刚地弓形虫抑制蛋白的原核表达与免疫反应性分析

发布时间：2019-02-21 16:05

【摘要】：目的克隆并表达刚地弓形虫抑制蛋白(Tg PRF)基因,并分析其免疫反应性。方法提取弓形虫RH株速殖子总RNA,根据Tg PRF的基因编码序列设计引物,RT-PCR扩增产物双酶切后连接入p ET30a(+)载体,重组质粒转化入大肠埃希菌(E.coli)DH5α,经双酶切、PCR和测序鉴定阳性菌落。p ET30a(+)-Tg PRF转化至E.coli BL21,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结合考马斯亮蓝染色检测表达产物。重组蛋白经镍亲和层析法纯化后,以兔抗弓形虫血清为一抗,蛋白质印迹(Western blotting)分析r Tg PRF蛋白的免疫反应性。结果 Tg PRF基因RT-PCR扩增产物约为492 bp。经双酶切、PCR和测序结果显示,重组质粒p ET30a(+)-Tg PRF构建成功。SDS-PAGE结果显示,经IPTG诱导获得相对分子质量(Mr)约35 000的可溶性重组蛋白。Western blotting结果显示,r Tg PRF蛋白能被兔抗弓形虫血清识别。结论本研究克隆的Tg PRF基因序列能在原核表达系统中表达,且具有免疫反应性。
[Abstract]:Objective to clone and express the (Tg PRF) gene of Toxoplasma gondii inhibitor and analyze its immunoreactivity. Methods the total RNA, of Toxoplasma gondii RH strain was extracted and primers were designed according to the coding sequence of Tg PRF. The amplified product of RT-PCR was digested by double enzyme and inserted into p ET30a () vector. The recombinant plasmid was transformed into E.coli DH5 伪 and digested by double enzyme. PCR and sequencing identified the positive colony. P ET30a ()-Tg PRF was transformed into E.coli BL21, and induced by isopropyl- 尾-D-galactoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with Coomassie brilliant blue staining was used to detect the expressed products. The recombinant protein was purified by nickel affinity chromatography and the rabbit anti-Toxoplasma gondii serum was used as the first antibody. Western blot (Western blotting) was used to analyze the immunoreactivity of r Tg PRF protein. Results the RT-PCR amplification product of Tg PRF gene was about 492 bp.. The results of double enzyme digestion, PCR and sequencing showed that the recombinant plasmid p ET30a ()-Tg PRF was successfully constructed. The results of SDS-PAGE showed that the soluble recombinant protein. Western blotting with relative molecular weight (Mr) of about 35 000 was obtained by IPTG induction. R Tg PRF protein can be recognized by rabbit antitoxoplasma serum. Conclusion the cloned Tg PRF gene sequence can be expressed in prokaryotic expression system and has immunoreactivity.
【作者单位】：徐州医学院解剖教研室;徐州医学院感染与免疫实验室;中国疾病预防控制中心寄生虫病预防控制所卫生部寄生虫病原与媒介生物学重点实验室世界卫生组织疟疾、血吸虫病和丝虫病合作中心;
【基金】：卫生部寄生虫病原与媒介生物学重点实验室开放课题(No.WSBKTKT201402) 徐州医学院科研课题(No.2014KJ07)~~
【分类号】：R382.5

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