,转化大肠埃希菌BL21(DE3)菌株,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,获得重组蛋白rAgB-GST。用谷胱甘肽琼脂糖树脂亲和层析柱(GST-sepharose4B)纯化,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析重组蛋白的表达情况。用蛋白质印迹(Western blotting)分析其免疫反应性,并用免疫胶体金包虫诊断试剂盒作为对照。结果PCR、双酶切及DNA测序结果均显示重组质粒pET41a-rAgB构建成功。SDS-PAGE结果表明,重组蛋白rAgB-GST的相对分子质量(Mr)为40800,纯化蛋白含量为78.4%。Westernblotting分析结果显示,重组蛋白rAgB-GST检测细粒棘球蚴病和多房棘球蚴病患者血清的阳性率分别为79.2%(95/120)和51.1%(23/45),但与卫氏并殖吸虫病患者、华支睾吸虫病患者血清以及健康人血清反应均为阴性。rAgB-GST的敏感性和特异性分别为79.2%(95/120)和81.0%(98/121),均略高于免疫胶体金棘球蚴病诊断试剂盒的敏感性(72.8%,75/103)和特异性(76.9%,30/39)。结论重组rAgB-GST蛋白可被细粒棘球蚴病和多房棘球蚴病患者血清识别,有较好的免疫反应性。
Objective To express the recombinant antigen B（rAgB）of Echinococcus granulosus（Eg）and investigate its immunoreactivity. Methods The rAgB gene fragments were inserted into pET41a（＋）prokaryotic vector. The recombinant plasmid was transformed into E. coli BL21（DE3）and followed by expression of the protein induced by isopropyl β-D-1-thiogalactopyranoside（IPTG）. The protein was purified with sepharose 4B by affinity chromatography, and tested by SDS-PAGE electrophoresis. Its immunoreactivity was examined by Western blotting, and a rapid diagnosis kit for human echinococcosis was used as control. Results The constructed recombinant plasmid pET41a-rAgB was identified by PCR, digestion with restriction enzyme and sequencing. The recombinant rAgB-GST was about Mr 40 800 with a purity of 78.4%. Western blotting showed that the positive rate of rAgB-GST reacting with sera of cystic echinococcosis（CE）, alveolar echinococcosis（AE）, paragonimiasis westermani and clonorchiasis sinensis patients, and healthy persons is 79.2%（95/120）, 51.1%（23/45）, 0（0/32）, 0（0/20）, and 0（0/24）, respectively. Its overall sensitivity and specificity were 79.2%（95/120） and 81.0%（98/121）, respectively, slightly higher than the sensitivity（72.8%, 75/103） and specificity （76.9%, 30/39）of the rapid diagnosis kit for human echinococcosis. Conclusion The rAgB-GST recombinant protein is recognized by the sera of CE and AE patients, showing a proper immunoreactivity.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2009年第2期107-110,共4页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家高技术研究发展计划(863计划)(No.2007AA02Z411)
新疆维吾尔自治区重点实验室开放课题(No.XJDX0202-2006-05
XJDX0202-2003-05)~~
关键词
细粒棘球绦虫
重组抗原B
免疫诊断
Echinococcus granulosus
Recombinant antigen B
Immunodiagnosis
