细粒棘球绦虫抗原B重组蛋白的免疫反应性

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-sepharose 4B）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40 800,纯化蛋白含量为78.4%。Western blotting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。

Immunoreactivity of the Recombinant Protein of Echinococcus granulosus Antigen B

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.