刚地弓形虫抑制蛋白的原核表达与免疫反应性分析  

Prokaryotic Expression and Immunoreactivity Analysis on Profilin of Toxoplasma gondii

作　　者：原飞[1] 刘转转[2] 张波[2] 曹建平[3] 郑葵阳[2] 王德广[1] 

机构地区：[1]徐州医学院解剖教研室,徐州221004 [2]徐州医学院感染与免疫实验室,徐州221004 [3]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,上海200025

出　　处：《中国寄生虫学与寄生虫病杂志》2015年第1期21-24,共4页Chinese Journal of Parasitology and Parasitic Diseases

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)~~

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。Objective To clone and express the profilin（PRF） gene of Toxoplasma gondii, and analyze the immunoreactivity. Methods Total RNA was extracted from tachyzoites of T. gondii RH strain. The coding region of TgPRF was amplified with a pair of specific primers. PCR product was digested with double restriction enzyme and ligated into pET30a（＋） vector. The recombinant pET30a（＋）-TgPRF plasmid was transformed into E. coli DH5α with positive clones confirmed by the double restriction enzyme digestion, PCR and sequencing. The correct plasmid was transformed into E. coli BL21 and induced by IPTG. The expressed proteins were purified with Ni-NTA affinity chromatography and analyzed by SDS-PAGE. Western blotting with rabbit anti-T. gondii serum was used to analyze its antigenicity. Results The product of RT-PCR was with 492 bp. pET30a-TgPRF was confirmed by the double restriction enzyme digestion, PCR and sequencing. SDS-PAGE analysis showed that the expressed product was a soluble protein with a relative molecular weight of 35 000. Western blotting assay revealed that rTgPRF was recognized by rabbit anti-T. gondii serum. Conclusion TgPRF gene has been expressed in prokaryotic expression system and shows immunoreactivity.

关 键 词：刚地弓形虫 抑制蛋白 基因克隆 原核表达 免疫反应性 

分 类 号：R382.5[医药卫生—医学寄生虫学]